WO2001062927A2 - Polypeptides and corresponding polynucleotides for diagnostics and therapeutics - Google Patents

Abstract

The present invention provides purified human polynucleotides for diagnostics and therapeutics (dithp). Also encompassed are the polypeptides (DITIIP) encoded by dithp. The invention also provides for the use of dithp, or complements, oligonucleotides, or fragments thereof in diagnostic assays. The invention further provides for vectors and host cells containing dithp for the expression of DITHP. The invention additionally provides for the use of isolated and purified DITHP to induce antibodies and to screen libraries of compounds and the use of anti-DITHP antibodies in diagnostic assays. Also provided are microarrays containing dithp and methods of use.

Description

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   MOLECULES FOR DIAGNOSTICS AND THERAPEUTICS 
TECHNICAL FIELD 
The present invention relates to human molecules and to the use of these sequences in the diagnosis, study, prevention, and treatment of diseases associated with, as well as effects of exogenous compounds on, the expression of human molecules. 



   BACKGROUND OF THE INVENTION 
The human genome is comprised of thousands of genes, many encoding gene products that function in the maintenance and growth of the various cells and tissues in the body. Aberrant expression or mutations in these genes and their products is the cause of, or is associated with, a variety of human diseases such as cancer and other cell proliferative disorders,   autoimmunelinflammatory   disorders, infections, developmental disorders, endocrine disorders, metabolic disorders, neurological disorders, gastrointestinal disorders, transport disorders, and connective tissue disorders. The identification of these genes and their products is the basis of an ever-expanding effort to find markers for early detection of diseases, and targets for their prevention and treatment.

   Therefore, these genes and their products are useful as diagnostics and therapeutics. These genes may encode, for example, enzyme molecules, molecules associated with growth and development, biochemical pathway molecules, extracellular information transmission molecules, receptor molecules, intracellular signaling molecules, membrane transport molecules, protein modification and maintenance molecules, nucleic acid synthesis and modification molecules, adhesion molecules, antigen recognition molecules, secreted and extracellular matrix molecules, cytoskeletal molecules, ribosomal molecules, electron transfer associated molecules, transcription factor molecules, chromatin molecules, cell membrane molecules, and organelle associated molecules. 



   For example, cancer represents a type of cell proliferative disorder that affects nearly every tissue in the body. A wide variety of molecules, either aberrantly expressed or mutated, cari be the cause of, or involved with, various cancers because tissue growth involves complex and ordered patterns of cell proliferation, cell differentiation, and apoptosis. Cell proliferation must be regulated to maintain both the number of cells and their spatial organization. This regulation depends upon the appropriate expression of proteins which control cell cycle progression in response to extracellular signals such as growth factors and other mitogens, and intracellular cues such as DNA damage or nutrient starvation.

   Molecules which directly or indirectly modulate cell cycle progression fall into several categories, including growth factors and their receptors, second messenger and signal transduction proteins, oncogene products, tumor-suppressor proteins, and mitosis-promoting factors. 

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   Aberrant expression or mutations in any of these gene products can result in cell proliferative disorders such as cancer. Oncogenes are genes generally derived from normal genes that, through abnormal expression or mutation, can effect the transformation of a normal cell to a malignant one (oncogenesis). 



   Oncoproteins, encoded by oncogenes, can affect cell proliferation in a variety of ways and include growth factors, growth factor receptors, intracellular signal transducers, nuclear transcription factors, and cell-cycle control proteins. In contrast, tumor-suppressor genes are involved in inhibiting cell proliferation. Mutations which cause reduced function or loss of function in tumor-suppressor genes result in aberrant cell proliferation and cancer. Although many different genes and their products have been found to be associated with cell proliferative disorders such as cancer, many more may exist that are yet to be discovered. 



   DNA-based arrays can provide a simple way to explore the expression of a single   polymorphic   gene or a large number of genes. When the expression of a single gene is explored, DNA-based arrays are employed to detect the expression of specific gene variants. For example, a   p53   tumor suppressor gene array is used to determine whether individuals are carrying mutations that predispose them to cancer.   A   cytochrome p450 gene array is useful to determine whether individuals have one of a number of specific mutations that could result in increased drug metabolism, drug resistance or drug toxicity. 



   DNA-based array technology is especially relevant for the rapid screening of expression of a large number of genes. There is a growing awareness that gene expression is affected in a global fashion. A genetic predisposition, disease or therapeutic treatment may affect, directly or indirectly, the expression of a large number of genes. In some cases the interactions may be expected, such as when the genes are part of the same signaling pathway. In other cases, such as when the genes participate in separate signaling pathways, the interactions may be totally unexpected. Therefore, DNA-based arrays can be used to investigate how genetic predisposition, disease, or therapeutic treatment affects the expression of a large number of genes. 



   Enzyme Molecules 
The cellular processes of biogenesis and biodegradation involve a number of key enzyme classes including   oxidoreductases,   transferases, hydrolases, lyases, isomerases, and ligases. These' enzyme classes are each comprised of numerous substrate-specific enzymes having precise and well regulated functions. These enzymes function by facilitating metabolic processes such as glycolysis, the tricarboxylic cycle, and fatty acid metabolism ; synthesis or degradation of amino acids, steroids, phospholipids, alcohols, etc. ; regulation of cell signalling, proliferation,   inflamation,   apoptosis, etc., and through catalyzing critical steps in DNA replication and repair, and the process of translation. 



   Oxidoreductases 

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Many pathways of biogenesis and biodegradation require oxidoreductase (dehydrogenase or reductase) activity, coupled to the reduction or oxidation of a donor or acceptor cofactor. Potential cofactors include cytochromes, oxygen, disulfide, iron-sulfur proteins, flavin adenine dinucleotide (FAD), and the   nicotinamide   adenine dinucleotides NAD and NADP   (Newsholme,   E. A. and A. R. 



  Leech (1983) Biochemistry for the Medical Sciences, John Wiley and Sons,   Chichester,   U. K., pp. 



  779-793). Reductase activity catalyzes the transfer of electrons between substrate (s) and cofactor (s) with concurrent oxidation of the cofactor. The reverse dehydrogenase reaction catalyzes the reduction of a cofactor and consequent oxidation of the substrate. Oxidoreductase enzymes are a broad superfamily of proteins that catalyze numerous reactions in all cells of organisms ranging from bacteria to plants to humans. These reactions include metabolism of sugar, certain detoxification reactions in the liver, and the synthesis or degradation of fatty acids, amino acids,   glucocorticoids,   estrogens, androgens, and prostaglandins. Different family. members are named according to the direction in which their reactions are typically catalyzed ; thus they may be referred to as   oxidoreductases,   oxidases, reductases, or dehydrogenases.

   In addition, family members often have distinct cellular localizations, including the cytosol, the plasma membrane, mitochondrial inner or outer membrane, and peroxisomes. 



   Short-chain alcohol dehydrogenases (SCADs) are a family of dehydrogenases that only share   15%   to 30% sequence identity, with similarity predominantly in the coenzyme binding domain and the substrate binding domain. In addition to the well-known role in detoxification of ethanol, SCADs are also involved in synthesis and degradation of fatty acids, steroids, and some prostaglandins, and are therefore implicated in a variety of disorders such as lipid storage disease, myopathy, SCAD deficiency, and certain genetic disorders. For example, retinol dehydrogenase is a SCAD-family member (Simon, A. et al. (1995) J. Biol. Chem. 270 : 1107-1112) that converts retinol to retinal, the precursor of retinoic acid.

   Retinoic acid, a regulator of differentiation and apoptosis, has been shown to down-regulate genes involved in cell proliferation and inflammation (Chai, X. et al. (1995) J. Biol. 



  Chem. 270 : 3900-3904). In addition, retinol dehydrogenase has been linked to hereditary eye diseases such as autosomal recessive childhood-onset severe retinal dystrophy (Simon, A. et   al ;   (1996) Genomics 36 : 424-430). 



   Propagation of nerve impulses, modulation of cell proliferation and differentiation, induction of the immune response, and tissue homeostasis involve neurotransmitter metabolism (Weiss, B. 



  (1991) Neurotoxicology 12 : 379-386 ; Collins, S. M. et al. (1992) Ann. N. Y. Acad. Sci. 664 : 415-424 ; Brown, J. K. and H. Imam (1991) J. Inherit. Metab. Dis. 14 : 436-458). Many pathways of neurotransmitter metabolism require oxidoreductase activity, coupled to reduction or oxidation of a cofactor, such as   NAD+/NADH   (Newsholme, E. A. and A. R. Leech (1983) Biochemistry for the 

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 Medical Sciences, John Wiley and Sons, Chichester, U. K. pp. 779-793). Degradation of catecholamines (epinephrine or norepinephrine) requires alcohol dehydrogenase (in the brain) or aldehyde dehydrogenase (in peripheral tissue).

   NAD-dependent aldehyde dehydrogenase oxidizes 5- hydroxyindole-3-acetate (the product of 5-hydroxytryptamine (serotonin) metabolism) in the brain, blood platelets, liver and pulmonary   endothelium     (Newsholme,   supra, p. 786). Other neurotransmitter degradation pathways that utilize NAD/NADH-dependent oxidoreductase activity include those of L-DOPA (precursor of dopamine, a neuronal excitatory compound), glycine (an inhibitory neurotransmitter in the brain and spinal cord), histamine (liberated from mast cells during the inflammatory response), and taurine (an inhibitory neurotransmitter of the brain stem, spinal cord and retina) (Newsholme, supra, pp. 790, 792).

   Epigenetic or genetic defects in neurotransmitter metabolic pathways can result in a spectrum of disease states in different tissues including Parkinson disease and inherited myoclonus (McCance, K. L. and S. E. Huether (1994)   Pathophvsiology,   Mosby- Year Book, Inc., St. Louis MO, pp. 402-404 ; Gundlach, A. L. (1990) FASEB J. 4 : 2761-2766). 



   Tetrahydrofolate is a derivatized glutamate molecule that acts as a carrier, providing activated one-carbon units to a wide variety of biosynthetic reactions, including synthesis of purines, pyrimidines, and the amino acid methionine. Tetrahydrofolate is generated by the activity of a holoenzyme complex called tetrahydrofolate synthase, which includes three enzyme activities : tetrahydrofolate dehydrogenase, tetrahydrofolate cyclohydrolase, and tetrahydrofolate synthetase. 



  Thus, tetrahydrofolate dehydrogenase plays an important role in generating building blocks for nucleic and amino acids, crucial to proliferating cells. 



   3-Hydroxyacyl-CoA dehydrogenase (3HACD) is involved in fatty acid metabolism. It catalyzes the reduction of 3-hydroxyacyl-CoA to 3-oxoacyl-CoA, with concomitant oxidation of NAD to NADH, in the mitochondria and peroxisomes of eukaryotic cells. In peroxisomes, 3HACD and enoyl-CoA hydratase form an enzyme complex called bifunctional enzyme, defects in which are associated with peroxisomal bifunctional enzyme deficiency. This interruption in fatty acid metabolism produces accumulation of very-long chain fatty acids, disrupting development of the brain, bone, and adrenal glands. Infants born with this deficiency typically die within 6 months (Watkins, P. et al. (1989) J. Clin. Invest. 83 : 771-777 ; Online Mendelian Inheritance in Man   (OMIM),   #261515).

   The neurodegeneration that is characteristic of Alzheimer's disease involves development of extracellular plaques in certain brain regions. A major protein component of these plaques is the peptide   amyloid-     (AP),   which is one of several cleavage products of amyloid precursor protein (APP). 3HACD has been shown to bind the   A p   peptide, and is overexpressed in neurons affected in Alzheimer's disease. In addition, an antibody against 3HACD can block the toxic effects of Ap in a cell culture model of Alzheimer's disease (Yan, S. et al. (1997) Nature 389 : 689-695 ; OMIM, 

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 #602057). 



   Steroids, such as estrogen, testosterone, corticosterone, and others, are generated from a common precursor, cholesterol, and are interconverted into one another. A wide variety of enzymes act upon cholesterol, including a number of dehydrogenases. Steroid dehydrogenases, such as the hydroxysteroid dehydrogenases, are involved in hypertension, fertility, and cancer (Duax, W. L. and D. Ghosh (1997) Steroids 62 : 95-100). One such dehydrogenase is   3-oxo-5-a-steroid   dehydrogenase (OASD), a microsomal membrane protein highly expressed in prostate and other androgen-responsive tissues. OASD catalyzes the conversion of testosterone into dihydrotestosterone, which is the most potent androgen.

   Dihydrotestosterone is essential for the formation of the male phenotype during embryogenesis, as well as for proper androgen-mediated growth of tissues such as the prostate and male genitalia. A defect in OASD that prevents the conversion of testosterone into dihydrotestosterone leads to a rare form of male pseudohermaphroditis, characterized by defective formation of the external genitalia (Andersson, S. et al. (1991) Nature 354 : 159-161 ; Labrie, F. et al. 



  (1992) Endocrinology 131 : 1571-1573 ; OMIM #264600). Thus, OASD plays a central role in sexual differentiation and androgen physiology. 



     17p-hydroxysteroid   dehydrogenase   (17 HSD6)   plays an important role in the regulation of the male reproductive hormone, dihydrotestosterone   (DHTT).     17 HSD6   acts to reduce levels of DHTT by oxidizing a precursor of DHTT, 3a-diol, to androsterone which is readily   glucuronidated   and removed from tissues.   17  HSD6   is active with both androgen and estrogen substrates when expressed in embryonic kidney 293 cells. At least five other isozymes of 17    HSD   have been identified that catalyze oxidation   and/or   reduction reactions in various tissues with preferences for different steroid substrates (Biswas, M. G. and D. W. Russell (1997) J. Biol. Chem. 272 : 15959- 15966).

   For example,   17 HSDl   preferentially reduces estradiol and is abundant in the ovary and placenta.   17HSD2   catalyzes oxidation of androgens and is present in the endometrium and placenta. 



    17 HSD3   is exclusively a reductive enzyme in the testis (Geissler, W. M. et al.   (1994)   Nat. Genet. 



  7 : 34-39). An excess of androgens such as DHTT can contribute to certain disease states such as benign prostatic hyperplasia and prostate cancer. 



   Oxidoreductases are components of the fatty acid metabolism pathways in mitochondria and peroxisomes. The main beta-oxidation pathway degrades both saturated and unsaturated fatty acids, while the auxiliary pathway performs additional steps required for the degradation of unsaturated fatty acids. The auxiliary beta-oxidation enzyme 2, 4-dienoyl-CoA reductase catalyzes the removal of even-numbered double bonds from unsaturated fatty acids prior to their entry into the main beta- oxidation pathway. The enzyme may also remove odd-numbered double bonds from unsaturated fatty acids (Koivuranta, K. T. et al. (1994) Biochem. J. 304 : 787-792 ; Smeland, T. E. et al. (1992) Proc. 

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  Natl. Acad. Sci. USA 89 : 6673-6677). 2, 4-dienoyl-CoA reductase is located in both mitochondria and peroxisomes. Inherited deficiencies in mitochondrial and peroxisomal beta-oxidation enzymes are associated with severe diseases, some of which manifest themselves soon after birth and lead to death within a few years. Defects in beta-oxidation are associated with Reye's syndrome, Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, acyl-CoA oxidase deficiency, and bifunctional protein deficiency (Suzuki, Y. et al. (1994) Am. J. Hum. Genet. 54 :   36-43   ; Hoefler, supra ; Cotran, R. S. et al. (1994) Robbins   Pathologic   Basis of Disease, W. B. Saunders Co., Philadelphia PA, p. 866).   Peroxisomal   beta-oxidation is impaired in cancerous tissue.

   Although neoplastic human breast epithelial cells have the same number of peroxisomes as do normal cells, fatty acyl-CoA oxidase activity is lower than in control tissue (el Bouhtoury, F. et al. (1992) J. Pathol. 



    166   : 27-35). Human colon carcinomas have fewer peroxisomes than normal colon tissue and have lower fatty-acyl-CoA oxidase and bifunctional enzyme (including enoyl-CoA hydratase) activities than normal tissue (Cable, S. et al. (1992) Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 62 : 221-   226).   Another important oxidoreductase is isocitrate dehydrogenase, which catalyzes the conversion of isocitrate to a-ketoglutarate, a substrate of the citric acid cycle. Isocitrate dehydrogenase can be either NAD or NADP dependent, and is found in the cytosol, mitochondria, and peroxisomes. 



  Activity of isocitrate dehydrogenase is regulated developmentally, and by hormones, neurotransmitters, and growth factors. 



   Hydroxypyruvate reductase (HPR), a peroxisomal 2-hydroxyacid dehydrogenase in the glycolate pathway, catalyzes the conversion of hydroxypyruvate to glycerate with the oxidation of both NADH and NADPH. The reverse dehydrogenase reaction reduces   NAD+ and NADP+.   HPR recycles nucleotides and bases back into pathways leading to the synthesis of ATP and GTP. ATP and GTP are used to produce DNA and RNA and to control various aspects of signal transduction and energy metabolism. Inhibitors of purine nucleotide biosynthesis have long been employed as antiproliferative agents to treat cancer and viral diseases. HPR also regulates biochemical synthesis of serine and cellular serine levels available for protein synthesis. 



   The mitochondrial electron transport (or respiratory) chain is a series of oxidoreductase-type enzyme complexes in the mitochondrial membrane that is responsible for the transport of electrons from NADH through a series of redox centers within these complexes to oxygen, and the coupling of this oxidation to the synthesis of ATP (oxidative phosphorylation). ATP then provides the primary source of energy for driving a cell's many energy-requiring reactions. The key complexes in the respiratory chain are NADH : ubiquinone oxidoreductase (complex I), succinate : ubiquinone oxidoreductase (complex II), cytochrome cl-b oxidoreductase (complex III), cytochrome c oxidase (complex IV), and ATP synthase (complex V) (Alberts, B. et al. (1994) Molecular Biology of the 

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 Cell, Garland Publishing, Inc., New York NY, pp. 677-678).

   All of these complexes are located on the inner matrix side of the mitochondrial membrane except complex   II,   which is on the cytosolic side. Complex II transports electrons generated in the citric acid cycle to the respiratory chain. The electrons generated by oxidation of succinate to fumarate in the citric acid cycle are transferred through electron carriers in complex II to membrane bound ubiquinone (Q). Transcriptional regulation of these nuclear-encoded genes appears to be the predominant means for controlling the biogenesis of respiratory enzymes. Defects and altered expression of enzymes in the respiratory chain are associated with a variety of disease conditions. 



   Other dehydrogenase activities using NAD as a cofactor are also important in mitochondrial function. 3-hydroxyisobutyrate dehydrogenase (3HBD), important in valine catabolism, catalyzes the NAD-dependent oxidation of 3-hydroxyisobutyrate to   methylmalonate     semialdehyde   within mitochondria. Elevated levels of 3-hydroxyisobutyrate have been reported in a number of disease states, including ketoacidosis,   methylmalonic   acidemia, and other disorders associated with deficiencies in methylmalonate   semialdehyde   dehydrogenase (Rougraff, P. M. et al. (1989) J. Biol. 



  Chem. 264 : 5899-5903). 



   Another mitochondrial dehydrogenase important in amino acid metabolism is the enzyme isovaleryl-CoA-dehydrogenase (IVD). IVD is involved in leucine metabolism and catalyzes the oxidation of isovaleryl-CoA to 3-methylcrotonyl-CoA. Human IVD is a tetrameric flavoprotein that is encoded in the nucleus and synthesized in the cytosol as a 45 kDa precursor with a mitochondrial import signal sequence. A genetic deficiency, caused by a mutation in the gene encoding IVD, results in the condition known as isovaleric acidemia. This mutation results in inefficient mitochondrial import and processing of the IVD precursor (Vockley, J. et al. (1992) J. Biol. Chem. 267 : 2494-2501). 



  Transferases 
Transferases are enzymes that catalyze the transfer of molecular groups. The reaction may involve an oxidation, reduction, or cleavage of covalent bonds, and is often specific to a substrate or to particular sites on a type of substrate. Transferases participate in reactions essential to such functions as synthesis and degradation of cell components, regulation of cell functions including cell signaling, cell proliferation, inflamation, apoptosis, secretion and excretion. Transferases are involved in key steps in disease processes involving these functions. Transferases are frequently classified according to the type of group transferred.

   For example, methyl transferases transfer one- carbon methyl groups, amino transferases transfer nitrogenous amino groups, and similarly denominated enzymes transfer aldehyde or ketone, acyl, glycosyl, alkyl or aryl, isoprenyl, saccharyl, phosphorous-containing, sulfur-containing, or selenium-containing groups, as well as small enzymatic groups such as Coenzyme A. 

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   Acyl transferases include peroxisomal carnitine octanoyl transferase, which is involved in the fatty acid beta-oxidation pathway, and mitochondrial carnitine palmitoyl transferases, involved in fatty acid metabolism and transport. Choline 0-acetyl transferase catalyzes the biosynthesis of the neurotransmitter acetylcholine. 



   Amino transferases play key roles in protein synthesis and degradation, and they contribute to other processes as well. For example, the amino transferase   5-aminolevulinic   acid synthase catalyzes the addition of succinyl-CoA to glycine, the first step in heme biosynthesis. Other amino transferases participate in pathways important for neurological function and metabolism. For example, glutamine- phenylpyruvate amino transferase, also known as glutamine transaminase K (GTK), catalyzes several reactions with a pyridoxal phosphate cofactor. GTK catalyzes the reversible conversion of L- glutamine and phenylpyruvate to 2-oxoglutaramate and L-phenylalanine. Other amino acid substrates for GTK include L-methionine, L-histidine, and L-tyrosine.

   GTK also catalyzes the conversion of   kynurenine   to kynurenic acid, a tryptophan metabolite that is an antagonist of the N-methyl-D- aspartate (NMDA) receptor in the brain and may exert a neuromodulatory function. Alteration of the   kynurenine   metabolic pathway may be associated with several neurological disorders. GTK also plays a role in the metabolism of halogenated xenobiotics conjugated to glutathione, leading to nephrotoxicity in rats and neurotoxicity in humans. GTK is expressed in kidney, liver, and brain. 



  Both human and rat GTKs contain a putative pyridoxal phosphate binding site (ExPASy ENZYME : EC 2. 6. 1. 64 ; Perry, S. J. et al. (1993) Mol. Pharmacol. 43 : 660-665 ; Perry, S. et al. (1995) FEBS Lett. 



  360 : 277-280 ; and Alberati-Giani, D. et al. (1995) J. Neurochem. 64 : 1448-1455). A second amino transferase associated with this pathway is   kynurenine/a-aminoadipate   amino transferase (AadAT). 



  AadAT catalyzes the reversible conversion of a-aminoadipate and a-ketoglutarate to a-ketoadipate and L-glutamate during lysine metabolism. AadAT also catalyzes the transamination of   kynurenine   to   kynurenic   acid. A cytosolic AadAT is expressed in rat kidney, liver, and brain   (Nakatani,     Y.   et al. 



  (1970) Biochim. Biophys. Acta 198 : 219-228 ; Buchli, R. et al. (1995) J. Biol. Chem. 270 : 29330- 29335). 



   Glycosyl transferases include the mammalian   UDP-glucouronosyl   transferases, a family of membrane-bound microsomal enzymes catalyzing the transfer of   glucouronic   acid to lipophilic substrates in reactions that play important roles in detoxification and excretion of drugs, carcinogens, and other foreign substances. Another mammalian glycosyl transferase, mammalian UDP-galactose- ceramide galactosyl transferase, catalyzes the transfer of galactose to ceramide in the synthesis of galactocerebrosides in myelin membranes of the nervous system. The   UDP-glycosyl   transferases share a conserved signature domain of about 50 amino acid residues (PROSITE : PDOC00359, http ://expasy. hcuge.   ch/sprot/prosite.   html). 

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   Methyl transferases are involved in a variety of pharmacologically important processes. 



  Nicotinamide N-methyl transferase catalyzes the N-methylation of nicotinamides and other pyridines, an important step in the cellular handling of drugs and other foreign compounds. 



    Phenylethanolamine   N-methyl transferase catalyzes the conversion of noradrenalin to adrenalin.   6-O-   methylguanine-DNA methyl transferase reverses DNA methylation, an important step in carcinogenesis.   Uroporphyrin-III   C-methyl transferase, which catalyzes the transfer of two methyl groups from   S-adenosyl-L-methionine   to uroporphyrinogen III, is the first specific enzyme in the biosynthesis of   cobalamin,   a dietary enzyme whose uptake is deficient in pernicious anemia. Protein- arginine methyl transferases catalyze the posttranslational methylation of arginine residues in proteins, resulting in the mono-and dimethylation of arginine on the guanidino group.

   Substrates include histones, myelin basic protein, and heterogeneous nuclear ribonucleoproteins involved in mRNA processing, splicing, and transport. Protein-arginine methyl transferase interacts with proteins upregulated by mitogens, with proteins involved in chronic lymphocytic leukemia, and with interferon, suggesting an important role for methylation in cytokine receptor signaling (Lin, W.-J. et al. (1996) J. Biol. Chem. 271 : 15034-15044 ; Abramovich, C. et al. (1997) EMBO J. 16 : 260-266 ; and Scott, H. S. et al. (1998) Genomics 48 : 330-340). 



   Phosphotransferases catalyze the transfer of high-energy phosphate groups and are important in energy-requiring and-releasing reactions. The metabolic enzyme creatine kinase catalyzes the reversible phosphate transfer between creatine/creatine phosphate and   ATP/ADP.   Glycocyamine kinase catalyzes phosphate transfer from ATP to guanidoacetate, and arginine kinase catalyzes phosphate transfer from ATP to arginine. A cysteine-containing active site is conserved in this family (PROSITE : PDOC00103). 



   Prenyl transferases are heterodimers, consisting of an alpha and a beta subunit, that catalyze the transfer of an isoprenyl group. An example of a prenyl transferase is the mammalian protein   farnesyl   transferase. The alpha subunit of farnesyl transferase consists of 5 repeats of 34 amino acids each, with each repeat containing an invariant tryptophan (PROSITE : PDOC00703). 



   Saccharyl transferases are glycating enzymes involved in a variety of metabolic processes. 



  Oligosacchryl transferase-48, for example, is a receptor for advanced glycation endproducts. 



  Accumulation of these endproducts is observed in vascular complications of diabetes, macrovascular disease, renal insufficiency, and Alzheimer's disease   (Thornalley,   P. J. (1998) Cell Mol. Biol. (Noisy- Le-Grand) 44 : 1013-1023). 



   Coenzyme A (CoA) transferase catalyzes the transfer of CoA between two carboxylic acids. 



  Succinyl CoA : 3-oxoacid CoA transferase, for example, transfers CoA from succinyl-CoA to a recipient such as acetoacetate. Acetoacetate is essential to the metabolism of ketone bodies, which 

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 accumulate in tissues affected by metabolic disorders such as diabetes (PROSITE : PDOC00980). 



  Hydrolases 
Hydrolysis is the breaking of a covalent bond in a substrate by introduction of a molecule of water. The reaction involves a   nucleophilic   attack by the water molecule's oxygen atom on a target bond in the substrate. The water molecule is split across the target bond, breaking the bond and generating two product molecules. Hydrolases participate in reactions essential to such functions as synthesis and degradation of cell components, and for regulation of cell functions including cell signaling, cell proliferation, inflamation, apoptosis, secretion and excretion. Hydrolases are involved in key steps in disease processes involving these functions. Hydrolytic enzymes, or hydrolases, may be grouped by substrate specificity into classes including phosphatases, peptidases, lysophospholipases, phosphodiesterases, glycosidases, and glyoxalases. 



   Phosphatases hydrolytically remove phosphate groups from proteins, an energy-providing step that regulates many cellular processes, including intracellular signaling pathways that in turn control cell growth and differentiation, cell-cell contact, the cell cycle, and oncogenesis. 



   Lysophospholipases (LPLs) regulate intracellular lipids by catalyzing the hydrolysis of ester bonds to remove an acyl group, a key step in lipid degradation. Small LPL isoforms, approximately 15-30 kD, function as hydrolases ; larger isoforms function both as hydrolases and transacylases. A particular substrate for   LPLs,   lysophosphatidylcholine, causes lysis of cell membranes. LPL activity is regulated by signaling molecules important in numerous pathways, including the inflammatory response. 



   Peptidases, also called proteases, cleave peptide bonds that form the backbone of peptide or protein chains. Proteolytic processing is essential to cell growth, differentiation, remodeling, and homeostasis as well as inflammation and immune response. Since typical protein half-lives range from hours to a few days, peptidases are continually cleaving precursor proteins to their active form, removing signal sequences from targeted proteins, and degrading aged or defective proteins. 



  Peptidases function in bacterial, parasitic, and viral invasion and replication within a host. Examples of peptidases include trypsin and chymotrypsin (components of the complement cascade and the blood-clotting cascade) lysosomal cathepsins,   calpains,   pepsin, renin, and chymosin (Beynon, R. J. and J. S. Bond (1994) Proteolytic Enzymes : A Practical Approach, Oxford University Press, New York NY, pp. 1-5). 



   The phosphodiesterases catalyze the hydrolysis of one of the two ester bonds in a phosphodiester compound. Phosphodiesterases are therefore crucial to a variety of cellular processes. 



  Phosphodiesterases include DNA and RNA endo-and exo-nucleases, which are essential to cell growth and replication as well as protein synthesis. Another phosphodiesterase is acid 

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 sphingomyelinase, which hydrolyzes the membrane phospholipid sphingomyelin to ceramide and phosphorylcholine. Phosphorylcholine is used in the synthesis of phosphatidylcholine, which is involved in numerous intracellular signaling pathways.   Ceramide   is an essential precursor for the generation of gangliosides, membrane lipids found in high concentration in neural tissue. Defective acid   sphingomyelinase   phosphodiesterase leads to a build-up of sphingomyelin molecules in lysosomes, resulting in   Niemann-Pick   disease. 



   Glycosidases catalyze the cleavage   of hemiacetyl   bonds of glycosides, which are compounds that contain one or more sugar. Mammalian lactase-phlorizin hydrolase, for example, is an intestinal enzyme that splits lactose. Mammalian beta-galactosidase removes the terminal galactose from gangliosides, glycoproteins, and   glycosaminoglycans,   and deficiency of this enzyme is associated with a gangliosidosis known as Morquio disease type B. Vertebrate lysosomal alpha-glucosidase, which hydrolyzes glycogen, maltose, and isomaltose, and vertebrate intestinal sucrase-isomaltase, which hydrolyzes sucrose, maltose, and isomaltose, are widely distributed members of this family with highly conserved sequences at their active sites. 



   The glyoxylase system is involved in gluconeogenesis, the production of glucose from storage compounds in the body. It consists of glyoxylase   I,   which catalyzes the formation of S-D- lactoylglutathione from   methyglyoxal,   a side product of triose-phosphate energy metabolism, and glyoxylase   II,   which hydrolyzes   S-D-lactoylglutathione   to D-lactic acid and reduced glutathione. 



  Glyoxylases are involved in hyperglycemia, non-insulin-dependent diabetes mellitus, the detoxification of bacterial toxins, and in the control of cell proliferation and microtubule assembly. 



  Lyases 
Lyases are a class of enzymes that catalyze the cleavage of C-C,   C-O,   C-N, C-S, C- (halide),   P-O   or other bonds without hydrolysis or oxidation to form two molecules, at least one of which contains a double bond (Stryer, L. (1995) Biochemistry W. H. Freeman and Co. New York, NY p. 620). Lyases are critical components of cellular biochemistry with roles in metabolic energy production including fatty acid metabolism, as well as other diverse enzymatic processes. Further classification of lyases reflects the type of bond cleaved as well as the nature of the cleaved group. 



   The group of C-C lyases include carboxyl-lyases (decarboxylases), aldehyde-lyases (aldolases), oxo-acid-lyases and others. The C-O lyase group includes hydro-lyases, lyases acting on polysaccharides and other lyases. The C-N lyase group includes ammonia-lyases,   amidine-lyases,   amine-lyases   (deaminases)   and other lyases. 



   Proper regulation of lyases is critical to normal physiology. For example, mutation induced deficiencies in the uroporphyrinogen decarboxylase can lead to photosensitive cutaneous lesions in the genetically-linked disorder familial porphyria cutanea tarda (Mendez, M. et al. (1998) Am. J. 

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  Genet. 63 : 1363-1375). It has also been shown that adenosine deaminase (ADA) deficiency stems from genetic mutations in the ADA gene, resulting in the disorder severe combined immunodeficiency disease (SCID) (Hershfield, M. S. (1998) Semin. Hematol. 35 : 291-298). 



  Isomerases 
Isomerases are a class of enzymes that catalyze geometric or structural changes within a molecule to form a single product. This class includes racemases and epimerases, cis-trans- isomerases, intramolecular   oxidoreductases,   intramolecular transferases (mutases) and intramolecular lyases. Isomerases are critical components of cellular biochemistry with roles in metabolic energy production including glycolysis, as well as other diverse enzymatic processes (Stryer, L. (1995) Biochemistry, W. H. Freeman and Co., New York NY, pp. 483-507). 



   Racemases are a subset of isomerases that catalyze inversion of a molecules configuration around the asymmetric carbon atom in a substrate having a single center of asymmetry, thereby interconverting two racemers. Epimerases are another subset of isomerases that catalyze inversion of configuration around an asymmetric carbon atom in a substrate with more than one center of symmetry, thereby interconverting two epimers. Racemases and epimerases can act on amino acids and derivatives, hydroxy acids and derivatives, as well as carbohydrates and derivatives. The interconversion of UDP-galactose and UDP-glucose is catalyzed by UDP-galactose-4'-epimerase. 



  Proper regulation and function of this epimerase is essential to the synthesis of glycoproteins and glycolipids. Elevated blood galactose levels have been correlated with UDP-galactose-4'-epimerase deficiency in screening programs of infants (Gitzelmann, R. (1972) Helv. Paediat. Acta 27 : 125-130). 



   Oxidoreductases can be isomerases as well. Oxidoreductases catalyze the reversible transfer of electrons from a substrate that becomes oxidized to a substrate that becomes reduced. This class of enzymes includes dehydrogenases, hydroxylases, oxidases, oxygenases, peroxidases, and reductases. 



  Proper maintenance of oxidoreductase levels is physiologically important. For example, genetically- linked deficiencies in lipoamide dehydrogenase can result in lactic acidosis (Robinson, B. H. et al. 



  (1977) Pediat. Res. 11 : 1198-1202). 



   Another subgroup of isomerases are the transferases (or mutases). Transferases transfer a chemical group from one compound (the donor) to another compound (the acceptor). The types of groups transferred by these enzymes include acyl groups, amino groups, phosphate groups (phosphotransferases or phosphomutases), and others. The transferase carnitine   palmitoyltransferase   is an important component of fatty acid metabolism. Genetically-linked deficiencies in this transferase can lead to myopathy (Scriver, C. R. et al. (1995) The Metabolic and Molecular Basis of Inherited Disease, McGraw-Hill, New York NY, pp.   1501-1533).   



   Yet another subgroup of isomerases are the topoisomersases. Topoisomerases are enzymes 

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 that affect the topological state of DNA. For example, defects in topoisomerases or their regulation can affect normal physiology. Reduced levels   of topoisomerase II   have been correlated with some of the DNA processing defects associated with the disorder ataxia-telangiectasia (Singh, S. P. et al. 



  (1988) Nucleic Acids Res. 16 : 3919-3929). 



  Ligases 
Ligases catalyze the formation of a bond between two substrate molecules. The process involves the hydrolysis of a pyrophosphate bond in ATP or a similar energy donor. Ligases are classified based on the nature of the type of bond they form, which can include carbon-oxygen, carbon-sulfur, carbon-nitrogen, carbon-carbon and phosphoric ester bonds. 



   Ligases forming carbon-oxygen bonds include the   aminoacyl-transfer   RNA   (tRNA)   synthetases which are important RNA-associated enzymes with roles in translation. Protein biosynthesis depends on each amino acid forming a linkage with the appropriate tRNA. The aminoacyl-tRNA synthetases are responsible for the activation and correct attachment of an amino acid with its cognate tRNA. The 20 aminoacyl-tRNA synthetase enzymes can be divided into two structural classes, and each class is characterized by a distinctive topology of the catalytic domain. 



  Class I enzymes contain a catalytic domain based on the nucleotide-binding Rossman fold. Class II enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel B-sheet motif, as well as N-and C-terminal regulatory domains. Class II enzymes are separated into two groups based on the heterodimeric or homodimeric structure of the enzyme ; the latter group is further subdivided by the structure of the N-and C-terminal regulatory domains (Hartlein, M. and S. Cusack (1995) J. Mol. Evol. 40 : 519-530). Autoantibodies against aminoacyl-tRNAs are generated by patients with dermatomyositis and polymyositis, and correlate strongly with complicating interstitial lung disease (ILD). These antibodies appear to be generated in response to viral infection, and coxsackie virus has been used to induce experimental viral myositis in animals. 



   Ligases forming carbon-sulfur bonds (Acid-thiol ligases) mediate a large number of cellular biosynthetic intermediary metabolism processes involve intermolecular transfer of carbon atom-containing substrates (carbon substrates). Examples of such reactions include the tricarboxylic acid cycle, synthesis of fatty acids and long-chain phospholipids, synthesis of alcohols and aldehydes, synthesis of intermediary metabolites, and reactions involved in the amino acid degradation pathways. Some of these reactions require input of energy, usually in the form of conversion of ATP to either ADP or AMP and pyrophosphate. 



   In many cases, a carbon substrate is derived from a small molecule containing at least two carbon atoms. The carbon substrate is often covalently bound to a larger molecule which acts as a carbon substrate carrier molecule within the cell. In the biosynthetic mechanisms described above, 

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 the carrier molecule is coenzyme A. Coenzyme A (CoA) is structurally related to derivatives of the nucleotide ADP and consists of 4'-phosphopantetheine linked via a phosphodiester bond to the alpha phosphate group of adenosine 3', 5'-bisphosphate. The terminal thiol group of 4'-phosphopantetheine acts as the site for carbon substrate bond formation. The predominant carbon substrates which utilize CoA as a carrier molecule during biosynthesis and intermediary metabolism in the cell are acetyl, succinyl, and propionyl moieties, collectively referred to as acyl groups.

   Other carbon substrates include enoyl lipid, which acts as a fatty acid oxidation intermediate, and carnitine, which acts as an acetyl-CoA flux regulator/mitochondrial acyl group transfer protein. Acyl-CoA and acetyl-CoA are synthesized in the cell by acyl-CoA synthetase and acetyl-CoA synthetase, respectively. 



   Activation of fatty acids is mediated by at least three forms of acyl-CoA synthetase activity : i) acetyl-CoA synthetase, which activates acetate and several other low molecular weight carboxylic acids and is found in muscle mitochondria and the cytosol of other tissues ; ii) medium-chain acyl-CoA synthetase, which activates fatty acids containing between four and eleven carbon atoms (predominantly from dietary sources), and is present only in liver mitochondria ; and iii) acyl CoA synthetase, which is specific for long chain fatty acids with between six and twenty carbon atoms, and is found in microsomes and the mitochondria. Proteins associated with acyl-CoA synthetase activity have been identified from many sources including bacteria, yeast, plants, mouse, and man.

   The activity of acyl-CoA synthetase may be modulated by phosphorylation of the enzyme by cAMP-dependent protein kinase. 



   Ligases forming carbon-nitrogen bonds include amide synthases such as glutamine synthetase (glutamate-ammonia ligase) that catalyzes the amination of glutamic acid to glutamine by ammonia using the energy of ATP hydrolysis. Glutamin is the primary source for the amino group in various amide transfer reactions involved in de novo pyrimidine nucleotide synthesis and in purine and pyrimidine ribonucleotide   interconversions.   Overexpression of glutamine synthetase has been observed in primary liver cancer (Christa, L. et al. (1994) Gastroent. 106 : 1312-1320). 



   Acid-amino-acid ligases (peptide synthases) are represented by the ubiquitin proteases which are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaryotic cells and some bacteria. The UCS mediates the elimination of abnormal proteins and regulates the half-lives of important regulatory proteins that control cellular processes such as gene transcription and cell cycle progression. In the UCS pathway, proteins targeted for degradation are conjugated to a ubiquitin (Ub), a small heat stable protein. Ub is first activated by a ubiquitin-activating enzyme   (E1),   and then transferred to one of several Ub- conjugating enzymes (E2). E2 then links the Ub molecule through its C-terminal glycine to an internal lysine (acceptor lysine) of a target protein.

   The ubiquitinated protein is then recognized and 

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 degraded by proteasome, a large, multisubunit proteolytic enzyme complex, and ubiquitin is released for reutilization by ubiquitin protease. The UCS is implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins, cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, A. (1994) Cell 79 : 13-21). A murine proto-oncogene, Unp, encodes a nuclear ubiquitin protease whose overexpression leads to oncogenic transformation of NIH3T3 cells, and the human homolog of this gene is consistently elevated in small cell tumors and adenocarcinomas of the lung (Gray, D. A. (1995) Oncogene 10 : 2179-2183). 



   Cyclo-ligases and other carbon-nitrogen ligases comprise various enzymes and enzyme complexes that participate in the de novo pathways to purine and pyrimidine biosynthesis. Because these pathways are critical to the synthesis of nucleotides for replication of both RNA and DNA, many of these enzymes have been the targets of clinical agents for the treatment of cell proliferative disorders such as cancer and infectious diseases. 



   Purine biosynthesis occurs de novo from the amino acids glycine and glutamine, and other small molecules. Three of the key reactions in this process are catalyzed by a trifunctional enzyme composed of glycinamide-ribonucleotide synthetase (GARS),   aminoimidazole   ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide transformylase   (GART).   Together these three enzymes combine   ribosylamine   phosphate with glycine to yield phosphoribosyl   aminoimidazole,   a precursor to both adenylate and guanylate nucleotides. This trifunctional protein has been implicated in the pathology of Downs syndrome (Aimi, J. et al. (1990) Nucleic Acid Res. 18 : 6665-6672). 



  Adenylosuccinate synthetase catalyzes a later step in purine biosynthesis that converts inosinic acid to adenylosuccinate, a key step on the path to ATP synthesis. This enzyme is also similar to another carbon-nitrogen ligase, argininosuccinate synthetase, that catalyzes a similar reaction in the urea cycle (Powell, S. M. et al. (1992) FEBS Lett. 303 : 4-10). 



   Like the de novo biosynthesis of purines, de novo synthesis of the pyrimidine nucleotides uridylate and cytidylate also arises from a common precursor, in this instance the nucleotide orotidylate derived from orotate and phosphoribosyl pyrophosphate (PPRP). Again a trifunctional enzyme comprising three carbon-nitrogen ligases plays a key role in the process. In this case the enzymes aspartate   transcarbamylase   (ATCase), carbamyl phosphate synthetase II, and dihydroorotase (DHOase) are encoded by a single gene called CAD. Together these three enzymes combine the initial reactants in pyrimidine biosynthesis, glutamine,   CO2   and ATP to form dihydroorotate, the precursor to orotate and orotidylate (Iwahana, H. et al. (1996) Biochem. Biophys. Res. Commun. 



  219 : 249-255). Further steps then lead to the synthesis of uridine nucleotides from orotidylate. 



  Cytidine nucleotides are derived from uridine-5'-triphosphate (UTP) by the amidation of UTP using 

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 glutamine as the amino donor and the enzyme CTP synthetase. Regulatory mutations in the human CTP synthetase are believed to confer multi-drug resistance to agents widely used in cancer therapy   (Yamauchi,   M. et al. (1990) EMBO J. 9 : 2095-2099). 



   Ligases forming carbon-carbon bonds include the carboxylases acetyl-CoA carboxylase and pyruvate carboxylase. Acetyl-CoA carboxylase catalyzes the carboxylation of acetyl-CoA from   CO2   and   H20   using the energy of ATP hydrolysis. Acetyl-CoA carboxylase is the rate-limiting step in the biogenesis of long-chain fatty acids. Two isoforms of acetyl-CoA carboxylase, types I and types   II,   are expressed in human in a tissue-specific manner (Ha, J. et al. (1994) Eur. J. Biochem. 219 : 297-   306). Pyruvate   carboxylase is a nuclear-encoded mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, a key intermediate in the citric acid cycle. 



   Ligases forming phosphoric ester bonds include the DNA ligases involved in both DNA replication and repair. DNA ligases seal phosphodiester bonds between two adjacent nucleotides in a DNA chain using the energy from ATP hydrolysis to first activate the free 5'-phosphate of one nucleotide and then react it with the 3'-OH group of the adjacent nucleotide. This resealing reaction is used in both DNA replication to join small DNA fragments called Okazaki fragments that are transiently formed in the process of replicating new DNA, and in DNA repair. DNA repair is the process by which accidental base changes, such as those produced by oxidative damage, hydrolytic attack, or uncontrolled methylation of DNA, are corrected before replication or transcription of the DNA can occur.

   Bloom's syndrome is an inherited human disease in which individuals are partially deficient in DNA ligation and consequently have an increased incidence of cancer (Alberts, B. et al. 



  (1994) The Molecular Biology of the Cell, Garland Publishing Inc., New York NY, p. 247). 



  Molecules Associated with Growth and Development
Human growth and development requires the spatial and temporal regulation of cell differentiation, cell proliferation, and apoptosis. These processes coordinately control reproduction, aging, embryogenesis, morphogenesis, organogenesis, and tissue repair and maintenance. At the cellular level, growth and development is governed by the cell's decision to enter into or exit from the cell division cycle and by the cell's commitment to a terminally differentiated state. These decisions are made by the cell in response to extracellular signals and other environmental cues it receives. The following discussion focuses on the molecular mechanisms of cell division, reproduction, cell differentiation and proliferation, apoptosis, and aging. 



  Cell Division
Cell division is the fundamental process by which all living things grow and reproduce. In unicellular organisms such as yeast and bacteria, each cell division doubles the number of organisms, 

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 while in multicellular species many rounds of cell division are required to replace cells lost by wear or by programmed cell death, and for cell differentiation to produce a new tissue or organ. Details of the cell division cycle may vary, but the basic process consists of three principle events. The first event, interphase, involves preparations for cell division, replication of the DNA, and production of essential proteins. In the second event, mitosis, the nuclear material is divided and separates to opposite sides of the cell. The final event, cytokinesis, is division and fission of the cell cytoplasm.

   The sequence and timing of cell cycle transitions is under the control of the cell cycle regulation system which controls the process by positive or negative regulatory circuits at various check points. 



   Regulated progression of the cell cycle depends on the integration of growth control pathways with the basic cell cycle machinery. Cell cycle regulators have been identified by selecting for human and yeast cDNAs that block or activate cell cycle arrest signals in the yeast mating pheromone pathway when they are overexpressed. Known regulators include human CPR (cell cycle progression restoration) genes, such as CPR8 and CPR2, and yeast CDC (cell division control) genes, including CDC91, that block the arrest signals. The CPR genes express a variety of proteins including cyclins, tumor suppressor binding proteins, chaperones, transcription factors, translation factors, and RNA-binding proteins (Edwards, M. C. et al.   (1997)   Genetics 147 : 1063-1076). 



   Several cell cycle transitions, including the entry and exit of a cell from mitosis, are dependent upon the activation and inhibition of cyclin-dependent kinases (Cdks). The Cdks are composed of a kinase subunit, Cdk, and an activating subunit, cyclin, in a complex that is subject to many levels of regulation. There appears to be a single Cdk in Saccharomyces cerevisiae and   Saccharomvces   pombe whereas mammals have a variety of specialized Cdks. Cyclins act by binding to and activating cyclin-dependent protein kinases which then phosphorylate and activate selected proteins involved in the mitotic process. The Cdk-cyclin complex is both positively and negatively regulated by phosphorylation, and by targeted degradation involving molecules such as CDC4 and CDC53.

   In addition, Cdks are further regulated by binding to inhibitors and other proteins such as   Sucl   that modify their specificity or accessibility to regulators (Patra, D. and W. G. Dunphy (1996) Genes Dev. 



  10 : 1503-1515 ; and Mathias, N. et al. (1996) Mol. Cell Biol. 16 : 6634-6643). 



  Reproduction 
The male and female reproductive systems are complex and involve many aspects of growth and development. The anatomy and physiology of the male and female reproductive systems are reviewed in (Guyton, A. C. (1991) Textbook of Medical   Physiology.   W. B. Saunders Co., Philadelphia PA, pp. 899-928). 



   The male reproductive system includes the process of spermatogenesis, in which the sperm are formed, and male reproductive functions are regulated by various hormones and their effects on 

 <Desc/Clms Page number 18> 

 accessory sexual organs, cellular metabolism, growth, and other bodily functions. 



   Spermatogenesis begins at puberty as a result of stimulation by gonadotropic hormones released from the anterior pituitary. Immature sperm   (spermatogonia)   undergo several mitotic cell divisions before undergoing meiosis and full maturation. The testes secrete several male sex hormones, the most abundant being testosterone, that is essential for growth and division of the immature sperm, and for the masculine characteristics of the male body. Three other male sex hormones, gonadotropin- releasing hormone (GnRH),   luteinizing   hormone (LH), and follicle-stimulating hormone (FSH) control sexual function. 



   The uterus, ovaries, fallopian tubes, vagina, and breasts comprise the female reproductive system. The ovaries and uterus are the source of ova and the location of fetal development, respectively. The fallopian tubes and vagina are accessory organs attached to the top and bottom of the uterus, respectively. Both the uterus and ovaries have additional roles in the development and loss of reproductive capability during a female's lifetime. The primary role of the breasts is lactation. 



  Multiple endocrine signals from the ovaries, uterus, pituitary, hypothalamus, adrenal glands, and other tissues coordinate reproduction and lactation. These signals vary during the monthly menstruation cycle and during the female's lifetime. Similarly, the sensitivity of reproductive organs to these endocrine signals varies during the female's lifetime. 



   A combination of positive and negative feedback to the ovaries, pituitary and hypothalamus glands controls physiologic changes during the monthly ovulation and endometrial cycles. The anterior pituitary secretes two major gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), regulated by negative feedback of steroids, most notably by ovarian estradiol. If fertilization does not occur, estrogen and progesterone levels decrease. This sudden reduction of the ovarian hormones leads to menstruation, the desquamation of the endometrium. 



   Hormones further govern all the steps of pregnancy, parturition, lactation, and menopause. 



  During pregnancy large quantities of human chorionic gonadotropin (hCG), estrogens, progesterone, and human chorionic   somatomammotropin   (hCS) are formed by the placenta. hCG, a glycoprotein similar to   luteinizing   hormone, stimulates the corpus luteum to continue producing more progesterone and estrogens, rather than to involute as occurs if the ovum is not fertilized. hCS is similar to growth hormone and is crucial for fetal nutrition. 



   The female breast also matures during pregnancy. Large amounts of estrogen secreted by the placenta trigger growth and branching of the breast milk ductal system while lactation is initiated by the secretion of prolactin by the pituitary gland. 



   Parturition involves several hormonal changes that increase uterine contractility toward the end of pregnancy, as follows. The levels of estrogens increase more than those of progesterone. Oxytocin 

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 is secreted by   the neurohypophysis.   Concomitantly, uterine sensitivity to oxytocin increases. The fetus itself secretes oxytocin, cortisol (from adrenal glands), and prostaglandins. 



   Menopause occurs when most of the ovarian follicles have degenerated. The ovary then produces less estradiol, reducing the negative feedback on the pituitary and hypothalamus glands. 



  Mean levels of circulating FSH and LH increase, even as ovulatory cycles continue. Therefore, the ovary is less responsive to gonadotropins, and there is an increase in the time between menstrual cycles. 



  Consequently, menstrual bleeding ceases and reproductive capability ends. 



  Cell Differentiation and Proliferation 
Tissue growth involves complex and ordered patterns of cell proliferation, cell differentiation, and apoptosis. Cell proliferation must be regulated to maintain both the number of cells and their spatial organization. This regulation depends upon the appropriate expression of proteins which control cell cycle progression in response to extracellular signals, such as growth factors and other mitogens, and intracellular cues, such as DNA damage or nutrient starvation. Molecules which directly or indirectly modulate cell cycle progression fall into several categories, including growth factors and their receptors, second messenger and signal transduction proteins, oncogene products, tumor-suppressor proteins, and mitosis-promoting factors. 



   Growth factors were originally described as serum factors required to promote cell proliferation. Most growth factors are large, secreted polypeptides that act on cells in their local environment. Growth factors bind to and activate specific cell surface receptors and initiate intracellular signal transduction cascades. Many growth factor receptors are classified as receptor tyrosine kinases which undergo autophosphorylation upon ligand binding. Autophosphorylation enables the receptor to interact with signal transduction proteins characterized by the presence of SH2 or SH3 domains (Src homology regions 2 or 3). These proteins then modulate the activity state of small G-proteins, such as Ras, Rab, and Rho, along with GTPase activating proteins (GAPs), guanine nucleotide releasing proteins (GNRPs), and other guanine nucleotide exchange factors.

   Small G proteins act as molecular switches that activate other downstream events, such as mitogen-activated protein kinase (MAP kinase) cascades. MAP kinases ultimately activate transcription of mitosis- promoting genes. 



   In addition to growth factors, small signaling peptides and hormones also influence cell proliferation. These molecules bind primarily to another class of receptor, the trimeric G-protein coupled receptor (GPCR), found predominantly on the surface of immune, neuronal and neuroendocrine cells. Upon ligand binding, the GPCR activates a trimeric G protein which in turn triggers increased levels of intracellular second messengers such as phospholipase C, Ca2+, and cyclic AMP. Most GPCR-mediated signaling pathways indirectly promote cell proliferation by causing the secretion or 

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 breakdown of other signaling molecules that have direct mitogenic effects. These signaling cascades often involve activation of kinases and phosphatases.

   Some growth factors, such as some members of the transforming growth factor beta   (TGF- )   family, act on some cells to stimulate cell proliferation and on other cells to inhibit it. Growth factors may also stimulate a cell at one concentration and inhibit the same cell at another concentration. Most growth factors also have a multitude of other actions besides the regulation of cell growth and division : they can control the proliferation, survival, differentiation, migration, or function of cells depending on the circumstance. For example, the tumor necrosis factor/nerve growth factor (TNF/NGF) family can activate or inhibit cell death, as well as regulate proliferation and differentiation.

   The cell response depends on the type of cell, its stage of differentiation and transformation status, which surface receptors are stimulated, and the types of stimuli acting on the cell (Smith, A. et al. (1994) Cell 76 : 959-962 ; and Nocentini, G. et al. (1997) Proc. 



  Natl. Acad. Sci. USA 94 : 6216-6221). 



   Neighboring cells in a tissue compete for growth factors, and when provided   with"unlimited"   quantities in a perfused system will grow to even higher cell densities before reaching density-dependent inhibition of cell division. Cells often demonstrate an anchorage dependence of cell division as well. 



  This anchorage dependence may be associated with the formation of focal contacts linking the cytoskeleton with the extracellular matrix (ECM). The expression of ECM components can be stimulated by growth factors. For example,   TGF-P   stimulates fibroblasts to produce a variety of ECM proteins, including fibronectin, collagen, and tenascin (Pearson, C. A. et al. (1988) EMBO J. 7 : 2677-   2981).   In fact, for some cell types specific ECM molecules, such as   laminin   or fibronectin, may act as growth factors. Tenascin-C and-R, expressed in developing and   lesioned neural   tissue, provide   stimulatory/anti-adhesive   or inhibitory properties, respectively, for axonal growth (Faissner, A. (1997) Cell Tissue Res. 290 : 331-341). 



   Cancers are associated with the activation of oncogenes which are derived from normal cellular genes. These oncogenes encode oncoproteins which convert normal cells into malignant cells. Some oncoproteins are mutant isoforms of the normal protein, and other oncoproteins are abnormally expressed with respect to location or amount of expression. The latter category of oncoprotein causes cancer by altering transcriptional control of cell proliferation. Five classes of oncoproteins are known to affect cell cycle controls. These classes include growth factors, growth factor receptors, intracellular signal transducers, nuclear transcription factors, and cell-cycle control proteins. Viral oncogenes are integrated into the human genome after infection of human cells by certain viruses. Examples of viral oncogenes include v-src, v-abl, and v-fps. 



   Many oncogenes have been identified and characterized. These include sis,   erbA,     erbB,   her-2, mutated Gs, src, abl, ras,   crk,   jun, fos, myc, and mutated tumor-suppressor genes such as RB, p53, 

 <Desc/Clms Page number 21> 

   mdm2,   Cipl,   pl6,   and cyclin D. Transformation of normal genes to oncogenes may also occur by chromosomal translocation. The Philadelphia chromosome, characteristic of chronic myeloid leukemia and a subset of acute   lymphoblastic   leukemias, results from a reciprocal translocation between chromosomes 9 and 22 that moves a truncated portion of the proto-oncogene   c-abl   to the breakpoint cluster region   (bcr)   on chromosome 22. 



   Tumor-suppressor genes are involved in regulating cell proliferation. Mutations which cause reduced or loss of function in tumor-suppressor genes result in uncontrolled cell proliferation. For example, the retinoblastoma gene product (RB), in a non-phosphorylated state, binds several early- response genes and suppresses their transcription, thus blocking cell division. Phosphorylation of RB causes it to dissociate from the genes, releasing the suppression, and allowing cell division to proceed. 



  Apoptosis 
Apoptosis is the genetically controlled process by which unneeded or defective cells undergo programmed cell death. Selective elimination of cells is as important for morphogenesis and tissue remodeling as is cell proliferation and differentiation. Lack of apoptosis may result in hyperplasia and other disorders associated with increased cell proliferation. Apoptosis is also a critical component of the immune response. Immune cells such as cytotoxic T-cells and natural killer cells prevent the spread of disease by inducing apoptosis in tumor cells and virus-infected cells. In addition, immune cells that fail to distinguish self molecules from foreign molecules must be eliminated by apoptosis to avoid an autoimmune response. 



   Apoptotic cells undergo distinct morphological changes. Hallmarks of apoptosis include cell shrinkage, nuclear and cytoplasmic condensation, and alterations in plasma membrane topology. 



  Biochemically, apoptotic cells are characterized by increased intracellular calcium concentration, fragmentation of chromosomal DNA, and expression of novel cell surface components. 



   The molecular mechanisms of apoptosis are highly conserved, and many of the key protein regulators and effectors of apoptosis have been identified. Apoptosis generally proceeds in response to a signal which is transduced intracellularly and results in altered patterns of gene expression and protein activity. Signaling molecules such as hormones and cytokines are known both to stimulate and to inhibit apoptosis through interactions with cell surface receptors. Transcription factors also play an important role in the onset of apoptosis. A number of downstream effector molecules, particularly proteases such as the cysteine proteases called caspases, have been implicated in the degradation of cellular components and the proteolytic activation of other apoptotic effectors. 



  Aging and Senescence 
Studies of the aging process or senescence have shown a number of characteristic cellular and molecular changes (Fauci et al. (1998) Harrison's Principles of Internal Medicine, McGraw-Hill, New 

 <Desc/Clms Page number 22> 

 York NY, p. 37). These characteristics include increases in chromosome structural abnormalities, DNA cross-linking, incidence of single-stranded breaks in DNA, losses in DNA methylation, and degradation of telomere regions. In addition to these DNA changes,   post-translational   alterations of proteins increase including,   deamidation,   oxidation, cross-linking, and nonenzymatic glycation. Still further molecular changes occur in the mitochondria of aging cells through deterioration of structure. These changes eventually contribute to decreased function in every organ of the body. 



  Biochemical Pathway Molecules 
Biochemical pathways are responsible for regulating metabolism, growth and development, protein secretion and   trafficking,   environmental responses, and ecological interactions including immune response and response to parasites. 



  DNA replication 
Deoxyribonucleic acid (DNA), the genetic material, is found in both the nucleus and mitochondria of human cells. The bulk of human DNA is nuclear, in the form of linear chromosomes, while mitochondrial DNA is circular. DNA replication begins at specific sites called origins of replication. Bidirectional synthesis occurs from the origin via two growing forks that move in opposite directions. Replication is semi-conservative, with each daughter duplex containing one old strand and its newly synthesized complementary partner. Proteins involved in DNA replication include DNA polymerases,   DNA primase,   telomerase, DNA helicase, topoisomerases, DNA ligases, replication factors, and DNA-binding proteins. 



  DNA Recombination and Repair 
Cells are constantly faced with replication errors and environmental assault (such as ultraviolet irradiation) that can produce DNA damage. Damage to DNA consists of any change that modifies the structure of the molecule. Changes to DNA can be divided into two general classes, single base changes and structural distortions. Any damage to DNA can produce a mutation, and the mutation may produce a disorder, such as cancer. 



   Changes in DNA are recognized by repair systems within the cell. These repair systems act to correct the damage and thus prevent any deleterious affects of a mutational event. Repair systems can be divided into three general types, direct repair, excision repair, and retrieval systems. Proteins involved in DNA repair include DNA polymerase, excision repair proteins, excision and cross link repair proteins, recombination and repair proteins, RAD51 proteins, and BLN and WRN proteins that are homologs of RecQ helicase. When the repair systems are eliminated, cells become exceedingly sensitive to environmental mutagens, such as ultraviolet irradiation. Patients with disorders associated with a loss in DNA repair systems often exhibit a high sensitivity to environmental mutagens. 

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  Examples of such disorders include xeroderma pigmentosum (XP), Bloom's syndrome (BS), and Werner's syndrome (WS) (Yamagata, K. et al. (1998) Proc. Natl. Acad. Sci. USA 95 : 8733-8738),   ataxiatelangiectasia,   Cockayne's syndrome,   andFanconi's anemia.   



   Recombination is the process whereby new DNA sequences are generated by the movements of large pieces of DNA. In homologous recombination, which occurs during meiosis and DNA repair, parent DNA duplexes align at regions of sequence similarity, and new DNA molecules form by the breakage and joining of homologous segments. Proteins involved include RAD51 recombinase. In site- specific recombination, two specific but not necessarily homologous DNA sequences are exchanged. In the immune system this process generates a diverse collection of antibody and T cell receptor genes. 



  Proteins involved in site-specific recombination in the immune system include recombination activating genes 1 and 2 (RAG1 and RAG2). A defect in immune system site-specific recombination causes severe combined immunodeficiency disease in mice. 



  RNA Metabolism 
Ribonucleic acid (RNA) is a linear single-stranded polymer of four nucleotides, ATP, CTP, UTP, and GTP. In most organisms, RNA is transcribed as a copy of DNA, the genetic material of the organism. In retroviruses RNA rather than DNA serves as the genetic material. RNA copies of the genetic material encode proteins or serve various structural, catalytic, or regulatory roles in organisms. 



  RNA is classified according to its cellular localization and function. Messenger RNAs (mRNAs) encode polypeptides. Ribosomal RNAs (rRNAs) are assembled, along with ribosomal proteins, into ribosomes, which are cytoplasmic particles that translate mRNA into polypeptides. Transfer RNAs (tRNAs) are cytosolic adaptor molecules that function in mRNA translation by recognizing both an mRNA codon and the amino acid that matches that codon. Heterogeneous nuclear RNAs (hnRNAs) include mRNA precursors and other nuclear RNAs of various sizes. Small nuclear RNAs (snRNAs) are a part of the nuclear spliceosome complex that removes intervening, non-coding sequences (introns) and rejoins exons in pre-mRNAs. 



  RNA Transcription 
The transcription process synthesizes an RNA copy of DNA. Proteins involved include multi- subunit RNA polymerases, transcription factors IIA, IIB, IID, IIE, IIF, IIH, and IIJ. Many transcription factors incorporate DNA-binding structural motifs which comprise either a-helices or    p-   sheets that bind to the major groove of DNA. Four well-characterized structural motifs are helix-turn- helix, zinc finger, leucine zipper, and helix-loop-helix. 



  RNA Processing 
Various proteins are necessary for processing of transcribed RNAs in the nucleus. Pre-mRNA processing steps include capping at the 5'end with methylguanosine, polyadenylating the 3'end, and 

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 splicing to remove introns. The   spliceosomal   complex is comprised of five small nuclear ribonucleoprotein particles (snRNPs) designated   Ul,   U2, U4, U5, and U6. Each snRNP contains a single species of snRNA and about ten proteins. The RNA components of some snRNPs recognize and base-pair with intron consensus sequences. The protein components mediate spliceosome assembly and the splicing reaction. Autoantibodies to snRNP proteins are found in the blood of patients with systemic lupus erythematosus (Stryer, L. (1995) Biochemistry W. H. Freeman and Company, New York NY, p. 863). 



   Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified that have roles in splicing, exporting of the mature RNAs to the cytoplasm, and mRNA translation (Biamonti, G. et al. 



  (1998) Clin. Exp.   Rheumatol.   16 : 317-326). Some examples of   hnRNPs   include the yeast proteins Hrplp, involved in cleavage and polyadenylation at the 3'end of the RNA ;   Cbp80p,   involved in capping the 5'end of the RNA ; and Npl3p, a homolog of mammalian hnRNP Al, involved in export of mRNA from the nucleus (Shen, E. C. et al. (1998) Genes Dev. 12 : 679-691). HnRNPs have been shown to be important targets of the autoimmune response in rheumatic diseases (Biamonti, supra). 



   Many snRNP proteins, hnRNP proteins, and alternative splicing factors are characterized by an RNA recognition motif (RRM). (Reviewed in Birney, E. et al. (1993) Nucleic Acids Res. 21 : 5803-   5816.)   The RRM is about 80 amino acids in length and forms four p-strands and two a-helices arranged in an   a/ (3 sandwich.   The RRM contains a core RNP-1 octapeptide motif along with surrounding conserved sequences. 



  RNA Stabilitv and Degradation 
RNA helicases alter and regulate RNA conformation and secondary structure by using energy derived from ATP hydrolysis to destabilize and unwind RNA duplexes. The most well-characterized and ubiquitous family of RNA helicases is the DEAD-box family, so named for the conserved B-type ATP-binding motif which is diagnostic of proteins in this family. Over 40 DEAD-box helicases have been identified in organisms as diverse as bacteria, insects, yeast, amphibians, mammals, and plants. 



  DEAD-box helicases function in diverse processes such as translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability. Some DEAD-box helicases play tissue-and stage- specific roles in spermatogenesis and embryogenesis. (Reviewed in Linder, P. et al. (1989) Nature 337 : 121-122.) 
Overexpression of the DEAD-box 1 protein   (DDX1)   may play a role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb) tumors. Other DEAD-box helicases have been implicated either directly or indirectly in ultraviolet light-induced tumors, B cell lymphoma, and myeloid malignancies. (Reviewed in Godbout, R. et al. (1998) J. Biol. Chem. 273 : 21161-21168.)   Ribonucleases   (RNases) catalyze the hydrolysis of phosphodiester bonds in RNA chains, thus 

 <Desc/Clms Page number 25> 

 cleaving the RNA.

   For example, RNase P is a ribonucleoprotein enzyme which cleaves the 5'end of pre-tRNAs as part of their maturation process. RNase H digests the RNA strand of an RNA/DNA hybrid. Such hybrids occur in cells invaded by retroviruses, and RNase H is an important enzyme in the retroviral replication cycle. RNase H domains are often found as a domain associated with reverse transcriptases. RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases (Schein, C. H. (1997) Nat. Biotechnol. 15 : 529-536). Regulation of RNase activity is being investigated as a means to control tumor angiogenesis, allergic reactions, viral infection and replication, and fungal infections. 



  Protein Translation 
The eukaryotic ribosome is composed of a 60S (large) subunit and a 40S (small) subunit, which together form the 80S ribosome. In addition to the 18S, 28S, 5S, and 5. 8S rRNAs, the ribosome also contains more than fifty proteins. The ribosomal proteins have a prefix which denotes the subunit to which they belong, either L (large) or S (small). Three important sites are identified on the ribosome. 



  The   aminoacyl-tRNA   site (A site) is where charged tRNAs (with the exception of the initiator-tRNA) bind on arrival at the ribosome. The peptidyl-tRNA site (P site) is where new peptide bonds are formed, as well as where the initiator tRNA binds. The exit site (E site) is where deacylated tRNAs bind prior to their release from the ribosome. (Translation is reviewed in Stryer, L. (1995) Biochemistry, W. H. Freeman and Company, New York NY, pp. 875-908 ; and Lodish, H. et al. (1995) Molecular Cell   Biology.   Scientific American Books, New York NY, pp. 119-138.) tRNA Charging 
Protein biosynthesis depends on each amino acid forming a linkage with the appropriate tRNA. 



  The   aminoacyl-tRNA   synthetases are responsible for the activation and correct attachment of an amino acid with its cognate tRNA. The 20   aminoacyl-tRNA   synthetase enzymes can be divided into two structural classes, Class I and Class II. Autoantibodies against   aminoacyl-tRNAs   are generated by patients with dermatomyositis and polymyositis, and correlate strongly with complicating interstitial lung disease (ILD). These antibodies appear to be generated in response to viral infection, and coxsackie virus has been used to induce experimental viral myositis in animals. 



  Translation Initiation 
Initiation of translation can be divided into three stages. The first stage brings an initiator transfer RNA (Met-tRNAf) together with the 40S ribosomal subunit to form the 43S preinitiation complex. The second stage binds the 43S preinitiation complex to the mRNA, followed by migration of the complex to the correct AUG initiation codon. The third stage brings the 60S ribosomal subunit to the 40S subunit to generate an 80S ribosome at the initiation codon. Regulation of translation primarily involves the first and second stage in the initiation process (Pain, V. M. (1996) Eur. J. Biochem. 

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  236 : 747-771). 



   Several initiation factors, many of which contain multiple subunits, are involved in bringing an initiator tRNA and 40S ribosomal subunit together. eIF2, a guanine nucleotide binding protein, recruits the initiator tRNA to the 40S ribosomal subunit. Only when eIF2 is bound to GTP does it associate with the initiator tRNA.   eIF2B,   a guanine nucleotide exchange protein, is responsible for converting eIF2 from the GDP-bound inactive form to the GTP-bound active form. Two other factors, eIFlA and eIF3 bind and stabilize the 40S subunit by interacting with 18S ribosomal RNA and specific ribosomal structural proteins. eIF3 is also involved in association of the 40S ribosomal subunit with mRNA. The Met-tRNAf,   eIFlA,   eIF3, and 40S ribosomal subunit together make up the 43S preinitiation complex (Pain, supra). 



   Additional factors are required for binding of the 43S preinitiation complex to an mRNA molecule, and the process is regulated at several levels. eIF4F is a complex consisting of three proteins : eIF4E, eIF4A, and eIF4G.   eIF4E   recognizes and binds to the mRNA   5'-terminal m'GTP   cap,   eIF4A   is a bidirectional RNA-dependent helicase, and   eIF4G   is a scaffolding polypeptide. eIF4G has three binding domains. The N-terminal third of   eIF4G   interacts with eIF4E, the central third interacts with eIF4A, and the C-terminal third interacts with eIF3 bound to the 43S preinitiation complex. Thus, eIF4G acts as a bridge between the 40S ribosomal subunit and the mRNA (Hentze, M. W. (1997) Science 275 : 500-501). 



   The ability of   eIF4F   to initiate binding of the 43S preinitiation complex is regulated by structural features of the mRNA. The mRNA molecule has an untranslated region (UTR) between the 5'cap and the AUG start codon. In some mRNAs this region forms secondary structures that impede binding of the 43S preinitiation complex. The helicase activity of eIF4A is thought to function in removing this secondary structure to facilitate binding of the 43S preinitiation complex (Pain, supra). 



  Translation Elongation 
Elongation is the process whereby additional amino acids are joined to the initiator methionine to form the complete polypeptide chain. The elongation factors   EFl a, EFl (3 y,   and EF2 are involved in elongating the polypeptide chain following initiation. EFla is a GTP-binding protein. In   EFla's   GTP-bound form, it brings an aminoacyl-tRNA to the ribosome's A site. The amino acid attached to the newly arrived   aminoacyl-tRNA   forms a peptide bond with the initiator methionine. The GTP on   EFla is hydrolyzed to   GDP, and   EFla-GDP   dissociates from the ribosome.   EFlpybindsEFla-GDP   and induces the dissociation of GDP from EFla, allowing EFla to bind GTP and a new cycle to begin. 



   As subsequent aminoacyl-tRNAs are brought to the ribosome, EF-G, another GTP-binding protein, catalyzes the translocation of tRNAs from the A site to the P site and finally to the E site of the ribosome. This allows the processivity of translation. 

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  Translation Termination 
The release factor eRF carries out termination of translation. eRF recognizes stop codons in the mRNA, leading to the release of the polypeptide chain from the ribosome. 



  Post-Translational Pathwavs 
Proteins may be modified after translation by the addition of phosphate, sugar, prenyl, fatty acid, and other chemical groups. These modifications are often required for proper protein activity. 



  Enzymes involved in post-translational modification include kinases, phosphatases, glycosyltransferases, and prenyltransferases. The conformation of proteins may also be modified after translation by the introduction and rearrangement of disulfide bonds (rearrangement catalyzed by protein disulfide isomerase), the isomerization of proline sidechains by prolyl isomerase, and by interactions with molecular chaperone proteins. 



   Proteins may also be cleaved by proteases. Such cleavage may result in activation, inactivation, or complete degradation of the protein. Proteases include serine proteases, cysteine proteases, aspartic proteases, and metalloproteases. Signal peptidase in the endoplasmic reticulum (ER) lumen cleaves the signal peptide from membrane or secretory proteins that are imported into the ER. Ubiquitin proteases are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaryotic cells and some bacteria. The UCS mediates the elimination of abnormal proteins and regulates the half-lives of important regulatory proteins that control cellular processes such as gene transcription and cell cycle progression. In the UCS pathway, proteins targeted for degradation are conjugated to a ubiquitin, a small heat stable protein.

   Proteins involved in the UCS include ubiquitin-activating enzyme, ubiquitin-conjugating enzymes, ubiquitin-ligases, and ubiquitin C-terminal hydrolases. The ubiquitinated protein is then recognized and degraded by the proteasome, a large, multisubunit proteolytic enzyme complex, and ubiquitin is released for reutilization by ubiquitin protease. 



  Lipid Metabolism 
Lipids are water-insoluble, oily or greasy substances that are soluble in nonpolar solvents such as chloroform or ether. Neutral fats (triacylglycerols) serve as major fuels and energy stores. Polar lipids, such as phospholipids,   sphingolipids,   glycolipids, and cholesterol, are key structural components of cell membranes. 



   Lipid metabolism is involved in human diseases and disorders. In the arterial disease atherosclerosis, fatty lesions form on the inside of the arterial wall. These lesions promote the loss of arterial flexibility and the formation of blood clots (Guyton, A. C. Textbook of Medical Physiology (1991) W. B. Saunders Company, Philadelphia PA, pp. 760-763). In Tay-Sachs disease, the   GM2   ganglioside (a sphingolipid) accumulates in lysosomes of the central nervous system due to a lack of the 

 <Desc/Clms Page number 28> 

 enzyme   N-acetylhexosaminidase.   Patients suffer nervous system degeneration leading to early death (Fauci, A. S. et al. (1998) Harrison's Principles of Internal Medicine McGraw-Hill, New York NY, p. 



    2171). TheNiemann-Pickdiseases arecausedby   defects in lipid metabolism. Niemann-Pick diseases types A and B are caused by accumulation of sphingomyelin (a   sphingolipid)   and other lipids in the central nervous system due to a defect in the enzyme   sphingomyelinase,   leading to neurodegeneration and lung disease. Niemann-Pick disease type C results from a defect in cholesterol transport, leading to the accumulation of sphingomyelin and cholesterol in lysosomes and a secondary reduction in   sphingomyelinase   activity. Neurological symptoms such as grand mal seizures, ataxia, and loss of previously learned speech, manifest 1-2 years after birth.

   A mutation in the NPC protein, which contains a putative cholesterol-sensing domain, was found in a mouse model of Niemann-Pick disease type C (Fauci, supra, p. 2175 ; Loftus, S. K. et al. (1997) Science 277 : 232-235). (Lipid metabolism is reviewed in Stryer, L. (1995)   Biochemistrv,   W. H. Freeman and Company, New York NY ; Lehninger, A. (1982) Principles of Biochemistry Worth Publishers, Inc., New York NY ; and ExPASy "Biochemical Pathways"index of Boehringer Mannheim World Wide Web site.) Fatty Acid Synthesis 
Fatty acids are long-chain organic acids with a single carboxyl group and a long non-polar hydrocarbon tail. Long-chain fatty acids are essential components of glycolipids, phospholipids, and cholesterol, which are building blocks for biological membranes, and of triglycerides, which are biological fuel molecules.

   Long-chain fatty acids are also substrates for eicosanoid production, and are important in the functional modification of certain complex carbohydrates and proteins. 16-carbon and 18-carbon fatty acids are the most common. 



   Fatty acid synthesis occurs in the cytoplasm. In the first step, acetyl-Coenzyme A (CoA) carboxylase (ACC) synthesizes malonyl-CoA from acetyl-CoA and bicarbonate. The enzymes which catalyze the remaining reactions are covalently linked into a single polypeptide chain, referred to as the multifunctional enzyme fatty acid synthase (FAS). FAS catalyzes the synthesis   of palmitate   from acetyl-CoA and malonyl-CoA. FAS contains acetyl transferase, malonyl transferase,   p-ketoacetyl   synthase, acyl carrier protein,   p-ketoacyl   reductase, dehydratase, enoyl reductase, and   thioesterase   activities. The final product of the FAS reaction is the 16-carbon fatty acid palmitate.

   Further elongation, as well as unsaturation, of   palmitate   by accessory enzymes of the ER produces the variety of long chain fatty acids required by the individual cell. These enzymes include a NADH-cytochrome b5 reductase, cytochrome   b5,   and a desaturase. 



  Phospholipid and Triacylglycerol Synthesis 
Triacylglycerols, also known as triglycerides and neutral fats, are major energy stores in animals. Triacylglycerols are esters of glycerol with three fatty acid chains. Glycerol-3-phosphate is 

 <Desc/Clms Page number 29> 

 produced from dihydroxyacetone phosphate by the enzyme glycerol phosphate dehydrogenase or from glycerol by glycerol kinase. Fatty acid-CoA's are produced from fatty acids by fatty acyl-CoA synthetases.   Glyercol-3-phosphate   is acylated with two fatty acyl-CoA's by the enzyme glycerol phosphate acyltransferase to give phosphatidate. Phosphatidate phosphatase converts phosphatidate to diacylglycerol, which is subsequently acylated to a triacylglyercol by the enzyme diglyceride acyltransferase.

   Phosphatidate phosphatase and diglyceride acyltransferase form a triacylglyerol synthetase complex bound to the ER membrane. 



   A major class of phospholipids are the phosphoglycerides, which are composed of a glycerol backbone, two fatty acid chains, and a phosphorylated alcohol. Phosphoglycerides are components of cell membranes. Principal phosphoglycerides are phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, and diphosphatidyl glycerol. Many enzymes involved in phosphoglyceride synthesis are associated with membranes (Meyers, R. A. (1995) Molecular Biology and Biotechnology, VCH Publishers Inc., New York NY, pp. 494-501). Phosphatidate is converted to   CDP-diacylglycerol   by the enzyme phosphatidate cytidylyltransferase (ExPASy ENZYME EC 2. 7. 7. 41).

   Transfer of the diacylglycerol group from CDP-diacylglycerol to serine to yield phosphatidyl serine, or to inositol to yield phosphatidyl inositol, is catalyzed by the enzymes CDP-diacylglycerol- serine   O-phosphatidyltransferase   and   CDP-diacylglycerol-inositol   3-phosphatidyltransferase, respectively (ExPASy ENZYME EC 2. 7. 8. 8 ; ExPASy ENZYME EC 2. 7. 8. 11). The enzyme phosphatidyl serine decarboxylase catalyzes the conversion of phosphatidyl serine to phosphatidyl   ethanolamine,   using a pyruvate cofactor (Voelker, D. R. (1997) Biochim. Biophys. Acta 1348 : 236-244). 



  Phosphatidyl choline is formed using diet-derived choline by the reaction of CDP-choline with 1, 2- diacylglycerol, catalyzed by diacylglycerol cholinephosphotransferase (ExPASy ENZYME 2. 7. 8. 2). 



  Sterol, Steroid, and Isoprenoid Metabolism 
Cholesterol, composed of four fused hydrocarbon rings with an alcohol at one end, moderates the fluidity of membranes in which it is incorporated. In addition, cholesterol is used in the synthesis of steroid hormones such as cortisol, progesterone, estrogen, and testosterone. Bile salts derived from cholesterol facilitate the digestion of lipids. Cholesterol in the skin forms a barrier that prevents excess water evaporation from the body.   Farnesyl   and   geranylgeranyl   groups, which are derived from cholesterol biosynthesis intermediates, are post-translationally added to signal transduction proteins such as ras and protein-targeting proteins such as rab. These modifications are important for the activities of these proteins (Guyton, supra ; Stryer, supra, pp. 279-280, 691-702, 934). 



   Mammals obtain cholesterol derived from both de novo biosynthesis and the diet. The liver is the major site of cholesterol biosynthesis in mammals. Two acetyl-CoA molecules initially condense to form acetoacetyl-CoA, catalyzed by a   thiolase.   Acetoacetyl-CoA condenses with a third acetyl-CoA to 

 <Desc/Clms Page number 30> 

 form   hydroxymethylglutaryl-CoA   (HMG-CoA), catalyzed by HMG-CoA synthase. Conversion of HMG-CoA to cholesterol is accomplished via a series of enzymatic steps known as the mevalonate pathway. The rate-limiting step is the conversion of HMG-CoA to mevalonate by HMG-CoA reductase. The drug lovastatin, a potent inhibitor of HMG-CoA reductase, is given to patients to reduce their serum cholesterol levels.

   Other mevalonate pathway enzymes include mevalonate kinase,   phosphomevalonate   kinase, diphosphomevalonate decarboxylase, isopentenyldiphosphate isomerase, dimethylallyl transferase, geranyl transferase,   farnesyl-diphosphate   farnesyltransferase, squalene monooxygenase, lanosterol synthase, lathosterol oxidase, and 7-dehydrocholesterol reductase. 



   Cholesterol is used in the synthesis of steroid hormones such as cortisol, progesterone, aldosterone, estrogen, and testosterone. First, cholesterol is converted to pregnenolone by cholesterol monooxygenases. The other steroid hormones are synthesized from pregnenolone by a series of enzyme-catalyzed reactions including   oxidations,   isomerizations, hydroxylations, reductions, and demethylations. Examples of these enzymes include steroid A-isomerase,   3p-hydroxy-A5-steroid   dehydrogenase, steroid   21-monooxygenase,   steroid 19-hydroxylase, and   3p-hydroxysteroid   dehydrogenase. Cholesterol is also the precursor to vitamin D. 



   Numerous compounds contain 5-carbon isoprene units derived from the mevalonate pathway intermediate isopentenyl pyrophosphate. Isoprenoid groups are found in vitamin K, ubiquinone, retinal, dolichol phosphate   (a   carrier of oligosaccharides needed for N-linked glycosylation), and   farnesyl   and   geranylgeranyl   groups that modify proteins. Enzymes involved include farnesyl transferase, polyprenyl transferases, dolichyl phosphatase, and dolichyl kinase. 



    Sphingolipid   Metabolism 
Sphingolipids are an important class of membrane lipids that contain   sphingosine,   a long chain amino alcohol. They are composed of one long-chain fatty acid, one polar head alcohol, and   sphingosine   or   sphingosine   derivative. The three classes of   sphingolipids   are   sphingomyelins,   cerebrosides, and gangliosides.   Sphingomyelins,   which contain phosphocholine or   phosphoethanolamine   as their head group, are abundant in the myelin sheath surrounding nerve cells. 



  Galactocerebrosides, which contain a glucose or galactose head group, are characteristic of the brain. 



  Other cerebrosides are found in nonneural tissues.   Gangliosides,   whose head groups contain multiple sugar units, are abundant in the brain, but are also found in nonneural tissues. 



   Sphingolipids are built on a   sphingosine   backbone.   Sphingosine   is acylated to ceramide by the enzyme sphingosine acetyltransferase.   Ceramide   and phosphatidyl choline are converted to sphingomyelin by the enzyme ceramide   choline phosphotransferase.   Cerebrosides are synthesized by the linkage of glucose or galactose to ceramide by a transferase. Sequential addition of sugar residues to ceramide by transferase enzymes yields gangliosides. 

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  Eicosanoid Metabolism 
Eicosanoids, including prostaglandins,   prostacyclin,   thromboxanes, and leukotrienes, are 20- carbon molecules derived from fatty acids. Eicosanoids are signaling molecules which have roles in pain, fever, and inflammation. The precursor of all eicosanoids is   arachidonate,   which is generated from phospholipids by phospholipase   A2   and from diacylglycerols by diacylglycerol lipase. 



  Leukotrienes are produced from   arachidonate   by the action of   lipoxygenases.   Prostaglandin synthase, reductases, and isomerases are responsible for the synthesis of the prostaglandins. Prostaglandins have roles in inflammation, blood flow, ion transport, synaptic transmission, and sleep. Prostacyclin and the thromboxanes are derived from a precursor prostaglandin by the action of prostacyclin synthase and thromboxane synthases, respectively. 



  Ketone Body Metabolism 
Pairs of acetyl-CoA molecules derived from fatty acid oxidation in the liver can condense to form acetoacetyl-CoA, which subsequently forms acetoacetate, D-3-hydroxybutyrate, and acetone. 



  These three products are known as ketone bodies. Enzymes involved in ketone body metabolism include HMG-CoA synthetase, HMG-CoA cleavage enzyme, D-3-hydroxybutyrate dehydrogenase, acetoacetate decarboxylase, and   3-ketoacyl-CoA transferase. Ketone   bodies are a normal fuel supply of the heart and renal cortex. Acetoacetate produced by the liver is transported to cells where the acetoacetate is converted back to acetyl-CoA and enters the citric acid cycle. In times of starvation, ketone bodies produced from stored triacylglyerols become an important fuel source, especially for the brain. Abnormally high levels of ketone bodies are observed in diabetics. Diabetic coma can result if ketone body levels become too great. 



  Lipid Mobilization 
Within cells, fatty acids are transported by cytoplasmic fatty acid binding proteins (Online Mendelian Inheritance in Man (OMIM) *134650 Fatty Acid-Binding Protein 1, Liver ;   FABP1).   



  Diazepam binding inhibitor (DBI), also known as endozepine and acyl CoA-binding protein, is an endogenous   y-aminobutyric   acid (GABA) receptor ligand which is thought to down-regulate the effects of GABA. DBI binds medium-and long-chain acyl-CoA esters with very high affinity and may function as an intracellular carrier of acyl-CoA esters (OMIM *125950 Diazepam Binding Inhibitor ; DBI ; PROSITE PDOC00686 Acyl-CoA-binding protein signature). 



   Fat stored in liver and adipose triglycerides may be released by hydrolysis and transported in the blood. Free fatty acids are transported in the blood by albumin. Triacylglycerols and cholesterol esters in the blood are transported in lipoprotein particles. The particles consist of a core of hydrophobic lipids surrounded by a shell of polar lipids and apolipoproteins. The protein components serve in the solubilization of hydrophobic lipids and also contain cell-targeting signals. Lipoproteins 

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 include chylomicrons,   chylomicron   remnants, very-low-density lipoproteins (VLDL), intermediate- density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). 



  There is a strong inverse correlation between the levels of plasma HDL and risk of premature coronary heart disease. 



   Triacylglycerols in   chylomicrons   and VLDL are hydrolyzed by lipoprotein lipases that line blood vessels in muscle and other tissues that use fatty acids. Cell surface LDL receptors bind LDL particles which are then internalized by endocytosis. Absence of the LDL receptor, the cause of the disease familial hypercholesterolemia, leads to increased plasma cholesterol levels and ultimately to atherosclerosis. Plasma cholesteryl ester transfer protein mediates the transfer of cholesteryl esters from HDL to apolipoprotein B-containing lipoproteins. Cholesteryl ester transfer protein is important in the reverse cholesterol transport system and may play a role in atherosclerosis (Yamashita, S. et al. 



  (1997) Curr. Opin. Lipidol. 8 : 101-110). Macrophage scavenger receptors, which bind and internalize modified lipoproteins, play a role in lipid transport and may contribute to atherosclerosis (Greaves, D. R. et al. (1998) Curr. Opin. Lipidol. 9 : 425-432). 



   Proteins involved in cholesterol uptake and biosynthesis are tightly regulated in response to cellular cholesterol levels. The sterol regulatory element binding protein (SREBP) is a sterol-responsive transcription factor. Under normal cholesterol conditions, SREBP resides in the ER membrane. When cholesterol levels are low, a regulated cleavage of SREBP occurs which releases the extracellular domain of the protein. This cleaved domain is then transported to the nucleus where it activates the transcription of the LDL receptor gene, and genes encoding enzymes of cholesterol synthesis, by binding the sterol regulatory element (SRE) upstream of the genes (Yang, J. et al. (1995) J. Biol. Chem. 



  270 : 12152-12161). Regulation of cholesterol uptake and biosynthesis also occurs via the oxysterol- binding protein (OSBP). OSBP is a high-affinity intracellular receptor for a variety of oxysterols that down-regulate cholesterol synthesis and stimulate cholesterol esterification (Lagace, T. A. et al. (1997) Biochem. J. 326 : 205-213). 



  Beta-oxidation 
Mitochondrial and peroxisomal beta-oxidation enzymes degrade saturated and unsaturated fatty acids by sequential removal of two-carbon units from CoA-activated fatty acids. The main beta- oxidation pathway degrades both saturated and unsaturated fatty acids while the auxiliary pathway performs additional steps required for the degradation of unsaturated fatty acids. 



   The pathways of mitochondrial and peroxisomal beta-oxidation use similar enzymes, but have different substrate specificities and functions. Mitochondria oxidize short-, medium-, and long-chain fatty acids to produce energy for cells. Mitochondrial beta-oxidation is a major energy source for cardiac and skeletal muscle. In liver, it provides ketone bodies to the peripheral circulation when 

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 glucose levels are low as in starvation, endurance exercise, and diabetes (Eaton, S. et al. (1996) Biochem. J. 320 : 345-357). Peroxisomes oxidize medium-, long-, and very-long-chain fatty acids, dicarboxylic fatty acids, branched fatty acids, prostaglandins, xenobiotics, and bile acid intermediates. 



  The chief roles of peroxisomal beta-oxidation are to shorten toxic lipophilic carboxylic acids to facilitate their excretion and to shorten very-long-chain fatty acids prior to mitochondrial beta-oxidation (Mannaerts, G. P. and P. P. van Veldhoven (1993) Biochimie 75 :   147-158).   



   Enzymes involved in beta-oxidation include acyl CoA synthetase, carnitine acyltransferase, acyl CoA dehydrogenases, enoyl CoA hydratases, L-3-hydroxyacyl CoA dehydrogenase,   p-ketothiolase,   2, 4-dienoyl CoA reductase, and isomerase. 



  Lipid Cleavage and Degradation 
Triglycerides are hydrolyzed to fatty acids and glycerol by lipases. Lysophospholipases (LPLs) are widely distributed enzymes that metabolize intracellular lipids, and occur in numerous isoforms. Small isoforms, approximately 15-30 kD, function as hydrolases ; large isoforms, those exceeding 60 kD, function both as hydrolases and transacylases. A particular substrate for   LPLs,   lysophosphatidylcholine, causes lysis of cell membranes when it is formed or imported into a cell. 



  LPLs are regulated by lipid factors including   acylcarnitine,   arachidonic acid, and phosphatidic acid. 



  These lipid factors are signaling molecules important in numerous pathways, including the inflammatory response. (Anderson, R. et al. (1994) Toxicol. Appl. Pharmacol. 125 : 176-183 ; Selle, H. et al. (1993) ; Eur. J. Biochem. 212 : 411-416.) 
The secretory phospholipase   A2   (PLA2) superfamily comprises a number of heterogeneous enzymes whose common feature is to hydrolyze the sn-2 fatty acid acyl ester bond of phosphoglycerides. Hydrolysis of the glycerophospholipids releases free fatty acids and lysophospholipids. PLA2 activity generates precursors for the biosynthesis of biologically active lipids, hydroxy fatty acids, and platelet-activating factor. PLA2 hydrolysis of the sn-2 ester bond in phospholipids generates free fatty acids, such as   arachidonic   acid and lysophospholipids. 



  Carbon and Carbohydrate Metabolism 
Carbohydrates, including sugars or saccharides, starch, and cellulose, are aldehyde or ketone compounds with multiple hydroxyl groups. The importance of carbohydrate metabolism is demonstrated by the sensitive regulatory system in place for maintenance of blood glucose levels. Two pancreatic hormones, insulin and glucagon, promote increased glucose uptake and storage by cells, and increased glucose release from cells, respectively. Carbohydrates have three important roles in mammalian cells. First, carbohydrates are used as energy stores, fuels, and metabolic intermediates. 



  Carbohydrates are broken down to form energy in glycolysis and are stored as glycogen for later use. 



  Second, the sugars deoxyribose and ribose form part of the structural support of DNA and RNA, 

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 respectively. Third, carbohydrate modifications are added to secreted and membrane proteins and lipids as they traverse the secretory pathway. Cell surface carbohydrate-containing macromolecules, including glycoproteins, glycolipids, and transmembrane proteoglycans, mediate adhesion with other cells and with components of the extracellular matrix. The extracellular matrix is comprised of diverse glycoproteins,   glycosaminoglycans   (GAGs), and carbohydrate-binding proteins which are secreted from the cell and assembled into an organized meshwork in close association with the cell surface. The interaction of the cell with the surrounding matrix profoundly influences cell shape, strength, flexibility, motility, and adhesion.

   These dynamic properties are intimately associated with signal transduction pathways controlling cell proliferation and differentiation, tissue construction, and embryonic development. 



   Carbohydrate metabolism is altered in several disorders including diabetes mellitus,   hyperglycemia,   hypoglycemia, galactosemia, galactokinase deficiency, and UDP-galactose-4-epimerase deficiency (Fauci, A. S. et al. (1998) Harrison's Principles of Internal Medicine, McGraw-Hill, New York NY, pp. 2208-2209). Altered carbohydrate metabolism is associated with cancer. Reduced GAG and proteoglycan expression is associated with human lung carcinomas (Nackaerts, K. et al. (1997) Int. 



  J. Cancer 74 : 335-345). The carbohydrate determinants sialyl Lewis A and sialyl Lewis X are frequently expressed on human cancer cells (Kannagi, R. (1997) Glycoconj. J. 14 : 577-584). 



  Alterations of the N-linked carbohydrate core structure of cell surface glycoproteins are linked to colon and pancreatic cancers (Schwarz, R. E. et al. (1996) Cancer Lett. 107 : 285-291). Reduced expression of the Sda blood group carbohydrate structure in cell surface glycolipids and glycoproteins is observed in gastrointestinal cancer (Dohi, T. et al. (1996) Int. J. Cancer 67 : 626-663). (Carbon and carbohydrate metabolism is reviewed in Stryer, L. (1995)   Biochemistrv   W. H. Freeman and Company, New York NY ; Lehninger,   A. L.   (1982) Principles of Biochemistry Worth Publishers Inc., New York NY ; and Lodish, H. et al. (1995) Molecular Cell Biology Scientific American Books, New York NY.)   Glycolysis   
Enzymes of the glycolytic pathway convert the sugar glucose to pyruvate while simultaneously producing ATP.

   The pathway also provides building blocks for the synthesis of cellular components such as long-chain fatty acids. After glycolysis, pyrvuate is converted to acetyl-Coenzyme A, which, in aerobic organisms, enters the citric acid cycle. Glycolytic enzymes include hexokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase, enolase, and pyruvate kinase. Of these, phosphofructokinase, hexokinase, and pyruvate kinase are important in regulating the rate of glycolysis. 



    Gluconeogenesis   

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Gluconeogenesis is the synthesis of glucose from noncarbohydrate precursors such as lactate and amino acids. The pathway, which functions mainly in times of starvation and intense exercise, occurs mostly in the liver and kidney. Responsible enzymes include pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose 1, 6-bisphosphatase, and glucose-6-phosphatase. 



  Pentose Phosphate   Pathwav   
Pentose phosphate pathway enzymes are responsible for generating the reducing agent NADPH, while at the same time oxidizing glucose-6-phosphate to ribose-5-phosphate. Ribose-5- phosphate and its derivatives become part of important biological molecules such as ATP, Coenzyme A, NAD+, FAD, RNA, and DNA. The pentose phosphate pathway has both oxidative and non- oxidative branches. The oxidative branch steps, which are catalyzed by the enzymes glucose-6- phosphate dehydrogenase, lactonase, and 6-phosphogluconate dehydrogenase, convert glucose-6- phosphate and NADP+ to ribulose-6-phosphate and NADPH. The non-oxidative branch steps, which are catalyzed by the enzymes phosphopentose isomerase, phosphopentose epimerase, transketolase, and transaldolase, allow the interconversion of three-, four-, five-, six-, and seven-carbon sugars. 



    Glucouronate   Metabolism 
Glucuronate is a monosaccharide which, in the form of D-glucuronic acid, is found in the GAGs chondroitin and dermatan. D-glucuronic acid is also important in the detoxification and excretion of foreign organic compounds such as phenol. Enzymes involved in glucuronate metabolism include UDP-glucose dehydrogenase and glucuronate reductase. 



  Disaccharide Metabolism 
Disaccharides must be hydrolyzed to monosaccharides to be digested. Lactose, a disaccharide found in milk, is hydrolyzed to galactose and glucose by the enzyme lactase. Maltose is derived from plant starch and is hydrolyzed to glucose by the enzyme maltase. Sucrose is derived from plants and is hydrolyzed to glucose and fructose by the enzyme sucrase. Trehalose, a disaccharide found mainly in insects and mushrooms, is hydrolyzed to glucose by the enzyme   trehalase   (OMIM   *275360   Trehalase ; Ruf, J. et al. (1990) J. Biol. Chem. 265 : 15034-15039). Lactase, maltase, sucrase,   andtrehalase   are bound to mucosal cells lining the small intestine, where they participate in the digestion of dietary disaccharides.

   The enzyme lactose synthetase, composed of the catalytic subunit galactosyltransferase and the modifier subunit   a-lactalbumin,   converts UDP-galactose and glucose to lactose in the mammary glands. 



  Glycogen, Starch, and Chitin Metabolism 
Glycogen is the storage form of carbohydrates in mammals. Mobilization of glycogen maintains glucose levels between meals and during muscular activity. Glycogen is stored mainly in the liver and in skeletal muscle in the form of cytoplasmic granules. These granules contain enzymes that 

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 catalyze the synthesis and degradation of glycogen, as well as enzymes that regulate these processes. 



  Enzymes that catalyze the degradation of glycogen include glycogen phosphorylase, a transferase,   a-   1, 6-glucosidase, and phosphoglucomutase. Enzymes that catalyze the synthesis of glycogen include UDP-glucose pyrophosphorylase, glycogen synthetase, a branching enzyme, and nucleoside diphosphokinase. The enzymes of glycogen synthesis and degradation are tightly regulated by the hormones insulin, glucagon, and epinephrine. Starch, a plant-derived polysaccharide, is hydrolyzed to maltose, maltotriose, and   a-dextrin by a-amylase,   an enzyme secreted by the salivary glands and pancreas. Chitin is a polysaccharide found in insects and crustacea. A   chitotriosidase   is secreted by macrophages and may play a role in the degradation of chitin-containing pathogens (Boot, R. G. et al. 



  (1995) J. Biol. Chem. 270 : 26252-26256). 



    Peptidoslvcans   and Glycosaminoglycans   Glycosaminoglycans   (GAGs) are anionic linear unbranched polysaccharides composed of repetitive disaccharide units. These repetitive units contain a derivative of an amino sugar, either   glucosamine   or   galactosamine.   GAGs exist free or as part of proteoglycans, large molecules composed of a core protein attached to one or more GAGs. GAGs are found on the cell surface, inside cells, and in the extracellular matrix. Changes in GAG levels are associated with several autoimmune diseases including autoimmune thyroid disease, autoimmune diabetes mellitus, and systemic lupus erythematosus (Hansen, C. et al. (1996) Clin. Exp.

   Rheum. 14 (Suppl. 15) :   S59-S67).   GAGs include chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, dermatan sulfate, and   hyaluronan.   



   The GAG hyaluronan (HA) is found in the extracellular matrix of many cells, especially in soft connective tissues, and is abundant in synovial fluid (Pitsillides, A. A. et al. (1993) Int. J. Exp. Pathol. 



  74 : 27-34). HA seems to play important roles in cell regulation, development, and differentiation (Laurent, T. C. and J. R.   Fraser   (1992) FASEB J. 6 : 2397-2404). Hyaluronidase is an enzyme that degrades HA to oligosaccharides. Hyaluronidases may function in cell adhesion, infection, angiogenesis, signal transduction, reproduction, cancer, and inflammation. 



   Proteoglycans, also known as peptidoglycans, are found in the extracellular matrix of connective tissues such as cartilage and are essential for distributing the load in weight-bearing joints. 



  Cell-surface-attached proteoglycans anchor cells to the extracellular matrix. Both extracellular and cell-surface proteoglycans bind growth factors, facilitating their binding to cell-surface receptors and subsequent triggering of signal transduction pathways. 



  Amino Acid and Nitrogen Metabolism   NH4+ is   assimilated into amino acids by the actions of two enzymes, glutamate dehydrogenase and glutamine synthetase. The carbon skeletons of amino acids come from the intermediates of glycolysis, the pentose phosphate pathway, or the citric acid cycle. Of the twenty 

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 amino acids used in proteins, humans can synthesize only thirteen (nonessential amino acids). The remaining nine must come from the diet (essential amino acids).

   Enzymes involved in nonessential amino acid biosynthesis include glutamate kinase dehydrogenase, pyrroline carboxylate reductase, asparagine synthetase, phenylalanine oxygenase, methionine adenosyltransferase, adenosylhomocysteinase,   cystathionine P-synthase,   cystathionine y-lyase, phosphoglycerate dehydrogenase, phosphoserine transaminase, phosphoserine phosphatase, serine hydroxylmethyltransferase, and glycine synthase. 



   Metabolism of amino acids takes place almost entirely in the liver, where the amino group is removed by   aminotransferases   (transaminases), for example, alanine aminotransferase. The amino group is transferred to   a-ketoglutarate   to form glutamate. Glutamate dehydrogenase converts glutamate to NH4+ and   o-ketoglutarate.   NH4+ is converted to urea by the urea cycle which is catalyzed by the enzymes arginase, ornithine transcarbamoylase, arginosuccinate synthetase, and arginosuccinase. Carbamoyl phosphate synthetase is also involved in urea formation.

   Enzymes involved in the metabolism of the carbon skeleton of amino acids include serine   dehydratase,   asparaginase, glutaminase, propionyl CoA carboxylase, methylmalonyl CoA mutase, branched-chain a-keto dehydrogenase complex, isovaleryl CoA dehydrogenase, (3-methylcrotonyl CoA carboxylase, phenylalanine hydroxylase,   p-hydroxylphenylpyruvate   hydroxylase, and homogentisate oxidase. 



   Polyamines, which include spermidine, putrescine, and spermine, bind tightly to nucleic acids and are abundant in rapidly proliferating cells. Enzymes involved in polyamine synthesis include ornithine decarboxylase. 



   Diseases involved in amino acid and nitrogen metabolism include hyperammonemia, carbamoyl phosphate synthetase deficiency, urea cycle enzyme deficiencies,   methylmalonic   aciduria, maple syrup disease, alcaptonuria, and phenylketonuria. 



  Energy Metabolism 
Cells derive energy from metabolism of ingested compounds that may be roughly categorized as carbohydrates, fats, or proteins. Energy is also stored in polymers such as triglycerides (fats) and glycogen (carbohydrates). Metabolism proceeds along separate reaction pathways connected by key intermediates such as acetyl coenzyme A (acetyl-CoA). Metabolic pathways feature anaerobic and aerobic degradation, coupled with the energy-requiring reactions such as phosphorylation of adenosine diphosphate (ADP) to the triphosphate (ATP) or analogous phosphorylations of guanosine   (GDP/GTP),   uridine   (UDPIUTP),   or cytidine (CDP/CTP). Subsequent dephosphorylation of the triphosphate drives reactions needed for cell maintenance, growth, and proliferation. 



   Digestive enzymes convert carbohydrates and sugars to glucose ; fructose and galactose are converted in the liver to glucose. Enzymes involved in these conversions include galactose-1- 

 <Desc/Clms Page number 38> 

 phosphate uridyl transferase and UDP-galactose-4 epimerase. In the cytoplasm, glycolysis converts glucose to pyruvate in a series of reactions coupled to ATP synthesis. 



   Pyruvate is transported into the mitochondria and converted to acetyl-CoA for oxidation via the citric acid cycle, involving pyruvate dehydrogenase components, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Enzymes involved in the citric acid cycle include : citrate synthetase, aconitases, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex including transsuccinylases, succinyl CoA synthetase, succinate dehydrogenase, fumarases, and malate dehydrogenase. Acetyl CoA is oxidized to   CO2   with concomitant formation of NADH, FADH2, and GTP. In oxidative phosphorylation, the transport of electrons from NADH and FADH2 to oxygen by dehydrogenases is coupled to the synthesis of ATP from ADP and Pi by the For ATPase complex in the mitochondrial inner membrane.

  *  Enzyme complexes responsible for electron transport and ATP synthesis include the FoFI ATPase complex, ubiquinone   (CoQ)-cytochrome   c reductase, ubiquinone reductase, cytochrome b, cytochrome   cl,   FeS protein, and cytochrome c oxidase. 



   Triglycerides are hydrolyzed to fatty acids and glycerol by lipases. Glycerol is then phosphorylated to glycerol-3-phosphate by glycerol kinase and glycerol phosphate dehydrogenase, and degraded by the glycolysis. Fatty acids are transported into the mitochondria as fatty acyl- carnitine esters and undergo oxidative degradation. 



   In addition to metabolic disorders such as diabetes and obesity, disorders of energy metabolism are associated with cancers (Dorward, A. et al. (1997) J.   Bioenerg.   Biomembr. 29 : 385- 392), autism (Lombard, J. (1998) Med. Hypotheses 50 : 497-500), neurodegenerative disorders   (Alexi,   T. et al. (1998) Neuroreport 9 : R57-64), and neuromuscular disorders (DiMauro, S. et al. (1998) Biochim. Biophys. Acta 1366 : 199-210). The myocardium is heavily dependent on oxidative metabolism, so metabolic dysfunction often leads to heart disease (DiMauro, S. and M. Hirano (1998) Curr. Opin. Cardiol. 13 : 190-197). 



   For a review of energy metabolism enzymes and intermediates, see Stryer, L. et al. (1995)   Biochemistrv,   W. H. Freeman and Co., San Francisco CA, pp. 443-652. For a review of energy metabolism regulation, see Lodish, H. et al. (1995) Molecular Cell Biology, Scientific American Books, New York NY, pp. 744-770. 



  Cofactor Metabolism 
Cofactors, including coenzymes and prosthetic groups, are small molecular weight inorganic or organic compounds that are required for the action of an enzyme. Many cofactors contain vitamins as a component. Cofactors include thiamine pyrophosphate, flavin adenine dinucleotide, flavin mononucleotide, nicotinamide adenine dinucleotide, pyridoxal phosphate, coenzyme A, tetrahydrofolate,   lipoamide,   and heme. The vitamins biotin and   cobalamin   are associated with 

 <Desc/Clms Page number 39> 

 enzymes as well. Heme, a prosthetic group found in myoglobin and hemoglobin, consists of protoporphyrin group bound to iron. Porphyrin groups contain four substituted pyrroles covalently joined in a ring, often with a bound metal atom.

   Enzymes involved in porphyrin synthesis include    5-   aminolevulinate synthase,   8-aminolevulinate   dehydrase, porphobilinogen deaminase, and cosynthase. 



  Deficiencies in heme formation cause porphyrias. Heme is broken down as a part of erythrocyte turnover. Enzymes involved in heme degradation include heme oxygenase and biliverdin reductase. 



   Iron is a required cofactor for many enzymes. Besides the heme-containing enzymes, iron is found in iron-sulfur clusters in proteins including aconitase, succinate dehydrogenase, and NADH-Q reductase. Iron is transported in the blood by the protein transferrin. Binding of transferrin to the transferrin receptor on cell surfaces allows uptake by receptor mediated endocytosis. Cytosolic iron is bound to ferritin protein. 



   A molybdenum-containing cofactor (molybdopterin) is found in enzymes including sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Molybdopterin biosynthesis is performed by two molybdenum cofactor synthesizing enzymes. Deficiencies in these enzymes cause mental retardation and lens dislocation. Other diseases caused by defects in cofactor metabolism include pernicious anemia and methylmalonic aciduria. 



  Secretion and Trafficking 
Eukaryotic cells are bound by a lipid bilayer membrane and subdivided into functionally distinct, membrane bound compartments. The membranes maintain the essential differences between the cytosol, the extracellular environment, and the lumenal space of each intracellular organelle. As lipid membranes are highly impermeable to most polar molecules, transport of essential nutrients, metabolic waste products, cell signaling molecules, macromolecules and proteins across lipid membranes and between organelles must be mediated by a variety of transport-associated molecules. 



  Protein Traffickin 
In eukaryotes, some proteins are synthesized on ER-bound ribosomes, co-translationally imported into the ER, delivered from the ER to the Golgi complex for post-translational processing and sorting, and transported from the Golgi to specific intracellular and extracellular destinations. All cells possess a constitutive transport process which maintains homeostasis between the cell and its environment. In many differentiated cell types, the basic machinery is modified to carry out specific transport functions. For example, in endocrine glands, hormones and other secreted proteins are packaged into secretory granules for regulated exocytosis to the cell exterior. In macrophage, foreign extracellular material is engulfed (phagocytosis) and delivered to lysosomes for degradation.

   In fat and muscle cells, glucose transporters are stored in vesicles which fuse with the plasma membrane only in response to insulin stimulation. 

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  The Secretorv   Pathwav   
Synthesis of most integral membrane proteins, secreted proteins, and proteins destined for the lumen of a particular organelle occurs on ER-bound ribosomes. These proteins are co-translationally imported into the ER. The proteins leave the ER via membrane-bound vesicles which bud off the ER at specific sites and fuse with each other (homotypic fusion) to form the ER-Golgi Intermediate Compartment (ERGIC). The ERGIC matures progressively through the cis,   niedial, and trans cisternal   stacks of the Golgi, modifying the enzyme composition by retrograde transport of specific Golgi enzymes. In this way, proteins moving through the Golgi undergo post-translational modification, such as glycosylation.

   The final Golgi compartment is the Trans-Golgi Network (TGN), where both membrane and lumenal proteins are sorted for their final destination. Transport vesicles destined for intracellular compartments, such as the lysosome, bud off the TGN. What remains is a secretory vesicle which contains proteins destined for the plasma membrane, such as receptors, adhesion molecules, and ion channels, and secretory proteins, such as hormones, neurotransmitters, and digestive enzymes. Secretory vesicles eventually fuse with the plasma membrane (Glick, B. S. and V. Malhotra (1998) Cell 95 : 883-889). 



   The secretory process can be constitutive or regulated. Most cells have a constitutive pathway for secretion, whereby vesicles derived from maturation of the TGN require no specific signal to fuse with the plasma membrane. In many cells, such as endocrine cells, digestive cells, and neurons, vesicle pools derived from the TGN collect in the cytoplasm and do not fuse with the plasma membrane until they are directed to by a specific signal. 



  Endocytosis 
Endocytosis, wherein cells internalize material from the extracellular environment, is essential for transmission of neuronal, metabolic, and proliferative signals ; uptake of many essential nutrients ; and defense against invading organisms. Most cells exhibit two forms of endocytosis. The first, phagocytosis, is an actin-driven process exemplified in macrophage and neutrophils. Material to be endocytosed contacts numerous cell surface receptors which stimulate the plasma membrane to extend and surround the particle, enclosing it in a membrane-bound phagosome. In the mammalian immune system, IgG-coated particles bind Fc receptors on the surface of phagocytic leukocytes. Activation of the Fc receptors initiates a signal cascade involving src-family cytosolic kinases and the monomeric GTP-binding (G) protein Rho.

   The resulting actin reorganization leads to phagocytosis of the particle. 



  This process is an important component of the humoral immune response, allowing the processing and presentation of bacterial-derived peptides to antigen-specific T-lymphocytes. 



   The second form of endocytosis, pinocytosis, is a more generalized uptake of material from the external milieu. Like phagocytosis, pinocytosis is activated by ligand binding to cell surface receptors. 

 <Desc/Clms Page number 41> 

 



  Activation of individual receptors stimulates an internal response that includes coalescence of the receptor-ligand complexes and formation of clathrin-coated pits. Invagination of the plasma membrane at clathrin-coated pits produces an endocytic vesicle within the cell cytoplasm. These vesicles undergo homotypic fusion to form an early endosomal (EE) compartment. The   tubulovesicular   EE serves as a sorting site for incoming material. ATP-driven proton pumps in the EE membrane lowers the pH of the EE lumen (pH 6. 3-6. 8). The acidic environment causes many ligands to dissociate from their receptors. 



  The receptors, along with membrane and other integral membrane proteins, are recycled back to the plasma membrane by budding off the tubular extensions of the EE in recycling vesicles (RV). This selective removal of recycled components produces a carrier vesicle containing ligand and other material from the external environment. The carrier vesicle fuses with TGN-derived vesicles which contain hydrolytic enzymes. The acidic environment of the resulting late endosome (LE) activates the hydrolytic enzymes which degrade the ligands and other material. As digestion takes place, the LE fuses with the lysosome where digestion is completed (Mellman,   1.     (1996)   Annu. Rev. Cell Dev. Biol. 



  12 : 575-625). 



   Recycling vesicles may return directly to the plasma membrane. Receptors internalized and returned directly to the plasma membrane have a turnover rate of 2-3 minutes. Some RVs undergo microtubule-directed relocation to a perinuclear site, from which they then return to the plasma membrane. Receptors following this route have a turnover rate of 5-10 minutes. Still other RVs are retained within the cell until an appropriate signal is received (Mellman, supra ; and James, D. E. et al. 



  (1994) Trends Cell Biol. 4 : 120-126). 



  Vesicle Formation 
Several steps in the transit of material along the secretory and endocytic pathways require the formation of transport vesicles. Specifically, vesicles form at the transitional endoplasmic reticulum (tER), the rim of Golgi cisternae, the face of the Trans-Golgi Network (TGN), the plasma membrane (PM), and tubular extensions of the endosomes. The process begins with the budding of a vesicle out of the donor membrane. The membrane-bound vesicle contains proteins to be transported and is surrounded by a protective coat made up of protein subunits recruited from the cytosol. The initial budding and coating processes are controlled by a cytosolic ras-like GTP-binding protein, ADP- ribosylating factor   (Arf),   and adapter proteins (AP). Different isoforms of both Arf and AP are involved at different sites of budding.

   Another small G-protein, dynamin, forms a ring complex around the neck of the forming vesicle and may provide the   mechanochemical   force to accomplish the final step of the budding process. The coated vesicle complex is then transported through the cytosol. During the transport process, Arf-bound GTP is hydrolyzed to GDP and the coat dissociates from the transport vesicle (West, M. A. et al. (1997) J. Cell Biol. 138 : 1239-1254). Two different classes of coat protein 

 <Desc/Clms Page number 42> 

 have also been identified. Clathrin coats form on the TGN and PM surfaces, whereas coatomer or COP coats form on the ER and Golgi. COP coats can further be distinguished as COPI, involved in retrograde traffic through the Golgi and from the Golgi to the ER, and COPII, involved in anterograde traffic from the ER to the Golgi (Mellman, supra).

   The COP coat consists of two major components, a G-protein (Arf or Sar) and coat protomer (coatomer). Coatomer is an equimolar complex of seven proteins, termed alpha-, beta-, beta'-, gamma-, delta-, epsilon-and zeta-COP. (Harter, C. and F. T. 



  Wieland (1998) Proc. Natl. Acad. Sci. USA 95 : 11649-11654.) Membrane Fusion 
Transport vesicles undergo homotypic or heterotypic fusion in the secretory and endocytotic pathways. Molecules required for appropriate targeting and fusion of vesicles with their target membrane include proteins incorporated in the vesicle membrane, the target membrane, and proteins recruited from the cytosol. During budding of the vesicle from the donor compartment, an integral membrane protein, VAMP (vesicle-associated membrane protein) is incorporated into the vesicle. Soon after the vesicle uncoats, a cytosolic prenylated GTP-binding protein, Rab (a member of the Ras superfamily), is inserted into the vesicle membrane. GTP-bound Rab proteins are directed into nascent transport vesicles where they interact with VAMP.

   Following vesicle transport, GTPase activating proteins (GAPs) in the target membrane convert Rab proteins to the GDP-bound form. A cytosolic protein, guanine-nucleotide dissociation inhibitor (GDI) helps return GDP-bound Rab proteins to their membrane of origin. Several Rab isoforms have been identified and appear to associate with specific compartments within the cell. Rab proteins appear to play a role in mediating the function of a viral gene, Rev, which is essential for replication of HIV-1, the virus responsible for AIDS (Flavell, R. A. et al. (1996) Proc. Natl. Acad. Sci. USA 93 : 4421-4424). 



   Docking of the transport vesicle with the target membrane involves the formation of a complex between the vesicle SNAP receptor (v-SNARE), target membrane (t-) SNAREs, and certain other membrane and cytosolic proteins. Many of these other proteins have been identified although their exact functions in the docking complex remain uncertain (Tellam, J. T. et al. (1995) J. Biol. Chem. 



  270 : 5857-5863 ;   and Hata,   Y. and T. C. Sudhof (1995) J. Biol. Chem. 270 : 13022-13028). 



    N-ethylmaleimide   sensitive factor (NSF) and soluble   NSF-attachment   protein (a-SNAP and   (3-SNAP)   are two such proteins that are conserved from yeast to man and function in most intracellular membrane fusion reactions. Secl represents a family of yeast proteins that function at many different stages in the secretory pathway including membrane fusion. Recently, mammalian homologs of   Secl,   called Munc-18 proteins, have been identified (Katagiri, H. et al. (1995)   J.   Biol. Chem. 270 : 4963-4966 ; Hata et al. supra). 



   The SNARE complex involves three SNARE molecules, one in the vesicular membrane and 

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 two in the target membrane.   Synaptotagmin   is an integral membrane protein in the synaptic vesicle which associates with the t-SNARE syntaxin in the docking complex.   Synaptotagmin   binds calcium in a complex with negatively charged phospholipids, which allows the cytosolic SNAP protein to displace synaptotagmin from syntaxin and fusion to occur. Thus, synaptotagmin is a negative regulator of fusion in the neuron (Littleton, J. T. et al. (1993) Cell 74 : 1125-1134). The most abundant membrane protein of synaptic vesicles appears to be the glycoprotein synaptophysin, a 38 kDa protein with four transmembrane domains. 



   Specificity between a vesicle and its target is derived from the   v-SNARE,   t-SNAREs, and associated proteins involved. Different isoforms of SNAREs and Rabs show distinct cellular and subcellular distributions. VAMP-1/synaptobrevin, membrane-anchored   synaptosome-associated   protein of 25 kDa (SNAP-25), syntaxin-1, Rab3A, Rabl5, and Rab23 are predominantly expressed in the brain and nervous system. Different syntaxin, VAMP, and Rab proteins are associated with distinct subcellular compartments and their vesicular carriers. 



  Nuclear Transport 
Transport of proteins and RNA between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs). NPC-mediated transport occurs in both directions through the nuclear envelope. All nuclear proteins are imported from the cytoplasm, their site of synthesis. tRNA and mRNA are exported from the nucleus, their site of synthesis, to the cytoplasm, their site of function. 



  Processing of small nuclear RNAs involves export into the cytoplasm, assembly with proteins and modifications such as hypermethylation to produce small nuclear ribonuclear proteins (snRNPs), and subsequent import of the snRNPs back into the nucleus. The assembly of ribosomes requires the initial import of ribosomal proteins from the cytoplasm, their incorporation with RNA into ribosomal subunits,   and export back to the cytoplasm. (Gorlich,   D.   and I.   W. Mattaj (1996) Science 271 : 1513- 1518.) 
The transport of proteins and mRNAs across the NPC is selective, dependent on nuclear localization signals, and generally requires association with nuclear transport factors. Nuclear localization signals (NLS) consist of short stretches of amino acids enriched in basic residues.

   NLS are found on proteins that are targeted to the nucleus, such as the glucocorticoid receptor. The NLS is recognized by the NLS receptor, importin, which then interacts with the monomeric GTP-binding protein Ran. This NLS   protein/receptor/Ran   complex navigates the nuclear pore with the help of the homodimeric protein nuclear transport factor 2 (NTF2). NTF2 binds the GDP-bound form of Ran and to multiple proteins of the nuclear pore complex containing FXFG repeat motifs, such as p62. 



  (Paschal, B. et al. (1997) J. Biol. Chem. 272 : 21534-21539 ;   and Wong,   D. H. et al. (1997) Mol. Cell Biol. 17 :   3755-3767).   Some proteins are dissociated before nuclear mRNAs are transported across the 

 <Desc/Clms Page number 44> 

 NPC while others are dissociated shortly after nuclear mRNA transport across the NPC and are reimported into the nucleus. 



  Disease Correlation 
The etiology of numerous human diseases and disorders can be attributed to defects in the transport or secretion of proteins. For example, abnormal hormonal secretion is linked to disorders such as diabetes insipidus (vasopressin), hyper-and hypoglycemia (insulin, glucagon), Grave's disease and goiter (thyroid hormone), and Cushing's and Addison's diseases (adrenocorticotropic hormone, ACTH). Moreover, cancer cells secrete excessive amounts of hormones or other biologically active peptides.

   Disorders related to excessive secretion of biologically active peptides by tumor cells include fasting hypoglycemia due to increased insulin secretion from insulinoma-islet cell tumors ; hypertension due to increased epinephrine and norepinephrine secreted from pheochromocytomas of the adrenal medulla and sympathetic paraganglia ; and carcinoid syndrome, which is characterized by abdominal cramps, diarrhea, and valvular heart disease caused by excessive amounts of vasoactive substances such as serotonin,   bradykinin,   histamine, prostaglandins, and polypeptide hormones, secreted from intestinal tumors.

   Biologically active peptides that are ectopically synthesized in and secreted from tumor cells include ACTH and vasopressin (lung and pancreatic cancers) ; parathyroid hormone (lung and bladder cancers) ; calcitonin (lung and breast cancers) ; and thyroid-stimulating hormone (medullary thyroid carcinoma). Such peptides may be useful as diagnostic markers for tumorigenesis (Schwartz, M. Z. (1997) Semin. Pediatr. Surg. 3 : 141-146 ; and Said, S. I. and G. R. Faloona (1975) N. Engl. J. Med. 



  293 : 155-160). 



   Defective nuclear transport may play a role in cancer. The   BRCA1   protein contains three potential NLSs which interact with importin alpha, and is transported into the nucleus by the   importin/NPC   pathway. In breast cancer cells the   BRCA1   protein is aberrantly localized in the cytoplasm. The   mislocation   of the   BRCA1   protein in breast cancer cells may be due to a defect in the NPC nuclear import pathway (Chen, C. F. et al. (1996) J. Biol. Chem. 271 : 32863-32868). 



   It has been suggested that in some breast cancers, the tumor-suppressing activity of p53 is inactivated by the sequestration of the protein in the cytoplasm, away from its site of action in the cell nucleus. Cytoplasmic   wild-type p53   was also found in human cervical carcinoma cell lines. (Moll, U. M. et al. (1992) Proc. Natl. Acad. Sci. USA 89 : 7262-7266 ;   and Liang,   X. H. et al. (1993) Oncogene 8 :   2645-2652.)   Environmental Responses 
Organisms respond to the environment by a number of pathways. Heat shock proteins, including hsp 70, hsp60, hsp90, and hsp 40, assist organisms in coping with heat damage to cellular proteins. 

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   Aquaporins (AQP) are channels that transport water and, in some cases,   nonionic   small solutes such as urea and glycerol. Water movement is important for a number of physiological processes including renal fluid filtration, aqueous humor generation in the eye, cerebrospinal fluid production in the brain, and appropriate hydration of the lung. Aquaporins are members of the major intrinsic protein   (MAP)   family of membrane transporters (King, L. S. and P. Agre (1996) Annu. Rev. Physiol. 58 : 619- 648 ;   Ishibashi,   K. et al. (1997) J. Biol. Chem. 272 : 20782-20786). The study of aquaporins may have relevance to understanding edema formation and fluid balance in both normal physiology and disease states   (King,   supra).

   Mutations in AQP2 cause autosomal recessive   nephrogenic   diabetes insipidus (OMIM *107777 Aquaporin 2 ; AQP2). Reduced AQP4 expression in skeletal muscle may be associated with Duchenne muscular dystrophy (Frigeri, A. et al. (1998) J. Clin. Invest. 102 : 695-703). 



  Mutations in AQPO cause autosomal dominant cataracts in the mouse (OMIM *154050 Major Intrinsic Protein of Lens Fiber ; MIP). 



   The metallothioneins (MTs) are a group of small (61 amino acids), cysteine-rich proteins that bind heavy metals such as cadmium, zinc, mercury, lead, and copper and are thought to play a role in metal detoxification or the metabolism and homeostasis of metals. Arsenite-resistance proteins have been identified in hamsters that are resistant to toxic levels of arsenite (Rossman, T. G. et al. (1997) Mutat. Res. 386 : 307-314). 



   Humans respond to light and odors by specific protein pathways. Proteins involved in light perception include rhodopsin, transducin, and cGMP phosphodiesterase. Proteins involved in odor perception include multiple olfactory receptors. Other proteins are important in human Circadian rhythms and responses to wounds. 



  Immunity and Host Defense 
All vertebrates have developed sophisticated and complex immune systems that provide protection from viral, bacterial, fungal and parasitic infections. Included in these systems are the processes of humoral immunity, the complement cascade and the inflammatory response (Paul, W. E. 



  (1993) Fundamental Immunology, Raven Press, Ltd., New York NY, pp.   1-20).   



   The cellular components of the humoral immune system include six different types of leukocytes : monocytes, lymphocytes, polymorphonuclear granulocytes (consisting of neutrophils,   eosinophils,   and basophils) and plasma cells. Additionally, fragments of megakaryocytes, a seventh type of white blood cell in the bone marrow, occur in large numbers in the blood as platelets. 



   Leukocytes are formed from two stem cell lineages in bone marrow. The myeloid stem cell line produces granulocytes and monocytes and, the lymphoid stem cell produces lymphocytes. 



  Lymphoid cells travel to the thymus, spleen and lymph nodes, where they mature and differentiate into lymphocytes. Leukocytes are responsible for defending the body against invading pathogens. 

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  Neutrophils and monocytes attack invading bacteria, viruses, and other pathogens and destroy them by phagocytosis. Monocytes enter tissues and differentiate into macrophages which are extremely phagocytic. Lymphocytes and plasma cells are a part of the immune system which recognizes specific foreign molecules and organisms and inactivates them, as well as signals other cells to attack the invaders. 



   Granulocytes and monocytes are formed and stored in the bone marrow until needed. 



  Megakaryocytes are produced in bone marrow, where they fragment into platelets and are released into the bloodstream. The main function of platelets is to activate the blood clotting mechanism. 



  Lymphocytes and plasma cells are produced in various lymphogenous organs, including the lymph nodes, spleen, thymus, and tonsils. 



   Both neutrophils and macrophages exhibit   chemotaxis   towards sites of inflammation. Tissue inflammation in response to pathogen invasion results in production of chemo-attractants for leukocytes, such as endotoxins or other bacterial products, prostaglandins, and products of leukocytes or platelets. 



   Basophils participate in the release of the chemicals involved in the inflammatory process. 



  The main function of basophils is secretion of these chemicals to such a degree that they have been referred to as"unicellular endocrine glands."A distinct aspect   of basophilic   secretion is that the contents of granules go directly into the extracellular environment, not into vacuoles as occurs with neutrophils,   eosinophils   and monocytes. Basophils have receptors for the Fc fragment of immunoglobulin E (IgE) that are not present on other leukocytes. Crosslinking of membrane IgE with anti-IgE or other ligands triggers degranulation. 



   Eosinophils are bi-or   multi-nucleated   white blood cells which contain eosinophilic granules. 



  Their plasma membrane is characterized by Ig receptors, particularly IgG and IgE. Generally,   eosinophils   are stored in the bone marrow until recruited for use at a site of inflammation or invasion. 



  They have specific functions in parasitic infections and allergic reactions, and are thought to detoxify some of the substances released by mast cells and basophils which cause inflammation. Additionally, they phagocytize antigen-antibody complexes and further help prevent spread of the inflammation. 



   Macrophages are monocytes that have left the blood stream to settle in tissue. Once monocytes have migrated into tissues, they do not re-enter the bloodstream. The mononuclear phagocyte system is comprised of precursor cells in the bone marrow, monocytes in circulation, and macrophages in tissues. The system is capable of very fast and extensive phagocytosis. A macrophage may phagocytize over 100 bacteria, digest them and extrude residues, and then survive for many more months. Macrophages are also capable of ingesting large particles, including red blood cells and malarial parasites. They increase several-fold in size and transform into macrophages 

 <Desc/Clms Page number 47> 

 that are characteristic of the tissue they have entered, surviving in tissues for several months. 



   Mononuclear phagocytes are essential in defending the body against invasion by foreign pathogens, particularly intracellular microorganisms such as M. tuberculosis, listeria, leishmania and   toxoplasma.   Macrophages can also control the growth of tumorous cells, via both phagocytosis and secretion of hydrolytic enzymes. Another important function of macrophages is that of processing antigen and presenting them in a biochemically modified form to lymphocytes. 



   The immune system responds to invading microorganisms in two major ways : antibody production and cell mediated responses. Antibodies are immunoglobulin proteins produced by B-lymphocytes which bind to specific antigens and cause inactivation or promote destruction of the antigen by other cells. Cell-mediated immune responses involve T-lymphocytes (T cells) that react   with foreign antigen on the surface of infected host cells. Depending on the type of T cell, the    infected cell is either killed or signals are secreted which activate macrophages and other cells to destroy the infected cell (Paul, supra). 



   T-lymphocytes originate in the bone marrow or liver in fetuses. Precursor cells migrate via the blood to the thymus, where they are processed to mature into T-lymphocytes. This processing is crucial because of positive and negative selection of T cells that will react with foreign antigen and not with self molecules. After processing, T cells continuously circulate in the blood and secondary lymphoid tissues, such as lymph nodes, spleen, certain epithelium-associated tissues in the gastrointestinal tract, respiratory tract and skin. When T-lymphocytes are presented with the complementary antigen, they are stimulated to proliferate and release large numbers of activated T cells into the lymph system and the blood system. These activated T cells can survive and circulate for several days.

   At the same time, T memory cells are created, which remain in the lymphoid tissue for months or years. Upon subsequent exposure to that specific antigen, these memory cells will respond more rapidly and with a stronger response than induced by the original antigen. This creates an"immunological memory"that can provide immunity for years. 



   There are two major types of T cells : cytotoxic T cells destroy infected host cells, and helper T cells activate other white blood cells via chemical signals. One class of helper cell,   TH1,   activates macrophages to destroy ingested microorganisms, while another,   TH2,   stimulates the production of antibodies by B cells. 



   Cytotoxic T cells directly attack the infected target cell. In virus-infected cells, peptides derived from viral proteins are generated by the proteasome. These peptides are transported into the ER by the transporter associated with antigen processing (TAP) (Pamer, E. and P. Cresswell (1998) Annu. Rev. Immunol. 16 : 323-358). Once inside the ER, the peptides bind MHC I chains, and the peptide/MHC I complex is transported to the cell surface. Receptors on the surface of T cells bind to 

 <Desc/Clms Page number 48> 

 antigen presented on cell surface MHC molecules. Once activated by binding to antigen, T cells secrete y-interferon, a signal molecule that induces the expression of genes necessary for presenting viral (or other) antigens to cytotoxic T cells. Cytotoxic T cells kill the infected cell by stimulating programmed cell death. 



   Helper T cells constitute up to   75%   of the total T cell population. They regulate the immune functions by producing a variety of lymphokines that act on other cells in the immune system and on bone marrow. Among these lymphokines are : interleukins-2, 3, 4, 5, 6 ; granulocyte-monocyte colony stimulating factor, and y-interferon. 



   Helper T cells are required for most B cells to respond to antigen. When an activated helper cell contacts a B cell, its centrosome and Golgi apparatus become oriented toward the B cell, aiding the directing of signal molecules, such as transmembrane-bound protein called CD40 ligand, onto the B cell surface to interact with the CD40 transmembrane protein. Secreted signals also help B cells to proliferate and mature and, in some cases, to switch the class of antibody being produced. 



   B-lymphocytes (B cells) produce antibodies which react with specific antigenic proteins presented by pathogens. Once activated, B cells become filled with extensive rough endoplasmic reticulum and are known as plasma cells. As with T cells, interaction of B cells with antigen stimulates proliferation of only those B cells which produce antibody specific to that antigen. There are five classes of antibodies, known as immunoglobulins, which together comprise about 20% of total plasma protein. Each class mediates a characteristic biological response after antigen binding. 



  Upon activation by specific antigen B cells switch from making membrane-bound antibody to secretion of that antibody. 



   Antibodies, or immunoglobulins   (Ig),   are the founding members of the Ig superfamily and the central components of the humoral immune response. Antibodies are either expressed on the surface of B cells or secreted by B cells into the circulation. Antibodies bind and neutralize blood-borne foreign antigens. The prototypical antibody is a tetramer consisting of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds. This arrangement confers the characteristic Y-shape to antibody molecules. 



  Antibodies are classified based on their H-chain composition. The five antibody classes, IgA, IgD, IgE, IgG and IgM, are defined by the   a,     8,   e, y, and   p   H-chain types. There are two types of L- chains, K   and i,   either of which may associate as a pair with any H-chain pair. IgG, the most common class of antibody found in the circulation, is tetrameric, while the other classes of antibodies are generally variants or multimers of this basic structure. 



   H-chains and L-chains each contain an N-terminal variable region and a C-terminal constant region. Both H-chains and L-chains contain repeated Ig domains. For example, a typical H-chain 

 <Desc/Clms Page number 49> 

 contains four Ig domains, three of which occur within the constant region and one of which occurs within the variable region and contributes to the formation of the antigen recognition site. Likewise, a typical L-chain contains two Ig domains, one of which occurs within the constant region and one of which occurs within the variable region. In addition, H chains such as   [i   have been shown to associate with other polypeptides during differentiation of the B cell. 



   Antibodies can be described in terms of their two main functional domains. Antigen recognition is mediated by the Fab (antigen binding fragment) region of the antibody, while effector functions are mediated by the Fc (crystallizable fragment) region. Binding of antibody to an antigen, such as a bacterium, triggers the destruction of the antigen by phagocytic white blood cells such as macrophages and neutrophils. These cells express surface receptors that specifically bind to the antibody Fc region and allow the phagocytic cells to engulf, ingest, and degrade the antibody-bound antigen. The Fc receptors expressed by phagocytic cells are single-pass transmembrane glycoproteins of about 300 to 400 amino acids (Sears, D.   W.   et al. (1990) J. Immunol. 144 : 371-378). The extracellular portion of the Fc receptor typically contains two or three Ig domains. 



   Diseases which cause over-or under-abundance of any one type of leukocyte usually result in the entire immune defense system becoming involved. A well-known autoimmune disease is AIDS (Acquired Immunodeficiency Syndrome) where the number of helper T cells is depleted, leaving the patient susceptible to infection by microorganisms and parasites. Another widespread medical condition attributable to the immune system is that of allergic reactions to certain antigens. Allergic reactions include : hay fever, asthma, anaphylaxis, and urticaria (hives). Leukemias are an excess production of white blood cells, to the point where a major portion of the body's metabolic resources are directed solely at proliferation of white blood cells, leaving other tissues to starve. Leukopenia or agranulocytosis occurs when the bone marrow stops producing white blood cells.

   This leaves the body unprotected against foreign microorganisms, including those which normally inhabit skin, mucous membranes, and gastrointestinal tract. If all white blood cell production stops completely, infection will occur within two days and death may follow only 1 to 4 days later. 



   Impaired phagocytosis occurs in several diseases, including monocytic leukemia, systemic lupus, and   granulomatous   disease. In such a situation, macrophages can phagocytize normally, but the enveloped organism is not killed. A defect in the plasma membrane enzyme which converts oxygen to lethally reactive forms results in abscess formation in liver, lungs, spleen, lymph nodes, and beneath the skin. Eosinophilia is an excess of   eosinophils   commonly observed in patients with allergies (hay fever, asthma), allergic reactions to drugs, rheumatoid arthritis, and cancers (Hodgkin's disease, lung, and liver cancer) (Isselbacher, K. J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, Inc., New York NY). 

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   Host defense is further augmented by the complement system. The complement system serves as an effector system and is involved in infectious agent recognition. It can function as an independent immune network or in conjunction with other humoral immune responses. The complement system is comprised of numerous plasma and membrane proteins that act in a cascade of reaction sequences whereby one component activates the next. The result is a rapid and amplified response to infection through either an inflammatory response or increased phagocytosis. 



   The complement system has more than 30 protein components which can be divided into functional groupings including modified serine proteases, membrane-binding proteins and regulators of complement activation. Activation occurs through two different pathways the classical and the alternative. Both pathways serve to destroy infectious agents through distinct triggering mechanisms that eventually merge with the involvement of the component C3. 



   The classical pathway requires antibody binding to infectious agent antigens. The antibodies serve to define the target and initiate the complement system cascade, culminating in the destruction of the infectious agent. In this pathway, since the antibody guides initiation of the process, the complement can be seen as an effector arm of the humoral immune system. 



   The alternative pathway of the complement system does not require the presence of pre- existing antibodies for targeting infectious agent destruction. Rather, this pathway, through low levels of an activated component, remains constantly primed and provides surveillance in the non- immune host to enable targeting and destruction of infectious agents. In this case foreign material triggers the cascade, thereby facilitating phagocytosis or lysis (Paul, supra, pp. 918-919). 



   Another important component of host defense is the process of inflammation. Inflammatory responses are divided into four categories on the basis of pathology and include allergic inflammation, cytotoxic antibody mediated inflammation, immune complex mediated inflammation and monocyte mediated inflammation. Inflammation manifests as a combination of each of these forms with one predominating. 



   Allergic acute inflammation is observed in individuals wherein specific antigens stimulate IgE antibody production. Mast cells and basophils are subsequently activated by the attachment of antigen-IgE complexes, resulting in the release of cytoplasmic granule contents such as histamine. 



  The products of activated mast cells can increase vascular permeability and constrict the smooth muscle of breathing passages, resulting in anaphylaxis or asthma. Acute inflammation is also mediated by cytotoxic antibodies and can result in the destruction of tissue through the binding of complement-fixing antibodies to cells. The responsible antibodies are of the IgG or IgM types. 



  Resultant clinical disorders include autoimmune hemolytic anemia and thrombocytopenia as associated with systemic lupus erythematosis. 

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   Immune complex mediated acute inflammation involves the IgG or IgM antibody types which combine with antigen to activate the complement cascade. When such immune complexes bind to neutrophils and macrophages they activate the respiratory burst to form protein-and vessel- damaging agents such as hydrogen peroxide, hydroxyl radical, hypochlorous acid, and chloramines. 



  Clinical manifestations include rheumatoid arthritis and systemic lupus erythematosus. 



   In chronic inflammation or delayed-type hypersensitivity, macrophages are activated and process antigen for presentation to T cells that subsequently produce lymphokines and monokines. 



  This type of inflammatory response is likely important for defense against intracellular parasites and certain viruses. Clinical associations include, granulomatous disease, tuberculosis, leprosy, and sarcoidosis (Paul, W. E., supra, pp.   1017-1018).   



  Extracellular Information Transmission Molecules 
Intercellular communication is essential for the growth and survival of multicellular organisms, and in particular, for the function of the endocrine, nervous, and immune systems. In addition, intercellular communication is critical for developmental processes such as tissue construction and organogenesis, in which cell proliferation, cell differentiation, and morphogenesis must be spatially and temporally regulated in a precise and coordinated manner. Cells communicate with one another through the secretion and uptake of diverse types of signaling molecules such as hormones, growth factors, neuropeptides, and cytokines. 



  Hormones 
Hormones are signaling molecules that coordinately regulate basic physiological processes from embryogenesis throughout adulthood. These processes include metabolism, respiration, reproduction, excretion, fetal tissue differentiation and organogenesis, growth and development, homeostasis, and the stress response. Hormonal secretions and the nervous system are tightly integrated and interdependent. Hormones are secreted by endocrine glands, primarily the hypothalamus and pituitary, the thyroid and parathyroid, the pancreas, the adrenal glands, and the ovaries and testes. 



   The secretion of hormones into the circulation is tightly controlled. Hormones are often secreted in diurnal, pulsatile, and cyclic patterns. Hormone secretion is regulated by perturbations in blood biochemistry, by other upstream-acting hormones, by neural impulses, and by negative feedback loops. Blood hormone concentrations are constantly monitored and adjusted to maintain optimal, steady-state levels. Once secreted, hormones act only on those target cells that express specific receptors. 



   Most disorders of the endocrine system are caused by either hyposecretion or hypersecretion of hormones. Hyposecretion often occurs when a hormone's gland of origin is damaged or otherwise 

 <Desc/Clms Page number 52> 

 impaired. Hypersecretion often results from the proliferation of tumors derived from hormone-secreting cells. Inappropriate hormone levels may also be caused by defects in regulatory feedback loops or in the processing of hormone precursors. Endocrine malfunction may also occur when the target cell fails to respond to the hormone. 



   Hormones can be classified biochemically as polypeptides, steroids, eicosanoids, or amines. 



  Polypeptides, which include diverse hormones such as insulin and growth hormone, vary in size and function and are often synthesized as inactive precursors that are processed intracellularly into mature, active forms. Amines, which include epinephrine and dopamine, are amino acid derivatives that function in neuroendocrine signaling. Steroids, which include the cholesterol-derived hormones estrogen and testosterone, function in sexual development and reproduction. Eicosanoids, which include prostaglandins and prostacyclins, are fatty acid derivatives that function in a variety of processes. Most polypeptides and some amines are soluble in the circulation where they are highly susceptible to proteolytic degradation within seconds after their secretion. Steroids and lipids are insoluble and must be transported in the circulation by carrier proteins.

   The following discussion will focus primarily on polypeptide hormones. 



   Hormones secreted by the hypothalamus and pituitary gland play a critical role in endocrine function by coordinately regulating hormonal secretions from other endocrine glands in response to neural signals. Hypothalamic hormones include thyrotropin-releasing hormone, gonadotropin-releasing hormone, somatostatin, growth-hormone releasing factor, corticotropin-releasing hormone, substance P, dopamine, and prolactin-releasing hormone. These hormones directly regulate the secretion of hormones from the anterior lobe of the pituitary.

   Hormones secreted by the anterior pituitary include adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone, somatotropic hormones such as growth hormone and prolactin, glycoprotein hormones such as thyroid-stimulating hormone,   luteinizing   hormone (LH), and follicle-stimulating hormone (FSH),   P-lipotropin,   and   P-endorphins.   



  These hormones regulate hormonal secretions from the thyroid, pancreas, and adrenal glands, and act directly on the reproductive organs to stimulate ovulation and spermatogenesis. The posterior pituitary synthesizes and secretes antidiuretic hormone (ADH, vasopressin) and oxytocin. 



   Disorders of the hypothalamus and pituitary often result from lesions such as primary brain tumors, adenomas, infarction associated with pregnancy, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immunological disorders, and complications due to head trauma. 



  Such disorders have profound effects on the function of other endocrine glands. Disorders associated with hypopituitarism include hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism. Disorders associated with   hyperpituitarism   include acromegaly, giantism, and syndrome of 

 <Desc/Clms Page number 53> 

 inappropriate ADH secretion (SIADH), often caused by benign adenomas. 



   Hormones secreted by the thyroid and parathyroid primarily control metabolic rates and the regulation of serum calcium levels, respectively. Thyroid hormones include calcitonin, somatostatin, and thyroid hormone. The parathyroid secretes parathyroid hormone. Disorders associated with hypothyroidism include goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism. Disorders associated with hyperthyroidism include thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic   multinodular   goiter, thyroid carcinoma, and Plummer's disease. Disorders associated with hyperparathyroidism include Conn disease (chronic   hypercalemia)   leading to bone resorption and parathyroid hyperplasia. 



   Hormones secreted by the pancreas regulate blood glucose levels by modulating the rates of carbohydrate, fat, and protein metabolism. Pancreatic hormones include insulin, glucagon,   amylin,     y-   aminobutyric acid, gastrin, somatostatin, and pancreatic polypeptide. The principal disorder associated with pancreatic dysfunction is diabetes mellitus caused by insufficient insulin activity. Diabetes mellitus is generally classified as either Type I (insulin-dependent, juvenile diabetes) or Type II (non- insulin-dependent, adult diabetes). The treatment of both forms by insulin replacement therapy is well known.

   Diabetes mellitus often leads to acute complications such as hypoglycemia (insulin shock), coma, diabetic ketoacidosis, lactic acidosis, and chronic complications leading to disorders of the eye, kidney, skin, bone, joint, cardiovascular system, nervous system, and to decreased resistance to infection. 



   The anatomy, physiology, and diseases related to hormonal function are reviewed in McCance, K. L. and S. E. Huether (1994)   Pathophysiology   : The Biological Basis for Disease in Adults and Children, Mosby-Year Book, Inc., St. Louis MO ; Greenspan, F. S. and J. D. Baxter (1994) Basic and Clinical   Endocrinology,   Appleton and Lange, East Norwalk CT. 



  Growth Factors 
Growth factors are secreted proteins that mediate intercellular communication. Unlike hormones, which travel great distances via the circulatory system, most growth factors are primarily local mediators that act on neighboring cells. Most growth factors contain a hydrophobic N-terminal signal peptide sequence which directs the growth factor into the secretory pathway. Most growth factors also undergo post-translational modifications within the secretory pathway. These modifications can include proteolysis, glycosylation, phosphorylation, and intramolecular disulfide bond formation. Once secreted, growth factors bind to specific receptors on the surfaces of neighboring target cells, and the bound receptors trigger intracellular signal transduction pathways. These signal transduction pathways elicit specific cellular responses in the target cells.

   These responses can include 

 <Desc/Clms Page number 54> 

 the modulation of gene expression and the stimulation or inhibition of cell division, cell differentiation, and cell motility. 



   Growth factors fall into at least two broad and overlapping classes. The broadest class includes the large polypeptide growth factors, which are wide-ranging in their effects. These factors include epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor-P   (TGF- ),   insulin-like growth factor (IGF), nerve growth factor (NGF), and platelet-derived growth factor   (PDGF),   each defining a family of numerous related factors. The large polypeptide growth factors, with the exception of NGF, act as mitogens on diverse cell types to stimulate wound healing, bone synthesis and remodeling, extracellular matrix synthesis, and proliferation of epithelial, epidermal, and connective tissues.

   Members of the   TGF- ,   EGF, and FGF families also function as inductive signals in the differentiation of embryonic tissue. NGF functions specifically as a   neurotrophic   factor, promoting neuronal growth and differentiation. 



   Another class of growth factors includes the hematopoietic growth factors, which are narrow in their target specificity. These factors stimulate the proliferation and differentiation of blood cells such as B-lymphocytes, T-lymphocytes, erythrocytes, platelets,   eosinophils,   basophils, neutrophils, macrophages, and their stem cell precursors. These factors include the colony-stimulating factors (G- CSF, M-CSF, GM-CSF, and   CSF1-3),   erythropoietin, and the cytokines. The cytokines are specialized hematopoietic factors secreted by cells of the immune system and are discussed in detail below. 



   Growth factors play critical roles in neoplastic transformation of cells in vitro and in tumor progression in vivo. Overexpression of the large polypeptide growth factors promotes the proliferation and transformation of cells in culture. Inappropriate expression of these growth factors by tumor cells in vivo may contribute to tumor vascularization and metastasis. Inappropriate activity of hematopoietic growth factors can result in anemias, leukemias, and lymphomas. Moreover, growth factors are both structurally and functionally related to oncoproteins, the potentially cancer-causing products   of proto-oncogenes. Certain   FGF and PDGF family members are themselves homologous to oncoproteins, whereas receptors for some members of the EGF, NGF, and FGF families are encoded by proto-oncogenes.

   Growth factors also affect the transcriptional regulation of both proto-oncogenes and oncosuppressor genes (Pimentel, E. (1994) Handbook of Growth Factors, CRC Press, Ann Arbor MI ; McKay,   1.   and I. Leigh, eds.   (1993)   Growth Factors : A Practical Approach, Oxford University Press, New York NY ; Habenicht, A., ed. (1990) Growth Factors, Differentiation Factors, and Cytokines, Springer-Verlag, New York NY). 



   In addition, some of the large polypeptide growth factors play crucial roles in the induction of the primordial germ layers in the developing embryo. This induction ultimately results in the formation of the embryonic mesoderm, ectoderm, and endoderm which in turn provide the framework for the 

 <Desc/Clms Page number 55> 

 entire adult body plan. Disruption of this inductive process would be catastrophic to embryonic development. 



  Small Peptide   Factors-Neuropeptides   and Vasomediators 
Neuropeptides and vasomediators   (NP/Vw   comprise a family of small peptide factors, typically of 20 amino acids or less. These factors generally function in neuronal excitation and inhibition of   vasoconstriction/vasodilation,   muscle contraction, and hormonal secretions from the brain and other endocrine tissues.

   Included in this family are neuropeptides and neuropeptide hormones such as bombesin, neuropeptide Y, neurotensin, neuromedin N, melanocortins, opioids,   galanin,   somatostatin, tachykinins, urotensin II and related peptides involved in smooth muscle stimulation, vasopressin, vasoactive intestinal peptide, and circulatory system-borne signaling molecules such as angiotensin, complement, calcitonin, endothelins, formyl-methionyl peptides, glucagon, cholecystokinin, gastrin, and many of the peptide hormones discussed above.   NP/VMs   can transduce signals directly, modulate the activity or release of other neurotransmitters and hormones, and act as catalytic enzymes in signaling cascades. The effects of NP/VMs range from extremely brief to long-lasting. (Reviewed in Martin, C. R. et al.

   (1985) Endocrine Physiology, Oxford University Press, New York NY, pp. 57-62.) C okies 
Cytokines comprise a family of signaling molecules that modulate the immune system and the inflammatory response. Cytokines are usually secreted by leukocytes, or white blood cells, in response to injury or infection. Cytokines function as growth and differentiation factors that act primarily on cells of the immune system such as B-and T-lymphocytes, monocytes, macrophages, and granulocytes. 



  Like other signaling molecules, cytokines bind to specific plasma membrane receptors and trigger intracellular signal transduction pathways which alter gene expression patterns. There is considerable potential for the use of cytokines in the treatment of inflammation and immune system disorders. 



   Cytokine structure and function have been extensively characterized in vitro. Most cytokines are small polypeptides of about 30 kilodaltons or less. Over 50 cytokines have been identified from human and rodent sources. Examples of cytokine subfamilies include the interferons   (IFN-a,-P,   and-   y),   the interleukins   (IL1-IL13),   the tumor necrosis factors   (TNF-a     and-P),   and the chemokines. Many cytokines have been produced using recombinant DNA techniques, and the activities of individual cytokines have been determined in vitro. These activities include regulation of leukocyte proliferation, differentiation, and motility. 



   The activity of an individual cytokine in vitro may not reflect the full scope of that cytokine's activity in vivo. Cytokines are not expressed individually in vivo but are instead expressed in combination with a multitude of other cytokines when the organism is challenged with a stimulus. 

 <Desc/Clms Page number 56> 

 



  Together, these cytokines collectively modulate the immune response in a manner appropriate for that particular stimulus. Therefore, the physiological activity of a cytokine is determined by the stimulus itself and by complex interactive networks among co-expressed cytokines which may demonstrate both synergistic and antagonistic relationships. 



   Chemokines comprise a cytokine subfamily with over 30 members. (Reviewed in Wells, T. 



  N. C. and M. C. Peitsch (1997) J. Leukoc. Biol. 61 : 545-550.) Chemokines were initially identified as chemotactic proteins that recruit monocytes and macrophages to sites of inflammation. Recent evidence indicates that chemokines may also play key roles in hematopoiesis and HIV-1 infection. Chemokines are small proteins which range from about 6-15 kilodaltons in molecular weight. Chemokines are further classified as C, CC, CXC, or CX3C based on the number and position of critical cysteine residues. The CC chemokines, for example, each contain a conserved motif consisting of two consecutive cysteines followed by two additional cysteines which occur downstream at 24-and 16- residue intervals, respectively (ExPASy PROSITE database, documents PS00472 and PDOC00434). 



  The presence and spacing of these four cysteine residues are highly conserved, whereas the intervening residues diverge significantly. However, a conserved tyrosine located about 15 residues downstream of the cysteine doublet seems to be important for chemotactic activity. Most of the human genes encoding CC chemokines are clustered on chromosome 17, although there are a few examples of CC chemokine genes that map elsewhere. Other chemokines include lymphotactin (C chemokine) ; macrophage chemotactic and activating factor   (MCAF/MCP-1   ; CC chemokine) ; platelet factor 4 and IL-8 (CXC chemokines) ; and fractalkine and   neurotractin     (CX3C   chemokines). (Reviewed in Luster, A. D. (1998) N. Engl. J.

   Med. 338 :   436-445.)   Receptor Molecules 
The term receptor describes proteins that specifically recognize other molecules. The category is broad and includes proteins with a variety of functions. The bulk of receptors are cell surface proteins which bind extracellular ligands and produce cellular responses in the areas of growth, differentiation, endocytosis, and immune response. Other receptors facilitate the selective transport of proteins out of the endoplasmic reticulum and localize enzymes to particular locations in the cell. The term may also be applied to proteins which act as receptors for ligands with known or unknown chemical composition and which interact with other cellular components. For example, the steroid hormone receptors bind to and regulate transcription of DNA. 



   Regulation of cell proliferation, differentiation, and migration is important for the formation and function of tissues. Regulatory proteins such as growth factors coordinately control these cellular processes and act as mediators in cell-cell signaling pathways. Growth factors are secreted proteins 

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 that bind to specific cell-surface receptors on target cells. The bound receptors trigger intracellular signal transduction pathways which activate various downstream effectors that regulate gene expression, cell division, cell differentiation, cell motility, and other cellular processes. 



   Cell surface receptors are typically integral plasma membrane proteins. These receptors recognize hormones such as   catecholamines   ; peptide hormones ; growth and differentiation factors ; small peptide factors such as thyrotropin-releasing hormone ;   galanin,   somatostatin, and tachykinins ; and circulatory system-borne signaling molecules. Cell surface receptors on immune system cells recognize antigens, antibodies, and major histocompatibility complex (MHC)-bound peptides. Other cell surface receptors bind ligands to be internalized by the cell.

   This receptor-mediated endocytosis functions in the uptake of low density lipoproteins (LDL), transferrin, glucose-or mannose-terminal glycoproteins, galactose-terminal glycoproteins, immunoglobulins,   phosphovitellogenins,   fibrin, proteinase-inhibitor complexes, plasminogen activators,   and tbrombospondin   (Lodish, H. et al. (1995) Molecular Cell   Biology.   Scientific American Books, New York NY, p. 723 ; Mikhailenko,   I.   et al. 



  (1997) J. Biol. Chem. 272 : 6784-6791). 



  Receptor Protein Kinases 
Many growth factor receptors, including receptors for epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, as well as the growth modulator a-thrombin, contain intrinsic protein kinase activities. When growth factor binds to the receptor, it triggers the autophosphorylation of a serine, threonine, or tyrosine residue on the receptor. These phosphorylated sites are recognition sites for the binding of other cytoplasmic signaling proteins. These proteins participate in signaling pathways that eventually link the initial receptor activation at the cell surface to the activation of a specific intracellular target molecule. In the case of tyrosine residue autophosphorylation, these signaling proteins contain a common domain referred to as a Src homology (SH) domain.

   SH2 domains and SH3 domains are found in phospholipase   C-y,   PI-3-K p85 regulatory subunit, Ras-GTPase activating protein,   and pp60C 5rs   (Lowenstein, E. J. et al. (1992) Cell 70 : 431-442). 



  The cytokine family of receptors share a different common binding domain and include transmembrane receptors for growth hormone (GH), interleukins, erythropoietin, and prolactin. 



   Other receptors and second messenger-binding proteins have intrinsic   serine/tbreorine   protein kinase activity. These include   activin/TGF-ss/BMP-superfamily   receptors, calcium-and diacylglycerol-   activated/phospholipid-dependant   protein kinase   (PK-C),   and RNA-dependant protein kinase (PK-R). 



  In addition, other serine/threonine protein kinases, including   nematode Twitchin, have fibronectin-like,   immunoglobulin C2-like domains. 



  G-Protein Coupled Receptors 
G-protein coupled receptors (GPCRs) are integral membrane proteins characterized by the 

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 presence of seven hydrophobic transmembrane domains which span the plasma membrane and form a bundle of antiparallel alpha (a) helices. These proteins range in size from under 400 to over 1000 amino acids (Strosberg, A. D. (1991) Eur. J. Biochem. 196 : 1-10 ;   Coughlin,   S. R. (1994) Curr. Opin. 



  Cell Biol. 6 : 191-197). The amino-terminus of the GPCR is extracellular, of variable length and often glycosylated ; the   carboxy-terminus   is cytoplasmic and generally phosphorylated. Extracellular loops of the GPCR alternate with intracellular loops and link the transmembrane domains. The most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops. The transmembrane domains account for structural and functional features of the receptor. In most cases, the bundle of a helices forms a binding pocket. In addition, the extracellular N-terminal segment or one or more of the three extracellular loops may also participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor.

   The activated receptor, in turn, interacts with an intracellular heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, generally the production of second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate, or interactions with ion channel proteins (Baldwin, J. M. (1994) Curr. Opin. Cell Biol. 6 : 180-190). 



   GPCRs include those for acetylcholine, adenosine, epinephrine and norepinephrine,   bombesin,     bradykinin,   chemokines, dopamine, endothelin, y-aminobutyric acid (GABA), follicle-stimulating hormone (FSH), glutamate, gonadotropin-releasing hormone (GnRH), hepatocyte growth factor, histamine, leukotrienes, melanocortins, neuropeptide Y, opioid peptides, opsins, prostanoids, serotonin, somatostatin, tachykinins, thrombin, thyrotropin-releasing hormone (TRH), vasoactive intestinal polypeptide family, vasopressin and oxytocin, and orphan receptors. 



   GPCR mutations, which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene. Rhodopsin is the retinal photoreceptor which is located within the discs of the eye rod cell. Parma, J. et al. (1993, Nature 365 :   649-651)   report that somatic activating mutations in the   thyrotropin   receptor cause hyperfunctioning thyroid adenomas and suggest that certain GPCRs susceptible to constitutive activation may behave as   protooncogenes.   



  Nuclear Receptors 
Nuclear receptors bind small molecules such as hormones or second messengers, leading to increased receptor-binding affinity to specific chromosomal DNA elements. In addition the affinity for other nuclear proteins may also be altered. Such binding and protein-protein interactions may regulate and modulate gene expression. Examples of such receptors include the steroid hormone receptors family, the retinoic acid receptors family, and the thyroid hormone receptors family. 



  Ligand-Gated Receptor Ion Channels 

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Ligand-gated receptor ion channels fall into two categories. The first category, extracellular ligand-gated receptor ion channels (ELGs), rapidly transduce neurotransmitter-binding events into electrical signals, such as fast synaptic neurotransmission. ELG function is regulated by post- translational modification. The second category, intracellular ligand-gated receptor ion channels (ILGs), are activated by many intracellular second messengers and do not require post-translational modification (s) to effect a channel-opening response. 



   ELGs depolarize excitable cells to the threshold of action potential generation. In non-excitable cells, ELGs permit a limited calcium ion-influx during the presence of agonist. ELGs include channels directly gated by neurotransmitters such as acetylcholine, L-glutamate, glycine, ATP, serotonin, GABA, and histamine. ELG genes encode proteins having strong structural and functional similarities. 



  ILGs are encoded by distinct and unrelated gene families and include receptors for cAMP, cGMP, calcium ions, ATP, and metabolites of   arachidonic   acid. 



  Macrophage Scavenger Receptors 
Macrophage scavenger receptors with broad ligand specificity may participate in the binding of low density lipoproteins (LDL) and foreign antigens. Scavenger receptors types I and II are trimeric membrane proteins with each subunit containing a small N-terminal intracellular domain, a transmembrane domain, a large extracellular domain, and a C-terminal cysteine-rich domain The extracellular domain contains a short spacer domain, an a-helical coiled-coil domain, and a triple helical collagenous domain. These receptors have been shown to bind a spectrum of ligands, including chemically modified lipoproteins and albumin, polyribonucleotides, polysaccharides, phospholipids, and asbestos (Matsumoto, A. et al. (1990) Proc. Natl. Acad. Sci. USA 87 : 9133-9137 ; Elomaa,   O.   et al. 



  (1995) Cell 80 : 603-609). The scavenger receptors are thought to play a key role in atherogenesis by mediating uptake of modified LDL in arterial walls, and in host defense by binding bacterial endotoxins, bacteria, and protozoa. 



  T-Cell Receptors 
T cells play a dual role in the immune system as effectors and regulators, coupling antigen recognition with the transmission of signals that induce cell death in infected cells and stimulate proliferation of other immune cells. Although a population of T cells can recognize a wide range of different antigens, an individual T cell can only recognize a single antigen and only when it is presented to the T cell receptor (TCR) as a peptide complexed with a major histocompatibility molecule (MHC) on the surface of an antigen presenting cell. The TCR on most T cells consists of immunoglobulin-like integral membrane glycoproteins containing two polypeptide subunits, a and, of similar molecular weight.

   Both TCR subunits have an extracellular domain containing both variable and constant regions, a transmembrane domain that traverses the membrane once, and a short intracellular domain 

 <Desc/Clms Page number 60> 

 (Saito, H. et al. (1984) Nature 309 : 757-762). The genes for the TCR subunits are constructed through somatic rearrangement of different gene segments. Interaction of antigen in the proper MHC context with the TCR initiates signaling cascades that induce the proliferation, maturation, and function of cellular components of the immune system (Weiss, A.   (1991) Annu.   Rev. Genet. 25 :   487-510).   



  Rearrangements in TCR genes and alterations in TCR expression have been noted in lymphomas, leukemias, autoimmune disorders, and immunodeficiency disorders (Aisenberg, A. C. et al. (1985) N. 



  Engl. J. Med. 313 : 529-533 ; Weiss, supra). 



    Intracellular   Signaling Molecules 
Intracellular signaling is the general process by which cells respond to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.) through a cascade of biochemical reactions that begins with the binding of a signaling molecule to a cell membrane receptor and ends with the activation of an intracellular target molecule. Intermediate steps in the process involve the activation of various cytoplasmic proteins by phosphorylation via protein kinases, and their deactivation by protein phosphatases, and the eventual translocation of some of these activated proteins to the cell nucleus where the transcription of specific genes is triggered.

   The intracellular signaling process regulates all types of cell functions including cell proliferation, cell differentiation, and gene transcription, and involves a diversity of molecules including protein kinases and phosphatases, and second messenger molecules, such as cyclic nucleotides,   calcium-calmodulin,   inositol, and various mitogens, that regulate protein phosphorylation. 



  Protein Phosphorylation 
Protein kinases and phosphatases play a key role in the intracellular signaling process by controlling the phosphorylation and activation of various signaling proteins. The high energy phosphate for this reaction is generally transferred from the adenosine triphosphate molecule   (ATP)   to a particular protein by a protein kinase and removed from that protein by a protein phosphatase. 



  Protein kinases are roughly divided into two groups : those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate serine or threonine residues   (serine/threonine   kinases, STK). A few protein kinases have dual specificity for serine/threonine and tyrosine residues. Almost all kinases contain a conserved 250-300 amino acid catalytic domain containing specific residues and sequence motifs characteristic of the kinase family (Hardie, G. and S. 



  Hanks (1995) The Protein Kinase Facts Books, Vol 1 : 7-20, Academic Press, San Diego CA). 



   STKs include the second messenger dependent protein kinases such as the cyclic-AMP dependent protein kinases (PKA), involved in mediating hormone-induced cellular responses ; calcium-calmodulin (CaM) dependent protein kinases, involved in regulation of smooth muscle 

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 contraction, glycogen breakdown, and neurotransmission ; and the mitogen-activated protein kinases (MAP) which mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, K. J. et al. 



  (1994) Harrison's Principles of Internal Medicine McGraw-Hill, New York NY, pp. 416-431, 1887). 



   PTKs are divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembrane PTKs are receptors for most growth factors. Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors. Receptors that function through non-receptor PTKs include those for cytokines and hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes. 



  Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells in which their activation was no longer subject to normal cellular controls. In fact, about one third of the known oncogenes encode PTKs, and it is well known that cellular transformation (oncogenesis) is often accompanied by increased tyrosine phosphorylation activity (Charbonneau, H. and N. K. Tonks (1992) Annu. Rev. Cell Biol. 8 : 463-493). 



   An additional family of protein kinases previously thought to exist only in procaryotes is the histidine protein kinase family (HPK). HPKs bear little homology with mammalian STKs or PTKs but have distinctive sequence motifs of their own (Davie, J. R. et al. (1995) J. Biol. Chem. 



  270 : 19861-19867). A histidine residue in the N-terminal half of the molecule (region   I)   is an autophosphorylation site. Three additional motifs located in the C-terminal half of the molecule include an invariant asparagine residue in region II and two glycine-rich loops characteristic of nucleotide binding domains in regions III and IV. Recently a branched chain alpha-ketoacid dehydrogenase kinase has been found with characteristics of HPK in rat (Davie, supra). 



   Protein phosphatases regulate the effects of protein kinases by removing phosphate groups from molecules previously activated by kinases. The two principal categories of protein phosphatases are the protein (serine/threonine) phosphatases (PPs) and the protein tyrosine phosphatases   (PTPs).   



  PPs dephosphorylate   phosphoserine/threonine   residues and are important regulators of many cAMP-mediated hormone responses (Cohen, P. (1989) Annu. Rev. Biochem. 58 : 453-508). PTPs reverse the effects of protein tyrosine kinases and play a significant role in cell cycle and cell signaling processes (Charbonneau, supra). As previously noted, many PTKs are encoded by oncogenes, and oncogenesis is often accompanied by increased tyrosine phosphorylation activity. It is therefore possible that PTPs may prevent or reverse cell transformation and the growth of various cancers by controlling the levels of tyrosine phosphorylation in cells.

   This hypothesis is supported by studies showing that overexpression of PTPs can suppress transformation in cells, and that specific 

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 inhibition of PTPs can enhance cell transformation (Charbonneau, supra). 



  Phospholipid and Inositol-Phosphate Signaling 
Inositol phospholipids (phosphoinositides) are involved in an intracellular signaling pathway that begins with binding of a signaling molecule to a G-protein linked receptor in the plasma membrane. This leads to the phosphorylation of phosphatidylinositol (PI) residues on the inner side of the plasma membrane to the biphosphate state   (PIP2)   by inositol kinases. Simultaneously, the G- protein linked receptor binding stimulates a trimeric G-protein which in turn activates a phosphoinositide-specific phospholipase   C-p.   Phospholipase C-then cleaves PIP2 into two products, inositol triphosphate   (IP3)   and diacylglycerol.

   These two products act as mediators for separate signaling events.   IP3   diffuses through the plasma membrane to induce calcium release from the endoplasmic reticulum (ER), while diacylglycerol remains in the membrane and helps activate protein kinase C, an STK that phosphorylates selected proteins in the target cell. The calcium response initiated by IP3 is terminated by the dephosphorylation of   IP3   by specific inositol phosphatases. Cellular responses that are mediated by this pathway are glycogen breakdown in the liver in response to vasopressin, smooth muscle contraction in response to acetylcholine, and thrombin-induced platelet aggregation. 



  Cyclic Nucleotide Signaling 
Cyclic nucleotides (cAMP and cGMP) function as intracellular second messengers to transduce a variety of extracellular signals including hormones, light, and neurotransmitters. In particular, cyclic-AMP dependent protein kinases (PKA) are thought to account for all of the effects of cAMP in most mammalian cells, including various hormone-induced cellular responses. Visual excitation and the   phototransmission   of light signals in the eye is controlled by cyclic-GMP regulated,   Ca2+-specific   channels. Because of the importance of cellular levels of cyclic nucleotides in mediating these various responses, regulating the synthesis and breakdown of cyclic nucleotides is an important matter.

   Thus adenylyl cyclase, which synthesizes cAMP from AMP, is activated to increase cAMP levels in muscle by binding of adrenaline   to P-andrenergic   receptors, while activation of guanylate cyclase and increased cGMP levels in photoreceptors leads to reopening of the   Ca2+-specific   channels and recovery of the dark state in the eye. In contrast, hydrolysis of cyclic nucleotides by cAMP and cGMP-specific phosphodiesterases   (PDEs)   produces the opposite of these and other effects mediated by increased cyclic nucleotide levels. PDEs appear to be particularly important in the regulation of cyclic nucleotides, considering the diversity found in this family of proteins.

   At least seven families of mammalian PDEs   (PDE1-7)   have been identified based on substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitory drugs (Beavo, J. A.   (1995)   Physiological Reviews 75 : 725-748). PDE inhibitors have been found to be particularly 

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 useful in treating various clinical disorders. Rolipram, a specific inhibitor of PDE4, has been used in the treatment of depression, and similar inhibitors are undergoing evaluation as anti-inflammatory agents. Theophylline is a nonspecific PDE inhibitor used in the treatment of bronchial asthma and other respiratory diseases (Banner, K. H. and C. P. Page (1995) Eur. Respir. J. 8 : 996-1000). 



  G-Protein Signaling 
Guanine nucleotide binding proteins (G-proteins) are critical mediators of signal transduction between a particular class of extracellular receptors, the G-protein coupled receptors (GPCR), and intracellular second messengers such as cAMP and   Ca2+.   G-proteins are linked to the cytosolic side of a GPCR such that activation of the GPCR by ligand binding stimulates binding of the G-protein to GTP, inducing an"active"state in the G-protein. In the active state, the G-protein acts as a signal to trigger other events in the cell such as the increase of cAMP levels or the release of Ca2+ into the cytosol from the ER, which, in turn, regulate phosphorylation and activation of other intracellular proteins. Recycling of the G-protein to the inactive state involves hydrolysis of the bound GTP to GDP by a GTPase activity in the G-protein.

   (See Alberts, B. et al. (1994) Molecular Biology of the Cell, Garland Publishing, Inc., New York NY, pp. 734-759.) Two structurally distinct classes of G- proteins are recognized : heterotrimeric G-proteins, consisting of three different subunits, and monomeric, low molecular weight (LMW), G-proteins consisting of a single polypeptide chain. 



   The three polypeptide subunits of heterotrimeric G-proteins are the   a,      ,   and y subunits. The a subunit binds and hydrolyzes GTP. The       and   Y   subunits form a tight complex that anchors the protein to the inner side of the plasma membrane. The   P   subunits, also known as   G-    proteins or        transducins, contain seven tandem repeats of the WD-repeat sequence motif, a motif found in many proteins with regulatory functions. Mutations and variant expression of   P   transducin proteins are linked with various disorders (Neer, E. J. et al. (1994) Nature 371 : 297-300 ; Margottin, F. et al. (1998) Mol. Cell 1 : 565-574). 



   LMW GTP-proteins are GTPases which regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. They consist of single polypeptides which, like the a subunit of the heterotrimeric G-proteins, are able to bind and hydrolyze GTP, thus cycling between an inactive and an active state. At least sixty members of the LMW G-protein superfamily have been identified and are currently grouped into the six subfamilies of ras, rho, arf, sarl, ran, and rab. 



  Activated ras genes were initially found in human cancers, and subsequent studies confirmed that ras function is critical in determining whether cells continue to grow or become differentiated. Other members of the LMW G-protein superfamily have roles in signal transduction that vary with the function of the activated genes and the locations of the G-proteins. 



   Guanine nucleotide exchange factors regulate the activities of LMW G-proteins by 

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 determining whether GTP or GDP is bound. GTPase-activating protein (GAP) binds to GTP-ras and induces it to hydrolyze GTP to GDP. In contrast, guanine nucleotide releasing protein (GNRP) binds to GDP-ras and induces the release of GDP and the binding of GTP. 



   Other regulators of G-protein signaling (RGS) also exist that act primarily by negatively regulating the G-protein pathway by an unknown mechanism (Druey, K.   M.   et al. (1996) Nature 379 : 742-746). Some 15 members of the RGS family have been identified. RGS family members are related structurally through similarities in an approximately 120 amino acid region termed the RGS domain and functionally by their ability to inhibit the interleukin (cytokine) induction of MAP kinase in cultured mammalian 293T cells (Druey, supra). 



  Calcium Signaling Molecules   Ca+2   is another second messenger molecule that is even more widely used as an intracellular mediator than cAMP. Two pathways exist by which   Ca+2   can enter the cytosol in response to extracellular signals : One pathway acts primarily in nerve signal transduction where   Ca+2   enters a nerve terminal through a voltage-gated Ca+2 channel. The second is a more ubiquitous pathway in which   Ca+2   is released from the ER into the cytosol in response to binding of an extracellular signaling molecule to a receptor.   Ca2+   directly activates regulatory enzymes, such as protein kinase C, which trigger signal transduction pathways.

   Ca2+ also binds to specific   Ca2+-binding   proteins (CBPs) such as calmodulin (CaM) which then activate multiple target proteins in the cell including enzymes, membrane transport pumps, and ion channels. CaM interactions are involved in a multitude of cellular processes including, but not limited to, gene regulation, DNA synthesis, cell cycle progression, mitosis, cytokinesis, cytoskeletal organization, muscle contraction, signal transduction, ion homeostasis, exocytosis, and metabolic regulation (Celio, M. R. et al. (1996) Guidebook to Calcium-binding Proteins, Oxford University Press, Oxford, UK, pp. 15-20). Some CBPs can serve as a storage depot for   Ca2+   in an inactive state. Calsequestrin is one such CBP that is expressed in isoforms specific to cardiac muscle and skeletal muscle.

   It is suggested that calsequestrin binds   Ca2+   in a rapidly exchangeable state that is released during   Ca2+-signaling   conditions (Celio, M. R. et al. 



  (1996) Guidebook to Calcium-binding Proteins, Oxford University Press, New York NY, pp. 222- 224). 



  Cyclins 
Cell division is the fundamental process by which all living things grow and reproduce. In most organisms, the cell cycle consists of three principle steps ; interphase, mitosis, and cytokinesis. 



  Interphase, involves preparations for cell division, replication of the DNA and production of essential proteins. In mitosis, the nuclear material is divided and separates to opposite sides of the cell. 



  Cytokinesis is the final division and fission of the cell cytoplasm to produce the daughter cells. 

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   The entry and exit of a cell from mitosis is regulated by the synthesis and destruction of a family of activating proteins called cyclins. Cyclins act by binding to and activating a group of cyclin-dependent protein kinases (Cdks) which then phosphorylate and activate selected proteins involved in the mitotic process. Several types of cyclins exist. (Ciechanover, A. (1994) Cell 79 : 13-21.) Two principle types are mitotic cyclin, or cyclin B, which controls entry of the cell into mitosis, and Gl cyclin, which controls events that drive the cell out of mitosis. 



  Signal Complex Scaffolding Proteins
Ceretain proteins in intracellular signaling pathways serve to link or cluster other proteins involved in the signaling cascade. A conserved protein domain called the PDZ domain has been identified in various membrane-associated signaling proteins. This domain has been implicated in receptor and ion channel clustering and in the targeting of multiprotein signaling complexes to specialized functional regions of the cytosolic face of the plasma membrane. (For a review of PDZ domain-containing proteins, see Ponting, C. P. et al. (1997) Bioessays 19 : 469-479.) A large proportion of PDZ domains are found in the eukaryotic MAGUK (membrane-associated guanylate kinase) protein family, members of which bind to the intracellular domains of receptors and channels. 



  However, PDZ domains are also found in diverse membrane-localized proteins such as protein tyrosine phosphatases,   serine/threonine   kinases, G-protein cofactors, and synapse-associated proteins such as syntrophins and neuronal nitric oxide synthase (nNOS). Generally, about one to three PDZ domains are found in a given protein, although up to nine PDZ domains have been identified in a single protein. 



  Membrane Transport Molecules
The plasma membrane acts as a barrier to most molecules. Transport between the cytoplasm and the extracellular environment, and between the cytoplasm and lumenal spaces of cellular organelles requires specific transport proteins. Each transport protein carries a particular class of molecule, such as ions, sugars, or amino acids, and often is specific to a certain molecular species of the class. A variety of human inherited diseases are caused by a mutation in a transport protein. For example, cystinuria is an inherited disease that results from the inability to transport cystine, the disulfide-linked dimer of cysteine, from the urine into the blood. Accumulation of cystine in the urine leads to the formation of cystine stones in the kidneys. 



   Transport proteins are multi-pass transmembrane proteins, which either actively transport molecules across the membrane or passively allow them to cross. Active transport involves directional pumping of a solute across the membrane, usually against an electrochemical gradient. 



  Active transport is tightly coupled to a source of metabolic energy, such as ATP hydrolysis or an 

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 electrochemically favorable ion gradient. Passive transport involves the movement of a solute down its electrochemical gradient. Transport proteins can be further classified as either carrier proteins or channel proteins. Carrier proteins, which can function in active or passive transport, bind to a specific solute to be transported and undergo a   conformational   change which transfers the bound solute across the membrane. Channel proteins, which only function in passive transport, form hydrophilic pores across the membrane. When the pores open, specific solutes, such as inorganic ions, pass through the membrane and down the electrochemical gradient of the solute. 



   Carrier proteins which transport a single solute from one side of the membrane to the other are called uniporters. In contrast, coupled transporters link the transfer of one solute with simultaneous or sequential transfer of a second solute, either in the same direction (symport) or in the opposite direction (antiport). For example, intestinal and kidney epithelium contains a variety of symporter systems driven by the sodium gradient that exists across the plasma membrane. Sodium moves into the cell down its electrochemical gradient and brings the solute into the cell with it. The sodium gradient that provides the driving force for solute uptake is maintained by the ubiquitous   Na+/K   ATPase. Sodium-coupled transporters include the mammalian glucose transporter   (SGLT1),   iodide transporter (NIS), and multivitamin transporter (SMVT).

   All three transporters have twelve putative transmembrane segments, extracellular glycosylation sites, and cytoplasmically-oriented   N-   and C-termini. NIS plays a crucial role in the evaluation, diagnosis, and treatment of various thyroid pathologies because it is the molecular basis for radioiodide thyroid-imaging techniques and for specific targeting of radioisotopes to the thyroid gland (Levy,   O.   et al. (1997) Proc. Natl. Acad. Sci. 



  USA 94 : 5568-5573). SMVT is expressed in the intestinal mucosa, kidney, and placenta, and is implicated in the transport of the water-soluble vitamins, e. g., biotin and pantothenate (Prasad, P. D. et al. (1998) J. Biol. Chem. 273 : 7501-7506). 



   Transporters play a major role in the regulation of pH, excretion of drugs, and the cellular   K+/Na   balance. Monocarboxylate anion transporters are proton-coupled symporters with a broad substrate specificity that includes L-lactate, pyruvate, and the ketone bodies acetate, acetoacetate, and beta-hydroxybutyrate. At least seven isoforms have been identified to date. The isoforms are predicted to have twelve transmembrane (TM) helical domains with a large intracellular loop between TM6 and TM7, and play a critical role in maintaining intracellular pH by removing the protons that are produced stoichiometrically with lactate during glycolysis. The best characterized H   (+)-monocarboxylate   transporter is that of the erythrocyte membrane, which transports L-lactate and a wide range of other aliphatic monocarboxylates.

   Other cells possess H (+)-linked monocarboxylate transporters with differing substrate and inhibitor selectivities. In particular, cardiac muscle and tumor cells have transporters that differ in their Km values for certain substrates, including stereoselectivity for L-over 

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 D-lactate, and in their sensitivity to inhibitors. There are Na (+)-monocarboxylate cotransporters on the   luminal   surface of intestinal and kidney epithelia, which allow the uptake of lactate, pyruvate, and ketone bodies in these tissues. In addition, there are specific and selective transporters for organic cations and organic anions in organs including the kidney, intestine and liver. Organic anion transporters are selective for hydrophobic, charged molecules with electron-attracting side groups. 



  Organic cation transporters, such as the ammonium transporter, mediate the secretion of a variety of drugs and endogenous metabolites, and contribute to the maintenance of intercellular pH. (Poole, R. C. and A. P. Halestrap (1993) Am. J. Physiol. 264 : C761-C782 ; Price, N. T. et al. (1998) Biochem. J. 



  329 : 321-328 ;   and Martinelle,   K. and   I.   Haggstrom (1993) J. Biotechnol. 30 : 339-350.) 
The largest and most diverse family of transport proteins known is the ATP-binding cassette (ABC) transporters. As a family, ABC transporters can transport substances that differ markedly in chemical structure and size, ranging from small molecules such as ions, sugars, amino acids, peptides, and phospholipids, to lipopeptides, large proteins, and complex hydrophobic drugs. ABC proteins consist of four modules : two nucleotide-binding domains (NBD), which hydrolyze ATP to supply the energy required for transport, and two membrane-spanning domains (MSD), each containing six putative transmembrane segments. These four modules may be encoded by a single gene, as is the case for the cystic fibrosis transmembrane regulator (CFTR), or by separate genes.

   When encoded by separate genes, each gene product contains a single NBD and MSD.   These"half-molecules"form   homo-and heterodimers, such as Tapl and Tap2, the endoplasmic reticulum-based major histocompatibility (MHC) peptide transport system. Several genetic diseases are attributed to defects in ABC transporters, such as the following diseases and their corresponding proteins : cystic fibrosis (CFTR, an ion channel), adrenoleukodystrophy (adrenoleukodystrophy protein, ALDP), Zellweger syndrome (peroxisomal membrane protein-70, PMP70), and hyperinsulinemic hypoglycemia (sulfonylurea receptor, SUR). Overexpression of the multidrug resistance (MDR) protein, another ABC transporter, in human cancer cells makes the cells resistant to a variety of cytotoxic drugs used in chemotherapy (Taglight, D. and S. Michaelis (1998) Meth. Enzymol. 292 : 131-163). 



   Transport of fatty acids across the plasma membrane can occur by diffusion, a high capacity, low affinity process. However, under normal physiological conditions a significant fraction of fatty acid transport appears to occur via a high affinity, low capacity protein-mediated transport process. 



  Fatty acid transport protein   (FATP),   an integral membrane protein with four transmembrane segments, is expressed in tissues exhibiting high levels of plasma membrane fatty acid flux, such as muscle, heart, and adipose. Expression   of FATP   is upregulated in   3T3-L1   cells during adipose conversion, and expression in COS7 fibroblasts elevates uptake of long-chain fatty acids (Hui, T. Y. et al. (1998) J. 



  Biol. Chem. 273 : 27420-27429). 

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  Ion Channels 
The electrical potential of a cell is generated and maintained by controlling the movement of ions across the plasma membrane. The movement of ions requires ion channels, which form an ion- selective pore within the membrane. There are two basic types of ion channels, ion transporters and gated ion channels. Ion transporters utilize the energy obtained from ATP hydrolysis to actively transport an ion against the ion's concentration gradient. Gated ion channels allow passive flow of an ion down the ion's electrochemical gradient under restricted conditions.

   Together, these types of ion channels generate, maintain, and utilize an electrochemical gradient that is used in 1) electrical impulse conduction down the axon of a nerve cell, 2) transport of molecules into cells against concentration gradients, 3) initiation of muscle contraction, and 4) endocrine cell secretion. 



   Ion transporters generate and maintain the resting electrical potential of a cell. Utilizing the energy derived from ATP hydrolysis, they transport ions against the ion's concentration gradient. 



  These transmembrane ATPases are divided into three families. The phosphorylated (P) class ion transporters, including   Na+-K+   ATPase,   Ca2+-ATPase,   and   H+-ATPase,   are activated by a phosphorylation event. P-class ion transporters are responsible for maintaining resting potential distributions such that cytosolic concentrations of Ne and   Ca2+   are low and cytosolic concentration of   K   is high. The vacuolar   (V)   class of ion transporters includes H+ pumps on intracellular organelles, such as lysosomes and Golgi. V-class ion transporters are responsible for generating the low pH within the lumen of these organelles that is required for function. The coupling factor (F) class consists of H+ pumps in the mitochondria.

   F-class ion transporters utilize a proton gradient to generate ATP from ADP and inorganic phosphate   (P,).   



   The resting potential of the cell is utilized in many processes involving carrier proteins and gated ion channels. Carrier proteins utilize the resting potential to transport molecules into and out of the cell. Amino acid and glucose transport into many cells is linked to sodium ion co-transport (symport) so that the movement of Na+ down an electrochemical gradient drives transport of the other molecule up a concentration gradient. Similarly, cardiac muscle links transfer of Ca2+ out of the cell with transport of Na+ into the cell (antiport). 



   Ion channels share common structural and mechanistic themes. The channel consists of four or five subunits or protein monomers that are arranged like a barrel in the plasma membrane. Each subunit typically consists of six potential transmembrane segments   (S1,   S2, S3, S4, S5, and S6). The center of the barrel forms a pore lined by a-helices or   (3-strands.   The side chains of the amino acid residues comprising the a-helices or strands establish the charge (cation or anion) selectivity of the channel. The degree of selectivity, or what specific ions are allowed to pass through the channel, depends on the diameter of the narrowest part of the pore. 

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   Gated ion channels control ion flow by regulating the opening and closing of pores. These channels are categorized according to the manner of regulating the gating function. Mechanically-gated channels open pores in response to mechanical stress, voltage-gated channels open pores in response to changes in membrane potential, and ligand-gated channels open pores in the presence of a specific ion, nucleotide, or neurotransmitter. 



   Voltage-gated Na+ and K+ channels are necessary for the function of electrically excitable cells, such as nerve and muscle cells. Action potentials, which lead to neurotransmitter release and muscle contraction, arise from large, transient changes in the permeability of the membrane to   Na+ and K+   ions. 



  Depolarization of the membrane beyond the threshold level opens voltage-gated Na+ channels. Sodium ions flow into the cell, further depolarizing the membrane and opening more voltage-gated Na+ channels, which propagates the depolarization down the length of the cell. Depolarization also opens voltage-gated potassium channels. Consequently, potassium ions flow outward, which leads to repolarization of the membrane. Voltage-gated channels utilize charged residues in the fourth transmembrane segment (S4) to sense voltage change. The open state lasts only about 1 millisecond, at which time the channel spontaneously converts into an inactive state that cannot be opened irrespective of the membrane potential. Inactivation is mediated by the channel's N-terminus, which acts as a plug that closes the pore.

   The transition from an inactive to a closed state requires a return to resting potential. 



   Voltage-gated Na+ channels are heterotrimeric complexes composed of a 260 kDa pore forming a subunit that associates with two smaller auxiliary subunits,   pi   and    2.   The   32   subunit is an integral membrane glycoprotein that contains an extracellular Ig domain, and its association with a and   pi   subunits correlates with increased functional expression of the channel, a change in its gating properties, and an increase in whole cell capacitance due to an increase in membrane surface area. 



  (Isom, L. L. et al. (1995) Cell 83 : 433-442.) 
Voltage-gated Ca 2+ channels are involved in presynaptic neurotransmitter release, and heart and skeletal muscle contraction. The voltage-gated Ca 2+ channels from skeletal muscle (L-type) and brain (N-type) have been purified, and though their functions differ dramatically, they have similar subunit compositions. The channels are composed of three subunits. The al subunit forms the membrane pore and voltage sensor, while the   cS and P   subunits modulate the voltage-dependence, gating properties, and the current amplitude of the channel. These subunits are encoded by at least six al, one a28, and four       genes. A fourth subunit,   y,   has been identified in skeletal muscle. (Walker, D. et al. (1998) J. Biol. Chem. 273 : 2361-2367 ; and Jay, S. D. et al.

   (1990) Science 248 : 490-492.) 
Chloride channels are necessary in endocrine secretion and in regulation of cytosolic and organelle pH. In secretory epithelial cells, Cl-enters the cell across a basolateral membrane through an 

 <Desc/Clms Page number 70> 

   Na +, K+/Cl-cotransporter,   accumulating in the cell above its electrochemical equilibrium concentration. 



  Secretion of Cl-from the apical surface, in response to hormonal stimulation, leads to flow of   Na + and   water into the secretory lumen. The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel encoded by the gene for cystic fibrosis, a common fatal genetic disorder in humans. 



  Loss of CFTR function decreases transepithelial water secretion and, as a result, the layers of mucus that coat the respiratory tree, pancreatic ducts, and intestine are dehydrated and difficult to clear. The resulting blockage of these sites leads to pancreatic insufficiency,"meconium ileus", and devastating "chronic   obstructivepulmonary disease"   (Al-Awqati, Q. et al. (1992) J. Exp. Biol. 172 : 245-266). 



   Many intracellular organelles contain H+-ATPase pumps that generate transmembrane pH and electrochemical differences by moving protons from the cytosol to the organelle lumen. If the membrane of the organelle is permeable to other ions, then the electrochemical gradient can be abrogated without affecting the pH differential. In fact, removal of the electrochemical barrier allows more H + to be pumped across the membrane, increasing the pH differential. Cl-is the sole counterion   of H + translocation   in a number of organelles, including   chromaffin   granules, Golgi vesicles, lysosomes, and endosomes.

   Functions that require a low vacuolar pH include uptake of small molecules such as   biogenic   amines in   chromaffm   granules, processing of vacuolar constituents such as pro-hormones by proteolytic enzymes, and protein degradation in lysosomes (Al-Awqati, supra). 



   Ligand-gated channels open their pores when an extracellular or intracellular mediator binds to the channel. Neurotransmitter-gated channels are channels that open when a neurotransmitter binds to their extracellular domain. These channels exist in the postsynaptic membrane of nerve or muscle cells. 



  There are two types of neurotransmitter-gated channels. Sodium channels open in response to excitatory neurotransmitters, such as acetylcholine, glutamate, and serotonin. This opening causes an influx of Na+ and produces the initial localized depolarization that activates the voltage-gated channels and starts the action potential. Chloride channels open in response to inhibitory neurotransmitters, such as   y-aminobutyric   acid (GABA) and glycine, leading to hyperpolarization of the membrane and the subsequent generation of an action potential. 



   Ligand-gated channels can be regulated by intracellular second messengers. Calcium-activated K+ channels are gated by internal calcium ions. In nerve cells, an influx of calcium during depolarization opens K+ channels to modulate the magnitude of the action potential   (Ishi,   T. M. et al. 



  (1997) Proc. Natl. Acad. Sci. USA 94 : 11651-11656). Cyclic nucleotide-gated (CNG) channels are gated by cytosolic cyclic nucleotides. The best examples of these are the cAMP-gated Na+ channels involved in olfaction and the cGMP-gated cation channels involved in vision. Both systems involve ligand-mediated activation of a G-protein coupled receptor which then alters the level of cyclic nucleotide within the cell. 

 <Desc/Clms Page number 71> 

 



   Ion channels are expressed in a number of tissues where they are implicated in a variety of processes. CNG channels, while abundantly expressed in photoreceptor and olfactory sensory cells, are also found in kidney, lung, pineal, retinal ganglion cells, testis, aorta, and brain.   Calcium-activated K+   channels may be responsible for the vasodilatory effects of   bradykinin   in the kidney and for shunting excess   K+ from   brain capillary endothelial cells into the blood. They are also implicated in repolarizing granulocytes after agonist-stimulated depolarization   (Ishi,   supra) Ion channels have been the target for many drug therapies. Neurotransmitter-gated channels have been targeted in therapies for treatment of insomnia, anxiety, depression, and schizophrenia.

   Voltage-gated channels have been targeted in therapies for arrhythmia,   ischemic   stroke, head trauma, and neurodegenerative disease (Taylor, C. P. and L. S.   Narasimhan   (1997) Adv. Pharmacol. 39 : 47-98). 



  Disease Correlation 
The etiology of numerous human diseases and disorders can be attributed to defects in the transport of molecules across membranes. Defects in the   trafficking   of membrane-bound transporters and ion channels are associated with several disorders, e. g. cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, von Gierke disease, and certain forms of diabetes mellitus. Single-gene defect diseases resulting in an inability to transport small molecules across membranes include, e. g., cystinuria,   iminoglycinuria,   Hartup disease, and Fanconi disease (van't Hoff, W. G. (1996) Exp.   Nephrol.   4 : 253-262 ; Talente, G. M. et al. (1994) Ann. Intern. Med. 120 : 218-226 ; and   Chillon,   M. et al. (1995) New Engl. J. Med. 332 : 1475-1480). 



  Protein Modification and Maintenance Molecules 
The cellular processes regulating modification and maintenance of protein molecules coordinate their conformation, stabilization, and degradation. Each of these processes is mediated by key enzymes or proteins such as proteases, protease inhibitors, transferases, isomerases, and molecular chaperones. 



  Proteases 
Proteases cleave proteins and peptides at the peptide bond that forms the backbone of the peptide and protein chain. Proteolytic processing is essential to cell growth, differentiation, remodeling, and homeostasis as well as inflammation and immune response. Typical protein half- lives range from hours to a few days, so that within all living cells, precursor proteins are being cleaved to their active form, signal sequences proteolytically removed from targeted proteins, and aged or defective proteins degraded by proteolysis. Proteases function in bacterial, parasitic, and viral invasion and replication within a host. Four principal categories of mammalian proteases have been identified based on active site structure, mechanism of action, and overall three-dimensional structure. 

 <Desc/Clms Page number 72> 

 



  (Beynon, R. J. and J. S. Bond (1994) Proteolytic Enzymes : A Practical Approach. Oxford University Press, New York NY, pp. 1-5). 



   The serine proteases (SPs) have a serine residue, usually within a conserved sequence, in an active site composed of the serine, an aspartate, and a histidine residue. SPs include the digestive enzymes trypsin and chymotrypsin, components of the complement cascade and the blood-clotting cascade, and enzymes that control extracellular protein degradation. The main SP sub-families are trypases, which cleave after arginine or lysine ; aspartases, which cleave after aspartate ; chymases, which cleave after phenylalanine or leucine ; metases, which cleavage after methionine ; and serases which cleave after serine. Enterokinase, the initiator of intestinal digestion, is a serine protease found in the intestinal brush border, where it cleaves the acidic propeptide from trypsinogen to yield active trypsin (Kitamoto, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 7588-7592). 



  Prolylcarboxypeptidase, a lysosomal serine peptidase that cleaves peptides such as angiotensin II and III and [des-Arg9]   bradykinin,   shares sequence homology with members of both the serine carboxypeptidase and prolylendopeptidase families (Tan, F. et al. (1993) J. Biol. Chem. 268 :   16631-   16638). 



   Cysteine proteases (CPs) have a cysteine as the major catalytic residue at an active site where catalysis proceeds via an intermediate thiol ester and is facilitated by adjacent histidine and aspartic acid residues. CPs are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation. Mammalian CPs include lysosomal cathepsins and cytosolic calcium activated proteases, calpains. CPs are produced by monocytes, macrophages and other cells of the immune system which migrate to sites of inflammation and secrete molecules involved in tissue repair. Overabundance of these repair molecules plays a role in certain disorders.

   In autoimmune diseases such as rheumatoid arthritis, secretion of the cysteine peptidase cathepsin C degrades collagen,   laminin,   elastin and other structural proteins found in the extracellular matrix of bones. 



   Aspartic proteases are members of the cathepsin family of lysosomal proteases and include pepsin A,   gastricsin,   chymosin, renin, and cathepsins D and E. Aspartic proteases have a pair of aspartic acid residues in the active site, and are most active in the pH 2-3 range, in which one of the aspartate residues is ionized, the other un-ionized. Aspartic proteases include bacterial penicillopepsin, mammalian pepsin, renin, chymosin, and certain fungal proteases. Abnormal regulation and expression of cathepsins is evident in various inflammatory disease states. In cells isolated from inflamed synovia, the mRNA for stromelysin, cytokines,   TIMP-1,   cathepsin, gelatinase, and other molecules is preferentially expressed.

   Expression of cathepsins L and D is elevated in synovial tissues from patients with rheumatoid arthritis and osteoarthritis.   Cathepsin L   expression may 

 <Desc/Clms Page number 73> 

 also contribute to the influx of mononuclear cells which exacerbates the destruction of the rheumatoid synovium. (Keyszer, G. M. (1995) Arthritis Rheum. 38 : 976-984.) The increased expression and differential regulation of the cathepsins are linked to the metastatic potential of a variety of cancers and as such are of therapeutic and prognostic interest (Chambers, A. F. et al. (1993) Crit. Rev. Oncog. 



  4 : 95-114). 



     Metalloproteases   have active sites that include two glutamic acid residues and one histidine residue that serve as binding sites for zinc. Carboxypeptidases A and B are the principal mammalian metalloproteases. Both are exoproteases of similar structure and active sites. Carboxypeptidase A, like chymotrypsin, prefers C-terminal aromatic and aliphatic side chains of hydrophobic nature, whereas carboxypeptidase B is directed toward basic arginine and lysine residues. Glycoprotease (GCP), or   O-sialoglycoprotein   endopeptidase, is a metallopeptidase which specifically cleaves   O-sialoglycoproteins   such as glycophorin A.

   Another metallopeptidase, placenta leucine aminopeptidase   (P-LAP)   degrades several peptide hormones such as oxytocin and vasopressin, suggesting a role in maintaining homeostasis during pregnancy, and is expressed in several tissues (Rogi, T. et al. (1996) J. Biol. Chem. 271 : 56-61). 



   Ubiquitin proteases are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaryotic cells and some bacteria. The UCS mediates the elimination of abnormal proteins and regulates the half-lives of important regulatory proteins that control cellular processes such as gene transcription and cell cycle progression. In the UCS pathway, proteins targeted for degradation are conjugated to a ubiquitin, a small heat stable protein. The ubiquitinated protein is then recognized and degraded by proteasome, a large, multisubunit proteolytic enzyme complex, and ubiquitin is released for reutilization by ubiquitin protease.

   The UCS is implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins, cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, A. (1994) Cell 79 : 13-21). A murine proto-oncogene, Unp, encodes a nuclear ubiquitin protease whose overexpression leads to oncogenic transformation of NIH3T3 cells, and the human homolog of this gene is consistently elevated in small cell tumors and adenocarcinomas of the lung (Gray, D. A. 



  (1995) Oncogene 10 : 2179-2183). 



  *Signal Peptidases 
The mechanism for the translocation process into the endoplasmic reticulum (ER) involves the recognition of an N-terminal signal peptide on the elongating protein. The signal peptide directs the protein and attached ribosome to a receptor on the ER membrane. The polypeptide chain passes through a pore in the ER membrane into the lumen while the N-terminal signal peptide remains 

 <Desc/Clms Page number 74> 

 attached at the membrane surface. The process is completed when signal peptidase located inside the ER cleaves the signal peptide from the protein and releases the protein into the lumen. 



  Protease Inhibitors 
Protease inhibitors and other regulators of protease activity control the activity and effects of proteases. Protease inhibitors have been shown to control pathogenesis in animal models of proteolytic disorders (Murphy, G. (1991) Agents Actions Suppl. 35 : 69-76). Low levels of the cystatins, low molecular weight inhibitors of the cysteine proteases, correlate with malignant progression of tumors. (Calkins, C. et al (1995) Biol. Biochem. Hoppe Seyler 376 : 71-80). Serpins are inhibitors of mammalian plasma serine proteases. Many serpins serve to regulate the blood clotting cascade and/or the complement cascade in mammals. Sp32 is a positive regulator of the mammalian acrosomal protease, acrosin, that binds the proenzyme, proacrosin, and thereby aides in packaging the enzyme into the acrosomal matrix (Baba, T. et al. (1994) J. Biol. Chem. 269 : 10133- 10140).

   The Kunitz family of serine protease inhibitors are characterized by one or more"Kunitz domains"containing a series of cysteine residues that are regularly spaced over approximately 50 amino acid residues and form three intrachain disulfide bonds. Members of this family include   aprotinin,   tissue factor pathway inhibitor   (TFPI-1   and TFPI-2), inter-a-trypsin inhibitor, and bikunin. 



  (Marlor, C. W. et al. (1997) J. Biol. Chem. 272 : 12202-12208.) Members of this family are potent inhibitors (in the nanomolar range) against serine proteases such as kallikrein and plasmin.   Aprotinin   has clinical utility in reduction of perioperative blood loss. 



   A major portion of all proteins synthesized in eukaryotic cells are synthesized on the cytosolic surface of the endoplasmic reticulum (ER). Before these immature proteins are distributed to other organelles in the cell or are secreted, they must be transported into the interior lumen of the ER where post-translational modifications are performed. These modifications include protein folding and the formation of disulfide bonds, and N-linked glycosylations. 



  Protein Isomerases 
Protein folding in the ER is aided by two principal types of protein isomerases, protein disulfide isomerase (PDI), and peptidyl-prolyl isomerase (PPI). PDI catalyzes the oxidation of free sulfhydryl groups in cysteine residues to form intramolecular disulfide bonds in proteins. PPI, an enzyme that catalyzes the isomerization of certain proline   imidic   bonds in oligopeptides and proteins, is considered to govern one of the rate limiting steps in the folding of many proteins to their final functional conformation. The   cyclophilins   represent a major class of PPI that was originally identified as the major receptor for the immunosuppressive drug cyclosporin A (Handschumacher, R. E. et al. (1984) Science 226 : 544-547). 



  Protein Glvcosylation 

 <Desc/Clms Page number 75> 

 
The glycosylation of most soluble secreted and membrane-bound proteins by oligosaccharides linked to asparagine residues in proteins is also performed in the ER. This reaction is catalyzed by a membrane-bound enzyme, oligosaccharyl transferase. Although the exact purpose of   this"N-linked"glycosylation   is unknown, the presence of oligosaccharides tends to make a glycoprotein resistant to protease digestion. In addition, oligosaccharides attached to cell-surface proteins called selectins are known to function in cell-cell adhesion processes (Alberts, B. et al. 



  (1994) Molecular Biology of the Cell, Garland Publishing Co., New York NY, p. 608)."O-linked" glycosylation of proteins also occurs in the ER by the addition of N-acetylgalactosamine to the hydroxyl group of a serine or threonine residue followed by the sequential addition of other sugar residues to the first. This process is catalysed by a series of glycosyltransferases each specific for a particular donor sugar nucleotide and acceptor molecule (Lodish, H. et al. (1995) Molecular Cell   Biology.     W.   H. Freeman and Co., New York NY, pp. 700-708). In many cases, both N-and 0-linked oligosaccharides appear to be required for the secretion of proteins or the movement of plasma membrane glycoproteins to the cell surface. 



   An additional glycosylation mechanism operates in the ER specifically to target lysosomal enzymes to lysosomes and prevent their secretion. Lysosomal enzymes in the ER receive an N-linked oligosaccharide, like plasma membrane and secreted proteins, but are then phosphorylated on one or two mannose residues. The phosphorylation of mannose residues occurs in two steps, the first step being the addition of an N-acetylglucosamine phosphate residue by   N-acetylglucosamine   phosphotransferase, and the second the removal of the   N-acetylglucosamine   group by phosphodiesterase. The phosphorylated mannose residue then targets the lysosomal enzyme to a mannose 6-phosphate receptor which transports it to a lysosome vesicle (Lodish, supra, pp. 708-711). 



  Chaperones 
Molecular chaperones are proteins that aid in the proper folding of immature proteins and refolding of improperly folded ones, the assembly of protein subunits, and in the transport of unfolded proteins across membranes. Chaperones are also called heat-shock proteins (hsp) because of their tendency to be expressed in dramatically increased amounts following brief exposure of cells to elevated temperatures. This latter property most likely reflects their need in the refolding of proteins that have become denatured by the high temperatures. Chaperones may be divided into several classes according to their location, function, and molecular weight, and include hsp60, TCP1, hsp70, hsp40 (also called DnaJ), and hsp90.

   For example, hsp90 binds to steroid hormone receptors, represses transcription in the absence of the ligand, and provides proper folding of the ligand-binding domain of the receptor in the presence of the hormone (Burston, S. G. and A. R. Clarke (1995) Essays Biochem. 29 : 125-136). Hsp60 and hsp70 chaperones aid in the transport and folding of newly 

 <Desc/Clms Page number 76> 

 synthesized proteins.   Hsp70   acts early in protein folding, binding a newly synthesized protein before it leaves the ribosome and transporting the protein to the mitochondria or ER before releasing the folded protein. Hsp60, along with hsplO, binds misfolded proteins and gives them the opportunity to refold correctly. All chaperones share an affinity for hydrophobic patches on incompletely folded proteins and the ability to hydrolyze ATP.

   The energy of ATP hydrolysis is used to release the hspbound protein in its properly folded state (Alberts, supra, pp 214, 571-572). 



  Nucleic Acid Synthesis and Modification Molecules Polvmerases
DNA and RNA replication are critical processes for cell replication and function. DNA and RNA replication are mediated by the enzymes DNA and RNA polymerase, respectively, by a "templating"process in which the nucleotide sequence of a DNA or RNA strand is copied by complementary base-pairing into a complementary nucleic acid sequence of either DNA or RNA. 



  However, there are fundamental'differences between the two processes. 



   DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotide to the 3'-OH end of a polynucleotide strand (the primer strand) that is paired to a second (template) strand. The new DNA strand therefore grows in the 5'to 3'direction (Alberts, B. et al. (1994) The Molecular Biology of the Cell, Garland Publishing Inc., New York NY, pp. 251-254). The substrates for the polymerization reaction are the corresponding   deoxynucleotide   triphosphates which must base-pair with the correct nucleotide on the template strand in order to be recognized by the polymerase. 



  Because DNA exists as a double-stranded helix, each of the two strands may serve as a template for the formation of a new complementary strand. Each of the two daughter cells of the dividing cell therefore inherits a new DNA double helix containing one old and one new strand. Thus, DNA is said to be replicated"semiconservatively"by DNA polymerase. In addition to the synthesis of new DNA, DNA polymerase is also involved in the repair of damaged DNA as discussed below under "Ligases."
In contrast to DNA polymerase, RNA polymerase uses a DNA template strand to"transcribe" DNA into RNA using ribonucleotide triphosphates as substrates. Like DNA polymerization, RNA polymerization proceeds in a 5'to 3'direction by addition of a   ribonucleoside     monophosphate   to the 3'-OH end of a growing RNA chain.

   DNA transcription generates messenger RNAs (mRNA) that carry information for protein synthesis, as well as the transfer, ribosomal, and other RNAs that have structural or catalytic functions. In eukaryotes, three discrete RNA polymerases synthesize the three different types of RNA (Alberts, supra, pp. 367-368). RNA polymerase I makes the large ribosomal RNAs, RNA polymerase II makes the mRNAs that will be translated into proteins, and RNA 

 <Desc/Clms Page number 77> 

 polymerase III makes a variety of small, stable RNAs, including 5S ribosomal RNA and the transfer RNAs (tRNA). In all cases, RNA synthesis is initiated by binding of the RNA polymerase to a promoter region on the DNA and synthesis begins at a start site within the promoter.

   Synthesis is completed at a broad, general stop or termination region in the DNA where both the polymerase and the completed RNA chain are released. 



  Ligases 
DNA repair is the process by which accidental base changes, such as those produced by oxidative damage, hydrolytic attack, or uncontrolled methylation of DNA are corrected before replication or transcription of the DNA can occur. Because of the efficiency of the DNA repair process, fewer than one in one thousand accidental base changes causes a mutation (Alberts, supra, pp.   245-249). The three   steps common to most types of DNA repair are (1) excision of the damaged or altered base or nucleotide by DNA nucleases, leaving a gap ; (2) insertion of the correct nucleotide in this gap by DNA polymerase using the complementary strand as the template ; and (3) sealing the break left between the inserted nucleotide (s) and the existing DNA strand by DNA ligase.

   In the last reaction, DNA ligase uses the energy from ATP hydrolysis to activate the 5'end of the broken phosphodiester bond before forming the new bond with the 3'-OH of the DNA strand. In Bloom's syndrome, an inherited human disease, individuals are partially deficient in DNA ligation and consequently have an increased incidence of cancer (Alberts, supra, p. 247). 



  Nucleases 
Nucleases comprise both enzymes that hydrolyze DNA (DNase) and RNA (RNase). They serve different purposes in nucleic acid metabolism. Nucleases hydrolyze the phosphodiester bonds between adjacent nucleotides either at internal positions (endonucleases) or at the terminal 3'or 5' nucleotide positions (exonucleases). A DNA exonuclease activity in DNA polymerase, for example, serves to remove improperly paired nucleotides attached to the 3'-OH end of the growing DNA strand by the polymerase and thereby serves a"proofreading"function. As mentioned above, DNA endonuclease activity is involved in the excision step of the DNA repair process. 



   RNases also serve a variety of functions. For example, RNase P is a ribonucleoprotein enzyme which cleaves the 5'end of pre-tRNAs as part of their maturation process. RNase H digests the RNA strand of an RNA/DNA hybrid. Such hybrids occur in cells invaded by retroviruses, and RNase H is an important enzyme in the retroviral replication cycle. Pancreatic RNase secreted by the pancreas into the intestine hydrolyzes RNA present in ingested foods. RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases (Schein, C. H. (1997) Nat. 



  Biotechnol. 15 : 529-536). Regulation of RNase activity is being investigated as a means to control tumor angiogenesis, allergic reactions, viral infection and replication, and fungal infections. 

 <Desc/Clms Page number 78> 

 



    Methvlases   
Methylation of specific nucleotides occurs in both DNA and RNA, and serves different functions in the two macromolecules. Methylation of cytosine residues to form 5-methyl cytosine in DNA occurs specifically at CG sequences which are base-paired with one another in the DNA double- helix. This pattern of methylation is passed from generation to generation during DNA replication by an enzyme called"maintenance methylase"that acts preferentially on those CG sequences that are base-paired with a CG sequence that is already methylated.

   Such methylation appears to distinguish active from inactive genes by preventing the binding of regulatory proteins that"turn on"the gene, but permit the binding of proteins that inactivate the gene (Alberts, supra, pp.   448-451).   In RNA   metabolism,"tRNA   methylase"produces one of several nucleotide modifications in tRNA that affect the conformation and base-pairing of the molecule and facilitate the recognition of the appropriate mRNA codons by specific tRNAs. The primary methylation pattern is the dimethylation of guanine residues to form   N,   N-dimethyl guanine. 



  Helicases and Single-Stranded Binding Proteins 
Helicases are enzymes that destabilize and unwind double helix structures in both DNA and RNA. Since DNA replication occurs more or less simultaneously on both strands, the two strands must first separate to generate a replication"fork"for DNA polymerase to act on. Two types of replication proteins contribute to this process, DNA helicases and single-stranded binding proteins. 



  DNA helicases hydrolyze ATP and use the energy of hydrolysis to separate the DNA strands. Single- stranded binding proteins   (SSBs)   then bind to the exposed DNA strands without covering the bases, thereby temporarily stabilizing them for templating by the DNA polymerase (Alberts, supra, pp. 255- 256). 



   RNA helicases also alter and regulate RNA conformation and secondary structure. Like the DNA helicases, RNA helicases utilize energy derived from ATP hydrolysis to destabilize and unwind RNA duplexes. The most well-characterized and ubiquitous family of RNA helicases is the DEAD- box family, so named for the conserved B-type ATP-binding motif which is diagnostic of proteins in this family. Over 40 DEAD-box helicases have been identified in organisms as diverse as bacteria, insects, yeast, amphibians, mammals, and plants. DEAD-box helicases function in diverse processes such as translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability. 



  Some DEAD-box helicases play tissue-and stage-specific roles in spermatogenesis and embryogenesis. Overexpression of the DEAD-box 1 protein   (DDX1)   may play a role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb) tumors (Godbout, R. et al. (1998) J. Biol. 



  Chem. 273 :   21161-21168).   These observations suggest that   DDX1   may promote or enhance tumor progression by altering the normal secondary structure and expression levels of RNA in cancer cells. 

 <Desc/Clms Page number 79> 

 



  Other DEAD-box helicases have been implicated either directly or indirectly in tumorigenesis (Discussed in Godbout, supra). For example, murine p68 is mutated in ultraviolet light-induced tumors, and human DDX6 is located at a chromosomal breakpoint associated with B-cell lymphoma. 



  Similarly, a chimeric protein comprised of   DDX10   and NUP98, a nucleoporin protein, may be involved in the pathogenesis of certain myeloid malignancies. 



  Topoisomerases 
Besides the need to separate DNA strands prior to replication, the two strands must be "unwound"from one another prior to their separation by DNA helicases. This function is performed by proteins known as DNA topoisomerases. DNA topoisomerase effectively acts as a reversible nuclease that hydrolyzes a phosphodiesterase bond in a DNA strand, permitting the two strands to rotate freely about one another to remove the strain of the helix, and then rejoins the original phosphodiester bond between the two strands. Two types of DNA topoisomerase exist, types I and II. 



  DNA Topoisomerase I causes a single-strand break in a DNA helix to allow the rotation of the two strands of the helix about the remaining phosphodiester bond in the opposite strand. DNA topoisomerase II causes a transient break in both strands of a DNA helix where two double helices cross over one another. This type of topoisomerase can efficiently separate two interlocked DNA circles (Alberts, supra, pp. 260-262). Type II topoisomerases are largely confined to proliferating cells in eukaryotes, such as cancer cells. For this reason they are targets for anticancer drugs. 



  Topoisomerase II has been implicated in multi-drug resistance (MDR) as it appears to aid in the repair of DNA damage inflicted by DNA binding agents such as doxorubicin and vincristine. 



  Recombinases 
Genetic recombination is the process of rearranging DNA sequences within an organism's genome to provide genetic variation for the organism in response to changes in the environment. 



  DNA recombination allows variation in the particular combination of genes present in an individual's genome, as well as the timing and level of expression of these genes (see Alberts, supra, pp. 263- 273). Two broad classes of genetic recombination are commonly recognized, general recombination and site-specific recombination. General recombination involves genetic exchange between any homologous pair of DNA sequences usually located on two copies of the same chromosome. The process is aided by enzymes called recombinases that"nick"one strand of a DNA duplex more or less randomly and permit exchange with the complementary strand of another duplex. The process does not normally change the arrangement of genes on a chromosome. In site-specific recombination, the recombinase recognizes specific nucleotide sequences present in one or both of the recombining molecules.

   Base-pairing is not involved in this form of recombination and therefore does not require DNA homology between the recombining molecules. Unlike general recombination, this form of 

 <Desc/Clms Page number 80> 

 recombination can alter the relative positions of nucleotide sequences in chromosomes. 



  Splicing Factors
Various proteins are necessary for processing of transcribed RNAs in the nucleus. PremRNA processing steps include capping at the 5'end with methylguanosine, polyadenylating the 3' end, and splicing to remove introns. The primary RNA transcript from DNA is a faithful copy of the gene containing both exon and intron sequences, and the latter sequences must be cut out of the RNA transcript to produce an mRNA that codes for a protein. This"splicing"of the mRNA sequence takes place in the nucleus with the aid of a large, multicomponent ribonucleoprotein complex known as a spliceosome. The   spliceosomal   complex is composed of five small nuclear ribonucleoprotein particles (snRNPs) designated   Ul,   U2, U4, U5, and U6, and a number of additional proteins. Each snRNP contains a single species of snRNA and about ten proteins.

   The RNA components of some snRNPs recognize and base pair with intron consensus sequences. The protein components mediate spliceosome assembly and the splicing reaction. Autoantibodies to snRNP proteins are found in the blood of patients with systemic lupus erythematosus (Stryer, L. (1995) Biochemistry, W. H. Freeman and Company, New York NY, p. 863). 



  Adhesion Molecules
The surface of a cell is rich in transmembrane proteoglycans, glycoproteins, glycolipids, and receptors. These macromolecules mediate adhesion with other cells and with components of the extracellular matrix (ECM). The interaction of the cell with its surroundings profoundly influences cell shape, strength, flexibility, motility, and adhesion. These dynamic properties are intimately associated with signal transduction pathways controlling cell proliferation and differentiation, tissue construction, and embryonic development. 



  Cadherins
Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. These proteins share multiple repeats of a cadherin-specific motif, and the repeats form the folding units of the cadherin extracellular domain. Cadherin molecules cooperate to form focal contacts, or adhesion plaques, between adjacent epithelial cells. The cadherin family includes the classical cadherins and protocadherins. Classical cadherins include the E-cadherin, N-cadherin, and P-cadherin subfamilies. 



  E-cadherin is present on many types of epithelial cells and is especially important for embryonic development. N-cadherin is present on nerve, muscle, and lens cells and is also critical for embryonic development. P-cadherin is present on cells of the placenta and epidermis. Recent studies report that protocadherins are involved in a variety of cell-cell interactions (Suzuki, S. T. (1996) J. Cell Sci. 

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  109 : 2609-2611). The intracellular anchorage of cadherins is regulated by their dynamic association with   catenins,   a family of cytoplasmic signal transduction proteins associated with the actin cytoskeleton. The anchorage of cadherins to the actin cytoskeleton appears to be regulated by protein tyrosine phosphorylation, and the cadherins are the target of phosphorylation-induced junctional disassembly (Aberle, H. et al. (1996) J. Cell. Biochem. 61 : 514-523). 



   Integrins 
Integrins are ubiquitous transmembrane adhesion molecules that link the ECM to the internal cytoskeleton. Integrins are composed of two noncovalently associated transmembrane glycoprotein subunits called a and   P.   Integrins function as receptors that play a role in signal transduction. For example, binding of integrin to its extracellular ligand may stimulate changes in intracellular calcium levels or protein kinase activity (Sjaastad, M. D. and W. J. Nelson (1997) BioEssays 19 : 47-55). At least ten cell surface receptors of the integrin family recognize the ECM component fibronectin, which is involved in many different biological processes including cell migration and embryogenesis (Johansson, S. et al. (1997) Front. Biosci. 2 :   D126-D146).   



  Lectins 
Lectins comprise a ubiquitous family of extracellular glycoproteins which bind cell surface carbohydrates specifically and reversibly, resulting in the agglutination of cells (reviewed in Drickamer, K. and M. E. Taylor (1993) Annu. Rev. Cell Biol. 9 : 237-264). This function is particularly important for activation of the immune response. Lectins mediate the agglutination and mitogenic stimulation of lymphocytes at sites of inflammation (Lasky, L. A. (1991) J. Cell. Biochem. 



  45 : 139-146 ; Paietta, E. et al. (1989) J. Immunol. 143 : 2850-2857). 



   Lectins are further classified into subfamilies based on carbohydrate-binding specificity and other criteria. The galectin subfamily, in particular, includes lectins that bind   P-galactoside   carbohydrate moieties in a thiol-dependent manner (reviewed in   Hadari,   Y. R. et al. (1998) J. Biol. 



  Chem. 270 : 3447-3453). Galectins are widely expressed and developmentally regulated. Because all galectins lack an N-terminal signal peptide, it is suggested that galectins are externalized through an atypical secretory mechanism. Two classes of galectins have been defined based on molecular weight and oligomerization properties. Small galectins form homodimers and are about 14 to 16 kilodaltons in mass, while large galectins are monomeric and about 29-37 kilodaltons. 



   Galectins contain a characteristic carbohydrate recognition domain (CRD). The CRD is about 140 amino acids and contains several stretches of about   1-10   amino acids which are highly conserved among all galectins. A particular 6-amino acid motif within the CRD contains conserved tryptophan and arginine residues which are critical for carbohydrate binding. The CRD of some galectins also contains cysteine residues which may be important for disulfide bond formation. 

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  Secondary structure predictions indicate that the CRD forms several   P-sheets.   



   Galectins play a number of roles in diseases and conditions associated with cell-cell and cellmatrix interactions. For example, certain galectins associate with sites of inflammation and bind to cell surface immunoglobulin E molecules. In addition, galectins may play an important role in cancer metastasis. Galectin overexpression is correlated with the metastatic potential of cancers in humans and mice. Moreover, anti-galectin antibodies inhibit processes associated with cell transformation, such as cell aggregation and anchorage-independent growth (See, for example, Su, Z.-Z. et al. (1996) Proc. Natl. Acad. Sci. USA 93 : 7252-7257). 



  Selectins
Selectins, or   LEC-CAMs,   comprise a specialized lectin subfamily involved primarily in inflammation and leukocyte adhesion (Reviewed in Lasky, supra). Selectins mediate the recruitment of leukocytes from the circulation to sites of acute inflammation and are expressed on the surface of vascular endothelial cells in response to cytokine signaling. Selectins bind to specific ligands on the leukocyte cell membrane and enable the leukocyte to adhere to and migrate along the endothelial surface. Binding of selectin to its ligand leads to polarized rearrangement of the actin cytoskeleton and stimulates signal transduction within the leukocyte (Brenner, B. et al. (1997) Biochem. Biophys. 



  Res. Commun. 231 : 802-807   ;     Hidari,   K. I. et al. (1997) J. Biol. Chem. 272 : 28750-28756). Members of the selectin family possess three characteristic motifs : a lectin or carbohydrate recognition domain ; an epidermal growth factor-like domain ; and a variable number of short consensus repeats   (scr   or "sushi"repeats) which are also present in complement regulatory proteins. The selectins include lymphocyte adhesion molecule-1   (Lam-1   or L-selectin), endothelial leukocyte adhesion molecule-1 (ELAM-1 or E-selectin), and granule membrane protein-140 (GMP-140 or P-selectin) (Johnston, G. I. et al. (1989) Cell 56 : 1033-1044). 



  Antigen Recognition Molecules
All vertebrates have developed sophisticated and complex immune systems that provide protection from viral, bacterial, fungal, and parasitic infections. A key feature of the immune system is its ability to distinguish foreign molecules, or antigens,   from"self'molecules.   This ability is mediated primarily by secreted and transmembrane proteins expressed by leukocytes (white blood cells) such as lymphocytes, granulocytes, and monocytes. Most of these proteins belong to the immunoglobulin   (Ig)   superfamily, members of which contain one or more repeats of a conserved structural domain. This Ig domain is comprised of antiparallel   p   sheets joined by a disulfide bond in an arrangement called the Ig fold.

   Members of the Ig superfamily include T-cell receptors, major histocompatibility (MHC) proteins, antibodies, and immune cell-specific surface markers such as 

 <Desc/Clms Page number 83> 

 CD4,   CD8,   and CD28. 



   MHC proteins are cell surface markers that bind to and present foreign antigens to T cells. 



  MHC molecules are classified as either class I or class II. Class I MHC molecules (MHC I) are expressed on the surface of almost all cells and are involved in the presentation of antigen to cytotoxic T cells. For example, a cell infected with virus will degrade intracellular viral proteins and express the protein fragments bound to MHC I molecules on the cell surface. The MHC I/antigen complex is recognized by cytotoxic T-cells which destroy the infected cell and the virus within. 



  Class II MHC molecules are expressed primarily on specialized antigen-presenting cells of the immune system, such as B-cells and macrophages. These cells ingest foreign proteins from the extracellular fluid and express MHC 11/antigen complex on the cell surface. This complex activates helper T-cells, which then secrete cytokines and other factors that stimulate the immune response. 



  MHC molecules also play an important role in organ rejection following transplantation. Rejection occurs when the recipient's T-cells respond to foreign MHC molecules on the transplanted organ in the same way as to self MHC molecules bound to foreign antigen. (Reviewed in Alberts, B. et al. 



  (1994) Molecular Biology of the Cell, Garland Publishing, New York NY, pp. 1229-1246.) 
Antibodies, or immunoglobulins, are either expressed on the surface of B-cells or secreted by B-cells into the circulation. Antibodies bind and neutralize foreign antigens in the blood and other extracellular fluids. The prototypical antibody is a tetramer consisting of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds. This arrangement confers the characteristic Y-shape to antibody molecules. 



  Antibodies are classified based on their H-chain composition. The five antibody classes, IgA, IgD, IgE, IgG and IgM, are defined by the   a,     8,     e, y, and    H-chain types. There are two types of L- chains,   x   and   A,   either of which may associate as a pair with any H-chain pair. IgG, the most common class of antibody found in the circulation, is tetrameric, while the other classes of antibodies are generally variants or multimers of this basic structure. 



   H-chains and L-chains each contain an N-terminal variable region and a C-terminal constant region. The constant region consists of about 110 amino acids in L-chains and about 330 or 440 amino acids in H-chains. The amino acid sequence of the constant region is nearly identical among H-or L-chains of a particular class. The variable region consists of about 110 amino acids in both H- and L-chains. However, the amino acid sequence of the variable region differs among H-or L-chains of a particular class. Within each H-or L-chain variable region are three hypervariable regions of extensive sequence diversity, each consisting of about 5 to 10 amino acids. In the antibody molecule, the H-and L-chain hypervariable regions come together to form the antigen recognition site. 



  (Reviewed in Alberts, supra, pp. 1206-1213 and 1216-1217.) 

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Both H-chains and L-chains contain repeated Ig domains. For example, a typical H-chain contains four Ig domains, three of which occur within the constant region and one of which occurs within the variable region and contributes to the formation of the antigen recognition site. Likewise, a typical L-chain contains two Ig domains, one of which occurs within the constant region and one of which occurs within the variable region. 



   The immune system is capable of recognizing and responding to any foreign molecule that enters the body. Therefore, the immune system must be armed with a full repertoire of antibodies against all potential antigens. Such antibody diversity is generated by somatic rearrangement of gene segments encoding variable and constant regions. These gene segments are joined together by site- specific recombination which occurs between highly conserved DNA sequences that flank each gene segment. Because there are hundreds of different gene segments, millions of unique genes can be generated combinatorially. In addition, imprecise joining of these segments and an unusually high rate of somatic mutation within these segments further contribute to the generation of a diverse antibody population. 



   T-cell receptors are both structurally and functionally related to antibodies. (Reviewed in Alberts, supra, pp. 1228-1229.) T-cell receptors are cell surface proteins that bind foreign antigens and mediate diverse aspects of the immune response. A typical T-cell receptor is a heterodimer comprised of two disulfide-linked polypeptide chains called   a   and. Each chain is about 280 amino acids in length and contains one variable region and one constant region. Each variable or constant region folds into an Ig domain. The variable regions from the a   and/3   chains come together in the heterodimer to form the antigen recognition site. T-cell receptor diversity is generated by somatic rearrangement of gene segments encoding the a and   13   chains.

   T-cell receptors recognize small peptide antigens that are expressed on the surface of antigen-presenting cells and pathogen-infected cells. These peptide antigens are presented on the cell surface in association with major histocompatibility proteins which provide the proper context for antigen recognition. 



  Secreted and Extracellular Matrix Molecules 
Protein secretion is essential for cellular function. Protein secretion is mediated by a signal peptide located at the amino terminus of the protein to be secreted. The signal peptide is comprised of about ten to twenty hydrophobic amino acids which target the nascent protein from the ribosome to the endoplasmic reticulum (ER). Proteins targeted to the ER may either proceed through the secretory pathway or remain in any of the secretory organelles such as the ER, Golgi apparatus, or lysosomes. 



  Proteins that transit through the secretory pathway are either secreted into the extracellular space or retained in the plasma membrane. Secreted proteins are often synthesized as inactive precursors that 

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 are activated by post-translational processing events during transit through the secretory pathway. 



  Such events include glycosylation, proteolysis, and removal of the signal peptide by a signal peptidase. 



  Other events that may occur during protein transport include chaperone-dependent unfolding and folding of the nascent protein and interaction of the protein with a receptor or pore complex. Examples of secreted proteins with amino terminal signal peptides include receptors, extracellular matrix molecules, cytokines, hormones, growth and differentiation factors, neuropeptides, vasomediators, ion channels, transporters/pumps, and proteases. (Reviewed in Alberts, B. et al. (1994) Molecular Biology of The Cell, Garland Publishing, New York NY, pp. 557-560, 582-592.) 
The extracellular matrix (ECM) is a complex network of glycoproteins, polysaccharides, proteoglycans, and other macromolecules that are secreted from the cell into the extracellular space. 



  The ECM remains in close association with the cell surface and provides a supportive meshwork that profoundly influences cell shape, motility, strength, flexibility, and adhesion. In fact, adhesion of a cell to its surrounding matrix is required for cell survival except in the case of metastatic tumor cells, which have overcome the need for cell-ECM anchorage. This phenomenon suggests that the ECM plays a critical role in the molecular mechanisms of growth control and metastasis. (Reviewed in Ruoslahti,   E.   (1996) Sci. Am. 275 : 72-77.) Furthermore, the ECM determines the structure and physical properties of connective tissue and is particularly important for morphogenesis and other processes associated with embryonic development and pattern formation. 



   The collagens comprise a family of ECM proteins that provide structure to bone, teeth, skin, ligaments, tendons, cartilage, blood vessels, and basement membranes. Multiple collagen proteins have been identified. Three collagen molecules fold together in a triple helix stabilized by interchain disulfide bonds. Bundles of these triple helices then associate to form fibrils. Collagen primary structure consists of hundreds of (Gly-X-Y) repeats where about a third of the X and Y residues are Pro. 



  Glycines are crucial to helix formation as the bulkier amino acid sidechains cannot fold into the triple helical conformation. Because of these strict sequence requirements, mutations in collagen genes have severe consequences. Osteogenesis imperfecta patients have brittle bones that fracture easily ; in severe cases patients die in utero or at birth.   Ehlers-Danlos   syndrome patients have hyperelastic skin, hypermobile joints, and susceptibility to aortic and intestinal rupture. Chondrodysplasia patients have short stature and ocular disorders. Alport syndrome patients   have hematuria,   sensorineural deafness, and eye lens deformation. (Isselbacher, K. J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, Inc., New York NY, pp. 2105-2117 ; and Creighton, T. E. (1984) Proteins, Structures and Molecular Principles, W. H.

   Freeman and Company, New York NY, pp. 191-197.) 
Elastin and related proteins confer elasticity to tissues such as skin, blood vessels, and lungs. 



  Elastin is a highly hydrophobic protein of about 750 amino acids that is rich in proline and glycine 

 <Desc/Clms Page number 86> 

 residues. Elastin molecules are highly cross-linked, forming an extensive extracellular network of fibers and sheets. Elastin fibers are surrounded by a sheath of   microSbrils   which are composed of a number of glycoproteins, including fibrillin. Mutations in the gene encoding fibrillin are responsible for Marfan's syndrome, a genetic disorder characterized by defects in connective tissue. In severe cases, the aortas of afflicted individuals are prone to rupture. (Reviewed in Alberts, supra, pp.   984-986.)   
Fibronectin is a large ECM glycoprotein found in all vertebrates. Fibronectin exists as a dimer of two subunits, each containing about 2, 500 amino acids.

   Each subunit folds into a rod-like structure containing multiple domains. The domains each contain multiple repeated modules, the most common of which is the type III fibronectin repeat. The type III fibronectin repeat is about 90 amino acids in length and is also found in other ECM proteins and in some plasma membrane and cytoplasmic proteins. Furthermore, some type III fibronectin repeats contain a characteristic tripeptide consisting of Arginine-Glycine-Aspartic acid   (RGD).   The RGD sequence is recognized by the integrin family of cell surface receptors and is also found in other ECM proteins. Disruption of both copies of the gene encoding fibronectin causes early embryonic lethality in mice.

   The mutant embryos display extensive morphological defects, including defects in the formation of the notochord, somites, heart, blood vessels, neural tube, and extraembryonic structures. (Reviewed in Alberts, supra, pp. 986-987.)   Laminin   is a major glycoprotein component of the basal lamina which underlies and supports epithelial cell sheets.   Laminin   is one of the first ECM proteins synthesized in the developing embryo. 



    Laminin   is an 850 kilodalton protein composed of three polypeptide chains joined in the shape of a cross by disulfide bonds.   Laminin   is especially important for angiogenesis and in particular, for guiding the formation of capillaries. (Reviewed in Alberts, supra, pp.   990-991.)   
There are many other types of proteinaceous ECM components, most of which can be classified as proteoglycans. Proteoglycans are composed of unbranched polysaccharide chains (glycosaminoglycans) attached to protein cores.

   Common proteoglycans include aggrecan, betaglycan, decorin, perlecan, serglycin, and   syndecan-1.   Some of these molecules not only provide mechanical support, but also bind to extracellular signaling molecules, such as fibroblast growth factor and transforming growth factor, suggesting a role for proteoglycans in cell-cell communication and cell growth. (Reviewed in Alberts, supra, pp. 973-978.) Likewise, the glycoproteins tenascin-C and tenascin-R are expressed in developing and lesioned neural tissue and provide stimulatory and anti- adhesive (inhibitory) properties, respectively, for axonal growth. (Faissner, A. (1997) Cell Tissue Res. 



  290 : 331-341.) Cytoskeletal Molecules 
The cytoskeleton is a cytoplasmic network of protein fibers that mediate cell shape, structure, 

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 and movement. The cytoskeleton supports the cell membrane and forms tracks along which organelles and other elements move in the cytosol. The cytoskeleton is a dynamic structure that allows cells to adopt various shapes and to carry out directed movements. Major cytoskeletal fibers include the microtubules, the microfilaments, and the intermediate filaments. Motor proteins, including myosin, dynein, and kinesin, drive movement of or along the fibers. The motor protein dynamin drives the formation of membrane vesicles. Accessory or associated proteins modify the structure or activity of the fibers while cytoskeletal membrane anchors connect the fibers to the cell membrane. 



    Tubulins   
Microtubules, cytoskeletal fibers with a diameter of about 24 nm, have multiple roles in the cell. Bundles of microtubules form cilia and flagella, which are whip-like extensions of the cell membrane that are necessary for sweeping materials across an epithelium and for swimming of sperm, respectively. Marginal bands of microtubules in red blood cells and platelets are important for these cells'pliability. Organelles, membrane vesicles, and proteins are transported in the cell along tracks of microtubules. For example, microtubules run through nerve cell axons, allowing bi- directional transport of materials and membrane vesicles between the cell body and the nerve terminal. Failure to supply the nerve terminal with these vesicles blocks the transmission of neural signals. Microtubules are also critical to chromosomal movement during cell division.

   Both stable and short-lived populations of microtubules exist in the cell. 



   Microtubules are polymers of GTP-binding tubulin protein subunits. Each subunit is a heterodimer of a-and   P-tubulin,   multiple isoforms of which exist. The hydrolysis of GTP is linked to the addition of tubulin subunits at the end of a microtubule. The subunits interact head to tail to form protofilaments ; the   protoSlaments   interact side to side to form a microtubule. A microtubule is polarized, one end ringed with   cc-tubulin   and the other with   p-tubulin,   and the two ends differ in their rates of assembly. Generally, each microtubule is composed of 13   protofilaments   although 11 or 15   protofilament-microtubules   are sometimes found. Cilia and flagella contain doublet microtubules. 



  Microtubules grow from specialized structures known as centrosomes or microtubule-organizing centers (MTOCs). MTOCs may contain one or two centrioles, which are pinwheel arrays of triplet microtubules. The basal body, the organizing center located at the base of a cilium or flagellum, contains one centriole. Gamma tubulin present in the MTOC is important for nucleating the polymerization of   a-and   p-tubulin heterodimers but does not polymerize into microtubules. 



  Microtubule-Associated Proteins   Microtubule-associated   proteins (MAPs) have roles in the assembly and stabilization of microtubules. One major family of MAPs, assembly MAPs, can be identified in neurons as well as 

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 non-neuronal cells. Assembly MAPs are responsible for cross-linking microtubules in the cytosol. 



  These MAPs are organized into two domains : a basic microtubule-binding domain and an acidic projection domain. The projection domain is the binding site for membranes, intermediate filaments, or other microtubules. Based on sequence analysis, assembly MAPs can be further grouped into two types : Type I and Type II. Type I MAPs, which include MAP1A and MAP1B, are large, filamentous molecules that co-purify with microtubules and are abundantly expressed in brain and testes. Type I MAPs contain several repeats of a positively-charged amino acid sequence motif that binds and neutralizes negatively charged tubulin, leading to stabilization of microtubules.   MAP1A   and MAP1B are each derived from a single precursor polypeptide that is subsequently proteolytically processed to generate one heavy chain and one light chain. 



   Another light chain, LC3, is a 16. 4 kDa molecule that binds MAP1A,   MAP1B,   and microtubules. It is suggested that LC3 is synthesized from a source other than the MAP1A or MAP1B transcripts, and that the expression of LC3 may be important in regulating the microtubule binding activity of MAP1A and MAP1B during cell proliferation (Mann, S. S. et al. (1994) J. Biol. Chem. 



  269 : 11492-11497). 



   Type   II   MAPs, which include MAP2a, MAP2b, MAP2c, MAP4, and Tau, are characterized by three to four copies of an 18-residue sequence in the microtubule-binding domain. MAP2a, MAP2b, and MAP2c are found only in dendrites, MAP4 is found in non-neuronal cells, and Tau is found in axons and dendrites of nerve cells. Alternative splicing of the Tau mRNA leads to the existence of multiple forms of Tau protein. Tau phosphorylation is altered in neurodegenerative disorders such as Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, and familial   frontotemporal   dementia and Parkinsonism linked to chromosome 17. The altered Tau phosphorylation leads to a collapse of the microtubule network and the formation of intraneuronal Tau aggregates   (Spillantini,   M. G. and M.

   Goedert (1998) Trends Neurosci. 21 : 428-433). 



   The protein pericentrin is found in the MTOC and has a role in microtubule assembly. 



  Actins 
Microfilaments, cytoskeletal filaments with a diameter of about 7-9   nm,   are vital to cell locomotion, cell shape, cell adhesion, cell division, and muscle contraction. Assembly and disassembly of the   microfilaments   allow cells to change their morphology.   MicroRlaments   are the polymerized form of actin, the most abundant intracellular protein in the eukaryotic cell. Human cells contain six isoforms of actin. The three a-actins are found in different kinds of muscle,   nonmuscle   (3- actin and   nonmuscle     y-actin   are found in   nonmuscle   cells, and another   y-actin   is found in intestinal smooth muscle cells.

   G-actin, the monomeric form of actin, polymerizes into polarized, helical F- actin filaments, accompanied by the hydrolysis of ATP to ADP. Actin filaments associate to form 

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 bundles and networks, providing a framework to support the plasma membrane and determine cell shape. These bundles and networks are connected to the cell membrane. In muscle cells, thin filaments containing actin slide past thick filaments containing the motor protein myosin during contraction. A family of actin-related proteins exist that are not part of the actin cytoskeleton, but rather associate with microtubules and dynein. 



  Actin-Associated Proteins 
Actin-associated proteins have roles in cross-linking, severing, and stabilization of actin filaments and in sequestering actin monomers. Several of the actin-associated proteins have multiple functions. Bundles and networks of actin filaments are held together by actin cross-linking proteins. 



  These proteins have two actin-binding sites, one for each filament. Short cross-linking proteins promote bundle formation while longer, more flexible cross-linking proteins promote network formation.   Calmodulin-like   calcium-binding domains in actin cross-linking proteins allow calcium regulation of cross-linking. Group I cross-linking proteins have unique actin-binding domains and include the 30 kD protein,   EF-la,   fascin, and scruin. Group II cross-linking proteins have a 7, 000- MW actin-binding domain and include villin and dematin. Group III cross-linking proteins have pairs of a 26, 000-MW actin-binding domain and include fimbrin, spectrin, dystrophin, ABP 120, and filamin. 



   Severing proteins regulate the length of actin filaments by breaking them into short pieces or by blocking their ends. Severing proteins include gCAP39, severin (fragmin), gelsolin, and villin. 



  Capping proteins can cap the ends of actin filaments, but cannot break filaments. Capping proteins include CapZ and tropomodulin. The proteins thymosin and profilin sequester actin monomers in the cytosol, allowing a pool of unpolymerized actin to exist. The actin-associated proteins tropomyosin, troponin, and caldesmon regulate muscle contraction in response to calcium. 



  Intermediate Filaments and Associated Proteins 
Intermediate filaments   (IFs)   are cytoskeletal fibers with a diameter of about 10   nm,   intermediate between that of microfilaments and microtubules. IFs serve structural roles in the cell, reinforcing cells and organizing cells into tissues. IFs are particularly abundant in epidermal cells and in neurons. IFs are extremely stable, and, in contrast to   microRlaments   and microtubules, do not function in cell motility. 



   Five types of IF proteins are known in mammals. Type I and Type II proteins are the acidic and basic keratins, respectively. Heterodimers of the acidic and basic keratins are the building blocks of keratin IFs. Keratins are abundant in soft epithelia such as skin and cornea, hard epithelia such as nails and hair, and in epithelia that line internal body cavities. Mutations in keratin genes lead to epithelial diseases including epidermolysis bullosa simplex, bullous congenital ichthyosiform 

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 erythroderma (epidermolytic hyperkeratosis), non-epidermolytic and epidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens,   pachyonychia   congenita, and white sponge nevus. Some of these diseases result in severe skin blistering. (See,   e. g.,   Wawersik, M. et al. (1997) J. Biol. Chem. 



  272 : 32557-32565 ;   andCordenL.   D.   and W.   H. McLean (1996) Exp. Dermatol. 5 : 297-307.) 
Type III IF proteins include desmin, glial fibrillary acidic protein, vimentin, and   peripherin.   



  Desmin filaments in muscle cells link myofibrils into bundles and stabilize sarcomeres in contracting muscle. Glial fibrillary acidic protein filaments are found in the glial cells that surround neurons and astrocytes. Vimentin filaments are found in blood vessel endothelial cells, some epithelial cells, and mesenchymal cells such as fibroblasts, and are commonly associated with microtubules. Vimentin filaments may have roles in keeping the nucleus and other organelles in place in the cell. Type IV IFs include the neurofilaments and nestin. Neurofilaments, composed of three polypeptides NF-L, NF-M, and NF-H, are frequently associated with microtubules in axons. Neurofilaments are responsible for the radial growth and diameter of an axon, and ultimately for the speed of nerve impulse transmission. 



  Changes in phosphorylation and metabolism of neurofilaments are observed in neurodegenerative diseases including   amyotrophic   lateral sclerosis, Parkinson's disease, and Alzheimer's disease (Julien, J.   P.   and W. E.   Mushynski   (1998) Prog. Nucleic Acid Res. Mol. Biol. 61 : 1-23). Type V IFs, the lamins, are found in the nucleus where they support the nuclear membrane. 



   IFs have a central a-helical rod region interrupted by short nonhelical linker segments. The rod region is bracketed, in most cases, by non-helical head and tail domains. The rod regions of intermediate filament proteins associate to form a coiled-coil dimer. A highly ordered assembly process leads from the dimers to the IFs. Neither ATP nor GTP is needed for IF assembly, unlike that of   microfilaments   and microtubules. 



   IF-associated proteins (IFAPs) mediate the interactions of IFs with one another and with other cell structures. IFAPs cross-link IFs into a bundle, into a network, or to the plasma membrane, and may cross-link IFs to the   microfilament   and microtubule cytoskeleton. Microtubules and IFs are in particular closely associated. IFAPs include   BPAG1,   plakoglobin, desmoplakin   I,   desmoplakin   II,   pectin, ankyrin, filaggrin, and lamin B receptor. 



  Cytoskeletal-Membrane Anchors 
Cytoskeletal fibers are attached to the plasma membrane by specific proteins. These attachments are important for maintaining cell shape and for muscle contraction. In erythrocytes, the spectrin-actin cytoskeleton is attached to cell membrane by three proteins, band 4. 1, ankyrin, and adducin. Defects in this attachment result in abnormally shaped cells which are more rapidly degraded by the spleen, leading to anemia. In platelets, the spectrin-actin cytoskeleton is also linked to the membrane by ankyrin ; a second actin network is anchored to the membrane by   filamin.   In 

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 muscle cells the protein dystrophin links actin filaments to the plasma membrane ; mutations in the dystrophin gene lead to Duchenne muscular dystrophy.

   In adherens junctions and adhesion plaques the peripheral membrane proteins   cc-actinin   and vinculin attach actin filaments to the cell membrane. 



   IFs are also attached to membranes by cytoskeletal-membrane anchors. The nuclear lamina is attached to the inner surface of the nuclear membrane by the lamin B receptor. Vimentin IFs are attached to the plasma membrane by ankyrin and pectin. Desmosome and   hemidesmosome   membrane junctions hold together epithelial cells of organs and skin. These membrane junctions allow shear forces to be distributed across the entire epithelial cell layer, thus providing strength and rigidity to the epithelium. IFs in epithelial cells are attached to the desmosome by plakoglobin and desmoplakins. The proteins that link IFs to   hemidesmosomes   are not known. Desmin IFs surround the sarcomere in muscle and are linked to the plasma membrane by paranemin, synemin, and ankyrin. 



  Myosin-related Motor Proteins   Myosins   are actin-activated ATPases, found in eukaryotic cells, that couple hydrolysis of ATP with motion. Myosin provides the motor function for muscle contraction and intracellular movements such as phagocytosis and rearrangement of cell contents during mitotic cell division (cytokinesis). The contractile unit of skeletal muscle, termed the sarcomere, consists of highly ordered arrays of thin actin-containing filaments and thick myosin-containing filaments. Crossbridges form between the thick and thin filaments, and the ATP-dependent movement of myosin heads within the thick filaments pulls the thin filaments, shortening the sarcomere and thus the muscle fiber. 



   Myosins are composed of one or two heavy chains and associated light chains. Myosin heavy chains contain an amino-terminal motor or head domain, a neck that is the site of light-chain binding, and a   carboxy-terminal   tail domain. The tail domains may associate to form an a-helical coiled coil. Conventional myosins, such as those found in muscle tissue, are composed of two myosin heavy-chain subunits, each associated with two light-chain subunits that bind at the neck region and play a regulatory role. Unconventional myosins, believed to function in intracellular motion, may contain either one or two heavy chains and associated light chains. There is evidence for about 25 myosin heavy chain genes in vertebrates, more than half of them unconventional. 



    Dynein-related   Motor Proteins 
Dyneins are (-) end-directed motor proteins which act on microtubules. Two classes of dyneins, cytosolic and axonemal, have been identified. Cytosolic dyneins are responsible for translocation of materials along cytoplasmic microtubules, for example, transport from the nerve terminal to the cell body and transport of endocytic vesicles to lysosomes. Cytoplasmic dyneins are also reported to play a role in mitosis.   Axonemal   dyneins are responsible for the beating of flagella and cilia. Dynein on one microtubule doublet walks along the adjacent microtubule doublet. This sliding 

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 force produces bending forces that cause the flagellum or cilium to beat.

   Dyneins have a native mass between 1000 and 2000 kDa and contain either two or three force-producing heads driven by the hydrolysis of ATP. The heads are linked via stalks to a basal domain which is composed of a highly variable number of accessory intermediate and light chains. 



  Kinesin-related Motor Proteins 
Kinesins are (+) end-directed motor proteins which act on microtubules. The prototypical kinesin molecule is involved in the transport of membrane-bound vesicles and organelles. This function is particularly important for axonal transport in neurons. Kinesin is also important in all cell types for the transport of vesicles from the Golgi complex to the endoplasmic reticulum. This role is critical for maintaining the identity and functionality of these secretory organelles. 



   Kinesins define a ubiquitous, conserved family of over 50 proteins that can be classified into at least 8 subfamilies based on primary amino acid sequence, domain structure, velocity of movement, and cellular function. (Reviewed in Moore, J. D. and S. A. Endow (1996) Bioessays 18 : 207-219 ; and   Hoyt,   A. M. (1994) Curr. Opin. Cell Biol. 6 : 63-68.) The prototypical kinesin molecule is a heterotetramer comprised of two heavy polypeptide chains   (KHCs)   and two light polypeptide chains   (KLCs).   The KHC subunits are typically referred to as"kinesin."KHC is about 1000 amino acids in length, and KLC is about 550 amino acids in length. Two KHCs dimerize to form a rod-shaped molecule with three distinct regions of secondary structure.

   At one end of the molecule is a globular motor domain that functions in ATP hydrolysis and microtubule binding. Kinesin motor domains are highly conserved and share over 70% identity. Beyond the motor domain is an a-helical coiled-coil region which mediates dimerization. At the other end of the molecule is a fan-shaped tail that associates with molecular cargo. The tail is formed by the interaction of the KHC C-termini with the two KLCs. 



   Members of the more divergent subfamilies of kinesins are called kinesin-related proteins (KRPs), many of which function during mitosis in eukaryotes (Hoyt, supra). Some KRPs are required for assembly of the mitotic spindle. In vivo and in vitro analyses suggest that these KRPs exert force on microtubules that comprise the mitotic spindle, resulting in the separation of spindle poles. 



  Phosphorylation of KRP is required for this activity. Failure to assemble the mitotic spindle results in abortive mitosis and chromosomal aneuploidy, the latter condition being characteristic of cancer cells. 



  In addition, a unique KRP, centromere protein E, localizes to the   kinetochore   of human mitotic chromosomes and may play a role in their segregation to opposite spindle poles. 



    Dynamin-related   Motor Proteins 
Dynamin is a large GTPase motor protein that functions as a"molecular pinchase," generating a mechanochemical force used to sever membranes. This activity is important in forming clathrin-coated vesicles from coated pits in endocytosis and in the biogenesis of synaptic vesicles in 

 <Desc/Clms Page number 93> 

 neurons. Binding of dynamin to a membrane leads to dynamin's self-assembly into spirals that may act to constrict a flat membrane surface into a tubule. GTP hydrolysis induces a change in conformation of the dynamin polymer that pinches the membrane tubule, leading to severing of the membrane tubule and formation of a membrane vesicle. Release of GDP and inorganic phosphate leads to dynamin disassembly.

   Following disassembly the dynamin may either dissociate from the membrane or remain associated to the vesicle and be transported to another region of the cell. Three homologous dynamin genes have been discovered, in addition to several dynamin-related proteins. 



  Conserved dynamin regions are the N-terminal GTP-binding domain, a central pleckstrin homology domain that binds membranes, a central coiled-coil region that may activate dynamin's GTPase activity, and a C-terminal proline-rich domain that contains several motifs that bind SH3 domains on other proteins. Some dynamin-related proteins do not contain the pleckstrin homology domain or the proline-rich domain. (See McNiven, M. A. (1998) Cell 94 : 151-154 ; Scaife, R. M. and R. L. Margolis (1997) Cell. Signal. 9 : 395-401.)
The cytoskeleton is reviewed in Lodish, H. et al. (1995) Molecular Cell Biology, Scientific American Books, New York NY. 



  Ribosomal Molecules
Ribosomal RNAs (rRNAs) are assembled, along with ribosomal proteins, into ribosomes, which are cytoplasmic particles that translate messenger RNA into polypeptides. The eukaryotic ribosome is composed of a 60S (large) subunit and a 40S (small) subunit, which together form the 80S ribosome. In addition to the 18S, 28S,   5S,   and 5.   8S   rRNAs, the ribosome also contains more than fifty proteins. The ribosomal proteins have a prefix which denotes the subunit to which they belong, either L (large) or S (small). Ribosomal protein activities include binding   rRNA   and organizing the conformation of the junctions between   rRNA   helices (Woodson, S. A. and N. B. 



  Leontis (1998) Curr. Opin.   Struct.   Biol. 8 : 294-300 ; Ramakrishnan, V. and S. W. White (1998) Trends Biochem. Sci. 23 : 208-212.) Three important sites are identified on the ribosome. The   aminoacyl-   tRNA site (A site) is where charged tRNAs (with the exception of the initiator-tRNA) bind on arrival at the ribosome. The   peptidyl-tRNA   site (P site) is where new peptide bonds are formed, as well as where the initiator tRNA binds. The exit site (E site) is where deacylated tRNAs bind prior to their release from the ribosome. (The ribosome is reviewed in Stryer, L. (1995) Biochemistry W. H. 



  Freeman and Company, New York NY, pp. 888-908 ; and Lodish, H. et al. (1995) Molecular Cell Biology Scientific American Books, New York NY. pp. 119-138.) Chromatin Molecules 

 <Desc/Clms Page number 94> 

 
The nuclear DNA of eukaryotes is organized into chromatin. Two types of chromatin are observed : euchromatin, some of which may be transcribed, and heterochromatin so densely packed that much of it is inaccessible to transcription. Chromatin packing thus serves to regulate protein expression in eukaryotes. Bacteria lack chromatin and the   chromatin-packing   level of gene regulation. 



   The fundamental unit of chromatin is the   nucleosome   of 200 DNA base pairs associated with two copies each of histones H2A, H2B, H3, and H4. Adjascent nucleosomes are linked by another class of histones,   H1.   Low molecular weight   non-histone   proteins called the high mobility group (HMG), associated with chromatin, may function in the unwinding of DNA and stabilization of single- stranded DNA.   Cbromodomain   proteins function in compaction of chromatin into its transcriptionally silent heterochromatin form. 



   During mitosis, all DNA is compacted into heterochromatin and transcription ceases. 



  Transcription in interphase begins with the activation of a region of chromatin. Active chromatin is decondensed.   Decondensation   appears to be accompanied by changes in binding coefficient, phosphorylation and acetylation states of chromatin histones. HMG proteins HMG13 and HMG17 selectively bind activated chromatin. Topoisomerases remove superhelical tension on DNA. The activated region decondenses, allowing gene regulatory proteins and transcription factors to assemble on the DNA. 



   Patterns of chromatin structure can be stably inherited, producing heritable patterns of gene expression. In mammals, one of the two X chromosomes in each female cell is inactivated by condensation to heterochromatin during zygote development. The inactive state of this chromosome is inherited, so that adult females are mosaics of clusters of paternal-X and maternal-X clonal cell groups. 



  The condensed X chromosome is reactivated in meiosis. 



   Chromatin is associated with disorders of protein expression such as thalassemia, a genetic anemia resulting from the removal of the locus control region (LCR) required for decondensation of the globin gene locus. 



   For a review of chromatin structure and function see Alberts, B. et al. (1994) Molecular Cell Biology, third edition, Garland Publishing, Inc., New York NY, pp. 351-354, 433-439. 



  Electron Transfer Associated Molecules 
Electron carriers such as cytochromes accept electrons from NADH or FADH2 and donate them to other electron carriers. Most electron-transferring proteins, except ubiquinone, are prosthetic groups such as flavins, heme, FeS clusters, and copper, bound to inner membrane proteins. 



  Adrenodoxin, for example, is an FeS protein that forms a complex with NADPH : adrenodoxin reductase and cytochrome p450. Cytochromes contain a heme prosthetic group, a porphyrin ring 

 <Desc/Clms Page number 95> 

 containing a tightly bound iron atom. Electron transfer reactions play a crucial role in cellular energy production. 



   Energy is produced by the oxidation of glucose and fatty acids. Glucose is initially converted to pyruvate in the cytoplasm. Fatty acids and pyruvate are transported to the mitochondria for complete oxidation to   CO2   coupled by enzymes to the transport of electrons from NADH and   FADH2   to oxygen and to the synthesis of ATP (oxidative phosphorylation) from ADP and Pi. 



   Pyruvate is transported into the mitochondria and converted to acetyl-CoA for oxidation via the citric acid cycle, involving pyruvate dehydrogenase components, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Enzymes involved in the citric acid cycle include : citrate synthetase, aconitases, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex including transsuccinylases, succinyl CoA synthetase, succinate dehydrogenase,   fumarases,   and malate dehydrogenase. Acetyl CoA is oxidized to   CO.,   with concomitant formation of NADH, FADH2, and GTP. In oxidative phosphorylation, the transfer of electrons from NADH and FADH2 to oxygen by dehydrogenases is coupled to the synthesis of ATP from ADP and Pi by the   FoFl   ATPase complex in the mitochondrial inner membrane.

   Enzyme complexes responsible for electron transport and ATP synthesis include the FFl ATPase complex, ubiquinone   (CoQ)-cytochrome   c reductase, ubiquinone reductase, cytochrome b, cytochrome cl, FeS protein, and cytochrome c oxidase. 



   ATP synthesis requires membrane transport enzymes including the phosphate transporter and the ATP-ADP antiport protein. The ATP-binding casette (ABC) superfamily has also been suggested as belonging to the mitochondrial transport group (Hogue, D. L. et al. (1999) J. Mol. Biol. 285 : 379- 389). Brown fat uncoupling protein dissipates oxidative energy as heat, and may be involved the fever response to infection and trauma (Cannon,   B.   et al. (1998) Ann. NY Acad. Sci. 856 : 171-187). 



   Mitochondria are oval-shaped organelles comprising an outer membrane, a tightly folded inner membrane, an intermembrane space between the outer and inner membranes, and a matrix inside the inner membrane. The outer membrane contains many porin molecules that allow ions and charged molecules to enter the intermembrane space, while the inner membrane contains a variety of transport proteins that transfer only selected molecules. Mitochondria are the primary sites of energy production in cells. 



   Mitochondria contain a small amount of DNA. Human mitochondrial DNA encodes 13 proteins, 22 tRNAs, and 2 rRNAs. Mitochondrial-DNA encoded proteins include NADH-Q reductase, a cytochrome reductase subunit, cytochrome oxidase subunits, and ATP synthase subunits. 



   Electron-transfer reactions also occur outside the mitochondria in locations such as the endoplasmic reticulum, which plays a crucial role in lipid and protein biosynthesis. Cytochrome b5 is a central electron donor for various reductive reactions occurring on the cytoplasmic surface of liver 

 <Desc/Clms Page number 96> 

 endoplasmic reticulum. Cytochrome b5 has been found in Golgi, plasma, endoplasmic reticulum (ER), and microbody membranes. 



   For a review of mitochondrial metabolism and regulation, see Lodish,   H.   et al. (1995) Molecular Cell Biology, Scientific American Books, New York NY, pp. 745-797 and Stryer (1995) Biochemistrv, W.   H.   Freeman and Co., San Francisco CA, pp 529-558, 988-989. 



   The majority of mitochondrial proteins are encoded by nuclear genes, are synthesized on cytosolic ribosomes, and are imported into the mitochondria. Nuclear-encoded proteins which are destined for the mitochondrial matrix typically contain positively-charged amino terminal signal sequences. Import of these preproteins from the cytoplasm requires a multisubunit protein complex in the outer membrane known as the translocase of outer mitochondrial membrane (TOM ; previously designated MOM ; Pfanner, N. et al. (1996) Trends Biochem. Sci. 21 : 51-52) and at least three inner membrane proteins which comprise the   translocase   of inner mitochondrial membrane (TIM ; previously designated MIM ; Pfanner, supra). An inside-negative membrane potential across the inner mitochondrial membrane is also required for preprotein import.

   Preproteins are recognized by surface receptor components of the TOM complex and are translocated through a proteinaceous pore formed by other TOM components. Proteins targeted to the matrix are then recognized by the import machinery of the TIM complex. The import systems of the outer and inner membranes can function independently (Segui-Real, B. et al. (1993) EMBO J. 12 : 2211-2218). 



   Once precursor proteins are in the mitochondria, the leader peptide is cleaved by a signal peptidase to generate the mature protein. Most leader peptides are removed in a one step process by a protease termed mitochondrial processing peptidase (MPP) (Paces, V. et al. (1993) Proc. Natl. Acad. 



  Sci. USA 90 : 5355-5358). In some cases a two-step process occurs in which MPP generates an intermediate precursor form which is cleaved by a second enzyme, mitochondrial intermediate peptidase, to generate the mature protein. 



   Mitochondrial dysfunction leads to impaired calcium buffering, generation of free radicals that may participate in deleterious intracellular and extracellular processes, changes in mitochondrial permeability and oxidative damage which is observed in several neurodegenerative diseases. 



  Neurodegenerative diseases linked to mitochondrial dysfunction include some forms of Alzheimer's disease, Friedreich's ataxia, familial amyotrophic lateral sclerosis, and Huntington's disease (Beal, M. F. (1998) Biochim. Biophys. Acta 1366 : 211-213). The myocardium is heavily dependent on oxidative metabolism, so mitochondrial dysfunction often leads to heart disease (DiMauro, S. and M. 



  Hirano (1998) Curr. Opin. Cardiol 13 : 190-197). Mitochondria are implicated in disorders of cell proliferation, since they play an important role in a cell's decision to proliferate or self-destruct through apoptosis. The oncoprotein   Bcl-2,   for example, promotes cell proliferation by stabilizing 

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 mitochondrial membranes so that apoptosis signals are not released (Susin, S. A. (1998) Biochim. 



  Biophys. Acta 1366 : 151-165). 



  Transcription Factor Molecules 
Multicellular organisms are comprised of diverse cell types that differ dramatically both in structure and function. The identity of a cell is determined by its characteristic pattern of gene expression, and different cell types express overlapping but distinctive sets of genes throughout development. Spatial and temporal regulation of gene expression is critical for the control of cell proliferation, cell differentiation, apoptosis, and other processes that contribute to   organismal   development. Furthermore, gene expression is regulated in response to extracellular signals that mediate cell-cell communication and coordinate the activities of different cell types. Appropriate gene regulation also ensures that cells function efficiently by expressing only those genes whose functions are required at a given time. 



   Transcriptional regulatory proteins are essential for the control of gene expression. Some of these proteins function as transcription factors that initiate, activate, repress, or terminate gene transcription. Transcription factors generally bind to the promoter, enhancer, and upstream regulatory regions of a gene in a sequence-specific manner, although some factors bind regulatory elements within or downstream of a gene's coding region. Transcription factors may bind to a specific region of DNA singly or as a complex with other accessory factors. (Reviewed in Lewin, B. (1990) Genes IV, Oxford University Press, New York NY, and Cell Press, Cambridge MA, pp. 554-570.) 
The double helix structure and repeated sequences of DNA create topological and chemical features which can be recognized by transcription factors.

   These features are hydrogen bond donor and acceptor groups, hydrophobic patches, major and minor grooves, and regular, repeated stretches of sequence which induce distinct bends in the helix. Typically, transcription factors recognize specific DNA sequence motifs of about 20 nucleotides in length. Multiple, adjacent transcription factor-binding motifs may be required for gene regulation. 



   Many transcription factors incorporate DNA-binding structural motifs which comprise either a helices or B sheets that bind to the major groove of DNA. Four well-characterized structural motifs are helix-turn-helix, zinc finger, leucine zipper, and helix-loop-helix. Proteins containing these motifs may act alone as monomers, or they may form homo-or heterodimers that interact with DNA. 



   The helix-turn-helix motif consists of two a helices connected at a fixed angle by a short chain of amino acids. One of the helices binds to the major groove. Helix-turn-helix motifs are exemplified by the homeobox motif which is present in homeodomain proteins. These proteins are critical for specifying the anterior-posterior body axis during development and are conserved 

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 throughout the animal kingdom. The Antennapedia and Ultrabithorax proteins of Drosophila   melanosaster   are prototypical homeodomain proteins (Pabo, C.   O.   and R. T. Sauer (1992) Annu. Rev. 



  Biochem. 61 : 1053-1095). 



   The zinc finger motif, which binds zinc ions, generally contains tandem repeats of about 30 amino acids consisting of periodically spaced cysteine and histidine residues. Examples of this sequence pattern, designated C2H2 and C3HC4 ("RING"finger), have been described (Lewin, supra). 



  Zinc finger proteins each contain an a helix and an antiparallel   13   sheet whose proximity and conformation are maintained by the zinc ion. Contact with DNA is made by the arginine prece ding the a helix and by the second, third, and sixth residues of the a helix. Variants of the zinc finger motif include poorly defined cysteine-rich motifs which bind zinc or other metal ions. These motifs may not contain histidine residues and are generally   nonrepetitive.   



   The leucine zipper motif comprises a stretch of amino acids rich in leucine which can form an   amphipathic   a helix. This structure provides the basis for dimerization of two leucine zipper proteins. The region adjacent to the leucine zipper is usually basic, and upon protein dimerization, is optimally positioned for binding to the major groove. Proteins containing such motifs are generally referred to as bZIP transcription factors. 



   The helix-loop-helix motif (HLH) consists of a short   a   helix connected by a loop to a longer a helix. The loop is flexible and allows the two helices to fold back against each other and to bind to DNA. The transcription factor Myc contains a prototypical HLH motif. 



   Most transcription factors contain characteristic DNA binding motifs, and variations on the above motifs and new motifs have been and are currently being characterized (Faisst, S. and S. Meyer (1992) Nucleic Acids Res. 20 : 3-26). 



   Many neoplastic disorders in humans can be attributed to inappropriate gene expression. 



  Malignant cell growth may result from either excessive expression of tumor promoting genes or insufficient expression of tumor suppressor genes (Cleary, M. L. (1992) Cancer Surv. 15 : 89-104). 



  Chromosomal translocations may also produce chimeric loci which fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. Such an arrangement likely results in inappropriate gene transcription, potentially contributing to malignancy. 



   In addition, the immune system responds to infection or trauma by activating a cascade of events that coordinate the progressive selection, amplification, and mobilization of cellular defense mechanisms. A complex and balanced program of gene activation and repression is involved in this process. However, hyperactivity of the immune system as a result of improper or insufficient regulation of gene expression may result in considerable tissue or organ damage. This damage is well documented in immunological responses associated with arthritis, allergens, heart attack, stroke, and 

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 infections (Isselbacher, K. J. et al. (1996) Harrison's Principles of Internal Medicine,   13/e,   McGraw Hill, Inc. and Teton Data Systems Software). 



   Furthermore, the generation of multicellular organisms is based upon the induction and coordination of cell differentiation at the appropriate stages of development. Central to this process is differential gene expression, which confers the distinct identities of cells and tissues throughout the body. Failure to regulate gene expression during development can result in developmental disorders. 



  Human developmental disorders caused by mutations in zinc finger-type transcriptional regulators include : urogenenital developmental abnormalities associated with WT1 ; Greig cephalopolysyndactyly, Pallister-Hall syndrome, and postaxial polydactyly type A   (GLI3)   ; and Townes-Brocks syndrome, characterized by anal, renal, limb, and ear abnormalities   (SALL1)   (Engelkamp, D. and V. van Heyningen (1996) Curr. Opin. Genet. Dev. 6 : 334-342 ; Kohlhase, J. et al. 



  (1999) Am. J. Hum. Genet. 64 : 435-445). 



  Cell Membrane Molecules 
Eukaryotic cells are surrounded by plasma membranes which enclose the cell and maintain an environment inside the cell that is distinct from its surroundings. In addition, eukaryotic organisms are distinct from prokaryotes in possessing many intracellular organelle and vesicle structures. Many of the metabolic reactions which distinguish eukaryotic biochemistry from prokaryotic biochemistry take place within these structures. The plasma membrane and the membranes surrounding organelles and vesicles are composed of phosphoglycerides, fatty acids, cholesterol, phospholipids, glycolipids, proteoglycans, and proteins. These components confer identity and functionality to the membranes with which they associate. 



  Integral Membrane Proteins 
The majority of known integral membrane proteins are transmembrane proteins (TM) which are characterized by an extracellular, a transmembrane, and an intracellular domain. TM domains are typically comprised of 15 to 25 hydrophobic amino acids which are predicted to adopt an a-helical conformation. TM proteins are classified as bitopic (Types I and   II)   and polytopic (Types III and IV) (Singer, S. J. (1990) Annu. Rev. Cell Biol. 6 : 247-296). Bitopic proteins span the membrane once while polytopic proteins contain multiple membrane-spanning segments. TM proteins function as cell-surface receptors, receptor-interacting proteins, transporters of ions or metabolites, ion channels, cell anchoring proteins, and cell type-specific surface antigens. 



   Many membrane proteins (MPs) contain amino acid sequence motifs that target these proteins to specific subcellular sites. Examples of these motifs include PDZ domains, KDEL, RGD, NGR, and GSL sequence motifs, von Willebrand factor A   (vWFA)   domains, and EGF-like domains. RGD, 

 <Desc/Clms Page number 100> 

 NGR, and GSL motif-containing peptides have been used as drug delivery agents in targeted cancer treatment of tumor vasculature (Arap, W. et al.   (1998)   Science 279 : 377-380). Furthermore,   MPs   may also contain amino acid sequence motifs, such as the carbohydrate recognition domain (CRD), that mediate interactions with extracellular or intracellular molecules. 



  G-Protein   Coupled Receptors   
G-protein coupled receptors (GPCR) are a superfamily of integral membrane proteins which transduce extracellular signals. GPCRs include receptors for   biogenic   amines, lipid mediators of inflammation, peptide hormones, and sensory signal mediators. The structure of these highly-conserved receptors consists of seven hydrophobic transmembrane regions, an extracellular N-terminus, and a cytoplasmic C-terminus. Three extracellular loops alternate with three intracellular loops to link the seven transmembrane regions. Cysteine disulfide bridges connect the second and third extracellular loops. The most conserved regions of GPCRs are the transmembrane regions and the first two cytoplasmic loops. A conserved, acidic-Arg-aromatic residue triplet present in the second cytoplasmic loop may interact with G proteins.

   A GPCR consensus pattern is characteristic of most proteins belonging to this superfamily (ExPASy PROSITE document PS00237 ; and Watson, S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego CA, pp. 2-6). Mutations and changes in transcriptional activation of GPCR-encoding genes have been associated with neurological disorders such as schizophrenia, Parkinson's disease, Alzheimer's disease, drug addiction, and feeding disorders. 



  Scavenger Receptors 
Macrophage scavenger receptors with broad ligand specificity may participate in the binding of low density lipoproteins (LDL) and foreign antigens. Scavenger receptors types I and II are trimeric membrane proteins with each subunit containing a small N-terminal intracellular domain, a transmembrane domain, a large extracellular domain, and a C-terminal cysteine-rich domain. The extracellular domain contains a short spacer region, an a-helical coiled-coil region, and a triple helical collagen-like region. These receptors have been shown to bind a spectrum of ligands, including chemically modified lipoproteins and albumin, polyribonucleotides, polysaccharides, phospholipids, and asbestos (Matsumoto, A. et al. (1990) Proc. Natl. Acad. Sci. USA 87 : 9133-9137 ;   andElomaa, O.   et al. (1995) Cell 80 : 603-609).

   The scavenger receptors are thought to play a key role in atherogenesis by mediating uptake of modified LDL in arterial walls, and in host defense by binding bacterial endotoxins, bacteria, and protozoa. 



    Tetraspan Familv   Proteins 
The transmembrane 4 superfamily   (TM4SF)   or tetraspan family is a multigene family encoding type III integral membrane proteins (Wright, M. D. and M. G. Tomlinson (1994) Immunol. 

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  Today 15 : 588-594). The TM4SF is comprised of membrane proteins which traverse the cell membrane four times. Members of the TM4SF include platelet and endothelial cell membrane proteins, melanoma-associated antigens, leukocyte surface glycoproteins, colonal carcinoma antigens, tumor-associated antigens, and surface proteins of the   schistosome   parasites (Jankowski, S. A.   (1994)   Oncogene 9 : 1205-1211). Members of the TM4SF share about 25-30% amino acid sequence identity with one another. 



   A number of TM4SF members have been implicated in signal transduction, control of cell adhesion, regulation of cell growth and proliferation, including development and oncogenesis, and cell motility, including tumor cell metastasis. Expression of TM4SF proteins is associated with a variety of tumors and the level of expression may be altered when cells are growing or activated. 



  Tumor Antigens 
Tumor antigens are cell surface molecules that are differentially expressed in tumor cells relative to normal cells. Tumor antigens distinguish tumor cells immunologically from normal cells and provide diagnostic and therapeutic targets for human cancers (Takagi, S. et al. (1995) Int. J. 



  Cancer 61 : 706-715 ; Liu, E. et al. (1992) Oncogene 7 : 1027-1032). 



  Leukocyte Antigens 
Other types of cell surface antigens include those identified on leukocytic cells of the immune system. These antigens have been identified using systematic, monoclonal antibody (mAb)-based "shot gun"techniques. These techniques have resulted in the production of hundreds of   mAbs   directed against unknown cell surface leukocytic antigens. These antigens have been grouped into "clusters of differentiation"based on common   immunocytochemical   localization patterns in various differentiated and undifferentiated leukocytic cell types. Antigens in a given cluster are presumed to identify a single cell surface protein and are assigned a"cluster of differentiation"or"CD" designation. Some of the genes encoding proteins identified by CD antigens have been cloned and verified by standard molecular biology techniques.

   CD antigens have been characterized as both transmembrane proteins and cell surface proteins anchored to the plasma membrane via covalent attachment to fatty acid-containing glycolipids such as glycosylphosphatidylinositol (GPI). 



  (Reviewed in Barclay, A. N. et al. (1995) The Leucocyte Antigen Facts Book, Academic Press, San Diego CA, pp. 17-20.) Ion Channels 
Ion channels are found in the plasma membranes of virtually every cell in the body. For example, chloride channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ions across epithelial membranes. Chloride channels also regulate the pH of organelles such as the Golgi apparatus and endosomes (see, e. g., Greger, R.   (1988)   

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 Annu. Rev. Physiol. 50 : 111-122).

   Electrophysiological and pharmacological properties of chloride channels, including ion conductance, current-voltage relationships, and sensitivity to modulators, suggest that different chloride channels exist in muscles, neurons, fibroblasts, epithelial cells, and lymphocytes. 



   Many ion channels have sites for phosphorylation by one or more protein kinases including protein kinase A, protein kinase C, tyrosine kinase, and casein kinase   II,   all of which regulate ion channel activity in cells. Inappropriate phosphorylation of proteins in cells has been linked to changes in cell cycle progression and cell differentiation. Changes in the cell cycle have been linked to induction of apoptosis or cancer. Changes in cell differentiation have been linked to diseases and disorders of the reproductive system, immune system, skeletal muscle, and other organ systems. 



  Proton Pumps 
Proton ATPases comprise a large class of membrane proteins that use the energy of ATP hydrolysis to generate an electrochemical proton gradient across a membrane. The resultant gradient may be used to transport other ions across the membrane (Na+,   K+,   or   Cl-)   or to maintain organelle pH. Proton ATPases are further subdivided into the mitochondrial F-ATPases, the plasma membrane ATPases, and the vacuolar ATPases. The vacuolar ATPases establish and maintain an acidic pH within various organelles involved in the processes of endocytosis and exocytosis (Mellman,   I.   et al. 



  (1986) Annu. Rev. Biochem. 55 : 663-700). 



   Proton-coupled, 12   membrane-spanning   domain transporters such as PEPT 1 and PEPT 2 are responsible for gastrointestinal absorption and for renal reabsorption of peptides using an electrochemical   H+   gradient as the driving force. Another type of peptide transporter, the TAP transporter, is a heterodimer consisting of TAP 1 and TAP 2 and is associated with antigen processing. Peptide antigens are transported across the membrane of the endoplasmic reticulum by TAP so they can be expressed on the cell surface in association with MHC molecules. Each TAP protein consists of multiple hydrophobic membrane spanning segments and a highly conserved ATP-binding cassette (Boll, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93 : 284-289).

   Pathogenic microorganisms, such as herpes simplex virus, may encode inhibitors of TAP-mediated peptide transport in order to evade immune surveillance (Marusina, K. and J. J Manaco (1996) Curr. Opin. 



  Hematol. 3 : 19-26). 



  ABC Transporters 
The ATP-binding cassette (ABC) transporters, also called   the"traffic   ATPases", comprise a superfamily of membrane proteins that mediate transport and channel functions in prokaryotes and eukaryotes (Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8 : 67-113). ABC proteins share a similar overall structure and significant sequence homology. All ABC proteins contain a conserved domain 

 <Desc/Clms Page number 103> 

 of approximately two hundred amino acid residues which includes one or more nucleotide binding domains. Mutations in ABC transporter genes are associated with various disorders, such as   hyperbilirubinemia     II/Dubin-Johnson   syndrome, recessive Stargardt's disease, X-linked adrenoleukodystrophy, multidrug resistance, celiac disease, and cystic fibrosis. 



  Peripheral and Anchored Membrane Proteins 
Some membrane proteins are not membrane-spanning but are attached to the plasma membrane via membrane anchors or interactions with integral membrane proteins. Membrane anchors are covalently joined to a protein post-translationally and include such moieties as prenyl, myristyl, and glycosylphosphatidyl inositol groups. Membrane localization of peripheral and anchored proteins is important for their function in processes such as receptor-mediated signal transduction. For example, prenylation of Ras is required for its localization to the plasma membrane and for its normal and oncogenic functions in signal transduction. 



  Vesicle Coat Proteins 
Intercellular communication is essential for the development and survival of multicellular organisms. Cells communicate with one another through the secretion and uptake of protein signaling molecules. The uptake of proteins into the cell is achieved by the endocytic pathway, in which the interaction of extracellular signaling molecules with plasma membrane receptors results in the formation of plasma membrane-derived vesicles that enclose and transport the molecules into the cytosol. These transport vesicles fuse with and mature into endosomal and lysosomal (digestive) compartments. The secretion of proteins from the cell is achieved by exocytosis, in which molecules inside of the cell proceed through the secretory pathway.

   In this pathway, molecules transit from the ER to the Golgi apparatus and finally to the plasma membrane, where they are secreted from the cell. 



   Several steps in the transit of material along the secretory and endocytic pathways require the formation of transport vesicles. Specifically, vesicles form at the transitional endoplasmic reticulum (tER), the rim of Golgi cisternae, the face of the Trans-Golgi Network (TGN), the plasma membrane   (Plus,   and tubular extensions of the endosomes. Vesicle formation occurs when a region of membrane buds off from the donor organelle. The membrane-bound vesicle contains proteins to be transported and is surrounded by a proteinaceous coat, the components of which are recruited from the cytosol. Two different classes of coat protein have been identified. Clathrin coats form on vesicles derived from the TGN and PM, whereas coatomer (COP) coats form on vesicles derived from the ER and Golgi.

   COP coats can be further classified as COPI, involved in retrograde traffic through the Golgi and from the Golgi to the ER, and COPII, involved in anterograde traffic from the ER to the Golgi (Mellman, supra). 



   In   clathrin-based   vesicle formation, adapter proteins bring vesicle cargo and coat proteins 

 <Desc/Clms Page number 104> 

 together at the surface of the budding membrane. Adapter protein-1 and-2 select cargo from the TGN and plasma membrane, respectively, based on molecular information encoded on the cytoplasmic tail of integral membrane cargo proteins. Adapter proteins also recruit   clathrin   to the bud site. Clathrin is a protein complex consisting of three large and three small polypeptide chains arranged in a three-legged structure called a triskelion. Multiple triskelions and other coat proteins appear to self-assemble on the membrane to form a coated pit. This assembly process may serve to deform the membrane into a budding vesicle. GTP-bound ADP-ribosylation factor (Arf) is also incorporated into the coated assembly.

   Another small G-protein, dynamin, forms a ring complex around the neck of the forming vesicle and may provide the   mechanochemical   force to seal the bud, thereby releasing the vesicle. The coated vesicle complex is then transported through the cytosol. 



  During the transport process, Arf-bound GTP is hydrolyzed to GDP, and the coat dissociates from the transport vesicle (West, M. A. et al. (1997) J. Cell Biol. 138 : 1239-1254). 



   Vesicles which bud from the ER and the Golgi are covered with a protein coat similar to the clathrin coat of endocytic and TGN vesicles. The coat protein (COP) is assembled from cytosolic precursor molecules at specific budding regions on the organelle. The COP coat consists of two major components, a G-protein (Arf or Sar) and coat protomer (coatomer). Coatomer is an   equimolar   complex of seven proteins, termed alpha-, beta-, beta'-, gamma-, delta-, epsilon-and zeta-COP. The coatomer complex binds to dilysine motifs contained on the cytoplasmic tails of integral membrane proteins. These include the KKXX retrieval motif of membrane proteins of the ER and   dibasic/diphenylamine   motifs of members of the p24 family. The p24 family of type I membrane proteins represent the major membrane proteins of COPI vesicles (Harter, C. and F. T.

   Wieland (1998) Proc. Natl. Acad. Sci. USA 95 : 11649-11654). 



  Organelle Associated Molecules
Eukaryotic cells are organized into various cellular organelles which has the effect of separating specific molecules and their functions from one another and from the cytosol. Within the cell, various membrane structures surround and define these organelles while allowing them to interact with one another and the cell environment through both active and passive transport processes. Important cell organelles include the nucleus, the Golgi apparatus, the endoplasmic reticulum, mitochondria, peroxisomes, lysosomes, endosomes, and secretory vesicles. 



  Nucleus
The cell nucleus contains all of the genetic information of the cell in the form of DNA, and the components and machinery necessary for replication of DNA and for transcription of DNA into RNA. (See Alberts,   B.   et al. (1994) Molecular Biology of the Cell, Garland Publishing Inc., New 

 <Desc/Clms Page number 105> 

 York NY, pp. 335-399.) DNA is organized into compact structures in the nucleus by interactions with various DNA-binding proteins such as histones and non-stone chromosomal proteins. 



  DNA-specific nucleases, DNAses, partially degrade these compacted structures prior to DNA replication or transcription. DNA replication takes place with the aid of DNA helicases which unwind the double-stranded DNA helix, and DNA polymerases that duplicate the separated DNA strands. 



   Transcriptional regulatory proteins are essential for the control of gene expression. Some of these proteins function as transcription factors that initiate, activate, repress, or terminate gene transcription. Transcription factors generally bind to the promoter, enhancer, and upstream regulatory regions of a gene in a sequence-specific manner, although some factors bind regulatory elements within or downstream of a gene's coding region. Transcription factors may bind to a specific region of DNA singly or as a complex with other accessory factors. (Reviewed in Lewin, B. (1990) Genes IV, Oxford University Press, New York NY, and Cell Press, Cambridge MA, pp. 554-570.) Many transcription factors incorporate DNA-binding structural motifs which comprise either a helices or 13 sheets that bind to the major groove of DNA.

   Four well-characterized structural motifs are helix-turn-helix, zinc finger, leucine zipper, and helix-loop-helix. Proteins containing these motifs may act alone as monomers, or they may form homo-or heterodimers that interact with DNA. 



   Many neoplastic disorders in humans can be attributed to inappropriate gene expression. 



  Malignant cell growth may result from either excessive expression of tumor promoting genes or insufficient expression of tumor suppressor genes (Cleary, M. L. (1992) Cancer Surv. 15 : 89-104). 



  Chromosomal translocations may also produce chimeric loci which fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. Such an arrangement likely results in inappropriate gene transcription, potentially contributing to malignancy. 



   In addition, the immune system responds to infection or trauma by activating a cascade of events that coordinate the progressive selection, amplification, and mobilization of cellular defense mechanisms. A complex and balanced program of gene activation and repression is involved in this process. However, hyperactivity of the immune system as a result of improper or insufficient regulation of gene expression may result in considerable tissue or organ damage. This damage is well documented in immunological responses associated with arthritis, allergens, heart attack, stroke, and infections (Isselbacher, K. J. et al. (1996) Harrison's Principles of Internal Medicine, 13/e, McGraw Hill, Inc. and Teton Data Systems Software). 



   Transcription of DNA into RNA also takes place in the nucleus catalyzed by RNA polymerases. Three types of RNA polymerase exist. RNA polymerase I makes large ribosomal RNAs, while RNA polymerase III makes a variety of small, stable RNAs including 5S ribosomal 

 <Desc/Clms Page number 106> 

 RNA and the transfer RNAs (tRNA). RNA polymerase II transcribes genes that will be translated into proteins. The primary transcript of RNA polymerase II is called heterogenous nuclear RNA (hnRNA), and must be further processed by splicing to remove non-coding sequences called introns. 



  RNA splicing is mediated by small nuclear ribonucleoprotein complexes, or snRNPs, producing mature messenger RNA (mRNA) which is then transported out of the nucleus for translation into proteins. 



  Nucleolus 
The nucleolus is a highly organized subcompartment in the nucleus that contains high concentrations of RNA and proteins and functions mainly in ribosomal RNA synthesis and assembly (Alberts, et al. supra, pp. 379-382). Ribosomal RNA (rRNA) is a structural RNA that is complexed with proteins to form ribonucleoprotein structures called ribosomes. Ribosomes provide the platform on which protein synthesis takes place. 



   Ribosomes are assembled in the nucleolus initially from a large,   45S rRNA   combined with a variety of proteins imported from the cytoplasm, as well as smaller, 5S rRNAs. Later processing of the immature ribosome results in formation of smaller ribosomal subunits which are transported from the nucleolus to the cytoplasm where they are assembled into functional ribosomes. 



    Endoplasmic   Reticulum 
In eukaryotes, proteins are synthesized within the endoplasmic reticulum (ER), delivered from the ER to the Golgi apparatus for post-translational processing and sorting, and transported from the Golgi to specific intracellular and extracellular destinations. Synthesis of integral membrane proteins, secreted proteins, and proteins destined for the lumen of a particular organelle occurs on the rough endoplasmic reticulum (ER). The rough ER is so named because of the rough appearance in electron micrographs imparted by the attached ribosomes on which protein synthesis proceeds.

   Synthesis of proteins destined for the ER actually begins in the cytosol with the synthesis of a specific signal peptide which directs the growing polypeptide and its attached ribosome to the ER membrane where the signal peptide is removed and protein synthesis is completed. Soluble proteins destined for the ER lumen, for secretion, or for transport to the lumen of other organelles pass completely into the ER lumen. Transmembrane proteins destined for the ER or for other cell membranes are   translocated   across the ER membrane but remain anchored in the lipid bilayer of the membrane by one or more membrane-spanning a-helical regions. 



   Translocated polypeptide chains destined for other organelles or for secretion also fold and assemble in the ER lumen with the aid of certain"resident"ER proteins. Protein folding in the ER is aided by two principal types of protein isomerases, protein disulfide isomerase (PDI), and peptidyl- prolyl isomerase (PPI). PDI catalyzes the oxidation of free   sulfhydryl   groups in cysteine residues to 

 <Desc/Clms Page number 107> 

 form intramolecular disulfide bonds in proteins. PPI, an enzyme that catalyzes the isomerization of certain proline imide bonds in oligopeptides and proteins, is considered to govern one of the rate limiting steps in the folding of many proteins to their final functional conformation.

   The cyclophilins represent a major class of PPI that was originally identified as the major receptor for the immunosuppressive drug cyclosporin A (Handschumacher, R. E. et al. (1984) Science 226 :   544-547).   



  Molecular"chaperones"such as BiP (binding protein) in the ER recognize incorrectly folded proteins as well as proteins not yet folded into their final form and bind to them, both to prevent improper aggregation between them, and to promote proper folding. 



   The"N-linked"glycosylation of most soluble secreted and membrane-bound proteins by oligosacchrides linked to asparagine residues in proteins is also performed in the ER. This reaction is catalyzed by a membrane-bound enzyme, oligosaccharyl transferase. 



  Golgi Apparatus 
The Golgi apparatus is a complex structure that lies adjacent to the ER in eukaryotic cells and serves primarily as a sorting and dispatching station for products of the ER (Alberts, et al. supra, pp. 



  600-610). Additional posttranslational processing, principally additional glycosylation, also occurs in the Golgi. Indeed, the Golgi is a major site of carbohydrate synthesis, including most of the   glycosaminoglycans   of the extracellular matrix. N-linked oligosaccharides, added to proteins in the ER, are also further modified in the Golgi by the addition of more sugar residues to form complex N- linked oligosaccharides."O-linked"glycosylation of proteins also occurs in the Golgi by the addition of N-acetylgalactosamine to the hydroxyl group of a serine or threonine residue followed by the sequential addition of other sugar residues to the first. This process is catalyzed by a series of glycosyltransferases each specific for a particular donor sugar nucleotide and acceptor molecule (Lodish, H. et al. (1995) Molecular Cell Biology,   W.   H.

   Freeman and Co., New York NY, pp. 700- 708). In many cases, both N-and 0-linked oligosaccharides appear to be required for the secretion of proteins or the movement of plasma membrane glycoproteins to the cell surface. 



   The terminal compartment of the Golgi is the Trans-Golgi Network (TGN), where both membrane and lumenal proteins are sorted for their final destination. Transport (or secretory) vesicles destined for intracellular compartments, such as lysosomes, bud off of the TGN. Other transport vesicles bud off containing proteins destined for the plasma membrane, such as receptors, adhesion molecules, and ion channels, and secretory proteins, such as hormones, neurotransmitters, and digestive enzymes. 



  Vacuoles 
The vacuole system is a collection of membrane bound compartments in eukaryotic cells that functions in the processes of endocytosis and exocytosis. They include phagosomes, lysosomes, 

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 endosomes, and secretory vesicles. Endocytosis is the process in cells of internalizing nutrients, solutes or small particles (pinocytosis) or large particles such as internalized receptors, viruses, bacteria, or bacterial toxins (phagocytosis). Exocytosis is the process of transporting molecules to the cell surface. 



  It facilitates placement or localization of membrane-bound receptors or other membrane proteins and secretion of hormones, neurotransmitters, digestive enzymes, wastes, etc. 



   A common property of all of these vacuoles is an acidic pH environment ranging from approximately pH 4. 5-5. 0. This acidity is maintained by the presence of a proton ATPase that uses the energy of ATP hydrolysis to generate an electrochemical proton gradient across a membrane (Mellman,   1.   et al. (1986) Annu. Rev. Biochem. 55 : 663-700). Eukaryotic vacuolar proton ATPase (vp-ATPase) is a multimeric enzyme composed of 3-10 different subunits. One of these subunits is a highly hydrophobic polypeptide of approximately 16 kDa that is similar to the proteolipid component of vp- ATPases from eubacteria, fungi, and plant vacuoles (Mandel, M. et al. (1988) Proc. Natl. Acad. Sci. 



  USA 85 :   5521-5524).   The 16 kDa proteolipid component is the major subunit of the membrane portion of vp-ATPase and functions in the transport of protons across the membrane. 



  Lysosomes 
Lysosomes are membranous vesicles containing various hydrolytic enzymes used for the controlled intracellular digestion of macromolecules. Lysosomes contain some 40 types of enzymes including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases, and sulfatases, all of which are acid hydrolases that function at a pH of about 5. Lysosomes are surrounded by a unique membrane containing transport proteins that allow the final products of macromolecule degradation, such as sugars, amino acids, and nucleotides, to be transported to the cytosol where they may be either excreted or reutilized by the cell. A vp-ATPase, such as that described above, maintains the acidic environment necessary for hydrolytic activity (Alberts, supra, pp. 610-611). 



  Endosomes 
Endosomes are another type of acidic vacuole that is used to transport substances from the cell surface to the interior of the cell in the process of endocytosis. Like lysosomes, endosomes have an acidic environment provided by a vp-ATPase (Alberts et al. supra, pp. 610-618). Two types of endosomes are apparent based on tracer uptake studies that distinguish their time of formation in the cell and their cellular location. Early endosomes are found near the plasma membrane and appear to function primarily in the recycling of internalized receptors back to the cell surface. Late endosomes appear later in the endocytic process close to the Golgi apparatus and the nucleus, and appear to be associated with delivery of endocytosed material to lysosomes or to the TGN where they may be recycled.

   Specific proteins are associated with particular transport vesicles and their target compartments that may provide selectivity in targeting vesicles to their proper compartments. A 

 <Desc/Clms Page number 109> 

 cytosolic prenylated GTP-binding protein, Rab, is one such protein. Rabs 4, 5, and 11 are associated with the early endosome, whereas Rabs 7 and 9 associate with the late endosome. 



  Mitochondria 
Mitochondria are oval-shaped organelles comprising an outer membrane, a tightly folded inner membrane, an intermembrane space between the outer and inner membranes, and a matrix inside the inner membrane. The outer membrane contains many porin molecules that allow ions and charged molecules to enter the intermembrane space, while the inner membrane contains a variety of transport proteins that transfer only selected molecules. Mitochondria are the primary sites of energy production in cells. 



   Energy is produced by the oxidation of glucose and fatty acids. Glucose is initially converted to pyruvate in the cytoplasm. Fatty acids and pyruvate are transported to the mitochondria for complete oxidation to   CO2   coupled by enzymes to the transport of electrons from NADH and FADH2 to oxygen and to the synthesis of ATP (oxidative phosphorylation) from ADP and Pi. 



   Pyruvate is transported into the mitochondria and converted to acetyl-CoA for oxidation via the citric acid cycle, involving pyruvate dehydrogenase components, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Enzymes involved in the citric acid cycle include : citrate synthetase, aconitases, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex including transsuccinylases, succinyl CoA synthetase, succinate dehydrogenase, fumarases, and malate dehydrogenase. Acetyl CoA is oxidized to   CO2   with concomitant formation of NADH, FADH2, and GTP. In oxidative phosphorylation, the transfer of electrons from NADH and FADH2 to oxygen by dehydrogenases is coupled to the synthesis of ATP from ADP and Pi by the FoFI ATPase complex in the mitochondrial inner membrane.

   Enzyme complexes responsible for electron transport and ATP synthesis include the   FoFl   ATPase   complex, ubiquinone (CoQ)-cytochrome   c reductase, ubiquinone reductase, cytochrome b, cytochrome   cl,   FeS protein, and cytochrome c oxidase. 



  Peroxisomes 
Peroxisomes, like mitochondria, are a major site of oxygen utilization. They contain one or more enzymes, such as catalase and urate oxidase, that use molecular oxygen to remove hydrogen atoms from specific organic substrates in an oxidative reaction that produces hydrogen peroxide (Alberts, supra, pp. 574-577). Catalase oxidizes a variety of substrates including phenols, formic acid, formaldehyde, and alcohol and is important in peroxisomes of liver and kidney cells for detoxifying various toxic molecules that enter the bloodstream.

   Another major function of oxidative reactions in peroxisomes is the breakdown of fatty acids in a process called P oxidation.   P   oxidation results in shortening of the alkyl chain of fatty acids by blocks of two carbon atoms that are converted to acetyl CoA and exported to the cytosol for reuse in biosynthetic reactions. 

 <Desc/Clms Page number 110> 

 



   Also like mitochondria, peroxisomes import their proteins from the cytosol using a specific signal sequence located near the C-terminus of the protein. The importance of this import process is evident in the inherited human disease Zellweger syndrome, in which a defect in importing proteins into perixosomes leads to a perixosomal deficiency resulting in severe abnormalities in the brain, liver, and kidneys, and death soon after birth. One form of this disease has been shown to be due to a mutation in the gene encoding a perixosomal integral membrane protein called peroxisome assembly factor-1. 



   The discovery of new human molecules satisfies a need in the art by providing new compositions which are useful in the diagnosis, study, prevention, and treatment of diseases associated with, as well as effects of exogenous compounds on, the expression of human molecules. 



   SUMMARY OF THE INVENTION 
The present invention relates to nucleic acid sequences comprising human diagnostic and therapeutic polynucleotides (dithp) as presented in the Sequence Listing. The dithp uniquely identify genes encoding human structural, functional, and regulatory molecules. 



   The invention provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO :   1-211   ;   c)   a polynucleotide sequence complementary to a) ; d) a polynucleotide sequence complementary to b) ; and e) an RNA equivalent of a) through d).

   In one alternative, the polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID   N0   :   1-211.   In another alternative, the polynucleotide comprises at least 60 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO :   1-211   ; b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO :   1-   211 ;   c)   a polynucleotide sequence complementary to a) ; d) a polynucleotide sequence complementary to b) ; and e) an RNA equivalent of a) through d).

   The invention further provides a composition for the detection of expression of human diagnostic and therapeutic polynucleotides comprising at least one isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO :   1-211   ; b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; c) a polynucleotide sequence complementary to a) ; d) a polynucleotide sequence complementary to b) ; and   e)   an RNA equivalent of a) through d) ; 

 <Desc/Clms Page number 111> 

 and a detectable label. 



   The invention also provides a method for detecting a target polynucleotide in a sample, said target polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO :   1-211   ; b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; c) a polynucleotide sequence complementary to a) ; d) a polynucleotide sequence complementary to b) ; and e) an RNA equivalent of a) through d). 



  The method comprises a) amplifying said target polynucleotide or a fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. 



   The invention also provides a method for detecting a target polynucleotide in a sample, said target polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; b) a naturally occurring polynucleotide sequence having at least   90%   sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; c) a polynucleotide sequence complementary to a) ; d) a polynucleotide sequence complementary to b) ; and e) an RNA equivalent of a) through d). 



  The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof. In one alternative, the probe comprises at least 30 contiguous nucleotides. In another alternative, the probe comprises at least 60 contiguous nucleotides. 



   The invention further provides a recombinant polynucleotide comprising a promoter sequence operably linked to an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1- 211 ; b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; c) a polynucleotide sequence complementary to a) ; d) a polynucleotide sequence complementary to b) ; and e) an RNA equivalent of a) through d). In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.

   In a further alternative, the invention provides a method for producing a human diagnostic and therapeutic polypeptide, the method comprising a) culturing a cell under conditions suitable for expression of the human diagnostic and therapeutic polypeptide, 

 <Desc/Clms Page number 112> 

 wherein said cell is transformed with the recombinant polynucleotide, and b) recovering the human diagnostic and therapeutic polypeptide so expressed. 



   The invention also provides a purified human diagnostic and therapeutic polypeptide (DITHP) encoded by at least one polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211. Additionally, the invention provides an isolated antibody which specifically binds to the human diagnostic and therapeutic polypeptide.

  *  The invention further provides a method of identifying a test compound which specifically binds to the human diagnostic and therapeutic polypeptide, the method comprising the steps of a) providing a test compound ; b) combining the human diagnostic and therapeutic polypeptide with the test compound for a sufficient time and under suitable conditions for binding ; and c) detecting binding of the human diagnostic and therapeutic polypeptide to the test compound, thereby identifying the test compound which specifically binds the human diagnostic and therapeutic polypeptide. 



   The invention further provides a microarray wherein at least one element of the microarray is an isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; c) a polynucleotide sequence complementary to a) ; d) a polynucleotide sequence complementary to b) ; and e) an RNA equivalent of a) through d). The invention also provides a method for generating a transcript image of a sample which contains polynucleotides.

   The method comprises a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample. 



   Additionally, the invention provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; c) a polynucleotide sequence complementary to a) ; d) a polynucleotide sequence complementary to b) ; and e) an RNA equivalent of a) through d).

   The method comprises a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. 



   The invention further provides a method for assessing toxicity of a test compound, said method 

 <Desc/Clms Page number 113> 

 comprising a) treating a biological sample containing nucleic acids with the test compound ; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of i) a polynucleotide sequence selected from the group consisting of SEQ ID NO :   1-   211 ; ii) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; iii) a polynucleotide sequence complementary to i), iv) a polynucleotide sequence complementary to ii), and v) an RNA equivalent of i)-iv).

   Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence selected from the group consisting of i) a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; ii) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211 ; iii) a polynucleotide sequence complementary to i), iv) a polynucleotide sequence complementary to ii), and v) an RNA equivalent of i)-iv), and alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i-v above ; c) quantifying the amount of hybridization complex ;

   and d) comparing the amount of hybridization complex in the treated biological sample with the amount t of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. 



   The invention further provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO : 212-422, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO : 212-422, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO : 212-422, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO : 212-422. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO : 212-422. 



   DESCRIPTION OF THE TABLES 
Table 1 shows the sequence identification numbers (SEQ ID NO : s) and template identification numbers (template IDs) corresponding to the polynucleotides of the present invention, along with their GenBank hits (GI Numbers), probability scores, and functional annotations corresponding to the GenBank hits. 



   Table 2 shows the sequence identification numbers (SEQ ID NO : s) and template identification 

 <Desc/Clms Page number 114> 

 numbers (template IDs) corresponding to the polynucleotides of the present invention, along with polynucleotide segments of each template sequence as defined by the   indicated"start"and"stop"   nucleotide positions. The reading frames of the polynucleotide segments and the Pfam hits, Pfam descriptions, and E-values corresponding to the polypeptide domains encoded by the polynucleotide segments are indicated. 



   Table 3 shows the sequence identification numbers (SEQ ID NO : s) and template identification numbers (template IDs) corresponding to the polynucleotides of the present invention, along with polynucleotide segments of each template sequence as defined by the indicated"start"and"stop" nucleotide positions. The reading frames of the polynucleotide segments are shown, and the polypeptides encoded by the polynucleotide segments constitute either signal peptide (SP) or transmembrane (TM) domains, as indicated. The membrane topology of the encoded polypeptide sequence is indicated, the N-terminus (N) listed as being oriented to either the cytosolic (in) or noncytosolic (out) side of the cell membrane or organelle. 



   Table 4 shows the sequence identification numbers (SEQ ID NO : s) corresponding to the polynucleotides of the present invention, along with component sequence identification numbers (component IDs) corresponding to each template. The component sequences, which were used to assemble the template sequences, are defined by the indicated"start"and"stop"nucleotide positions along each template. 



   Table 5 shows the tissue distribution profiles for the templates of the invention. 



   Table 6 shows the sequence identification numbers (SEQ ID NO : s) corresponding to the polypeptides of the present invention, along with the reading frames used to obtain the polypeptide segments, the lengths of the polypeptide segments, the"start"and"stop"nucleotide positions of the polynucleotide sequences used to define the encoded polypeptide segments, the GenBank hits (GI Numbers), probability scores, and functional annotations corresponding to the GenBank hits. 



   Table 7 summarizes the bioinformatics tools which are useful for analysis of the polynucleotides of the present invention. The first column of Table 7 lists analytical tools, programs, and algorithms, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences). 



  DETAILED DESCRIPTION OF THE INVENTION
Before the nucleic acid sequences and methods are presented, it is to be understood that this 

 <Desc/Clms Page number 115> 

 invention is not limited to the particular machines, methods, and materials described. Although particular embodiments are described, machines, methods, and materials similar or equivalent to these embodiments may be used to practice the invention. The preferred machines, methods, and materials set forth are not intended to limit the scope of the invention which is limited only by the appended claims. 



   The singular   forms"a","an",     and"the"include   plural reference unless the context clearly dictates otherwise. All technical and scientific terms have the meanings commonly understood by one of ordinary skill in the art. All publications are incorporated by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are presented and which might be used in connection with the invention. Nothing in the specification is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. 



  Definitions 
As used herein, the lower case"dithp"refers to a nucleic acid sequence, while the upper case "DITHP"refers to an amino acid sequence encoded by dithp. A"full-length"dithp refers to a nucleic acid sequence containing the entire coding region of a gene endogenously expressed in human tissue. 



     "Adjuvants"are   materials such as Freund's adjuvant, mineral gels (aluminum hydroxide), and surface active substances (lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet   hemocyanin,   and dinitrophenol) which may be administered to increase a host's immunological response. 



   "Allele"refers to an alternative form of a nucleic acid sequence. Alleles result from a "mutation,"a change or an alternative reading of the genetic code. Any given gene may have none, one, or many allelic forms. Mutations which give rise to alleles include deletions, additions, or substitutions of nucleotides. Each of these changes may occur alone, or in combination with the others, one or more times in a given nucleic acid sequence. The present invention encompasses allelic dithp. 



   "Amino acid   sequence"refers   to a peptide, a polypeptide, or a protein of either natural or synthetic origin. The amino acid sequence is not limited to the complete, endogenous amino acid sequence and may be a fragment, epitope, variant, or derivative of a protein expressed by a nucleic acid sequence. 



     "Amplification"refers   to the production of additional copies of a sequence and is carried out using polymerase chain reaction (PCR) technologies well known in the art. 



   "Antibody"refers to intact molecules as well as to fragments thereof, such as Fab, F (ab') 2, and Fv fragments, which are capable of binding the epitopic determinant. Antibodies that bind DITHP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of 

 <Desc/Clms Page number 116> 

 interest as the immunizing antigen. The polypeptide or peptide used to immunize an animal (e. g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin,   thyroglobulin,   and keyhole limpet   hemocyanin   (KLH). The coupled peptide is then used to immunize the animal. 



   "Antisense   sequence"refers   to a sequence capable of specifically hybridizing to a target sequence. The antisense sequence may include DNA, RNA, or any nucleic acid mimic or analog such as peptide nucleic acid (PNA) ; oligonucleotides having modified backbone linkages such as   phosphorothioates,   methylphosphonates, or benzylphosphonates ; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars ; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. 



   "Antisense sequence'refers to a sequence capable of specifically hybridizing to a target sequence. The antisense sequence can be DNA, RNA, or any nucleic acid mimic or analog. 



   "Antisense technology"refers to any technology which relies on the specific hybridization of an antisense sequence to a target sequence. 



   A"bin"is a portion of computer memory space used by a computer program for storage of data, and bounded in such a manner that data stored in a bin may be retrieved by the program. 



   "Biologically   active"refers   to an amino acid sequence having a structural, regulatory, or biochemical function of a naturally occurring amino acid sequence. 



   "Clone joining"is a process for combining gene bins based upon the bins'containing sequence . information from the same clone. The sequences may assemble into a primary gene transcript as well as one or more splice variants. 



   "Complementary"describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing   (5'-A-G-T-3'pairs   with its complement 3'-T-C-A-5'). 



   A"component   sequence"is   a nucleic acid sequence selected by a computer program such as 
PHRED and used to assemble a consensus or template sequence from one or more component sequences. 



   A"consensus sequence"or"template   sequence"is   a nucleic acid sequence which has been assembled from overlapping sequences, using a computer program for fragment assembly such as the 
GELVIEW fragment assembly system (Genetics Computer Group (GCG), Madison WI) or using a relational database management system (RDMS). 



   "Conservative amino acid   substitutions"are   those substitutions that, when made, least interfere with the properties of the original protein, i. e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids 

 <Desc/Clms Page number 117> 

 which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions. 



   Original Residue Conservative Substitution 
Ala Gly, Ser 
Arg His, Lys 
Asn Asp, Gln, His 
Asp Asn, Glu 
Cys Ala, Ser 
Gln Asn, Glu, His 
Glu Asp, Gln, His 
Gly Ala 
His Asn, Arg, Gln, Glu 
Ile Leu, Val 
Leu Ile, Val 
Lys Arg, Gln, Glu 
Met Leu, Ile 
Phe His, Met, Leu, Trp, Tyr 
Ser   Cys, Thr     Thr   Ser, Val 
Trp Phe, Tyr 
Tyr His, Phe, Trp   Val Ile,   Leu, Thr 
Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. 



     "Deletion"refers   to a change in either a nucleic or amino acid sequence in which at least one nucleotide or amino acid residue, respectively, is absent. 



     "Derivative"refers   to the chemical modification of a nucleic acid sequence, such as by replacement of hydrogen by an alkyl, acyl, amino, hydroxyl, or other group. 



   The   terms"element"and"array   element"refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray. 



     "E-value"refers   to the statistical probability that a match between two sequences occurred by chance. 



   A"fragment"is a unique portion of dithp or DITHP which is identical in sequence to but shorter in length than the parent sequence. A fragment may comprise up to the entire length of the defined sequence, minus one   nucleotide/amino   acid residue. For example, a fragment may comprise from 10 to 1000 contiguous amino acid residues or nucleotides. A fragment used as a probe, primer, 

 <Desc/Clms Page number 118> 

 antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous amino acid residues or nucleotides in length. 



  Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first   250   or   500 amino   acids (or first   25%   or 50%) of a polypeptide as shown in a certain defined sequence. 



  Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing and the figures, may be encompassed by the present embodiments. 



   A fragment of dithp comprises a region of unique polynucleotide sequence that specifically identifies dithp, for example, as distinct from any other sequence in the same genome. A fragment of dithp is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish dithp from related polynucleotide sequences. The precise length of a fragment of dithp and the region of dithp to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment. 



   A fragment of DITHP is encoded by a fragment of dithp. A fragment of DITHP comprises a region of unique amino acid sequence that specifically identifies DITHP. For example, a fragment of DITHP is useful as an immunogenic peptide for the development of antibodies that specifically recognize DITHP. The precise length of a fragment of DITHP and the region of DITHP to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment. 



   A"full   length"nucleotide   sequence is one containing at least a start site for translation to a protein sequence, followed by an open reading frame and a stop site, and encoding a"full length" polypeptide. 



   "Hit"refers to a sequence whose annotation will be used to describe a given template. Criteria for selecting the top hit are as follows : if the template has one or more exact nucleic acid matches, the top hit is the exact match with highest percent identity. If the template has no exact matches but has significant protein hits, the top hit is the protein hit with the lowest E-value. If the template has no significant protein hits, but does have significant non-exact nucleotide hits, the top hit is the nucleotide hit with the lowest E-value. 



   "Homology"refers to sequence similarity either between a reference nucleic acid sequence and at least a fragment of a dithp or between a reference amino acid sequence and a fragment of a DITHP. 



     "Hybridization"refers   to the process by which a strand of nucleotides anneals with a complementary strand through base pairing. Specific hybridization is an indication that two nucleic acid sequences share a high degree of identity. Specific hybridization complexes form under defined annealing conditions, and remain hybridized after the"washing"step. The defined hybridization 

 <Desc/Clms Page number 119> 

 conditions include the annealing conditions and the washing step (s), the latter of which is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i. e., binding between pairs of nucleic acid probes that are not perfectly matched.

   Permissive conditions for annealing of nucleic acid sequences are routinely determinable and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency. 



   Generally, stringency of hybridization is expressed with reference to the temperature under which the wash step is carried out. Generally, such wash temperatures are selected to be about   5 C   to   20 C   lower than the thermal melting point (T, for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating   Tm   and conditions for nucleic acid hybridization is well known and can be found in Sambrook et al., 1989, Molecular   Cloning : ALaboratorv Manual, 2nd   ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY ; specifically see volume 2, chapter   9.   



   High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of   68 C   in the presence of about 0. 2 x SSC and about 0. 1% SDS, for 1 hour. 



  Alternatively, temperatures of about   65 C,     60 C,   or   55 C may be   used. SSC concentration may be varied from about 0. 2 to 2 x SSC, with SDS being present at about 0. 1%. Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, denatured salmon sperm DNA at about 100-200   llg/ml.   Useful variations on these conditions will be readily apparent to those skilled in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their resultant proteins. 



   Other parameters, such as temperature, salt concentration ; and detergent concentration may be varied to achieve the desired stringency. Denaturants, such as formamide at a concentration of about   35-50% v/v, may also be used under particular circumstances, such as RNA : DNA hybridizations.   



  Appropriate hybridization conditions are routinely determinable by one of ordinary skill in the art. 



     "Immunogenic"describes   the potential for a natural, recombinant, or synthetic peptide, epitope, polypeptide, or protein to induce antibody production in appropriate animals, cells, or cell lines. 



   "Insertion"or"addition"refers to a change in either a nucleic or amino acid sequence in which at least one nucleotide or residue, respectively, is added to the sequence. 



   "Labeling"refers to the covalent or noncovalent joining of a polynucleotide, polypeptide, or antibody with a reporter molecule capable of producing a detectable or measurable signal. 

 <Desc/Clms Page number 120> 

 



     "Microarray"is   any arrangement of nucleic acids, amino acids, antibodies, etc., on a substrate. 



  The substrate may be a solid support such as beads, glass, paper, nitrocellulose, nylon, or an appropriate membrane. 



   "Linkers"are short stretches of nucleotide sequence which may be added to a vector or a dithp to create restriction endonuclease sites to facilitate   cloning."Polylinkers"are   engineered to incorporate multiple restriction enzyme sites and to provide for the use of enzymes which leave 5'or 3'overhangs (e. g.,   BamHI,     EcoRI,   and   HindIII)   and those which provide blunt ends (e. g., EcoRV,   SnaBI,   and StuI). 



   "Naturally occurring"refers to an endogenous polynucleotide or polypeptide that may be isolated from viruses or prokaryotic or eukaryotic cells. 



   "Nucleic acid   sequence"refers   to the specific order of nucleotides joined by phosphodiester bonds in a linear,   polymeric   arrangement. Depending on the number of nucleotides, the nucleic acid sequence can be considered an oligomer, oligonucleotide, or polynucleotide. The nucleic acid can be DNA, RNA, or any nucleic acid analog, such as PNA, may be of genomic or synthetic origin, may be either double-stranded or single-stranded, and can represent either the sense or antisense (complementary) strand. 



   "Oligomer"refers to a nucleic acid sequence of at least about 6 nucleotides and as many as about 60 nucleotides, preferably about 15 to 40 nucleotides, and most preferably between about 20 and 30 nucleotides, that may be used in hybridization or amplification technologies. Oligomers may be used as, e. g., primers for PCR, and are usually chemically synthesized. 



   "Operably   linked"refers   to the situation in which a first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame. 



   "Peptide nucleic acid" (PNA) refers to a DNA mimic in which nucleotide bases are attached to a pseudopeptide backbone to increase stability.   PNAs,   also designated antigene agents, can prevent gene expression by targeting complementary messenger RNA. 



   The phrases"percent identity"and"% identity", as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. 



   Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3. 12e sequence 

 <Desc/Clms Page number 121> 

 alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison   WI).   CLUSTAL V is described in Higgins,   D. G.   and Sharp, P. M. (1989) CABIOS 5 : 151-153   and in Higgins,   D. G. et al. (1992) CABIOS 8 : 189-191. 



  For pairwise alignments of polynucleotide sequences, the default parameters are set as follows :   Ktuple=2,   gap penalty=5, window=4, and"diagonals   saved"=4. The"weighted"residue   weight table is selected as the default. Percent identity is reported by CLUSTAL V as the"percent similarity"between aligned polynucleotide sequence pairs. 



   Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215 : 403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http   ://www.   ncbi.   im.   nih. gov/BLAST/. The BLAST software suite includes various sequence analysis programs including"blastn,"that is used to determine alignment between a known polynucleotide sequence and other sequences on a variety of databases.

   Also available is a tool called"BLAST 2 Sequences"that is used for direct pairwise comparison of two nucleotide sequences."BLAST 2 Sequences"can be accessed and used interactively at http   ://www.   ncbi.   nlm.   nih.   gov/gorf/bl2/.   The "BLAST 2 Sequences"tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with   the"BLAST   2 Sequences"tool Version 2. 0. 9 (May-07-1999) set at default parameters.

   Such default parameters may be, for example :    Matrx : BLOSUM62   
Rewardfor match : 1 
Penalty for mismatch :-2 
Open   Gap : 5 and Extension Gap : 2 penalties   
Gap x drop-off: 50 
Expect: 10 
Word Size :   11   
Filter : on 
Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least   50,   at least 70, at least 100, or at least 200 contiguous nucleotides.

   Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences 

 <Desc/Clms Page number 122> 

 shown herein, in figures or Sequence Listings, may be used to describe a length over which percentage identity may be measured. 



   Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein. 



   The phrases"percent identity"and"% identity", as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the hydrophobicity and acidity of the substituted residue, thus preserving the structure (and therefore function) of the folded polypeptide 
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3. 12e sequence alignment program (described and referenced above).

   For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows : Ktuple=l, gap penalty=3, window=5, and "diagonals   saved"=5.   The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the"percent similarity" between aligned polypeptide sequence pairs. 



   Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the"BLAST 2 Sequences"tool Version 2. 0. 9 (May-07-1999) with blastp set at default parameters. Such default parameters may be, for example :    Matrix :

   BLOSUM62   
Open Gap :   Il   and Extension Gap :   1   penalty 
Gap x drop-off.    50      Expect : 10   
Word Size : 3 
Filter : on 
Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.

   Such lengths are exemplary only, and it is understood that any fragment length 

 <Desc/Clms Page number 123> 

 supported by the sequences shown herein, in figures or Sequence Listings, may be used to describe a length over which percentage identity may be measured. 



   "Post-translational   modification"of   a DITHP may involve lipidation, glycosylation, phosphorylation, acetylation,   racemization,   proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu and the DITHP. 



     "Probe"refers   to dithp or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands,   chemiluminescent   agents, and enzymes."Primers"are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e. g., by the polymerase chain reaction (PCR). 



   Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the figures and Sequence Listing, may be used. 



   Methods for preparing and using probes and primers are described in the references, for example Sambrook et al., 1989, Molecular Cloning : A Laboratory Manual,   2nd   ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY ; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New York NY ; Innis et al., 1990, PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0. 5,   1991, Whitehead   Institute for Biomedical Research, Cambridge MA). 



   Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4. 06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5, 000 nucleotides from an input polynucleotide sequence of up to 32   kilobases.   Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the   PrimOU   primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.

   The Primer3 primer selection 

 <Desc/Clms Page number 124> 

 program (available to the public from the Whitehead   Institute/MIT   Center for Genome Research, Cambridge MA) allows the user to input   a"mispriming   library,"in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays.

   (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. 



  Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above. 



   "Purified"refers to molecules, either polynucleotides or polypeptides that are isolated or separated from their natural environment and are at least 60% free, preferably at least   75 %   free, and most preferably at least 90% free from other compounds with which they are naturally associated. 



   A"recombinant nucleic   acid"is   a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. 



  This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e. g., by genetic engineering techniques such as those described in Sambrook, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. 



  Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell. 



   Alternatively, such recombinant nucleic acids may be part of a viral vector, e. g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal. 



   "Regulatory element"refers to a nucleic acid sequence from nontranslated regions of a gene, and includes enhancers, promoters, introns, and 3'untranslated regions, which interact with host proteins to carry out or regulate transcription or translation. 



   "Reporter"molecules are chemical or biochemical moieties used for labeling a nucleic acid, an amino acid, or an antibody. They include radionuclides ; enzymes ; fluorescent,   chemiluminescent,   or 

 <Desc/Clms Page number 125> 

   chromogenic   agents ; substrates ; cofactors ; inhibitors ; magnetic particles ; and other moieties known in the art. 



   An"RNA equivalent,"in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose. 



   "Samples used in its broadest sense. Samples may contain nucleic or amino acids, antibodies, or other materials, and may be derived from any source (e. g., bodily fluids including, but not limited to, saliva, blood, and urine ; chromosome (s), organelles, or membranes isolated from a cell ; genomic DNA, RNA, or cDNA in solution or bound to a substrate ; and cleared cells or tissues or blots or imprints from such cells or tissues). 



   "Specific   binding"or"specifically   binding"refers to the interaction between a protein or peptide and its agonist, antibody, antagonist, or other binding partner. The interaction is dependent upon the presence of a particular structure of the protein, e. g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope"A,"the presence of a polypeptide containing epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody. 



     "Substitution"refers   to the replacement of at least one nucleotide or amino acid by a different nucleotide or amino acid. 



     "Substrate"refers   to any suitable rigid or semi-rigid support including, e. g., membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles or capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound. 



   A"transcript image"refers to the collective pattern of gene expression by a particular tissue or cell type under given conditions at a given time. 



   "Transformation"refers to a process by which exogenous DNA enters a recipient cell. 



  Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed. 



   "Transformants"include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as cells which transiently express inserted DNA or RNA. 

 <Desc/Clms Page number 126> 

 



   A"transgenic organism,"as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by   microinjection   or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, and plants and animals.

   The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra. 



   A"variant"of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 25 % sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the"BLAST 2 Sequences"tool Version 2. 0. 9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or even at least 98% or greater sequence identity over a certain defined length. The variant may result in"conservative"amino acid changes which do not affect structural and/or chemical properties.

   A variant may be described as, for example,   an"allelic"   (as defined   above),"splice,""species,"or"polymorphic"variant.   A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A   polymorphic   variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.

   Polymorphic variants also may encompass"single nucleotide   polymorphisms"   (SNPs) in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state. 



   In an alternative, variants of the polynucleotides of the present invention may be generated through recombinant methods. One possible method is a DNA shuffling technique such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA ; described in U. S. Patent Number 5, 837, 458 ; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17 : 793-797 ; Christians, F. C. et al. (1999) Nat. 

 <Desc/Clms Page number 127> 

 



  Biotechnol. 17 : 259-264 ; and Crameri, A. et al. (1996) Nat. Biotechnol. 14 : 315-319) to alter or improve the biological properties of DITHP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and   selection/screening.   Thus, genetic diversity is created   through"artificial"breeding   and rapid molecular evolution.

   For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner. 



   A"variant"of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the"BLAST 2 Sequences"tool Version 2. 0. 9 (May-07- 1999) set at default parameters. Such a pair of polypeptides may show, for example, at least   50%,   at least   60. %,   at least 70%, at least 80%, at least 90%, at least   95 %,   or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides. 



  THE INVENTION 
In a particular embodiment, cDNA sequences derived from human tissues and cell lines were aligned based on nucleotide sequence identity and assembled into"consensus"or"template"sequences which are designated by the template identification numbers (template IDs) in column 2 of Table 1. 



  The sequence identification numbers (SEQ ID NO : s) corresponding to the template IDs are shown in column 1. The template sequences have similarity to GenBank sequences, or"hits,"as designated by the GI Numbers in column 3. The statistical probability of each GenBank hit is indicated by a probability score in column 4, and the functional annotation corresponding to each GenBank hit is listed in column 5. 



   The invention incorporates the nucleic acid sequences of these templates as disclosed in the Sequence Listing and the use of these sequences in the diagnosis and treatment of disease states characterized by defects in human molecules. The invention further utilizes these sequences in hybridization and amplification technologies, and in particular, in technologies which assess gene expression patterns correlated with specific cells or tissues and their responses in vivo or in vitro to 

 <Desc/Clms Page number 128> 

 pharmaceutical agents, toxins, and other treatments. In this manner, the sequences of the present invention are used to develop a transcript image for a particular cell or tissue. 



  Derivation of Nucleic Acid Sequences cDNA was isolated from libraries constructed using RNA derived from normal and diseased human tissues and cell lines. The human tissues and cell lines used for cDNA library construction were selected from a broad range of sources to provide a diverse population of cDNAs representative of gene transcription throughout the human body. Descriptions of the human tissues and cell lines used for cDNA library construction are provided in the LIFESEQ database (Incyte Genomics, Inc. (Incyte), Palo Alto CA). Human tissues were broadly selected from, for example, cardiovascular, dermatologic, endocrine, gastrointestinal,   hematopoietic/immune   system, musculoskeletal, neural, reproductive, and urologic sources. 



   Cell lines used for cDNA library construction were derived from, for example, leukemic cells, teratocarcinomas, neuroepitheliomas, cervical carcinoma, lung fibroblasts, and endothelial cells. Such cell lines include, for example, THP-1, Jurkat, HUVEC,   hNT2,     WI38,   HeLa, and other cell lines commonly used and available from public depositories (American Type Culture Collection, Manassas VA). Prior to mRNA isolation, cell lines were untreated, treated with a pharmaceutical agent such as   5'-aza-2'-deoxycytidine,   treated with an activating agent such as lipopolysaccharide in the case of leukocytic cell lines, or, in the case of endothelial cell lines, subjected to shear stress. 



  Sequencing of the cDNAs 
Methods for DNA sequencing are well known in the art. Conventional enzymatic methods employ the   Klenow   fragment of   DNA polymerase I, SEQ {1ENASE DNA polymerase   (U. S. 



  Biochemical Corporation, Cleveland OH), Taq polymerase (Applied Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia Biotech, Inc. (Amersham Pharmacia Biotech), Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in   the ELONGASE amplification   system (Life Technologies Inc. (Life Technologies), Gaithersburg MD), to extend the nucleic acid sequence from an oligonucleotide primer annealed to the DNA template of interest. Methods have been developed for the use of both single-stranded and double-stranded templates. Chain termination reaction products may be electrophoresed on urea-polyacrylamide gels and detected either by autoradiography (for radioisotope-labeled nucleotides) or by fluorescence (for fluorophore-labeled nucleotides).

   Automated methods for mechanized reaction preparation, sequencing, and analysis using fluorescence detection methods have been developed. Machines used to prepare cDNAs for sequencing can include the MICROLAB 2200 liquid transfer system   (Hamilton   Company 

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 (Hamilton), Reno   NV),   Peltier thermal cycler (PTC200 ; MJ Research, Inc. (MJ Research), Watertown MA), and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing can be carried out using, for example, the ABI 373 or 377 (Applied Biosystems) or   MEGABACE   1000 (Molecular Dynamics, Inc. (Molecular Dynamics), Sunnyvale CA) DNA sequencing systems, or other automated and manual sequencing systems well known in the art. 



   The nucleotide sequences of the Sequence Listing have been prepared by current, state-of-the- art, automated methods and, as such, may contain occasional sequencing errors or unidentified nucleotides. Such unidentified nucleotides are designated by an N. These infrequent unidentified bases do not represent a hindrance to practicing the invention for those skilled in the art. Several methods employing standard recombinant techniques may be used to correct errors and complete the missing sequence information. (See, e. g., those described in Ausubel, F. M. et al. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY ; and Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Press, Plainview NY.) Assembly of cDNA Sequences 
Human polynucleotide sequences may be assembled using programs or algorithms well known in the art.

   Sequences to be assembled are related, wholly or in part, and may be derived from a single or many different transcripts. Assembly of the sequences can be performed using such programs as PHRAP (Phils Revised Assembly Program) and the GELVIEW fragment assembly system (GCG), or other methods known in the art. 



   Alternatively, cDNA sequences are used as"component"sequences that are assembled into   "template"or"consensus"sequences   as follows. Sequence chromatograms are processed, verified, and quality scores are obtained using PHRED. Raw sequences are edited using an editing pathway known as Block 1 (See, e. g., the LIFESEQ Assembled User Guide, Incyte Genomics, Palo Alto, CA). A series of BLAST comparisons is performed and low-information segments and repetitive elements (e.   g.,   dinucleotide repeats, Alu repeats, etc.) are replaced by"n's", or masked, to prevent spurious matches. 



  Mitochondrial and ribosomal RNA sequences are also removed. The processed sequences are then loaded into a relational database management system (RDMS) which assigns edited sequences to existing templates, if available. When additional sequences are added into the RDMS, a process is initiated which modifies existing templates or creates new templates from works in progress (i. e.,   nonfinal   assembled sequences) containing queued sequences or the sequences themselves. After the new sequences have been assigned to templates, the templates can be merged into bins. If multiple templates exist in one bin, the bin can be split and the templates   reannotated.   

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   Once gene bins have been generated based upon sequence alignments, bins are"clone joined" based upon clone information. Clone joining occurs when the 5'sequence of one clone is present in one bin and the 3'sequence from the same clone is present in a different bin, indicating that the two bins should be merged into a single bin. Only bins which share at least two different clones are merged. 



   A resultant template sequence may contain either a partial or a full length open reading frame, or all or part of a genetic regulatory element. This variation is due in part to the fact that the full length cDNAs of many genes are several hundred, and sometimes several thousand, bases in length. With current technology, cDNAs comprising the coding regions of large genes cannot be cloned because of vector limitations, incomplete reverse transcription of the mRNA, or incomplete"second strand" synthesis. Template sequences may be extended to include additional contiguous sequences derived from the parent RNA transcript using a variety of methods known to those of skill in the art. Extension may thus be used to achieve the full length coding sequence of a gene. 



  Analysis of the cDNA Sequences 
The cDNA sequences are analyzed using a variety of programs and algorithms which are well known in the art. (See, e. g., Ausubel, 1997, supra, Chapter 7. 7 ; Meyers, R. A. (Ed.) (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853 ; and Table 7.) These analyses comprise both reading frame determinations, e. g., based on triplet codon periodicity for particular organisms (Fickett, J. W. (1982) Nucleic Acids Res. 10 : 5303-5318) ; analyses of potential start and stop codons ; and homology searches. 



   Computer programs known to those of skill in the art for performing computer-assisted searches for amino acid and nucleic acid sequence similarity, include, for example, Basic Local Alignment Search Tool (BLAST ; Altschul, S. F. (1993) J. Mol. Evol. 36 : 290-300 ; Altschul, S. F. et al. 



  (1990) J. Mol. Biol. 215 : 403-410). BLAST is especially useful in determining exact matches and comparing two sequence fragments of arbitrary but equal lengths, whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user (Karlin, S. et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 841-845). Using an appropriate search tool (e. g., BLAST or HMM), GenBank, SwissProt, BLOCKS, PFAM and other databases may be searched for sequences containing regions of homology to a query dithp or DITHP of the present invention. 



   Other approaches to the identification, assembly, storage, and display of nucleotide and polypeptide sequences are provided in"Relational Database for Storing Biomolecule Information," U. S. S. N. 08/947, 845, filed October 9, 1997 ;"Project-Based Full-Length Biomolecular Sequence   Database,"U.   S.   S.   N. 08/811, 758, filed March 6, 1997 ; and"Relational Database and System for 

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 Storing Information Relating to Biomolecular Sequences,"U. S. S. N. 09/034, 807, filed March 4, 1998, all of which are incorporated by reference herein in their entirety. 



   Protein hierarchies can be assigned to the putative encoded polypeptide based on, e. g., motif, BLAST, or biological analysis. Methods for assigning these hierarchies are described, for example, in "Database System Employing Protein Function Hierarchies for Viewing Biomolecular Sequence Data," U. S. S. N. 08/812, 290, filed March 6, 1997, incorporated herein by reference. 



  Identification of Human Diagnostic and Therapeutic Molecules Encoded by dithp 
The identities of the DITHP encoded by the dithp of the present invention were obtained by analysis of the assembled cDNA sequences. 



   SEQ ID NO : 212, SEQ ID NO : 213, SEQ ID NO : 214, SEQ ID NO : 215, SEQ ID NO : 216, SEQ ID NO : 217, SEQ ID NO : 218, SEQ ID NO : 219, SEQ ID NO : 220, SEQ ID NO : 221, SEQ ID NO : 222, and SEQ ID NO : 223, encoded by SEQ ID NO : 1, SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO :   5,   SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO :   11,   and SEQ ID NO : 12, respectively, are, for example, human enzyme molecules. 



   SEQ ID NO : 224, SEQ ID NO : 225, SEQ ID NO : 226, SEQ ID NO : 227, and SEQ ID NO : 228, encoded by SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, and SEQ ID NO : 17, respectively, are, for example, receptor molecules. 



   SEQ ID NO : 229, SEQ ID   NO :   230, SEQ ID NO : 231, SEQ ID NO : 232, SEQ ID NO : 233, SEQ ID NO : 234, SEQ ID NO : 235, SEQ ID NO : 236, SEQ ID NO : 237, SEQ ID NO : 238, SEQ ID NO : 239, SEQ ID NO : 240, and SEQ ID NO : 241, encoded by SEQ ID NO : 18, SEQ ID NO :   19,   SEQ ID NO : 20, SEQ ID NO   : 21,   SEQ ID NO : 22, SEQ ID NO : 23, SEQ ID NO : 24, SEQ ID NO : 25, SEQ ID NO : 26, SEQ ID NO ; 27, SEQ ID NO : 28, SEQ ID NO : 29, and SEQ ID NO : 30, respectively, are, for example, intracellular signaling molecules. 



   SEQ ID NO : 242, SEQ ID NO : 243, SEQ ID NO : 244, SEQ ID NO : 245, SEQ ID NO : 246, SEQ ID NO : 247, SEQ ID NO : 248, SEQ ID NO : 249, SEQ ID NO : 250, SEQ ID NO : 251, SEQ ID NO : 252, SEQ ID NO : 253, SEQ ID NO : 254, SEQ ID NO : 255, SEQ ID NO : 256, SEQ ID NO : 257, SEQ ID NO : 258, SEQ   ID   NO : 259, SEQ ID NO : 260, SEQ ID NO : 261, SEQ ID NO : 262, SEQ ID NO : 263, SEQ ID NO : 264, SEQ ID NO : 265, SEQ ID NO : 266, SEQ ID NO : 267, SEQ ID NO : 268, SEQ ID NO : 269, SEQ ID NO : 270, SEQ ID NO : 271, SEQ ID NO : 272, SEQ ID NO : 273, SEQ ID NO : 274, SEQ ID NO : 275, SEQ ID NO : 276, SEQ ID NO : 277, SEQ ID NO : 278, SEQ ID NO : 279, SEQ ID NO : 280, SEQ ID NO : 281, SEQ ID NO : 282, SEQ ID NO : 283, SEQ ID NO : 284, SEQ ID NO : 285, SEQ ID NO : 286, SEQ ID NO : 287, SEQ ID NO : 288, SEQ ID NO : 289, SEQ ID NO : 290, SEQ ID NO : 291, SEQ ID NO :

   292, SEQ ID NO : 293, SEQ ID NO : 294, SEQ ID NO : 295, SEQ ID 

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 NO : 296, SEQ ID NO : 297, SEQ ID NO : 298, SEQ ID NO : 299, SEQ ID NO : 300, SEQ ID NO : 301, SEQ ID NO : 302, SEQ ID NO : 303, SEQ ID NO : 304, SEQ ID NO : 305, SEQ ID NO : 306, SEQ ID NO : 307, SEQ ID NO : 308, and SEQ ID NO : 309, encoded by SEQ   ID   NO : 31, SEQ ID NO : 32, SEQ ID NO : 33, SEQ ID NO : 34, SEQ ID NO : 35, SEQ ID NO : 36, SEQ ID NO : 37, SEQ ID NO : 38, SEQ ID NO : 39, SEQ ID NO : 40, SEQ ID NO : 41, SEQ ID NO : 42, SEQ ID NO : 43, SEQ ID NO : 44, SEQ ID NO : 45, SEQ ID NO : 46, SEQ ID NO : 47, SEQ ID NO : 48, SEQ ID NO : 49, SEQ ID NO : 50, SEQ ID NO : 51, SEQ ID NO : 52, SEQ ID NO : 53, SEQ ID NO : 54, SEQ ID NO : 55, SEQ ID NO : 56, SEQ ID NO : 57, SEQ ID NO : 58, SEQ ID NO : 59, SEQ ID NO : 60, SEQ ID NO : 61, SEQ ID NO : 62, SEQ ID NO :

   63, SEQ ID NO : 64, SEQ ID NO : 65, SEQ ID NO : 66, SEQ ID NO : 67, SEQ ID NO :   68,   SEQ ID NO :   69,   SEQ ID NO : 70, SEQ ID NO : 71, SEQ ID NO : 72, SEQ ID NO : 73, SEQ ID NO : 74, SEQ ID NO : 75, SEQ ID NO : 76, SEQ ID NO : 77, SEQ ID NO : 78, SEQ ID NO : 79, SEQ ID NO : 80, SEQ ID NO :   81,   SEQ ID NO : 82, SEQ ID NO : 83, SEQ ID NO : 84, SEQ ID NO : 85, SEQ ID NO : 86, SEQ ID NO : 87, SEQ ID NO : 88, SEQ ID NO : 89, SEQ ID NO : 90, SEQ ID NO : 91, SEQ ID NO : 92, SEQ ID NO : 93, SEQ ID NO : 94, SEQ ID NO : 95, SEQ ID NO : 96, SEQ ID NO : 97, and SEQ ID NO : 98, respectively, are, for example, transcription factor molecules. 



     SEQ ID N0   :   310, SEQ ID N0   :   311, SEQ ID N0   : 312,   SEQ ID N0   : 313,   SEQ ID N0   : 314, SEQ ID NO :   315,   SEQ ID NO : 316, and SEQ ID NO : 317, encoded by SEQ ID NO : 99, SEQ ID NO : 100, SEQ ID NO : 101, SEQ ID NO : 102, SEQ ID NO : 103, SEQ ID NO : 104, SEQ ID NO : 105, and SEQ ID NO : 106, respectively, are, for example, membrane transport molecules. 



   SEQ ID NO : 318, SEQ ID NO : 319, SEQ ID NO : 320, SEQ ID NO : 321, SEQ ID NO : 322, SEQ ID NO : 323, SEQ ID NO : 324, SEQ ID NO : 325, SEQ ID NO : 326, and SEQ ID NO : 327, encoded by SEQ ID NO : 107, SEQ ID NO :   108,   SEQ ID NO : 109, SEQ ID NO :   110,   SEQ ID NO :   lull,   SEQ ID NO : 112, SEQ ID NO : 113, SEQ ID NO : 114, SEQ   ID   NO : 115, and SEQ ID NO : 116, respectively, are, for example, protein modification and maintenance molecules. 



   SEQ ID NO : 328, SEQ ID NO : 329, SEQ ID NO : 330, SEQ ID   NO : 331,   SEQ ID NO : 332, SEQ ID NO : 333, SEQ ID NO : 334, SEQ ID NO : 335, SEQ ID NO : 336, SEQ ID NO : 337, SEQ ID NO : 338, SEQ ID NO : 339, SEQ ID NO : 340, and SEQ ID NO : 341, encoded by SEQ ID NO :   117,   SEQ ID NO :   118, SEQ ID NO   :   119, SEQ ID N0   : 120,   SEQ ID NO   : 121,   SEQ ID N0   : 122,   SEQ ID N0   : 123, SEQ   ID   NO : 124, SEQ ID NO : 125, SEQ ID NO : 126, SEQ ID NO : 127, SEQ ID NO : 128, SEQ ID NO : 129, and SEQ ID NO : 130, respectively, are, for example, nucleic acid synthesis and modification molecules. 



   SEQ ID NO : 342, encoded by SEQ ID   NO :   131 is, for example, an adhesion molecule. 



   SEQ ID NO : 343, SEQ ID NO : 344, SEQ ID NO : 345, SEQ ID NO : 346, SEQ ID NO : 347, SEQ ID NO : 348, and SEQ ID NO : 349, encoded by SEQ ID NO : 132, SEQ ID NO : 133, SEQ ID 

 <Desc/Clms Page number 133> 

 NO : 134,   SEQ ID N0   : 135,   SEQ ID N0   : 136,   SEQ ID N0   : 137, and SEQ ID NO : 138, respectively, are, for example, antigen recognition molecules. 



   SEQ ID NO : 350, SEQ   ID   NO : 351,   SEQ ID N0   : 352, and   SEQ ID N0   : 353, encoded by SEQ ID NO : 139,   SEQ ID N0   : 140, SEQ ID NO :   141,   and   SEQ ID N0   : 142, respectively, are, for example, electron transfer associated molecules. 



   SEQ ID NO : 354, SEQ ID NO : 355, SEQ ID NO : 356,   SEQ ID N0   : 357, SEQ ID NO : 358, and SEQ ID NO : 359, encoded by SEQ ID NO :   143,     SEQ ID NO   : 144, SEQ ID NO : 145,   SEQ ID N0   :   146,   SEQ ID NO : 147, and   SEQID NO   : 148, respectively, are, for example, secreted/extracellular matrix molecules. 



   SEQ ID NO : 360, SEQ ID NO : 361, SEQ ID NO : 362, SEQ ID NO : 363, SEQ ID NO : 364,   SEQ ID N0   : 365, SEQ ID NO : 366, SEQ ID NO : 367,   SEQ ID N0   : 368, and SEQ ID NO : 369, encoded by   SEQ ID N0   : 149, SEQ ID NO : 150, SEQ ID NO :   151,   SEQ ID NO : 152, SEQ ID NO : 153, SEQ ID NO : 154, SEQ ID NO : 155, SEQ ID NO : 156, SEQ ID NO : 157, and SEQ ID N0 : 158, respectively, are, for example, cytoskeletal molecules. 



   SEQ ID NO : 370, SEQ ID NO :   371,   SEQ ID NO : 372, and SEQ ID NO : 373, encoded by SEQ ID NO : 159, SEQ ID NO : 160, SEQ ID NO :   161,   and SEQ ID NO :   162,   respectively, are, for example, cell membrane molecules. 



   SEQ ID NO : 374, SEQ ID NO : 375, SEQ ID NO : 376, SEQ ID NO : 377, SEQ ID NO : 378, SEQ ID NO : 379, SEQ ID NO : 380, SEQ ID NO : 381, SEQ ID N0 : 382,   SEQ ID N0   : 383,   SEQ ID   NO : 384, SEQ ID NO : 385, SEQ ID NO : 386, SEQ ID NO : 387, SEQ ID NO : 388, SEQ ID NO : 389, SEQ ID NO : 390, SEQ ID NO : 391, and SEQ ID NO : 392, encoded by SEQ ID NO : 163,   SEQ ID   NO : 164, SEQ ID NO : 165, SEQ ID NO : 166, SEQ ID NO : 167, SEQ ID NO : 168, SEQ ID NO :   169,   SEQ ID NO : 170, SEQ ID NO :   171,   SEQ ID NO : 172, SEQ ID NO : 173,   SEQ ID NO   : 174,   SEQ ID   NO : 175, SEQ ID NO : 176, SEQ ID NO : 177, SEQ ID NO : 178, SEQ ID NO : 179, SEQ ID NO : 180, and SEQ ID NO :

     181,   respectively, are, for example, ribosomal molecules. 



   SEQ ID NO : 393, SEQ ID NO : 394, SEQ ID NO : 395, SEQ ID NO : 396, SEQ ID NO : 397, SEQ ID NO : 398, SEQ ID NO : 399, SEQ ID NO : 400, SEQ ID NO : 401, SEQ ID NO : 402, and SEQ ID NO : 403, encoded by SEQ ID NO : 182, SEQ ID NO : 183,   SEQ ID NO   : 184, SEQ ID NO : 185, SEQ ID NO : 186,   SEQ ID NO   : 187,   SEQ ID NO   : 188, SEQ ID NO : 189,   SEQ ID NO   :   190,     SEQ ID NO   :   191,     and SEQ ID NO   : 192, respectively, are, for example, organelle associated molecules. 



   SEQ ID NO : 404, SEQ ID NO :   405,   SEQ ID NO : 406, SEQ ID NO : 407, SEQ ID NO : 408, SEQ ID NO : 409, SEQ ID NO : 410, SEQ ID NO :   411, SEQ ID N0   : 412, SEQ ID NO : 413, and SEQ ID NO : 414, encoded by SEQ ID NO : 193, SEQ ID NO : 194, SEQ ID NO : 195, SEQ ID NO : 196, SEQ ID 

 <Desc/Clms Page number 134> 

 NO : 197,   SEQ ID NO   : 198,   SEQ ID N0   :   199,   SEQ   ID N0   : 200, SEQ   ID N0   : 201,   SEQ ID N0   : 202, and   SEQ ID N0   : 203, respectively, are ; for example, biochemical pathway molecules. 



     SEQ ID N0   : 415,   SEQ ID N0   :   416,     SEQ ID N0   :   417,     SEQ ID N0   :   418,     SEQ ID N0   : 419,   SEQ ID N0   : 420,   SEQ ID N0   : 421, and   SEQ ID N0   : 422, encoded by   SEQ ID N0   : 204, SEQ ID NO : 205,   SEQ ID N0   : 206,   SEQ ID N0   : 207,   SEQ ID N0   : 208,   SEQ ID N0   : 209, SEQ ID NO : 210, and   SEQ ID N0   :   211,   respectively, are, for example, molecules associated with growth and development. 



  Sequences of Human Diagnostic and Therapeutic Molecules 
The dithp of the present invention may be used for a variety of diagnostic and therapeutic purposes. For example, a dithp may be used to diagnose a particular condition, disease, or disorder associated with human molecules. Such conditions, diseases, and disorders include, but are not limited to, a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary   thrombocythemia,   and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,

   gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus ; an autoimmune/inflammatory disorder, such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis,   glomerulonephritis,   Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis,

     hypereosinophilia,   irritable bowel syndrome, episodic lymphopenia with   lymphocytotoxins,   mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or   pericardial   inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,   scleroderma,     Sjogren's   syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia,   thrombocytopenic   purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, trauma, and hematopoietic cancer including lymphoma, leukemia, and myeloma ;

   an infection caused by a viral agent classified 

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 as   adenovirus,   arenavirus, bunyavirus, calicivirus, coronavirus,   filovirus,   hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, or togavirus ;

   an infection caused by a bacterial agent classified as pneumococcus, staphylococcus, streptococcus, bacillus, corynebacterium, clostridium,   meningococcus,   gonococcus, listeria,   moraxella,   kingella, haemophilus, legionella,   bordetella,   gram- negative enterobacterium including shigella, salmonella, or campylobacter, pseudomonas, vibrio, brucella, francisella,   yersinia,   bartonella, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia, or mycoplasma ; an infection caused by a fungal agent classified as aspergillus, blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, or other mycosis- causing fungal agent ;

   and an infection caused by a parasite classified as plasmodium or malaria- causing, parasitic   entamoeba,   leishmania, trypanosoma, toxoplasma, pneumocystis   carinii,   intestinal protozoa such as giardia, trichomonas, tissue nematode such as trichinella, intestinal nematode such as ascaris, lymphatic filarial nematode, trematode such as schistosoma, and cestrode such as tapeworm ;

   a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms'tumor, aniridia,   genitourinary   abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary   keratodermas,   hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy,   spina bifida,   anencephaly,   craniorachischisis,   congenital glaucoma, cataract, and sensorineural hearing loss ;

   an endocrine disorder such as a disorder of the hypothalamus and/or pituitary resulting from lesions such as a primary brain tumor, adenoma, infarction associated with pregnancy, hypophysectomy, aneurysm, vascular malformation, thrombosis, infection, immunological disorder, and complication due to head trauma ; a disorder associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease,   Hand-Schuller-Christian   disease, Letterer- Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism ; a disorder associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma ;

   a disorder associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism ; a disorder associated with hyperthyroidism including   thyrotoxicosis   and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease ; a disorder associated with hyperparathyroidism including Conn disease (chronic hypercalemia) ; a pancreatic disorder such as Type I or Type II diabetes mellitus and associated complications ;

   a disorder associated 

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 with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis,   amyloidosis,   hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease ; a disorder associated with gonadal steroid hormones such as : in women, abnormal prolactin production, infertility, endometriosis, perturbation of the menstrual cycle, polycystic ovarian disease,   hyperprolactinemia,   isolated gonadotropin deficiency,   amenorrhea,   galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteoporosis ;

   and, in men, Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, a hypergonadal disorder associated with Leydig cell tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 a-reductase, and gynecomastia ; a metabolic disorder such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty hepatocirrhosis, fructose-1, 6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal storage diseases, mannosidosis,   neuraminidase   deficiency, obesity,

   pentosuria   phenylketonuria,   pseudovitamin D-deficiency rickets ; disorders of carbohydrate metabolism such as congenital type II   dyserythropoietic   anemia, diabetes, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, fructose-1, 6-diphosphatase deficiency, galactosemia, glucagonoma, hereditary fructose intolerance, hypoglycemia, mannosidosis,   neuraminidase   deficiency, obesity, galactose epimerase deficiency, glycogen storage diseases, lysosomal storage diseases, fructosuria, pentosuria, and inherited abnormalities of pyruvate metabolism ;

   disorders of lipid metabolism such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palmitoyltransferase deficiency,   myoadenylate   deaminase deficiency,   hypertriglyceridemia,   lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy,   GME2   gangliosidosis, and ceroid   lipofuscinosis,     abetalipoproteinemia,   Tangier disease, hyperlipoproteinemia, diabetes mellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary   hypoalphalipoproteinemia,

     hypothyroidism, renal disease, liver disease, lecithin : cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis,   sitosterolemia,   hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, and obesity ; and disorders of copper metabolism such as Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type IX ;

   a neurological 

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 disorder such as epilepsy,   ischemic   cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease,   Huntington's   disease, dementia, Parkinson's disease and other extrapyramidal disorders,   amyotrophic   lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy,   retinitis   pigmentosa, hereditary ataxias, multiple sclerosis and other   demyelinating   diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative   intracranial     thrombophlebitis,   myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease,

   and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorder of the central nervous system, cerebral palsy, a neuroskeletal disorder, an   autonomic nervous   system disorder, a cranial nerve disorder, a spinal cord disease, muscular dystrophy and other neuromuscular disorder, a peripheral nervous system disorder, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathy, myasthenia gravis, periodic paralysis, a mental disorder including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias,

   paranoid psychoses, postherpetic neuralgia,   and Tourette's   disorder ; a gastrointestinal disorder including ulcerative colitis, gastric and duodenal ulcers, cystinuria,   dibasicaminoaciduria,     hypercystinuria,   lysinuria, hartnup disease, tryptophan malabsorption, methionine malabsorption, histidinuria, iminoglycinuria,   dicarboxylicaminoaciduria,     cystinosis,   renal glycosuria, hypouricemia, familial   hypophophatemic   rickets, congenital chloridorrhea, distal renal tubular acidosis, Menkes'disease, Wilson's disease, lethal diarrhea, juvenile pernicious anemia, folate malabsorption, adrenoleukodystrophy, hereditary myoglobinuria, and Zellweger syndrome ;

   a transport disorder such as akinesia,   amyotrophic   lateral sclerosis, ataxia telangiectasia, cystic fibrosis, Becker's muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease, diabetes mellitus, diabetes insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic paralysis, normokalemic periodic paralysis, Parkinson's disease, malignant hyperthermia, multidrug resistance, myasthenia gravis, myotonic dystrophy, catatonia, tardive dyskinesia, dystonias, peripheral neuropathy, cerebral neoplasms, prostate cancer, cardiac disorders associated with transport, e.

   g., angina,   bradyarrythmia,     tachyarrythmia,   hypertension, Long QT syndrome, myocarditis, cardiomyopathy, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy,   thyrotoxic   myopathy, ethanol myopathy, dermatomyositis, inclusion body myositis, infectious myositis, and polymyositis, neurological disorders associated with transport, e. g., Alzheimer's disease, amnesia, bipolar disorder, dementia, depression, epilepsy, Tourette's disorder, paranoid psychoses, and schizophrenia, and other disorders associated with transport, e.

   g., 

 <Desc/Clms Page number 138> 

 neurofibromatosis, postherpetic neuralgia,   trigeminal   neuropathy, sarcoidosis, sickle cell anemia, cataracts, infertility, pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia, Grave's disease, goiter, glucose-galactose malabsorption syndrome, hypercholesterolemia, Cushing's disease, and Addison's disease ;

   and a connective tissue disorder such as osteogenesis imperfecta, Ehlers-Danlos syndrome, chondrodysplasias, Marfan syndrome, Alport syndrome, familial aortic aneurysm, achondroplasia, mucopolysaccharidoses, osteoporosis, osteopetrosis, Paget's disease, rickets, osteomalacia, hyperparathyroidism, renal osteodystrophy, osteonecrosis, osteomyelitis, osteoma, osteoid osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondroma, chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous cortical defect, nonossifying fibroma, fibrous dysplasia, fibrosarcoma, malignant fibrous   histiocytoma,   Ewing's sarcoma, primitive neuroectodermal tumor, giant cell tumor, osteoarthritis, rheumatoid arthritis, ankylosing spondyloarthritis, Reiter's syndrome, psoriatic arthritis, enteropathic arthritis, infectious arthritis, gout, gouty arthritis,

   calcium pyrophosphate crystal deposition disease, ganglion, synovial cyst,   villonodular   synovitis, systemic sclerosis, Dupuytren's contracture, hepatic fibrosis, lupus erythematosus, mixed connective tissue disease, epidermolysis bullosa simplex, bullous congenital ichthyosiform erythroderma (epidermolytic hyperkeratosis), non-epidermolytic and epidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens,   pachyonychia   congenita, and white sponge nevus. The dithp can be used to detect the presence of, or to quantify the amount of, a dithp-related polynucleotide in a sample. This information is then compared to information obtained from appropriate reference samples, and a diagnosis is established.

   Alternatively, a polynucleotide complementary to a given dithp can inhibit or inactivate a therapeutically relevant gene related to the dithp. 



  Analysis of dithp Expression Patterns 
The expression of dithp may be routinely assessed by hybridization-based methods to determine, for example, the tissue-specificity, disease-specificity, or developmental stage-specificity of dithp expression. For example, the level of expression of dithp may be compared among different cell types or tissues, among diseased and normal cell types or tissues, among cell types or tissues at different developmental stages, or among cell types or tissues undergoing various treatments.

   This type of analysis is useful, for example, to assess the relative levels of dithp expression in fully or partially differentiated cells or tissues, to determine if changes in dithp expression levels are correlated with the development or progression of specific disease states, and to assess the response of a cell or tissue to a specific therapy, for example, in pharmacological or toxicological studies. Methods for the analysis of dithp expression are based on hybridization and amplification technologies and include membrane- 

 <Desc/Clms Page number 139> 

 based procedures such as northern blot analysis, high-throughput procedures that utilize, for example, microarrays, and PCR-based procedures. 



  Hybridization and Genetic Analysis 
The dithp, their fragments, or complementary sequences, may be used to identify the presence of and/or to determine the degree of similarity between two (or more) nucleic acid sequences. The dithp may be hybridized to naturally occurring or recombinant nucleic acid sequences under appropriately selected temperatures and salt concentrations. Hybridization with a probe based on the nucleic acid sequence of at least one of the dithp allows for the detection of nucleic acid sequences, including genomic sequences, which are identical or related to the dithp of the Sequence Listing. Probes may be selected from non-conserved or unique regions of at least one of the polynucleotides of SEQ ID NO :   1-   211 and tested for their ability to identify or amplify the target nucleic acid sequence using standard protocols. 



   Polynucleotide sequences that are capable of hybridizing, in particular, to those shown in SEQ ID NO :   1-211   and fragments thereof, can be identified using various conditions of stringency. (See, e. g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152 : 399-407 ; Kimmel, A. R. (1987) Methods Enzymol. 152 : 507-511.) Hybridization conditions are discussed   in"Definitions."   
A probe for use in Southern or northern hybridization may be derived from a fragment of a dithp sequence, or its complement, that is up to several hundred nucleotides in length and is either single-stranded or double-stranded. Such probes may be hybridized in solution to biological materials such as plasmids, bacterial, yeast, or human artificial chromosomes, cleared or sectioned tissues, or to artificial substrates containing dithp.

   Microarrays are particularly suitable for identifying the presence of and detecting the level of expression for multiple genes of interest by examining gene expression correlated with, e. g., various stages of development, treatment with a drug or compound, or disease progression. An array analogous to a dot or slot blot may be used to arrange and link polynucleotides to the surface of a substrate using one or more of the following : mechanical (vacuum), chemical, thermal, or UV bonding procedures. Such an array may contain any number of dithp and may be produced by hand or by using available devices, materials, and machines. 



   Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e. g., Brennan, T. M. et al. (1995) U. S. Patent No. 5, 474, 796 ; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 



  USA 93 : 10614-10619 ; Baldeschweiler et al. (1995) PCT application W095/251116 ; Shalon, D. et al. 



  (1995) PCT application   W095135505   ; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA 94 : 2150- 2155 ; and Heller, M. J. et al. (1997) U. S. Patent No. 5, 605, 662.) 
Probes may be labeled by either PCR or enzymatic techniques using a variety of commercially 

 <Desc/Clms Page number 140> 

 available reporter molecules. For example, commercial kits are available for radioactive and   chemiluminescent   labeling (Amersham Pharmacia Biotech) and for alkaline phosphatase labeling (Life Technologies). Alternatively, dithp may be cloned into commercially available vectors for the production of RNA probes. Such probes may be transcribed in the presence of at least one labeled nucleotide (e.   g.,     32P-ATP,   Amersham Pharmacia Biotech). 



   Additionally the polynucleotides of SEQ ID NO : 1-211 or suitable fragments thereof can be used to isolate full length cDNA sequences utilizing hybridization and/or amplification procedures well known in the art,   e. g., cDNA   library screening, PCR amplification, etc. The molecular cloning of such full length cDNA sequences may employ the method of cDNA library screening with probes using the hybridization, stringency, washing, and probing strategies described above and in Ausubel, supra, Chapters 3, 5, and 6. These procedures may also be employed with genomic libraries to isolate genomic sequences of dithp in order to analyze, e. g., regulatory elements. 



  Genetic Mapping 
Gene identification and mapping are important in the investigation and treatment of almost all conditions, diseases, and disorders. Cancer, cardiovascular disease, Alzheimer's disease, arthritis, diabetes, and mental illnesses are of particular interest. Each of these conditions is more complex than the single gene defects of sickle cell anemia or cystic fibrosis, with select groups of genes being predictive of predisposition for a particular condition, disease, or disorder. For example, cardiovascular disease may result from malfunctioning receptor molecules that fail to clear cholesterol from the bloodstream, and diabetes may result when a particular individual's immune system is activated by an infection and attacks the insulin-producing cells of the pancreas.

   In some studies, Alzheimer's disease has been linked to a gene on chromosome 21 ; other studies predict a different gene and location. Mapping of disease genes is a complex and reiterative process and generally proceeds from genetic linkage analysis to physical mapping. 



   As a condition is noted among members of a family, a genetic linkage map traces parts of chromosomes that are inherited in the same pattern as the condition. Statistics link the inheritance of particular conditions to particular regions of chromosomes, as defined by RFLP or other markers. 



  (See, for example, Lander, E. S. and Botstein, D. (1986) Proc. Natl. Acad. Sci. USA 83 : 7353-7357.) Occasionally, genetic markers and their locations are known from previous studies. More often, however, the markers are simply stretches of DNA that differ among individuals. Examples of genetic linkage maps can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. 



   In another embodiment of the invention, dithp sequences may be used to generate hybridization 

 <Desc/Clms Page number 141> 

 probes useful in chromosomal mapping of naturally occurring genomic sequences. Either coding or noncoding sequences of dithp may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a dithp coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e. g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries. (See, e. g., Harrington, J. J. et al.

   (1997) Nat. 



  Genet. 15 : 345-355 ; Price, C. M. (1993) Blood Rev. 7 : 127-134 ;   and Trask,   B. J. (1991) Trends Genet. 



  7 : 149-154.) 
Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e. g., Meyers, supra, pp. 965-968.) Correlation between the location of dithp on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder. 



  The dithp sequences may also be used to detect   polymorphisms   that are genetically linked to the inheritance of a particular condition, disease, or disorder. 



   In situ hybridization of chromosomal preparations and genetic mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending existing genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of the corresponding human chromosome is not known. These new marker sequences can be mapped to human chromosomes and may provide valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques.

   Once a disease or syndrome has been crudely correlated by genetic linkage with a particular genomic region, e.   g.,   ataxia-telangiectasia to   11q22-23,   any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e. g., Gatti, R. A. et al. (1988) Nature 336 : 577-580.) The nucleotide sequences of the subject invention may also be used to detect differences in chromosomal architecture due to translocation, inversion, etc., among normal, carrier, or affected individuals. 



   Once a disease-associated gene is mapped to a chromosomal region, the gene must be cloned in order to identify mutations or other alterations (e. g., translocations or inversions) that may be correlated with disease. This process requires a physical map of the chromosomal region containing the disease- gene of interest along with associated markers. A physical map is necessary for determining the nucleotide sequence of and order of marker genes on a particular chromosomal region. Physical mapping techniques are well known in the art and require the generation of overlapping sets of cloned 

 <Desc/Clms Page number 142> 

 DNA fragments from a particular organelle, chromosome, or genome. These clones are analyzed to reconstruct and catalog their order.

   Once the position of a marker is determined, the DNA from that region is obtained by consulting the catalog and selecting clones from that region. The gene of interest is located through positional cloning techniques using hybridization or similar methods. 



  Diagnostic Uses 
The dithp of the present invention may be used to design probes useful in diagnostic assays. 



  Such assays, well known to those skilled in the art, may be used to detect or confirm conditions, disorders, or diseases associated with abnormal levels of dithp expression. Labeled probes developed from dithp sequences are added to a sample under hybridizing conditions of desired stringency. In some instances, dithp, or fragments or oligonucleotides derived from dithp, may be used as primers in amplification steps prior to hybridization. The amount of hybridization complex formed is quantified and compared with standards for that cell or tissue. If dithp expression varies significantly from the standard, the assay indicates the presence of the condition, disorder, or disease.

   Qualitative or quantitative diagnostic methods may include northern, dot blot, or other membrane or dip-stick based technologies or multiple-sample format technologies such as PCR, enzyme-linked immunosorbent assay   (ELISA)-like,   pin, or chip-based assays. 



   The probes described above may also be used to monitor the progress of conditions, disorders, or diseases associated with abnormal levels of dithp expression, or to evaluate the efficacy of a particular therapeutic treatment. The candidate probe may be identified from the dithp that are specific to a given human tissue and have not been observed in GenBank or other genome databases. Such a probe may be used in animal studies, preclinical tests, clinical trials, or in monitoring the treatment of an individual patient. In a typical process, standard expression is established by methods well known in the art for use as a basis of comparison, samples from patients affected by the disorder or disease are combined with the probe to evaluate any deviation from the standard profile, and a therapeutic agent is administered and effects are monitored to generate a treatment profile.

   Efficacy is evaluated by determining whether the expression progresses toward or returns to the standard normal pattern. 



  Treatment profiles may be generated over a period of several days or several months. Statistical methods well known to those skilled in the art may be use to determine the significance of such therapeutic agents. 



   The polynucleotides are also useful for identifying individuals from minute biological samples, for example, by matching the RFLP pattern of a sample's DNA to that of an individual's DNA. The polynucleotides of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR 

 <Desc/Clms Page number 143> 

 primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, an individual can be identified through a unique set of DNA sequences. Once a unique ID database is established for an individual, positive identification of that individual can be made from extremely small tissue samples. 



   In a particular aspect, oligonucleotide primers derived from the dithp of the invention may be used to detect single nucleotide   polymorphisms   (SNPs). SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans. Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from dithp are used to amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.

   In   fSCCP,   the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high- throughput equipment such as DNA sequencing machines. Additionally, sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying   polymorphisms   by comparing the sequences of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA). 



   DNA-based identification techniques are critical in forensic technology. DNA sequences taken from very small biological samples such as tissues, e. g., hair or skin, or body fluids, e. g., blood, saliva, semen, etc., can be amplified using, e. g., PCR, to identify individuals. (See, e. g., Erlich, H. (1992)   PCR Technologv, Freeman   and Co., New York, NY). Similarly, polynucleotides of the present invention can be used as polymorphic markers. 



   There is also a need for reagents capable of identifying the source of a particular tissue. 



  Appropriate reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention that are specific for particular tissues. Panels of such reagents can identify tissue by species   and/or   by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination. 



   The polynucleotides of the present invention can also be used as molecular weight markers on nucleic acid gels or Southern blots, as diagnostic probes for the presence of a specific mRNA in a particular cell type, in the creation of subtracted cDNA libraries which aid in the discovery of novel 

 <Desc/Clms Page number 144> 

 polynucleotides, in selection and synthesis of oligomers for attachment to an array or other support, and as an antigen to elicit an immune response. 



  Disease Model Systems Using dithp 
The dithp of the invention or their mammalian homologs may be"knocked out"in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e. g., U. S. 



  Patent Number 5, 175, 383 and U. S. Patent Number 5, 767, 337.) For example, mouse ES cells, such as the mouse   129/SvJ   cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e. g., the   neomycinphosphotransferase gene   (neo ;   Capecchi,   M. R. (1989) Science 244 : 1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. 



  Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue-or developmental stage-specific manner (Marth, J. D. (1996) Clin. Invest. 97 : 1999- 2002 ; Wagner, K. U. et al. (1997) Nucleic Acids Res. 25 : 4323-4330). Transformed ES cells are identified and microinjected into mouse cell   blastocysts   such as those from the C57BL/6 mouse strain. 



  The   blastocysts   are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents. 



   The dithp of the invention may also be manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.   A.   et al. (1998) Science 282 : 1145-1147). 



   The dithp of the invention can also be used to   create"knockin"humanized   animals (pigs) or transgenic animals (mice or rats) to model human disease. With   knockin   technology, a region of dithp is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. 



  Transformed cells are injected into blastulae, and the blastulae are implanted as described above. 



  Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease. Alternatively, a mammal inbred to overexpress dithp, resulting, e. g., in the secretion of DITHP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4 : 55-74). 



  Screening Assays 

 <Desc/Clms Page number 145> 

 
DITHP encoded by polynucleotides of the present invention may be used to screen for molecules that bind to or are bound by the encoded polypeptides. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the bound molecule. Examples of such molecules include antibodies, oligonucleotides, proteins (e. g., receptors), or small molecules. 



   Preferably, the molecule is closely related to the natural ligand of the polypeptide, e. g., a ligand or fragment thereof, a natural substrate, or a structural or functional mimetic. (See, Coligan et al., (1991) Current Protocols in Immunology 1 (2) : Chapter 5.) Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or to at least a fragment of the receptor, e. g., the active site. In either case, the molecule can be rationally designed using known techniques. 



  Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide or cell membrane fractions which contain the expressed polypeptide are then contacted with a test compound and binding, stimulation, or inhibition of activity of either the polypeptide or the molecule is analyzed. 



   An assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. Alternatively, the assay may assess binding in the presence of a labeled competitor. 



   Additionally, the assay can be carried out using cell-free preparations,   polypeptide/molecule   affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring   polypeptide/molecule   activity or binding, and comparing the   polypeptide/molecule   activity or binding to a standard. 



   Preferably, an ELISA assay using, e. g., a monoclonal or polyclonal antibody, can measure polypeptide level in a sample. The antibody can measure polypeptide level by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate. 



   All of the above assays can be used in a diagnostic or prognostic context. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e. g., blood vessel growth) by activating or inhibiting the   polypeptide/molecule.   Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. 



  Transcript   Imaging   and Toxicological Testing 
Another embodiment relates to the use of dithp to develop a transcript image of a tissue or cell 

 <Desc/Clms Page number 146> 

 type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"U. S. Patent Number 5, 840, 484, expressly incorporated by reference herein.) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type.

   In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity pertaining to human molecules for diagnostics and therapeutics. 



   Transcript images which profile dithp expression may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect dithp expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line. 



   Transcript images which profile dithp expression may also be used in conjunction with in vitro model systems and   preclinical   evaluation   of pharmaceuticals,   as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E. F. et al. (1999) Mol.   Carcinog.   24 :   153-   159 ; Steiner, S. and Anderson, N. L. (2000) Toxicol. Lett. 112-113 :   467-71,   expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.

   These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. 



  Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity.

   (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http   ://www.   niehs. nih. gov/oc/news/toxchip. htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences. 



   In one embodiment, the toxicity of a test compound is assessed by treating a biological sample 

 <Desc/Clms Page number 147> 

 containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample. 



   Another particular embodiment relates to the use of DITHP encoded by polynucleotides of the present invention to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.

   In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample.

   The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially   sequenced   using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. 



  The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification. 



     A proteomic   profile may also be generated using antibodies specific for DITHP to quantify the levels of DITHP expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270 : 103-11 ; Mendoze, L. G. et al. (1999) Biotechniques 27 : 778-88). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol-or amino- reactive fluorescent compound and detecting the amount of fluorescence bound at each array element. 



     Toxicant   signatures at the proteome level are also useful for toxicological screening, and should 

 <Desc/Clms Page number 148> 

 be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N. L. and Seilhamer, J. (1997) Electrophoresis 18 : 533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the   proteomic   profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so   proteomic   profiling may be more reliable and informative in such cases. 



   In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the DITHP encoded by polynucleotides of the present invention. 



   In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the DITHP encoded by polynucleotides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. 



   Transcript images may be used to profile dithp expression in distinct tissue types. This process can be used to determine human molecule activity in a particular tissue type relative to this activity in a different tissue type. Transcript images may be used to generate a profile of dithp expression characteristic of diseased tissue. Transcript images of tissues before and after treatment may be used for diagnostic purposes, to monitor the progression of disease, and to monitor the efficacy of drug treatments for diseases which affect the activity of human molecules. 



   Transcript images of cell lines can be used to assess human molecule activity   and/or   to identify cell lines that lack or misregulate this activity. Such cell lines may then be treated with pharmaceutical agents, and a transcript image following treatment may indicate the efficacy of these agents in restoring desired levels of this activity. A similar approach may be used to assess the toxicity of pharmaceutical agents as reflected by undesirable changes in human molecule activity. Candidate pharmaceutical agents may be evaluated by comparing their associated transcript images with those of pharmaceutical agents of known effectiveness. 

 <Desc/Clms Page number 149> 

 



  Antisense Molecules 
The polynucleotides of the present invention are useful in antisense technology. Antisense technology or therapy relies on the modulation of expression of a target protein through the specific binding of an antisense sequence to a target sequence encoding the target protein or directing its expression. (See, e. g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ ;   Alama,   A. et al. (1997) Pharmacol. Res. 36 (3) : 171-178 ; Crooke, S. T. (1997) Adv. Pharmacol. 



  40 : 1-49 ; Sharma, H. W.   and R.   Narayanan (1995) Bioessays 17 (12) : 1055-1063 ;   and Lavrosky,   Y. et al. (1997) Biochem. Mol. Med. 62   (1)   : 11-22.) An antisense sequence is a polynucleotide sequence capable of specifically hybridizing to at least a portion of the target sequence. Antisense sequences bind to cellular mRNA and/or genomic DNA, affecting translation and/or transcription. Antisense sequences can be DNA, RNA, or nucleic acid mimics and analogs. (See, e. g., Rossi, J. J. et al. (1991) Antisense Res. Dev. 1 (3) : 285-288 ; Lee, R. et al. (1998) Biochemistry 37 (3) : 900-1010 ; Pardridge, W. M. et al. (1995) Proc. Natl. Acad. Sci. USA 92 (12) : 5592-5596 ;   and Nielsen,   P. E. and Haaima, G. 



  (1997) Chem. Soc. Rev. 96 : 73-78.) Typically, the binding which results in modulation of expression occurs through hybridization or binding of complementary base pairs. Antisense sequences can also bind to DNA duplexes through specific interactions in the major groove of the double helix. 



   The polynucleotides of the present invention and fragments thereof can be used as antisense sequences to modify the expression of the polypeptide encoded by dithp. The antisense sequences can be produced ex vivo, such as by using any of the ABI nucleic acid synthesizer series (Applied Biosystems) or other automated systems known in the art. Antisense sequences can also be produced biologically, such as by transforming an appropriate host cell with an expression vector containing the sequence of interest. (See, e. g., Agrawal, supra.) 
In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used.

  *  Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See,   e. g.,   Slater, J. E., et al. (1998) J. Allergy Clin. Immunol. 102 (3) : 469-475 ; and Scanlon, K. J., et al. (1995) 9 (13) :   1288-1296.)   Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e. g., Miller, A. D. (1990) Blood 76 : 271 ; Ausubel, F. M. et al. (1995) Current Protocols in Molecular   Biology.   John Wiley & Sons, New York NY ; Uckert, W. and W. Walther (1994) Pharmacol.

   Ther. 63 (3) : 323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See,   e. g.,   Rossi, J. J. (1995) Br. Med. Bull.   51     (1)   : 217-225 ; Boado, R. J. et al. (1998) J. Pharm. Sci.   87   (11) :   1308-   1315 ; and Morris, M. C. et al. (1997) Nucleic Acids Res. 25 (14) : 2730-2736.) 

 <Desc/Clms Page number 150> 

 Expression 
In order to express a biologically active DITHP, the nucleotide sequences encoding DITHP or fragments thereof may be inserted into an appropriate expression vector, i. e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.

   Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding DITHP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e. g., Sambrook,   supra.   Chapters 4, 8, 16, and 17 ;   andAusubel, supra,   Chapters 9, 10, 13, and 16.) 
A variety of expression vector/host systems may be utilized to contain and express sequences encoding DITHP.

   These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors ; yeast transformed with yeast expression vectors ; insect cell systems infected with viral expression vectors (e. g., baculovirus) ; plant cell systems transformed with viral expression vectors (e. g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus,   TMV)   or with bacterial expression vectors (e. g., Ti or pBR322 plasmids) ; or animal (mammalian) cell systems. (See, e. g., Sambrook, supra ; Ausubel, 1995, supra, Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264 : 5503-5509 ; Bitter, G. A. et al. (1987) Methods Enzymol. 



  153 : 516-544 ; Scorer, C. A. et al. (1994) Bio/Technology 12 : 181-184 ; Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 3224-3227 ; Sandig, V. et al. (1996) Hum. Gene Ther. 7 : 1937-1945 ; Takamatsu, N. (1987) EMBO J. 6 : 307-311 ; Coruzzi, G. et al. (1984) EMBO J. 3 :   1671-1680   ; Broglie, R. et al. (1984) Science 224 : 838-843 ; Winter, J. et al. (1991) Results Probl. Cell Differ. 17 : 85-105 ; The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 



  191-196 ; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81 : 3655-3659 ; and Harrington, J. J. et al. (1997) Nat. Genet. 15 : 345-355.) Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e. g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5 (6) : 350-356 ; Yu, M. et al., (1993) Proc. Natl. Acad. Sci. USA 90 (13) : 6340-6344 ; Buller, R. M. et al. (1985) Nature 317 (6040) :   813-815   ; McGregor, D. P. et al. (1994) Mol. Immunol. 



  31 (3) : 219-226 ; and Verma, I. M.   and N. Somia   (1997) Nature 389 : 239-242.) The invention is not limited by the host cell employed. 



   For long term production of recombinant proteins in mammalian systems, stable expression of DITHP in cell lines is preferred. For example, sequences encoding DITHP can be transformed into cell lines using expression vectors which may contain viral origins of replication   and/or   endogenous expression elements and a selectable marker gene on the same or on a separate vector. Any number of 

 <Desc/Clms Page number 151> 

 selection systems may be used to recover transformed cell lines. (See, e. g., Wigler, M. et al. (1977) Cell 11 : 223-232 ; Lowy,   I.   et al. (1980) Cell 22 : 817-823. ; Wigler, M. et al. (1980) Proc. Natl. Acad. 



  Sci. USA 77 :   3567-3570   ; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150 : 1-14 ; Hartman, S. C. and R.   C. Mulligan   (1988) Proc. Natl. Acad. Sci. USA 85 : 8047-8051 ; Rhodes, C. A. (1995) Methods Mol. 



  Biol. 55 : 121-131.) Therapeutic Uses of dithp 
The dithp of the invention may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e. g., in the cases of severe combined immunodeficiency   (SCID)-X1   disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288 : 669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R. M. et al. (1995) Science 270 : 475-480 ; Bordignon, C. et al. (1995) Science 270 : 470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75 : 207- 216 ; Crystal, R. G. et al. (1995) Hum. Gene Therapy 6 : 643-666 ; Crystal, R. G. et al. (1995) Hum.

   Gene Therapy 6 : 667-703), thalassemias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX deficiencies (Crystal, R. G. (1995) Science 270 : 404-410 ; Verma, I. M. and Somia, N. 



  (1997) Nature 389 : 239-242)), (ii) express a conditionally lethal gene product (e. g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e. g., against human retroviruses, such as human immunodeficiency virus   (HIV)   (Baltimore, D. (1988) Nature 335 : 395-396 ; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93 : 11395-11399), hepatitis B or C virus (HBV, HCV) ; fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis ; and protozoan parasites such as Plasmodium   falciparum   and   Trvpanosoma   cruzi).

   In the case where a genetic deficiency in dithp expression or regulation causes disease, the expression of dithp from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency. 



   In a further embodiment of the invention, diseases or disorders caused by deficiencies in dithp are treated by constructing mammalian expression vectors comprising dithp and introducing these vectors by mechanical means into dithp-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA   microinjection   into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R. A. and Anderson, W. F. (1993) Annu. Rev. Biochem. 62 : 191-217 ;   Ivics,   Z. (1997) Cell 91 :   501-510   ; Boulay, J-L.   and Recipon,   H. (1998) Curr. Opin. Biotechnol. 9 : 445- 450). 



   Expression vectors that may be effective for the expression of dithp include, but are not limited 

 <Desc/Clms Page number 152> 

 to, the PCDNA 3. 1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,   PTET-ON,   PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). The dithp of the invention may be expressed using (i) a constitutively active promoter, (e.   g.,     from cytomegalovirus (CMV),   Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or p-actin genes), (ii) an inducible promoter (e. g., the tetracycline-regulated promoter (Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. 



  U. S. A. 89 :   5547-5551   ; Gossen, M. et al., (1995) Science 268 : 1766-1769 ; Rossi, F. M. V. and Blau, H. M. (1998) Curr. Opin. Biotechnol. 9 : 451-456), commercially available in the   T-REX plasmid   (Invitrogen) ; the   ecdysone-inducible   promoter (available in the plasmids PVGRXR and PIND ; Invitrogen) ; the   FK506/rapamycin   inducible promoter ; or the RU486/mifepristone inducible promoter (Rossi, F. M. V. and Blau, H. M. supra), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding DITHP from a normal individual. 



   Commercially available liposome transformation kits (e. g., the PERFECT LIPID   TRANSFECTION   KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F. L. and Eb, A. J. (1973) Virology 52 : 456-467), or by electroporation   (Neumann,   E. et al. 



  (1982) EMBO J. 1 : 841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols. 



   In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to dithp expression are treated by constructing a retrovirus vector consisting of (i) dithp under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation. 



  Retrovirus vectors (e. g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92 : 6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as   VSVg   (Armentano, D. et al. (1987) J. Virol. 61 : 1647-1650 ; Bender, M. A. et al. (1987) J. Virol. 61 : 1639-1646 ; Adam, M. A. and Miller, A. D. (1988) J. Virol. 62 : 3802-3806 ; Dull, T. et al. 



  (1998) J. Virol. 72 : 8463-8471 ; Zufferey, R. et al. (1998) J. Virol. 72 : 9873-9880). U. S. Patent Number 5, 910, 434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of 

 <Desc/Clms Page number 153> 

 cells   (e. g., CD4+ T-cells),   and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga,   U.   et al. (1997) J. 



  Virol. 71 : 7020-7029 ; Bauer, G. et al. (1997) Blood 89 : 2259-2267 ;   Bonyhadi,   M. L. (1997) J. Virol. 



  71 : 4707-4716 ; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95 : 1201-1206 ; Su, L. (1997) Blood 89 : 2283-2290). 



   In the alternative, an adenovirus-based gene therapy delivery system is used to deliver dithp to cells which have one or more genetic abnormalities with respect to the expression of dithp. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M. E. et al. (1995) Transplantation 27 : 263-268). Potentially useful adenoviral vectors are described in U. S. Patent Number 5, 707, 618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P. A. et al. (1999) Annu. Rev. Nutr. 19 : 511-544 and Verma, I. M.   and Somia,   N.

   (1997) Nature 18 : 389 : 239-242, both incorporated by reference herein. 



   In another alternative, a herpes-based, gene therapy delivery system is used to deliver dithp to target cells which have one or more genetic abnormalities with respect to the expression of dithp. The use of herpes simplex virus   (HSV)-based   vectors may be especially valuable for introducing dithp to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169 : 385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U. S.

   Patent Number 5, 804, 413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U. S. Patent Number 5, 804, 413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W. F. et al. 1999 J. 



  Virol. 73 : 519-532 and Xu, H. et al., (1994) Dev. Biol. 163 : 152-161, hereby incorporated by reference. 



  The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art. 



   In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver dithp to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus   (SFV),   has 

 <Desc/Clms Page number 154> 

 been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff,   H.   and Li, K-J. (1998) Curr. Opin. Biotech. 9 :   464-469).   During alphavirus RNA replication, a   subgenomic   RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full-length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e. g., protease and polymerase).

   Similarly, inserting dithp into the alphavirus genome in place of the capsid-coding region results in the production of a large number of dithp RNAs and the synthesis of high levels of DITHP in vector transduced cells. 



  While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S. A. et al. (1997) Virology 228 : 74-83). The wide host range of alphaviruses will allow the introduction of dithp into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction. The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art. 



  Antibodies   Anti-DITHP   antibodies may be used to analyze protein expression levels. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, and Fab fragments. For descriptions of and protocols of antibody technologies, see,   e. g.,   Pound J. D.   (1998)     Immunochemical   Protocols, Humana Press, Totowa, NJ. 



   The amino acid sequence encoded by the dithp of the Sequence Listing may be analyzed by appropriate software (e. g., LASERGENE NAVIGATOR software, DNASTAR) to determine regions   of high immunogenicity.   The optimal sequences for immunization are selected from the C-terminus, the N-terminus, and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the polypeptide is in its natural conformation. Analysis used to select appropriate epitopes is also described by Ausubel (1997, supra, Chapter 11. 7). Peptides used for antibody induction do not need to have biological activity ; however, they must be antigenic.

   Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids, and most preferably at least 15 amino acids. A peptide which mimics an antigenic fragment of the natural polypeptide may be fused with another protein such as keyhole limpet   hemocyanin     (KLH     ;   Sigma, St. Louis MO) for antibody production. A peptide encompassing an antigenic region may be expressed from a dithp, synthesized as described above, or 

 <Desc/Clms Page number 155> 

 purified from human cells. 



   Procedures well known in the art may be used for the production of antibodies. Various hosts including mice, goats, and rabbits, may be immunized by injection with a peptide. Depending on the host species, various adjuvants may be used to increase immunological response. 



   In one procedure, peptides about 15 residues in length may be synthesized using an ABI 431A peptide synthesizer (Applied Biosystems) using   fmoc-chemistry   and coupled to KLH (Sigma) by reaction with   M-maleimidobenzoyl-N-hydroxysuccinimide   ester (Ausubel, 1995, supra). Rabbits are immunized with the   peptide-KLH   complex in complete Freund's adjuvant. The resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with   1 %   bovine serum albumin (BSA), reacting with rabbit antisera, washing, and reacting with radioiodinated goat anti-rabbit IgG. 



  Antisera with antipeptide activity are tested for anti-DITHP activity using protocols well known in the art, including ELISA, radioimmunoassay (RIA), and immunoblotting. 



   In another procedure, isolated and purified peptide may be used to immunize mice (about 100   lug ouf   peptide) or rabbits (about 1 mg of peptide). Subsequently, the peptide is radioiodinated and used to screen the immunized animals'B-lymphocytes for production of antipeptide antibodies. Positive cells are then used to produce hybridomas using standard techniques. About 20 mg of peptide is sufficient for labeling and screening several thousand clones. Hybridomas of interest are detected by screening with radioiodinated peptide to identify those fusions producing peptide-specific monoclonal antibody.

   In a typical protocol, wells of a multi-well plate (FAST, Becton-Dickinson, Palo Alto, CA) are coated with affinity-purified, specific rabbit-anti-mouse (or suitable anti-species IgG) antibodies at   10 mg/ml.   The coated wells are blocked with 1% BSA and washed and exposed to supernatants from hybridomas. After incubation, the wells are exposed to radiolabeled peptide at 1 mg/ml. 



   Clones producing antibodies bind a quantity of labeled peptide that is detectable above background. Such clones are expanded and subjected to 2 cycles of cloning. Cloned hybridomas are injected into pristane-treated mice to produce ascites, and monoclonal antibody is purified from the ascitic fluid by affinity chromatography on protein A (Amersham Pharmacia Biotech). Several procedures for the production of monoclonal antibodies, including in vitro production, are described in Pound   (supra).   Monoclonal antibodies with antipeptide activity are tested for   anti-DITHP   activity using protocols well known in the art, including ELISA, RIA, and immunoblotting. 



   Antibody fragments containing specific binding sites for an epitope may also be generated. For example, such fragments include, but are not limited to, the F (ab') 2 fragments produced by pepsin digestion of the antibody molecule, and the Fab fragments generated by reducing the disulfide bridges of the F (ab') 2 fragments. Alternatively, construction of Fab expression libraries in   filamentous   bacteriophage allows rapid and easy identification of monoclonal fragments with desired specificity 

 <Desc/Clms Page number 156> 

 (Pound, supra, Chaps. 45-47). Antibodies generated against polypeptide encoded by dithp can be used to purify and characterize full-length DITHP protein and its activity, binding partners, etc. 



  Assays Using Antibodies   Anti-DITHP   antibodies may be used in assays to quantify the amount of DITHP found in a particular human cell. Such assays include methods utilizing the antibody and a label to detect expression level under normal or disease conditions. The peptides and antibodies of the invention may be used with or without modification or labeled by joining them, either covalently or noncovalently, with a reporter molecule. 



   Protocols for detecting and measuring protein expression using either polyclonal or monoclonal antibodies are well known in the art. Examples include ELISA, RIA, and fluorescent activated cell sorting (FACS). Such immunoassays typically involve the formation of complexes between the DITHP and its specific antibody and the measurement of such complexes. These and other assays are described in Pound   (supra).   



   Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. 



   The disclosures of all patents, applications, and publications mentioned above and below, including U. S. Ser. No. 60/184, 777, U. S. Ser. No. 60/184, 797, U. S. Ser. No. 60/184, 698, U. S. Ser. 



  No. 60/184, 770, U. S. Ser. No. 60/184, 774, U. S. Ser. No. 60/184, 693, U. S. Ser. No. 60/184, 771, U. S. 



  Ser. No. 60/184, 813, U. S. Ser. No. 60/184, 773, U. S. Ser. No. 60/184, 776, U. S. Ser. No. 60/184, 769, U. S. Ser. No.   60/184,   768, U. S. Ser. No. 60/184, 837, U. S. Ser. No. 60/184, 697, U. S. Ser. No. 



  60/184, 841, U. S. Ser. No.   60/184,   772, U. S. Ser. No. 60/185, 213, U. S. Ser. No. 60/185, 216, U. S. Ser. 



  No. 60/204, 863, U. S. Ser. No. 60/205, 221, U. S. Ser. No. 60/204, 815, U. S. Ser. No. 60/203, 785, U. S. 



  Ser.   No.   60/204, 821, U. S. Ser. No. 60/204, 908, U. S. Ser. No. 60/204, 226, U. S. Ser. No. 60/204, 525, U. S. Ser. No. 60/205, 285, U. S. Ser. No. 60/205, 232, U. S. Ser. No. 60/205, 323, U. S. Ser. No. 



  60/205, 287, U. S. Ser. No. 60/205, 324, and U. S. Ser. No. 60/205, 286, are hereby expressly incorporated by reference. 



   EXAMPLES   I.   Construction of cDNA Libraries 
RNA was purchased from CLONTECH Laboratories, Inc. (Palo Alto CA) or isolated from various tissues. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others 

 <Desc/Clms Page number 157> 

 were homogenized and lysed in phenol or in a suitable mixture of   denaturants,   such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated with either isopropanol or sodium acetate and ethanol, or by other routine methods. 



   Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In most cases, RNA was treated with DNase. For most libraries, poly (A+) RNA was isolated using oligo d (T)-coupled paramagnetic particles (Promega Corporation (Promega), Madison WI), OLIGOTEX latex particles (QIAGEN, Inc. (QIAGEN), Valencia CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e. g., the POLY (A) PURE mRNA purification kit (Ambion, Inc., Austin TX). 



   In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene Cloning Systems, Inc. (Stratagene), La Jolla CA) or   SUPERSCRIPT   plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e. g., Ausubel, 1997, supra, Chapters 5.   1   through 6. 6.) Reverse transcription was initiated using oligo d (T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.

   For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE   CL2B,   or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e. g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2. 1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or   pINCY   (Incyte Genomics, Palo Alto CA), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including   XLl-Blue,   XLl-BlueMRF, or SOLR from Stratagene or   DHSa, DH 1 OB,   or ElectroMAX DH 1 OB from Life Technologies. 



  II. Isolation of cDNA Clones 
Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following : the Magic or WIZARD Minipreps DNA purification system (Promega) ; the AGTC   Miniprep   purification kit (Edge BioSystems, Gaithersburg MD) ; and the QIAWELL 8, QIAWELL 8 Plus, and QIAWELL 8 Ultra plasmid purification systems or the R. E. A. L. PREP 96 plasmid purification kit (QIAGEN). Following precipitation, plasmids were resuspended in 0. 1 ml of distilled water and stored, with or without   lyophilization, at 4 C.   

 <Desc/Clms Page number 158> 

 



   Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format. (Rao, V. B. (1994) Anal. Biochem. 216 : 1-14.) Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384- well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Inc. (Molecular Probes), Eugene OR) and a   FLUOROSKAN   II fluorescence scanner (Labsystems Oy, Helsinki, Finland). 



  III. Sequencing and Analysis cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 thermal cycler (Applied Biosystems) or the PTC- 200 thermal cycler (MJ Research) in conjunction with the HYDRA   microdispenser   (Robbins Scientific Corp., Sunnyvale CA) or the MICROLAB 2200 liquid transfer system   (Hamilton).   cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).

   Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics) ; the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software ; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, Chapter 7. 7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII. 



  IV. Assembly and Analysis of Sequences 
Component sequences from chromatograms were subject to PHRED analysis and assigned a quality score. The sequences having at least a required quality score were subject to various pre- processing editing pathways to eliminate, e. g., low quality 3'ends, vector and linker sequences,   polyA   tails, Alu repeats, mitochondrial and ribosomal sequences, bacterial contamination sequences, and sequences smaller than 50 base pairs. In particular, low-information sequences and repetitive elements (e. g., dinucleotide repeats, Alu repeats, etc.) were replaced by"n's", or masked, to prevent spurious matches. 



   Processed sequences were then subject to assembly procedures in which the sequences were assigned to gene bins (bins). Each sequence could only belong to one bin. Sequences in each gene bin were assembled to produce consensus sequences (templates). Subsequent new sequences were added to existing bins using   BLASTn   (v. 1. 4 WashU) and CROSSMATCH. Candidate pairs were identified as 

 <Desc/Clms Page number 159> 

 all BLAST hits having a quality score greater than or equal to 150. Alignments of at least 82% local identity were accepted into the bin. The component sequences from each bin were assembled using a version of PHRAP. Bins with several overlapping component sequences were assembled using DEEP PHRAP. The orientation (sense or antisense) of each assembled template was determined based on the number and orientation of its component sequences.

   Template sequences as disclosed in the sequence listing correspond to sense strand sequences   (the"forward"reading   frames), to the best determination. 



  The complementary (antisense) strands are inherently disclosed herein. The component sequences which were used to assemble each template consensus sequence are listed in Table 4, along with their positions along the template nucleotide sequences. 



   Bins were compared against each other and those having local similarity of at least 82% were combined and reassembled. Reassembled bins having templates of insufficient overlap (less than 95% local identity) were   re-split.   Assembled templates were also subject to analysis by   STITCHER/EXON   MAPPER algorithms which analyze the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced genes across tissue types or disease states, etc. These resulting bins were subject to several rounds of the above assembly procedures. 



   Once gene bins were generated based upon sequence alignments, bins were clone joined based upon clone information. If the 5'sequence of one clone was present in one bin and the 3'sequence from the same clone was present in a different bin, it was likely that the two bins actually belonged together in a single bin. The resulting combined bins underwent assembly procedures to regenerate the consensus sequences. 



   The final assembled templates were subsequently annotated using the following procedure. 



  Template sequences were analyzed using BLASTn (v2. 0, NCBI) versus gbpri (GenBank version 120). 



  "Hits"were defined as an exact match having from 95 % local identity over 200 base pairs through 100% local identity over 100 base pairs, or a homolog match having an E-value, i. e. a probability score, of   :     1   x   10-8.   The hits were subject to   frameshift     FASTx   versus GENPEPT (GenBank version 120). (See Table 7). In this analysis, a homolog match was defined as having   an E-value   of   <1 x 10-8.   



  The assembly method used above was described in"System and Methods for Analyzing Biomolecular Sequences,"U. S. S. N. 09/276, 534, filed March 25, 1999, and the LIFESEQ Gold user manual (Incyte) both incorporated by reference herein. 



   Following assembly, template sequences were subjected to motif, BLAST, and functional analyses, and categorized in protein hierarchies using methods described in, e. g.,"Database System Employing Protein Function Hierarchies for Viewing Biomolecular Sequence Data,"U. S. S. N. 



  08/812, 290, filed March 6, 1997 ;"Relational Database for Storing Biomolecule Information," 

 <Desc/Clms Page number 160> 

 U. S. S.   N.   08/947, 845, filed October 9, 1997 ;"Project-Based Full-Length Biomolecular Sequence Database,"U. S. S. N. 08/811, 758, filed March 6, 1997 ; and"Relational Database and System for Storing Information Relating to Biomolecular Sequences,"U. S. S. N. 09/034, 807, filed March 4, 1998, all of which are incorporated by reference herein. 



   The template sequences were further analyzed by translating each template in all three forward reading frames and searching each translation against the Pfam database of hidden Markov model- based protein families and domains using the HMMER software package (available to the public from Washington University School of Medicine, St. Louis MO). Regions of templates which, when translated, contain similarity to Pfam consensus sequences are reported in Table 2, along with descriptions of Pfam protein domains and families. Only those Pfam hits with an E-value of   <     1     x     10-3   are reported.

   (See also World Wide Web site http ://pfam. wustl. edu/ for detailed descriptions of Pfam protein domains and families.) 
Additionally, the template sequences were translated in all three forward reading frames, and each translation was searched against hidden Markov models for signal peptides using the HMMER software package. Construction of hidden Markov models and their usage in sequence analysis has been described. (See, for example, Eddy, S. R. (1996) Curr. Opin. Str. Biol. 6 : 361-365.) Only those signal peptide hits with a cutoff score of 11 bits or greater are reported. A cutoff score of 11 bits or greater corresponds to at least about 91-94% true-positives in signal peptide prediction.

   Template sequences were also translated in all three forward reading frames, and each translation was searched against TMAP, a program that uses weight matrices to delineate transmembrane segments on protein sequences and determine orientation, with respect to the cell cytosol (Persson, B. and P. Argos (1994) J. 



  Mol. Biol. 237 : 182-192 ; Persson, B. and P. Argos (1996) Protein Sci. 5 : 363-371). Regions of templates which, when translated, contain similarity to signal peptide or transmembrane consensus sequences are reported in Table 3. 



   The results of HMMER analysis as reported in Tables 2 and 3 may support the results of BLAST analysis as reported in Table 1 or may suggest alternative or additional properties of template- encoded polypeptides not previously uncovered by BLAST or other analyses. 



   Template sequences are further analyzed using the bioinformatics tools listed in Table 7, or using sequence analysis software known in the art such as MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Template sequences may be further queried against public databases such as the GenBank rodent, mammalian, vertebrate, prokaryote, and eukaryote databases. 



   The template sequences were translated to derive the corresponding longest open reading frame as presented by the polypeptide sequences. Alternatively, a polypeptide of the invention may begin at 

 <Desc/Clms Page number 161> 

 any of the methionine residues within the full length translated polypeptide. Polypeptide sequences were subsequently analyzed by querying against the GenBank protein database (GENPEPT, (GenBank version   121)).   Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).

   Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences. 



   Table 6 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (GENPEPT) database. Column 1 shows the polypeptide sequence identification number (SEQ ID NO :) for the polypeptide segments of the invention. Column 2 shows the reading frame used in the translation of the polynucleotide sequences encoding the polypeptide segments. Column 3 shows the length of the translated polypeptide segments. Columns 4 and 5 show the start and stop nucleotide positions of the polynucleotide sequences encoding the polypeptide segments. Column 6 shows the GenBank identification number (GI Number) of the nearest GenBank homolog. Column 7 shows the probability score for the match between each polypeptide and its GenBank homolog. Column 8 shows the annotation of the GenBank homolog. 



  V. Analysis of Polynucleotide Expression 
Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e. g., Sambrook, supra, ch. 7 ; Ausubel, 1995, supra, ch. 4 and 16.) 
Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. 



  The basis of the search is the product score, which is defined as : 
BLAST Score x Percent Identity 
5 x minimum {length (Seq. 1), length (Seq.   2)}   The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows : the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by 

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 assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and-4 for every mismatch. Two sequences may share more than one HSP (separated by gaps).

   If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap : VI. Tissue Distribution Profiling 
A tissue distribution profile is determined for each template by compiling the cDNA library tissue classifications of its component cDNA sequences. Each component sequence, is derived from a cDNA library constructed from a human tissue.

   Each human tissue is classified into one of the following categories : cardiovascular system ; connective tissue ; digestive system ; embryonic structures ; endocrine system ; exocrine glands ; genitalia, female ; genitalia, male ; germ cells ; hemic and immune system ; liver ; musculoskeletal system ; nervous system ; pancreas ; respiratory system ; sense organs ; skin ;   stomatognathic   system ; unclassified/mixed ; or urinary tract. Template sequences, component sequences, and cDNA   library/tissue   information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). 



   Table 5 shows the tissue distribution profile for the templates of the invention. For each template, the three most frequently observed tissue categories are shown in column 3, along with the percentage of component sequences belonging to each category. Only tissue categories with percentage values of   210%   are shown. A tissue distribution of"widely distributed"in column 3 indicates percentage values of <10% in all tissue categories. 



  VII. Transcript Image Analysis 
Transcript images are generated as described in Seilhamer et al.,"Comparative Gene Transcript Analysis,"U. S. Patent Number 5, 840, 484, incorporated herein by reference. 



  VIII. Extension of Polynucleotide Sequences and Isolation of a Full-length cDNA 
Oligonucleotide primers designed using a dithp of the Sequence Listing are used to extend the nucleic acid sequence. One primer is synthesized to initiate 5'extension of the template, and the other primer, to initiate 3'extension of the template. The initial primers may be designed using OLIGO 4. 06 software (National Biosciences, Inc. (National Biosciences), Plymouth MN), or another appropriate 

 <Desc/Clms Page number 163> 

 program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about   68  C   to about   72 C.   Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations are avoided.

   Selected human cDNA libraries are used to extend the sequence. If more than one extension is necessary or desired, additional or nested sets of primers are designed. 



   High fidelity amplification is obtained by PCR using methods well known in the art. PCR is performed in 96-well plates using the PTC-200 thermal cycler (MJ Research). The reaction mix contains DNA template, 200 nmol of each primer, reaction buffer containing   Mg2+,     (NH4) 2SO4,   and B- mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A   and PCI   B : Step 1 :   94 C,   3 min ; Step 2 :   94 C,   15 sec ; Step 3 :   60 C, 1   min ; Step 4 :   68 C,   2 min ; Step 5 : Steps 2, 3, and 4 repeated 20 times ; Step 6 : 68  C, 5 min ;

   Step 7 : storage at   4  C.   In the alternative, the parameters for primer pair T7 and SK+ are as follows : Step 1 :   94 C,   3 min ; Step 2 :   94 C,   15 sec ; Step 3 :   57 C, 1   min ; Step 4 :   68  C,   2 min ; Step 5 : Steps 2, 3, and 4 repeated 20 times ; Step 6 : 68 C, 5 min ; Step 7 : storage at   4 C.   



   The concentration of DNA in each well is determined by dispensing 100 il PICOGREEN quantitation reagent (0.   25%   (v/v) ; Molecular Probes) dissolved in   1X   Tris-EDTA (TE) and 0. 5   81   of undiluted PCR product into each well of an opaque fluorimeter plate (Corning   Incorporated (Corning),   Corning   NY),   allowing the DNA to bind to the reagent. The plate is scanned in a   FLUOROSKAN   II (Labsystems Oy) to measure the fluorescence of the sample and to quantify the concentration of DNA. 



  A 5   j-il   to 10   Ill   aliquot of the reaction mixture is analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions are successful in extending the sequence. 



   The extended nucleotides are desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides are separated on low concentration (0. 6 to 0.   8 %)   agarose gels, fragments are excised, and agar digested with AGAR ACE   (Promega).   Extended clones are religated using T4 ligase (New England Biolabs, Inc., Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells.

   Transformed cells are selected on antibiotic-containing media, individual colonies are picked and cultured overnight at   37  C   in 384-well plates in   LB/2x   carbenicillin liquid media. 



   The cells are lysed, and DNA is amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters : Step 1 : 

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   94 C,   3 min ; Step 2 :   94 C,   15 sec ; Step 3 :   60 C,     1   min ; Step   : 72 C, 2 min   ; Step 5 : steps 2, 3, and4 repeated 29 times ; Step 6 :   72 C,   5 min ; Step 7 : storage at   4 C.   DNA is quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries are reamplified using the same conditions as described above.

   Samples are diluted with 20%   dimethysulfoxide   (1 : 2,   v/v),   and sequenced using DYNAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems). \ 
In like manner, the dithp is used to obtain regulatory sequences (promoters, introns, and enhancers) using the procedure above, oligonucleotides designed for such extension, and an appropriate genomic library. 



  IX. Labeling of Probes and Southern Hybridization Analyses 
Hybridization probes derived from the dithp of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA. The labeling of probe nucleotides between 100 and 1000 nucleotides in length is specifically described, but, essentially the same procedure may be used with larger cDNA fragments. Probe sequences are labeled at room temperature for 30 minutes using a T4   polynucleotide kinase, v32P-ATP,   and   0.   5X   One-Phor-AIl   Plus (Amersham Pharmacia Biotech) buffer and purified using a ProbeQuant G-50 Microcolumn (Amersham Pharmacia Biotech). The probe mixture is diluted to   107   dpm/g/ml hybridization buffer and used in a typical membrane-based hybridization analysis. 



   The DNA is digested with a restriction endonuclease such as Eco RV and is electrophoresed through a 0. 7% agarose gel. The DNA fragments are transferred from the agarose to nylon membrane (NYTRAN Plus, Schleicher & Schuell, Inc., Keene NH) using procedures specified by the manufacturer of the membrane. Prehybridization is carried out for three or more hours at   68   C,   and hybridization is carried out overnight at   68   C.   To remove non-specific signals, blots are sequentially washed at room temperature under increasingly stringent conditions, up to 0. Ix saline sodium citrate (SSC) and   0.     5 %   sodium dodecyl sulfate.

   After the blots are placed in a PHOSPHORIMAGER cassette (Molecular Dynamics) or are exposed to autoradiography film, hybridization patterns of standard and experimental lanes are compared. Essentially the same procedure is employed when screening RNA. 



  X. Chromosome Mapping of dithp 
The cDNA sequences which were used to assemble SEQ ID NO : 1-211 are compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that match SEQ 

 <Desc/Clms Page number 165> 

    i   ID NO : 1-211 are assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as PHRAP (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and   G n thon   are used to determine if any of the clustered sequences have been previously mapped.

   Inclusion of a mapped sequence in a cluster will result in the assignment of all sequences of that cluster, including its particular SEQ ID NO :, to that map location. The genetic map locations of SEQ ID NO : 1-211 are described as ranges, or intervals, of human chromosomes. The map position of an interval, in   centiMorgans,   is measured relative to the terminus of the chromosome's p- arm. (The   centiMorgan   (cM) is a unit of measurement based on recombination frequencies between chromosomal markers.

   On average,   1   cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by   G n thon   which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. 



  XI. Microarray Analysis Probe Preparation from Tissue or Cell Samples 
Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and polyA+ RNA is purified using the oligo (dT) cellulose method. Each polyA+ RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0. 05   pg/lll   oligo-dT primer   (21mer),   1X first strand buffer, 0. 03   units/pl   RNase inhibitor, 500 uM dATP, 500 MM dGTP, 500 uM dTTP,   40   uM   dCTP,   40 uM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng polyA+ RNA with GEMBRIGHT kits (Incyte). Specific control   polar   RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA (W. Lei, unpublished).

   As quantitative controls, the control mRNAs at 0. 002 ng, 0. 02 ng, 0. 2 ng, and 2 ng are diluted into reverse transcription reaction at ratios of 1 : 100, 000, 1 : 10, 000, 1 : 1000, 1 : 100 (w/w) to sample mRNA respectively. The control mRNAs are diluted into reverse transcription reaction at ratios of 1 : 3, 3 : 1, 1 : 10, 10 : 1, 1 : 25, 25 : 1 (w/w) to sample mRNA differential expression patterns. After incubation at 37  C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2. 5   ml   of   0.   5M sodium hydroxide and incubated for 20 minutes at   85    C to the stop the reaction and degrade the RNA. Probes are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.

   (CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1   ml   of glycogen   (1     mg/n),   60   ml   sodium acetate, and 300 ml of 100% ethanol. The probe is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14   ul SX   SSC/0. 2% 

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 SDS. 



    Microarrav   Preparation 
Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts. PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5   ug.   



  Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech). 



   Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.   1 %   SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester, PA), washed extensively in distilled water, and coated with 0.   05% aminopropyl   silane (Sigma) in 95% ethanol. Coated slides are cured in a   110 C   oven. 



   Array elements are applied to the coated glass substrate using a procedure described in US Patent No. 5, 807,   522,   incorporated herein by reference.   1     ul   of the array element DNA, at an average concentration of 100   ng/pl,   is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per slide. 



   Microarrays are UV-crosslinked using a   STRATALINKER   UV-crosslinker (Stratagene). 



  Microarrays are washed at room temperature once in 0. 2% SDS and three times in distilled water. 



  Non-specific binding sites are blocked by incubation of microarrays in 0. 2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford, MA) for 30 minutes at   60  C   followed by washes in 0. 2% SDS and distilled water as before. 



  Hybridization 
Hybridization reactions contain 9 1 of probe mixture consisting of 0. 2   Hg   each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0. 2% SDS hybridization buffer. The probe mixture is heated to   65    C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1. 8 cm2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of   140     nl   of   5x   SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6. 5 hours at 60  C.

   The arrays are washed for 10 min at   45  C   in a first   wash buffer (1X   SSC, 0. 1% SDS), three times for 10 minutes each at 45  C in a second wash buffer (0. 1X SSC), and dried. 



  Detection 

 <Desc/Clms Page number 167> 

 
Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of   Cy5.   The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville   NY).   The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective. The 1. 8 cm x 1. 8 cm array used in the present example is scanned with a resolution of 20 micrometers. 



   In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially. 



  Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for   Cy5.   Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously. 



   The sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the probe mix at a known concentration. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1 : 100, 000. When two probes from different sources   (e. g.,   representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture. 



   The output of the photomultiplier tube is digitized using a   12-bit   RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood, MA) installed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum. 



   A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). 

 <Desc/Clms Page number 168> 

 



   XII. Complementary Nucleic Acids 
Sequences complementary to the dithp are used to detect, decrease, or inhibit expression of the naturally occurring nucleotide. The use of oligonucleotides comprising from about 15 to 30 base pairs is typical in the art. However, smaller or larger sequence fragments can also be used. Appropriate oligonucleotides are designed from the dithp using OLIGO 4. 06 software (National Biosciences) or other appropriate programs and are synthesized using methods standard in the art or ordered from a commercial supplier. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5'sequence and used to prevent transcription factor binding to the promoter sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding and processing of the transcript. 



  XIII. Expression of DITHP 
Expression and purification of DITHP is accomplished using bacterial or virus-based expression systems. For expression of DITHP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the   trp-lac (tac)   hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts,   e.   g., BL21 (DE3). Antibiotic resistant bacteria express DITHP upon induction with isopropyl beta-D-   thiogalactopyranoside   (IPTG).

   Expression of DITHP in eukaryotic cells is achieved by infecting insect or mammalian cell lines   with recombinant Autographica   californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding DITHP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect   Spodoptera frugiperda (Sf9)   insect cells in most cases, or human hepatocytes, in some cases. 



  Infection of the latter requires additional genetic modifications to baculovirus. (See e. g., Engelhard, supra ; and Sandig, supra.) 
In most expression systems, DITHP is synthesized as a fusion protein with, e. g., glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from   Schistosoma   japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from DITHP at 

 <Desc/Clms Page number 169> 

 specifically engineered sites.

   FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak Company, Rochester   NY).   6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, Chapters 10 and 16). Purified DITHP obtained by these methods can be used directly in the following activity assay. 



  XIV. Demonstration of DITHP Activity 
DITHP activity is demonstrated through a variety of specific assays, some of which are outlined below. 



     Oxidoreductase   activity of   DITHP is   measured by the increase in extinction coefficient of NAD (P) H coenzyme at 340 nmfor the measurement of oxidation activity, or the decrease in extinction coefficient of NAD (P) H coenzyme at 340 nmfor the measurement of reduction activity (Dalziel, K. 



  (1963) J. Biol. Chem. 238 : 2850-2858). One of three substrates may be used :   Asn-0Gal, biocytidine,   or ubiquinone-10. The respective subunits of the enzyme reaction, for example, cytochtome   cl-b   oxidoreductase and cytochrome c, are reconstituted. The reaction mixture contains a) 1-2 mg/ml DITHP ; and b) 15 mM substrate, 2.   4 mM NAD (P) +   in 0. 1 M phosphate buffer, pH 7. 1 (oxidation reaction), or 2. 0 mM NAD (P) H, in 0. 1 M   Na2HPO4   buffer, pH 7. 4 (reduction reaction) ; in a total volume of 0. 1 ml. Changes in absorbance at 340 nm (A340) are measured at 23. 5       C using a recording spectrophotometer   (Shimadzu   Scientific Instruments, Inc., Pleasanton   CA).

   The   amount of NAD (P) H is stoichiometrically equivalent to the amount of substrate initially present, and the change in   A340   is a direct measure of the amount of NAD (P) H produced ;   AA340   = 6620 [NADH].   Oxidoreductase   activity of DITHP activity is proportional to the amount of NAD (P) H present in the assay. 



     Transferase   activity of DITHP is measured through assays such as a methyl transferase assay in which the transfer of radiolabeled methyl groups between a donor substrate and an acceptor substrate is measured (Bokar, J. A. et al. (1994) J. Biol. Chem. 269 : 17697-17704). Reaction mixtures   (50 p1 final   volume) contain 15 mM HEPES, pH 7. 9, 1. 5 mM MgCl, 10 mM dithiothreitol,   3%   polyvinylalcohol,   1.     5     Ci   [methyl-3H] AdoMet (0. 375 uM AdoMet) (DuPont-NEN), 0. 6   llg   DITHP, and acceptor substrate (0. 4   g     [35S]   RNA or 6-mercaptopurine (6-MP) to 1 mM final concentration). 



  Reaction mixtures are incubated at   30 C   for 30 minutes, then 65    C   for 5 minutes. The products are separated by chromatography or electrophoresis and the level of methyl transferase activity is determined by quantification of methyl recovery. 



   DITHP hydrolase activity is measured by the hydrolysis of appropriate synthetic peptide substrates conjugated with various chromogenic molecules in which the degree of hydrolysis is 

 <Desc/Clms Page number 170> 

 quantified by spectrophotometric (or fluorometric) absorption of the released chromophore. (Beynon, R. J. and J. S. Bond (1994) Proteolytic Enzymes : A Practical Approach, Oxford University Press, New York NY, pp. 25-55) Peptide substrates are designed according to the category of protease activity as endopeptidase (serine, cysteine, aspartic proteases),   animopeptidase   (leucine aminopeptidase), or carboxypeptidase (Carboxypeptidase A and B, procollagen C-proteinase). 



   DITHP isomerase activity such as peptidyl prolyl cisltrans isomerase activity can be assayed by an enzyme assay described by Rahfeld, J. U., et al. (1994) (FEBS Lett. 352 : 180-184). The assay is performed at   10 C   in 35 mM HEPES buffer, pH 7. 8, containing chymotrypsin (0. 5   mg/ml)   and DITHP at a variety of concentrations. Under these assay conditions, the substrate, Suc-Ala-Xaa-Pro- Phe-4-NA, is in equilibrium with respect to the prolyl bond, with   80-95%   in trans and 5-20% in cis conformation. An aliquot (2 ul) of the substrate dissolved in dimethyl sulfoxide (10 mg/ml) is added to the reaction mixture described above. Only the cis isomer of the substrate is a substrate for cleavage by chymotrypsin.

   Thus, as the substrate is isomerized by DITHP, the product is cleaved by chymotrypsin to produce   4-nitroanilide,   which is detected by it's absorbance at 390 nm. 4-   Nitroanilide   appears in a time-dependent and a DITHP concentration-dependent manner. 



   An assay for DITHP activity associated with growth and development measures cell proliferation as the amount of newly initiated DNA synthesis in Swiss mouse 3T3 cells. A plasmid containing polynucleotides encoding DITHP is transfected into quiescent 3T3 cultured cells using methods well known in the art. The transiently transfected cells are then incubated in the presence of [3H] thymidine, a radioactive DNA precursor. Where applicable, varying amounts of DITHP ligand are added to the transfected cells. Incorporation of [3H] thymidine into acid-precipitable DNA is measured over an appropriate time interval, and the amount incorporated is directly proportional to the amount of newly synthesized DNA. 



   Growth factor activity of DITHP is measured by the stimulation of DNA synthesis in Swiss mouse 3T3 cells (McKay,   1.   and   I.   Leigh, eds. (1993) Growth Factors : A Practical Approach, Oxford University Press, New York NY). Initiation of DNA synthesis indicates the cells'entry into the mitotic cycle and their commitment to undergo later division. 3T3 cells are competent to respond to most growth factors, not only those that are mitogenic, but also those that are involved in embryonic induction. This competence is possible because the in vivo specificity demonstrated by some growth factors is not necessarily inherent but is determined by the responding tissue. In this assay, varying amounts of DITHP are added to quiescent 3T3 cultured cells in the presence of   [3H] thymidine,   a radioactive DNA precursor.

   DITHP for this assay can be obtained by recombinant means or from biochemical preparations. Incorporation of   [3H]   thymidine into acid-precipitable DNA is measured over an appropriate time interval, and the amount incorporated is directly proportional to the amount of 

 <Desc/Clms Page number 171> 

 newly synthesized DNA. A linear dose-response curve over at least a hundred-fold DITHP concentration range is indicative of growth factor activity. One unit of activity per milliliter is defined as the concentration of DITHP producing a 50% response level, where 100% represents maximal incorporation of [3H] thymidine into acid-precipitable DNA. 



   Alternatively, an assay for cytokine activity of DITHP measures the proliferation of leukocytes. In this assay, the amount of tritiated thymidine incorporated into newly synthesized DNA is used to estimate proliferative activity. Varying amounts of DITHP are added to cultured leukocytes, such as granulocytes, monocytes, or lymphocytes, in the presence of   [3H]   thymidine, a radioactive DNA precursor. DITHP for this assay can be obtained by recombinant means or from biochemical preparations. Incorporation of [3H] thymidine into acid-precipitable DNA is measured over an appropriate time interval, and the amount incorporated is directly proportional to the amount of newly synthesized DNA. A linear dose-response curve over at least a hundred-fold DITHP concentration range is indicative of DITHP activity.

   One unit of activity per milliliter is conventionally defined as the concentration of DITHP producing a 50% response level, where 100% represents maximal incorporation of [3H] thymidine into acid-precipitable DNA. 



   An alternative assay for DITHP cytokine activity utilizes a Boyden micro chamber (Neuroprobe, Cabin John MD) to measure leukocyte chemotaxis (Vicari, supra). In this assay, about   105   migratory cells such as macrophages or monocytes are placed in cell culture media in the upper compartment of the chamber. Varying dilutions of DITHP are placed in the lower compartment. The two compartments are separated by a 5 or 8 micron pore polycarbonate filter (Nucleopore, Pleasanton CA). After incubation at 37    C   for 80 to 120 minutes, the filters are fixed in methanol and stained with appropriate labeling agents. Cells which migrate to the other side of the filter are counted using standard microscopy.

   The chemotactic index is calculated by dividing the number of migratory cells counted when DITHP is present in the lower compartment by the number of migratory cells counted when only media is present in the lower compartment. The chemotactic index is proportional to the activity of DITHP. 



   Alternatively, cell lines or tissues transformed with a vector containing dithp can be assayed for DITHP activity by immunoblotting. Cells are denatured in SDS in the presence   of p-mercaptoethanol,   nucleic acids removed by ethanol precipitation, and proteins purified by acetone precipitation. Pellets are resuspended in 20 mM tris buffer at pH 7.   5   and incubated with Protein G-Sepharose pre-coated with an antibody specific for DITHP. After washing, the Sepharose beads are boiled in electrophoresis sample buffer, and the eluted proteins subjected to SDS-PAGE.

   The SDS-PAGE is transferred to a nitrocellulose membrane for immunoblotting, and the DITHP activity is assessed by visualizing and quantifying bands on the blot using the antibody specific for DITHP as the primary antibody   and 1251¯   

 <Desc/Clms Page number 172> 

 labeled IgG specific for the primary antibody as the secondary antibody. 



   DITHP kinase activity is measured by phosphorylation of a protein substrate using y-labeled   [32P]-ATP   and quantitation of the incorporated radioactivity using a radioisotope counter. DITHP is incubated with the protein substrate,   [32P]-ATP,   and an appropriate kinase buffer. The   [32p]   incorporated into the product is separated from free   [32P3-ATP   by electrophoresis and the incorporated [32P] is counted. The amount of [32P] recovered is proportional to the kinase activity of DITHP in the assay. A determination of the specific amino acid residue phosphorylated is made by phosphoamino acid analysis of the hydrolyzed protein. 



   In the alternative, DITHP activity is measured by the increase in cell proliferation resulting from transformation of a mammalian cell line such as COS7, HeLa or CHO with an eukaryotic expression vector encoding DITHP. Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression of DITHP. Phase microscopy is then used to compare the mitotic index of transformed versus control cells. An increase in the mitotic index indicates DITHP activity. 



   In a further alternative, an assay for DITHP signaling activity is based upon the ability of GPCR family proteins to modulate G protein-activated second messenger signal transduction pathways (e. g., cAMP ; Gaudin, P. et al. (1998) J. Biol. Chem. 273 : 4990-4996). A plasmid encoding full length DITHP is transfected into a mammalian cell line (e. g., Chinese hamster ovary (CHO) or human embryonic kidney (HEK-293) cell lines) using methods well-known in the art. Transfected cells are grown in 12-well trays in culture medium for 48 hours, then the culture medium is discarded, and the attached cells are gently washed with PBS. The cells are then incubated in culture medium with or without ligand for 30 minutes, then the medium is removed and cells lysed by treatment with 1 M   perchloric   acid.

   The cAMP levels in the lysate are measured by radioimmunoassay using methods well-known in the art. Changes in the levels of cAMP in the lysate from cells exposed to ligand compared to those without ligand are proportional to the amount of DITHP present in the transfected cells. 



   Alternatively, an assay for DITHP protein phosphatase activity measures the hydrolysis of P- nitrophenyl phosphate (PNPP). DITHP is incubated together with PNPP in HEPES buffer pH 7. 5, in the presence of 0.   1 % (3-mercaptoethanol   at   37   C   for 60 min. The reaction is stopped by the addition of 6   ml   of 10 N   NaOH,   and the increase in light absorbance of the reaction mixture at   410 nm   resulting from the hydrolysis of PNPP is measured using a spectrophotometer. The increase in light absorbance is proportional to the phosphatase activity of DITHP in the assay (Diamond, R. H. et al (1994) Mol Cell Biol 14 : 3752-3762). 

 <Desc/Clms Page number 173> 

 



   An alternative assay measures DITHP-mediated G-protein signaling activity by monitoring the mobilization of   Ca++ as   an indicator of the signal transduction pathway stimulation. (See, e. g.,   Grynkievicz,   G. et al. (1985) J. Biol. Chem. 260 : 3440 ; McColl, S. et al. (1993) J. Immunol. 



  150 : 4550-4555 ; and Aussel, C. et al. (1988) J. Immunol. 140 : 215-220). The assay requires preloading neutrophils or T cells with a fluorescent dye such as FURA-2 or BCECF (Universal Imaging Corp, Westchester PA) whose emission characteristics are altered by   Ce-binding.   When the cells are exposed to one or more activating stimuli artificially (e. g., anti-CD3 antibody ligation of the T cell receptor) or physiologically (e. g., by allogeneic stimulation),   Ca++   flux takes place. This flux can be observed and quantified by assaying the cells in a fluorometer or fluorescent activated cell sorter. Measurements of   Ca++   flux are compared between cells in their normal state and those transfected with DITHP.

   Increased   Ca++   mobilization attributable to increased DITHP concentration is proportional to DITHP activity. 



   DITHP transport activity is assayed by measuring uptake of labeled substrates into   Xenopus   laevis oocytes. Oocytes at stages V and VI are injected with DITHP mRNA (10 ng per oocyte) and incubated for 3 days at   18 C   in OR2 medium (82. 5mM NaCl, 2.   5   mM   KC1,     ImM     CaCl2,     1mM   MgCl2,   lmM Na2HPO4,   5 mM Hepes, 3. 8 mM   NaOH,     50g/ml   gentamycin, pH 7. 8) to allow expression of DITHP protein. Oocytes are then transferred to standard uptake medium   (lOOmM   NaCl, 2 mM   KC1,     1mM   CaCl2,   1mM   MgCl2, 10 mM Hepes/Tris pH 7. 5).

   Uptake of various substrates (e. g., amino acids, sugars, drugs, ions, and neurotransmitters) is initiated by adding labeled substrate (e. g. radiolabeled with 3H, fluorescently labeled with rhodamine, etc.) to the oocytes. After incubating for 30 minutes, uptake is terminated by washing the oocytes three times in   Na+-free   medium, measuring the incorporated label, and comparing with controls. DITHP transport activity is proportional to the level of internalized labeled substrate. 



   DITHP transferase activity is demonstrated by a test for galactosyltransferase activity. This can be determined by measuring the transfer of radiolabeled galactose from UDP-galactose to a   GlcNAc-terminated   oligosaccharide chain (Kolbinger,   F.   et al. (1998) J. Biol. Chem. 273 : 58-65). The sample is incubated with 14   pl   of assay stock solution (180 mM sodium cacodylate, pH 6. 5, 1 mg/ml bovine serum albumin, 0. 26 mM UDP-galactose, 2   ul ofUDP-pH]   galactose),   1     1     of MnCl2   (500 mM), and 2.   5   ul of   GlcNAc,  O-(CH2) 8-CO2Me   (37 mg/ml in dimethyl sulfoxide) for 60 minutes at   37  C.

   The reaction is   quenched by the addition of 1   ml   of water and loaded on a   C18   Sep-Pak cartridge (Waters), and the column is washed twice with 5 ml of water to remove unreacted UDP-   [Hjgalactose.   The   [3H] galactosylated GlcNAc (30- (CHZ) $-COZMe   remains bound to the column during the water washes and is eluted with 5   ml   of methanol. Radioactivity in the eluted material is measured by liquid scintillation counting and is proportional to galactosyltransferase activity in the starting 

 <Desc/Clms Page number 174> 

 sample. 



   In the alternative, DITHP induction by heat or toxins may be demonstrated using primary cultures of human fibroblasts or human cell lines such as CCL-13,   HEK293,   or HEP G2 (ATCC). To heat induce DITHP expression, aliquots of cells are incubated at   42  C   for 15, 30, or 60 minutes. 



  Control aliquots are incubated at   37  C   for the same time periods. To induce DITHP expression by toxins, aliquots of cells are treated with 100   pM   arsenite or 20 mM azetidine-2-carboxylic acid for 0, 3, 6, or 12 hours. After exposure to heat, arsenite, or the amino acid analogue, samples of the treated cells are harvested and cell lysates prepared for analysis by western blot. Cells are lysed in lysis buffer containing   1%   Nonidet P-40, 0. 15 M NaCl, 50 mM   Tris-HCl,   5 mM EDTA, 2 mM N-ethylmaleimide, 2 mM   phenylmethylsulfonyl   fluoride,   1   mg/ml leupeptin, and 1   mg/ml   pepstatin. 



  Twenty micrograms of the cell lysate is separated on an 8% SDS-PAGE gel and transferred to a membrane. After blocking with 5% nonfat dry milk/phosphate-buffered saline for   1   h, the membrane is incubated overnight at   4 C   or at room temperature for 2-4 hours with a 1 : 1000 dilution of anti-DITHP serum in 2% nonfat dry   milk/phosphate-buffered   saline. The membrane is then washed and incubated with a 1 : 1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG in 2% dry   milk/phosphate-buffered   saline. After washing with 0.   1 %   Tween 20 in phosphate-buffered saline, the DITHP protein is detected and compared to controls using   chemiluminescence.   



   Alternatively, DITHP protease activity is measured by the hydrolysis of appropriate synthetic peptide substrates conjugated with various   chromogenic   molecules in which the degree of hydrolysis is quantified by spectrophotometric (or fluorometric) absorption of the released chromophore (Beynon, R. J. and J. S. Bond (1994) Proteolytic Enzymes : A Practical Approach, Oxford University Press, New York, NY, pp. 25-55). Peptide substrates are designed according to the category of protease activity as endopeptidase (serine, cysteine, aspartic proteases, or metalloproteases), aminopeptidase (leucine aminopeptidase), or carboxypeptidase (carboxypeptidases A and B, procollagen C-proteinase). Commonly used chromogens are   2-naphthylamine,   4-nitroaniline, and furylacrylic acid.

   Assays are performed at ambient temperature and contain an aliquot of the enzyme and the appropriate substrate in a suitable buffer. Reactions are carried out in an optical cuvette, and the increase/decrease in absorbance of the chromogen released during hydrolysis of the peptide substrate is measured. The change in absorbance is proportional to the DITHP protease activity in the assay. 



   In the alternative, an assay for DITHP protease activity takes advantage of fluorescence resonance energy transfer (FRET) that occurs when one donor and one acceptor fluorophore with an appropriate spectral overlap are in close proximity. A flexible peptide linker containing a cleavage site specific for PRTS is fused between a red-shifted variant (RSGFP4) and a blue variant   (BFP5)   of 

 <Desc/Clms Page number 175> 

 Green Fluorescent Protein. This fusion protein has spectral properties that suggest energy transfer is occurring from BFP5 to RSGFP4. When the fusion protein is incubated with DITHP, the substrate is cleaved, and the two fluorescent proteins dissociate. This is accompanied by a marked decrease in energy transfer which is quantified by comparing the emission spectra before and after the addition of DITHP (Mitra, R.

   D. et al (1996) Gene 173 :   13-17).   This assay can also be performed in living cells. 



  In this case the fluorescent substrate protein is expressed constitutively in cells and DITHP is introduced on an inducible vector so that FRET can be monitored in the presence and absence of DITHP (Sagot,   I.   et al (1999) FEBS Lett. 447 : 53-57). 



   A method to determine the nucleic acid binding activity of DITHP involves a polyacrylamide gel mobility-shift assay. In preparation for this assay, DITHP is expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with a eukaryotic expression vector containing DITHP cDNA. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of DITHP. Extracts containing solubilized proteins can be prepared from cells expressing DITHP by methods well known in the art. 



  Portions of the extract containing DITHP are added to   [32P]-labeled   RNA or DNA. Radioactive nucleic acid can be synthesized in vitro by techniques well known in the art. The mixtures are incubated at   25  C   in the presence of RNase-and DNase-inhibitors under buffered conditions for 5-10 minutes. After incubation, the samples are analyzed by polyacrylamide gel electrophoresis followed by autoradiography. The presence of a band on the autoradiogram indicates the formation of a complex between DITHP and the radioactive transcript. A band of similar mobility will not be present in samples prepared using control extracts prepared from untransformed cells. 



   In the alternative, a method to determine the methylase activity of a DITHP measures transfer of radiolabeled methyl groups between a donor substrate and an acceptor substrate. Reaction mixtures   (50 fol   final volume) contain 15 mM HEPES, pH 7. 9, 1. 5 mM MgCl 2, 10 mM   dithiothreitol,   3% polyvinylalcohol, 1. 5 pCi   [metliyl.-3H]   AdoMet (0. 375 uM AdoMet) (DuPont-NEN), 0. 6   ig   DITHP, and acceptor substrate (e. g., 0. 4   ug     [35S]   RNA, or 6-mercaptopurine (6-MP) to 1 mM final concentration). Reaction mixtures are incubated at   30 C   for 30 minutes, then   65  C   for 5 minutes. 



  Analysis of   [methyl-3H]   RNA is as follows : 1) 50 pi of 2 x loading buffer (20 mM   Tris-HCI,   pH 7. 6, 1 M   LiCI,     1   mM EDTA,   1%   sodium dodecyl sulphate (SDS)) and   50 pi oligo   d (T)-cellulose (10 mg/ml in 1 x loading buffer) are added to the reaction mixture, and incubated at ambient temperature with shaking for 30 minutes. 2) Reaction mixtures are transferred to a 96-well filtration plate attached to a vacuum apparatus. 3) Each sample is washed sequentially with three 2. 4 ml aliquots of 1 x oligo d (T) loading buffer containing 0. 5% SDS, 0. 1% SDS, or no SDS.

     and 4) RNA is eluted   with 300 pi of water into a 96-well collection plate, transferred to scintillation vials containing liquid scintillant, and 

 <Desc/Clms Page number 176> 

 radioactivity determined. Analysis of   [niethyl-3H] 6-UP   is as follows : 1) 500 pi 0. 5 M borate buffer, pH 10. 0, and then 2. 5 rnl of 20% (v/v) isoamyl alcohol in toluene are added to the reaction mixtures. 



  2) The samples mixed by vigorous   vortexing   for ten seconds. 3) After centrifugation at 700g for 10 minutes, 1. 5 ml of the organic phase is transferred to scintillation vials containing 0. 5 ml absolute ethanol and liquid scintillant, and radioactivity determined. and 4) Results are corrected for the extraction of 6-MP into the organic phase (approximately   41%).   



   An assay for adhesion activity of DITHP measures the disruption of cytoskeletal filament networks upon overexpression of DITHP in cultured cell lines   (Rezniczek,   G. A. et al. (1998) J. Cell Biol. 141 : 209-225). cDNA encoding DITHP is subcloned into a mammalian expression vector that drives high levels of cDNA expression. This construct is transfected into cultured cells, such as rat kangaroo PtK2 or rat bladder carcinoma 804G cells. Actin filaments and intermediate filaments such as keratin and vimentin are visualized by immunofluorescence microscopy using antibodies and techniques well known in the art. The configuration and abundance of cytoskeletal filaments can be assessed and quantified using confocal imaging techniques. In particular, the bundling and collapse of cytoskeletal filament networks is indicative of DITHP adhesion activity. 



   Alternatively, an assay for DITHP activity measures the expression of DITHP on the cell surface. cDNA encoding DITHP is transfected into a non-leukocytic cell line. Cell surface proteins are labeled with biotin (de la Fuente, M. A. et al. (1997) Blood 90 : 2398-2405). Immunoprecipitations are performed using DITHP-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled   immunoprecipitant   is proportional to the amount of DITHP expressed on the cell surface. 



   Alternatively, an assay for DITHP activity measures the amount of cell aggregation induced by overexpression of DITHP. In this assay, cultured cells such as NIH3T3 are transfected with cDNA encoding DITHP contained within a suitable mammalian expression vector under control of a strong promoter. Cotransfection with cDNA encoding a fluorescent marker protein, such as Green Fluorescent Protein (CLONTECH), is useful for identifying stable transfectants. The amount of cell agglutination, or clumping, associated with transfected cells is compared with that associated with untransfected cells. The amount of cell agglutination is a direct measure of DITHP activity. 



   DITHP may recognize and precipitate antigen from serum. This activity can be measured by the quantitative precipitin reaction (Golub, E. S. et al. (1987) Immunology : A Synthesis. Sinauer Associates, Sunderland MA, pages 113-115). DITHP is isotopically labeled using methods known in the art. Various serum concentrations are added to constant amounts of labeled DITHP. DITHP- antigen complexes precipitate out of solution and are collected by centrifugation. The amount of precipitable DITHP-antigen complex is proportional to the amount of radioisotope detected in the 

 <Desc/Clms Page number 177> 

 precipitate. The amount of precipitable DITHP-antigen complex is plotted against the serum concentration.

   For various serum concentrations, a characteristic precipitation curve is obtained, in which the amount of precipitable DITHP-antigen complex initially increases proportionately with increasing serum concentration, peaks at the equivalence point, and then decreases proportionately with further increases in serum concentration. Thus, the amount of precipitable DITHP-antigen complex is a measure of DITHP activity which is characterized by sensitivity to both limiting and excess quantities of antigen. 



   A microtubule motility assay for DITHP measures motor protein activity. In this assay, recombinant DITHP is immobilized onto a glass slide or similar substrate.   Taxol-stabilized   bovine brain microtubules (commercially available) in a solution containing ATP and cytosolic extract are perfused onto the slide. Movement of microtubules as driven by DITHP motor activity can be visualized and quantified using video-enhanced light microscopy and image analysis techniques. 



  DITHP motor protein activity is directly proportional to the frequency and velocity of microtubule movement. 



   Alternatively, an assay for DITHP measures the formation of protein filaments in vitro. A solution of DITHP at a concentration greater than the"critical concentration"for polymer assembly is applied to carbon-coated grids. Appropriate nucleation sites may be supplied in the solution. The grids are negative stained with   0.   7% (w/v) aqueous uranyl acetate and examined by electron microscopy. The appearance of filaments of approximately 25 nm (microtubules), 8 nm (actin), or 10 nm (intermediate filaments) is a demonstration of protein activity. 



   DITHP electron transfer activity is demonstrated by oxidation or reduction of NADP. 



  Substrates such as   Asn-pGal,   biocytidine, or ubiquinone-10 may be used. The reaction mixture contains 1-2   mg/rnl   HORP, 15 mM substrate, and 2. 4 mM NAD (P)   +   in 0. 1 M phosphate buffer, pH 7. 1 (oxidation reaction), or 2. 0 mM NAD (P) H, in 0. 1 M   Na2HPO4   buffer, pH 7. 4 (reduction reaction) ; in a total volume of 0. 1   ml.   FAD may be included with NAD, according to methods well known in the art. Changes in absorbance are measured using a recording spectrophotometer. The amount of NAD (P) H is stoichiometrically equivalent to the amount of substrate initially present, and the change in A340 is a direct measure of the amount of NAD (P) H produced ;   AA340   = 6620 [NADH]. DITHP activity is proportional to the amount of NAD (P) H present in the assay.

   The increase in extinction coefficient of NAD (P) H coenzyme at 340 nm is a measure of oxidation activity, or the decrease in extinction coefficient of NAD (P) H coenzyme at 340 nm is a measure of reduction activity (Dalziel, K. (1963) J. 



  Biol. Chem. 238 :   2850-2858).   



   DITHP transcription factor activity is measured by its ability to stimulate transcription of a reporter gene (Liu, H.   Y.   et al. (1997) EMBO J. 16 : 5289-5298). The assay entails the use of a well 

 <Desc/Clms Page number 178> 

 characterized reporter gene construct,   LexAOp-LacZ,   that consists of LexA DNA transcriptional control elements   (LexA)   fused to sequences encoding the E. coli LacZ enzyme. The methods for constructing and expressing fusion genes, introducing them into cells, and measuring LacZ enzyme activity, are well known to those skilled in the art. Sequences encoding DITHP are cloned into a plasmid that directs the synthesis of a fusion protein, LexA-DITHP, consisting of DITHP and a DNA binding domain derived from the LexA transcription factor.

   The resulting plasmid, encoding a LexA-DITHP fusion protein, is introduced into yeast cells along with a plasmid containing the   LexAOp-LacZ   reporter gene. The amount of LacZ enzyme activity associated with LexA-DITHP transfected cells, relative to control cells, is proportional to the amount of transcription stimulated by the DITHP. 



   Chromatin activity of DITHP is demonstrated by measuring sensitivity to DNase I (Dawson, B. A. et al. (1989) J. Biol. Chem. 264 : 12830-12837). Samples are treated with DNase   I,   followed by insertion of a cleavable biotinylated nucleotide analog,   5-[(N-biotinamido) hexanoamido-ethyl-1,   3-   thiopropionyl-3-aminoallyl]-2'-deoxyuridine   5'-triphosphate using nick-repair techniques well known to those skilled in the art. Following purification and digestion with   EcoRI   restriction endonuclease, biotinylated sequences are affinity isolated by sequential binding to streptavidin and biotincellulose. 



   Another specific assay demonstrates the ion conductance capacity of DITHP using an electrophysiological assay. DITHP is expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with a eukaryotic expression vector encoding DITHP. Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art. A small amount of a second plasmid, which expresses any one of a number of marker genes such as   (3-galactosidase,   is co-transformed into the cells in order to allow rapid identification of those cells which have taken up and expressed the foreign DNA.

   The cells are incubated for   48-72   hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of DITHP and   P-galactosidase.   Transformed cells expressing    -   galactosidase are stained blue when a suitable colorimetric substrate is added to the culture media under conditions that are well known in the art. Stained cells are tested for differences in membrane conductance due to various ions by electrophysiological techniques that are well known in the art. 



  Untransformed cells, and/or cells transformed with either vector sequences alone   or-galactosidase   sequences alone, are used as controls and tested in parallel. The contribution of DITHP to cation or anion conductance can be shown by incubating the cells using antibodies specific for either DITHP The respective antibodies will bind to the extracellular side of DITHP, thereby blocking the pore in the ion channel, and the associated conductance. 



  XV. Functional Assays 

 <Desc/Clms Page number 179> 

 
DITHP function is assessed by expressing dithp at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression. Vectors of choice include   pCMV   SPORT (Life Technologies) and pCR3. 1 (Invitrogen Corporation, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 Mg of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or   electroporation.     1-2 ig   of an additional plasmid containing sequences encoding a marker protein are   co-transfected.   



   Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. 



  Marker proteins of choice include, e. g., Green Fluorescent Protein (GFP ; CLONTECH), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. 



   FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide ; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies ; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow   Cvtometry,   Oxford, New York NY. 



   The influence of DITHP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding DITHP and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Inc., Lake Success NY). mRNA can be purified from the cells using methods well known by those of skill in the art. 



  Expression of mRNA encoding DITHP and other genes of interest can be analyzed by northern analysis or microarray techniques. 



   XVI. Production of Antibodies 
DITHP substantially purified using polyacrylamide gel electrophoresis (PAGE ; see, e. g., 

 <Desc/Clms Page number 180> 

 Harrington, M. G. (1990) Methods Enzymol. 182 : 488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols. 



   Alternatively, the DITHP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high   immunogenicity,   and a corresponding peptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art.

   (See, e. g., Ausubel, 1995, supra, Chapter 11.) 
Typically, peptides 15 residues in length are synthesized using an ABI   431A   peptide synthesizer (Applied Biosystems) using   fmoc-chemistry   and coupled to KLH (Sigma) by reaction with   N-maleimidobenzoyl-N-hydroxysuccinimide   ester (MBS) to increase   immunogenicity.   (See, e. g., Ausubel, supra.) Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with   1%   BSA, reacting with rabbit antisera, washing, and reacting with radio- iodinated goat anti-rabbit IgG.

   Antisera with antipeptide activity are tested for anti-DITHP activity using protocols well known in the art, including ELISA, RIA, and immunoblotting. 



  XVII. Purification of Naturally Occurring DITHP Using Specific Antibodies 
Naturally occurring or recombinant DITHP is substantially purified by immunoaffinity chromatography using antibodies specific for DITHP. An immunoaffinity column is constructed by covalently coupling   anti-DITHP   antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions. 



   Media containing DITHP are passed over the   immunoaffinity   column, and the column is washed under conditions that allow the preferential absorbance of DITHP (e. g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/DITHP binding (e. g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and DITHP is collected. 



  XVIII. Identification of Molecules Which Interact with DITHP 
DITHP, or biologically active fragments thereof, are labeled   with'2'1   Bolton-Hunter reagent. 



  (See, e. g., Bolton, A. E.   and W.   M. Hunter (1973) Biochem. J. 133 : 529-539.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled DITHP, washed, and any wells with labeled DITHP complex are assayed. Data obtained using different concentrations of DITHP are used to calculate values for the number, affinity, and association of DITHP with the 

 <Desc/Clms Page number 181> 

 candidate molecules. 



   Alternatively, molecules interacting with DITHP are analyzed using the yeast two-hybrid system as described in Fields, S. and   O.   Song (1989) Nature 340 : 245-246, or using commercially available kits based on the two-hybrid system, such as the   MATCHMAKER   system (CLONTECH). 



   DITHP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U. S. Patent No. 6, 057, 101). 



   All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. 



  Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. 



  Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims. 

 <Desc/Clms Page number 182> 

 



    TABLE 1 SEQ ID NO: Template ID GI Number Probability Score Annotation 1 LG:1040582.1:2000FEB18 g178480 1.00E-92 Human aldehyde reductase mRNA, complete cds. 



  2 LG:453570.1:2000FEB18 g2909424 2.40E-65 Glyoxalase I 3 LG:408751.3:2000FEB18 g3608122 4.40E-74 dihydropyrimidinase 4 LI:090574.1:2000FEB01 g29600 5.00E-85 carbonic anhydrase I (AA 1-261) 5 LI:229932.2:2000FEB01 g1835116 2.00E-63 acetyl-CoA synthetase 6 LI:332176.1:2000FEB01 g2104689 0 alpha glucosidase II, alpha subunit 7 LI:403248.2:2000FEB01 g63713 4.00E-23 ornithine decarboxylase 8 LG:220992.1:2000MAY19 g10435462 0 unnamed protein product (Homo saplens) 9 LG:1094571.1:2000MAY19 g7023634 4.00E-92 unnamed protein product (Homo sapiens) 10 LI:350754.4:2000MAY01 g307504 0 transglutaminase E3 (Homo sapiens) 11 LI:255828.29:2000MAY01 g189998 9.00E-65 M2-type pyruvate kinase (Homo sapiens) 12 LI:1190263.1:2000MAY01 g2576305 1.00E-172 arylsulphatase (Homo sapiens) 13 LG:270916.2:2000FEB18 g2088668 1.20E-11 similar to Achlya amblsexualis antheridiol steroid receptor (NID:

  g166306) Human chromosome 3, olfactory receptor pseudogene cluster 1,     complete sequence, and myosin light chain kinase (MLCK) 14 LG:999414.3:2000FEB18 g3861482 0 @seudodene, partial seduence. 



  Human T-cell receptor alpha delta locus from bases 501613 to 752736 15 LG:429446.1:2000FEB18 g2358042 0 (section 3 of 5) of the Complete Nucleotide Sequence. 



  16 LI:057229.1:2000FEB01 g10439739 2.00E-19 unnamed protein product (Homo sapiens) Human T-cell receptor alpha de@ta locus from bases 501613 to 752736 17 LI:351965.1:2000FEB01 g2358042 0 (section 3 of 5) of the Complete Nucleotide Sequence. 



  18 LG:068682.1:2000FEB18 g404634 1.10E-31 serine/threonine kinase 19 LG:242665.1:2000FEB18 g2117166 1.00E-160 Ras like GTPase (Homo sapiens) 20 LG:241743.1:2000FEB18 g5763838 9.70E-49 dJ593C16.1 (ras GTPase activating protein) 21 LI:034212.1:2000FEB01 g1469876 0 The KIAA0147 gene product is related to adenylyl cyclase. 



  22 LG:344886.1:2000MAY19 g7008402 2.00E-89 kappa B-ras 1 (Homo sapiens) 23 LG:228930.1:2000MAY19 g206218 4.50E-87 phospholipase C-1 24 LG:338927.1:2000MAY19 g3599940 3.00E-57 faclogenital dysplasia protein 2 (Mus musculus) 25 LG:898771.1:2000MAY19 g508528 5.00E-58 myocyte nuclear factor (Mus musculus) Human guanine nucleotide-binding protein alpha-subunit gene (G-s- 26 LI:257664.67:2000MAY01 g183399 1.00E-142 alpha), exon 3. 



  27 LI:001496.2:2000MAY01 g3005085 1.00E-177 hook@ protein (Homo sapiens) 28 LI:1085273.2:2000MAY01 g1781037 0 neuronal tyrosine threonine phosphatase 1 (Mus musculus) 29 LI:333138.2:2000MAY01 g2077934 1.00E-164 Protein Kinase (Raffus norvegicus)   

 <Desc/Clms Page number 183> 

   30 LI:338927.1:2000MAY01 g3599940 6.00E-45 faclogenital dysplasia protein 2 (Mus musculus) 31 LG:335558.1:2000FEB18 g1181619 5.00E-97 a variant of TSC-22 (Gallus gallus) 32 LG:998283.7:2000FEB18 g6683492 1.00E-105 bromodomain PHD finger transcription factor (Homo sapiens) 33 LI:402739.1:2000FEB01 g4164151 6.00E-34 AhR repressor 34 LI:175223.1:2000FEB01 g2745892 2.00E-11 Y box transcription factor supported by Genscan and several ESTs:

   C83049 (NID:g3062006), AA823760 (NID:g2893628), AA215791 (NID:g1815572), AI095488 35 LG:981076.2:2000MAY19 g3924670 1.00E-59 (NID:a3434464), and AA969095 (NID:a3544275) (Homo sapiens) 36 LI:1008973.1:2000MAY01 g6939732 2.00E-52 transcription factor Elongin A2 (Homo sapiens) 37 LI:1190250.1:2000MAY01 g3757892 4.00E-66 enhancer of polycomb (Mus musculus) 38 LG:021371.3:2000FEB18 g984814 1.40E-60 zinc finger protein 39 LG:475404.1:2000FEB18 g487784 5.00E-36 Human zinc finger protein ZNF136. 



  40 LG:979406.2:2000FEB18 g4325310 3.60E-11 zinc-finger protein 7 41 LG:410726.1:2000FEB18 g6002480 2.60E-39 BWSCR2 associated zinc-finger protein BAZ2 42 LG:200005.1:2000FEB18 g1504006 3.20E-25 similarto human ZFY protein. 



  43 LG:1076828.1:2000FEB18 g498720 2.00E-33 Human HZF10 mRNA for zinc finger protein. 



  44 LG:1076931.1:2000FEB18 g498151 2.00E-52 Human mRNA for KIAA0065 gene, partial cds. 



  45 LG:1078121.1:2000FEB18 g186773 2.00E-47 Human Kruppel related zinc finger protein (HTF10) mRNA, complete cds. 



  46 LG:10792031:2000FEB18 g1017721 0 Human repressor transcriptional factor (ZNF85) mRNA, complete cds. 



  47 LG:1082586.1:2000FEB18 g7959207 1.00E-19 KIAA1473 protein (Homo sapiens) 48 LG:1082774.1:2000FEB18 g184451 0 Human Krueppel-related DNA-binding protein (TF9 PF4) mRNA, 5' cds. 



  49 LG:1082775.1:2000FEB18 g506502 3.50E-36 NK10 50 LG:1083120.1:2000FEB18 g7023216 1.00E-14 unnamed protein product (Homo sapiens) 51 LG:1087707.1:2000FEB18 g347905 2.00E-40 Human zinc finger protein (ZNF141) mRNA, complete cds. 



  52 LG:1090915.1:2000FEB18 g347905 2.00E-24 Human zinc finger protein (ZNF141) mRNA, complete cds. 



  53 LG:1094230.1:2000FEB18 g454818 1.00E-98 Human Krueppel-related DNA-binding protein (PF4) mRNA, 5' end. 



  54 LG:474848.3:2000FEB18 g498152 4.00E-16 ha0946 protein is Kruppel-related. 



  55 LI:251656.1:2000FEB01 g55471 0 Zfp-29 56 LI:021371.1:2000FEB01 g984814 2.00E-96 zinc finger protein 57 LI:133095.1:2000FEB01 g453376 8.00E-42 zinc finger protein PZF 58 LI:236654.2:2000FEB01 g498721 2.00E-22 zinc finger protein 59 LI:200009.1:2000FEB01 g498719 4.00E-24 zinc finger protein 60 LI:758502.1:2000FEB01 g200407 0 pMLZ-4 61 LI:344772.1:2000FEB01 g4062983 3.00E-67 Eos protein 62 LI:789445.1:2000FEB01 g1049301 2.00E-26 KRAB zinc finger protein; Method: conceptual translation supplied by 63 LI:789657.1:2000FEB01 g1020145 1.00E-53 DNA binding protein   

 <Desc/Clms Page number 184> 

   64 LI:789808.1:2000FEB01 g288424 0 Human ZNF37A mRNA for zinc finger protein. 



  65 LI:792919.1:2000FEB01 g2232012 0 Human zinc finger protein (FDZF2) mRNA, complete cds. 



  66 LI:793949.1:2000FEB01 g1017721 3.00E-53 Human repressor transcriptional factor (ZNF85) mRNA, complete cds. 



  67 LI:794389.1:2000FEB01 g5640017 4.00E-45 zinc finger protein ZFP113 68 LI:796010.1:2000FEB01 g288424 0 Human ZNF37A mRNA for zinc finger protein. 



  69 LI:796324.1:2000FEB01 g288424 0 Human ZNF37A mRNA for zinc finger protein. 



  70 LI:796373.1:2000FEB01 g1020145 9.00E-36 DNA binding protein 71 LI:796415.1:2000FEB01 g498151 4.00E-28 Human mRNA for KIAA0065 gene, partial cds. 



  72 LI:798636.1:2000FEB01 g2970037 0 Human HKL1 mRNA, complete cds. 



  73 LI:800045.1:2000FEB01 g538413 2.00E-55 zinc finger protein 74 LI:800680.1:2000FEB01 g7023216 7.00E-18 unnarmed protein product (Homo sapiens) 75 LI:800894.1:2000FEB01 g3342001 0 Human hematopoietic cell derived zinc finger protein mRNA, complete 76 LI:801015.1:2000FEB01 g487785 4.00E-16 zinc finger protein ZNF136 (Homo sapiens) 77 LI:801236.1:2000FEB01 g488555 4.00E-48 zinc finger protein ZNF135 78 LI:803335.1:2000FEB01 g498152 1.00E-20 ha0946 protein is Kruppel-related. 



  79 LI:803998.1:2000FEB01 g1017722 1.00E-53 repressor transcriptional factor 80 LI:478757.1:2000FEB01 g498151 9.00E-27 Human mRNA for KIAA0065 gene, partial cds. 



  81 LI:808532.1:2000FEB01 g2232012 0 Human zinc finger protein (FDZF2) mRNA, complete cds. 



  82 LI:443073.1:2000FEB01 g4567179 3.00E-33 BC37295¯1 (Homo sapiens) 83 LI:479671.1:2000FEB01 g487784 3.00E-38 Human zinc finger protein ZNF136. 



  84 LI:810078.1:2000FEB01 g498718 0 Human HZF1 mRNA for zinc finger protein. 



  85 LI:810224.1:2000FEB01 g288424 0 Human ZNF37A mRNA for zinc finger protein. 



  86 LI:817052.2:2000Feb01 g1020145 1.00E-51 DNA binding protein 87 LG:892274.1:2000MAY19 g6650686 2.00E-95 Human Y-linked zinc finger protein (ZFY) gene, complete cds. 



  88 LG:1080959.1:2000MAY19 g5262560 2.00E-40 hypothetical protein (Homo sapiens) 89 LG:1054900.1:2000MAY19 g5262560 3.00E-35 hypothetical protein (Homo sapiens) 90 LG:1077357.1:2000MAY19 g10047297 2.00E-23 KIAA1611 protein (Homo sapiens) 91 LG:1084051.1:2000MAY19 g5931821 8.00E-79 dJ228H13.3 (zinc finger protein) (Homo sapiens) 92 LG:1076853.1:2000MAY19 g506502 1.00E-141 NK10 (Mus musculus) 93 LG:481631.10:2000MAY19 g7023216 1.00E-142 unnamed protein product (Homo sapiens) 94 LG:1088431.2:2000MAY19 g7023216 7.00E-18 unnamed protein product (Homo sapiens) 95 LI:401619.10:2000MAY01 g7959865 1.00E-18 PRO2032 (Homo sapiens) putative kruppel-related zinc finger protein NY-REN-23 antigen (Homo 96 LI:1144007.1:2000MAY01 g5360097 0 sapiens) 97 LI:331074.1:2000MAY01 g2149792 0 Roaz (Rattus norvegicus) 98 LI:1170349.1:

  2000MAY01 g487787 1.00E-45 zinc finger protein ZNF140 (Homo sapiens)   

 <Desc/Clms Page number 185> 

   99 LG:335097.1:2000FEB18 g7020440 6.00E-25 unnamed protein product (Homo sapiens) Human hereditary haemochromatosis region, histone 2A-like protein gene, hereditary haemochromatosis (HLA-H) gene, RoRet gene, and 100 LG:1076451.1:2000FEB18 g2088550 0 sodium phosphate transporter (NPT3) @ene. complete cds. 



  Human hereditary haemochromatosis region, histone 2A-like protein gene, hereditary haemochromatosis (HLA-H) gene, RoRet gene, and 101 LI:805478.1:2000FEB01 g2088550 0 sodium phosphate transporter (NPT3) @ene, complete cds. 



  102 LG:101269.1:2000MAY19 g3953533 2.10E-56 inwardly rectifying potassium channel Kir5.1 103 LI:331087.1:2000MAY01 94186073 3.00E-41 calcium channel alpha-2-de@ta-C subunit (Mus musculus) 104 LI:410188.1:2000MAY01 g4838145 0 tetrodotoxin-resistant voltage-gated sodium channel (Homo sapiens) 105 LI:1188288.1:2000MAY01 g204220 0 beta-alanine-sensitive neuronal GABA transporter (Rattus norveg@cus) 106 LI:427997.4:2000MAY01 g6996442 1.00E-48 CTL1 protein (Homo sapiens) ESTs AU058081(E30812).AU058365(E50679), AU030138(E50679) correspond to a region of the predicted gene.; Similar to Spinacia oleracea mRNA 107 LG:451682.1:2000FEB18 g5091520 3.00E-75 for proteasome 37kD subunit.(X96974) 108 LG:1077283.1:2000FEB18 g2565302 0 Rhesus monkey cyclophilin A mRNA, complete cds. 



  *Similarity to B.subtilis DNAJ protein (SW:DNAJ¯BACSU); cDNA EST 109 LG:481436.5:2000FEB18 g3873707 2.60E-34 yk437a1.5 comes from this gene Method: conceptual translation supplied by author; putative hybrid protein similar to HERV-H protease and HERV-E integrase (Human 110 LI:793701.1:2000FEB01 g1049231 4.00E-33 endogenous retrovirus) 111 LI:373637.1:2000FEB01 g2286123 3.00E-50 testis specific DNAj-homolog 112 LG:239368.2:2000MAY19 g4981382 1.00E-11 dnaJ protein (Thermotoga maritim@) 113 LI:053826.1:2000MAY01 g2943716 4.00E-67 25 kDa trypsin inhibitor (Homo sapiens) 114 LI:449393.1:2000MAY01 g6957716 1.00E-128 putative chaperonin (Arabidopsis thalian@) similar to Homo sapiens mRNA for KIAA0723 protein with GenBank 115 LI:1071427.96:2000MAY01 g9956070 1.00E-144 Accession Number AB018266.1 0 116 LI:336338.8:2000MAY01 g9296929 2.00E-16 protease PC6 isoform A (Homo sapiens) 117 LG:345527.1:

  2000FEB18 g805296 3.40E-176 lymphocyte specific helicase ORF derived from D1 leader region and integrase coding region (Homo 118 LG:1089383.1:2000FEB18 g2104910 3.00E-23 sapiens) 119 LG:1092522.1:2000FEB18 g1263080 0 Human marinerl transposase gene, complete consensus sequence. 



  ORF derived from D1 leader region and integrase coding region (Homo 120 LG:1093216.1:2000FEB18 g2104910 1.00E-23 sapiens) similar to mitochondrial RNA splicing MSR4 like protein; cDNA EST 121 LI:270318.3:2000FEB01 g3880433 3.00E-12 EMBL:C09217 comes from this gene   

 <Desc/Clms Page number 186> 

   122 LI:335671.2:2000FEB01 g805296 1.00E-83 lymphocyte specific helicase ORF derived from D1 leader region and integrase coding region (Homo 123 LI:793758.1:2000FEB01 g2104910 4.00E-26 sapiens) ORF derived from D1 leader region and integrase coding region (Homo 124 LI:803718.1:2000FEB01 g2104910 3.00E-23 sapiens) 125 LI:412179.1:2000FEB01 g1263080 4.00E-93 Human mariner@ transposase gene, complete consensus sequence. 



  126 LI:815679.1:2000FEB01 g7020440 3.00E-12 unnamed protein product (Homo sapiens) 127 LI:481361.3:2000FEB01 g3776011 5.00E-25 RNA helicase dJ620E11.1a(novel Helicase C-terminal domain and SNF2 N-temrinal 128 LG:247388.1:2000MAY19 g6016932 3.00E-127 domains containing protein, similar to KIAA0308) 129 LG:255789.10:2000MAY19 g37542 2.00E-57 Human mRNA for Ul small nuclear RNP-specific C protein. 



  130 LI:787618.1:2000MAY01 g7020440 3.00E-12 unnamed protein product (Homo sapiens) 131 LI:331610.2:2000MAY01 g2599502 0 protocadherin 68 (Homo sapiens) 132 LG:982697.1:2000FEB18 g10436424 1.00E-25 unnamed protein product (Homo sapiens) Human genomic DNA, chromosome 6p21.3, HLA Class I region, section 133 LG:1080896.1:2000FEB18 g9526696 0 8/20. 



  Human genomic DNA, chromosome 6p21.3, HLA Class I region, section 134 LI:811341.1:2000FEB01 g5926696 0 8/20. 



  Human genomic DNA, chromosome 6p21.3, HLA Class I region, section 135 LI:903225.1:2000FEB01 g5926710 0 20/20. 



  Human genomic DNA, chromosome 6p21.3, HLA Class I region, section 136 LI:242079.2:2000FEB01 g5926703 0 15/20. 



  137 LG:979580.1:2000MAY19 g9280152 7.00E-23 unn@med portein product (Macaca fascicularis) 138 LI:1169865.1:2000MAY01 g673417 1.00E-112 class II antigen (Homo sapiens) 139 LG:337818.2:2000FEB18 g404777 4.80E-84 cytochrome P-450 2B-Bx 140 LI:337818.1:2000FEB01 g203759 4.00E-58 cytochrome P-450(1) 141 LG:241577.4:2000MAY19 g2809498 1.50E-29 cytochrome c oxidase subunit IV 142 LG:344786.4:2000MAY19 g164981 2.00E-06 cytochrome P-450p-2 (oryctolagus cuniculus) 143 LI:414307.1:2000FEB01 g30095 9.00E-48 collagen subunit (alpha-1 (X)) 3 144 LI:202943.2:2000FEB01 g391663 7.00E-06 hikaru genki type1 product 145 LI:246194.2:2000FEB01 g1405821 1.00E-05 SULFATED SURFACE GLYCOPROTEIN 185 146 LI:815961.1:2000FEB01 g292045 0 Human mucin mRNA, partial cds. 



  147 LG:120744.1:2000MAY19 g4582324 1.00E-168 dJ708F5.1 (PUTATIVE novel Collagen alpha 1 LIKE protein) (Homo 148 LI:757520.1:2000MAY01 g7161771 0 keratin (Homo sapiens) 149 LG:160570.1:2000FEB18 g466548 1.00E-46 NBL4 150 LI:350398.3:2000FEB01 g3724141 1.00E-06 myosin I   

 <Desc/Clms Page number 187> 

   151 LI:221285.1:2000FEB01 g18218 2.00E-74 spoke protein 152 LI:401605.2:2000FEB01 g1755049 1.00E-15 myosin X 153 LI:329017.1:2000FEB01 g1813638 2.00E-51 PF20 154 LI:401322.1:2000FEB01 g38076 2.00E-30 Macaque mRNA for alpha-@ubulin. 



  155 LG:403409.1:2000MAY19 g7303061 0 Khc-73 gene product (Drosophila melanogaster) 156 LG:233933.5:2000MAY19 g738511 2.00E-18 ankyrin 1 (Bos taurus) 157 LI:290344.1:2000MAY01 g1353782 0 dystrophin-related protein 2 (Homo sapiens) 158 LI:410742.1:2000MAY01 g2290200 0 desmoglein 3 (Mus musculus) 159 LG:406568.1:2000MAY19 g28969 5.30E-44 64 Kd autoantigen The KIAA0143 gene product is related to a putative C.elegans gene 160 LI:283762.1:2000MAY01 g1469868 0 encoded on cosmid C32D5.

   (Homo sapiens) 161 LI:347687.113:2000MAY01 g387514 1.00E-123 DM-20 protein (Mus musculus) 162 LI:1146510.1:2000MAY01 g2149291 3.00E-24 defender against death 1 protein (Homo sapiens) 163 LG:451710.1:2000FEB18 g5816996 2.40E-42 ribosomal protein L23-like protein 164 LG:455771.1:2000FEB18 g643074 1.70E-59 putative 40S ribosomal protein s12 165 LG:452089.1:2000FEB18 g463252 1.90E-62 RL5 ribosomal protein 166 LG:246415.1:2000FEB18 g296451 0 Human mRNA for ribosomal protein S26. 



  167 LG:414144.10:2000FEB18 g200785 1.80E-16 ribosomal protein L7 168 LG:1101445.1:2000FEB18 g1800114 0 Human ribosomal protein L7 antisense mRNA gene, partial sequence. 



  169 LG:452134.1:2000FEB18 g550024 0 Human ribosomal protein S10 mRNA, complete cds. 



  170 LI:903021.1:2000FEB01 g36139 0 Human mRNA for ribosomal protein L7. 



  171 LI:246422.1:2000FEB01 g409069 0 Human mRNA for HBp15/L22, complete cds. 



  172 LG:449404.1:2000MAY19 g4886269 5.00E-66 putative ribosomal protein S14 (Arabldopsis thaliana) 173 LG:449413.1:2000MAY19 g643074 1.00E-70 putative 40S ribosomal protein s12 (Fragaria x ananassa) 174 LG:450105.1:2000MAY19 g643074 6.00E-76 putative 40S ribosomal protein s12 (Fragaria x ananassa) 175 LG:460809.1:2000MAY19 g36129 4.00E-54 Human mRNA for ribosomal protein L31. 



  176 LG:481781.1:2000MAY19 g2331301 1.00E-130 ribosomal protein S4 type I (Zea mays) 177 LG:1101153.1:2000MAY19 g2668748 2.00E-95 ribosomal protein L17 (Zea mays) 178 LI:257695.20:2000MAY01 g57714 1.00E-62 ribosomal protein S16 (AA 1-146) (Rattus rattus) 179 LI:455771.1:2000MAY01 g643074 6.00E-76 putative 40S ribosomal protein s12 (Fragaria x ananassa) 180 LI:274551.1:2000MAY01 g36145 2.00E-59 Human 40S ribosomal protein s12 (Fragaria x ananassa) 181 LI:035973.1:2000MAY01 g57121 8.00E-29 ribosomal protein L37 (Rattus norvegicus) 182 LG:978427.5:2000FEB18 9545998 2.50E-67 tricarboxylate carrier (rats, liver, Peptide Mitochondrial Partial, 357 aa) 183 LG:247781.2:2000FEB18 g2352427 9.40E-29 peroxisomal CA-dependent solute carrier 184 LI:03483.1:

  2000FEB01 g5815141 0 nuclear body associated kinase 1b   

 <Desc/Clms Page number 188> 

   selected as a weak suppressor of a mutant of the subunit AC40 of DNA 185 LI:333307.2:2000FEB01 g295671 0.0003 dependant RNA polymerase I and III 186 LI:814710.2:2000FEB01 g178281 1.00E-46 AHNAK nucleoprotein 187 LG:14732.1:2000MAY19 g183233 2.00E-54 beta-glucuronidase precursor (EC 3.2.1.31) 188 LG:413910.6:2000MAY19 g7022046 1.00E-109 unnamed protein product (Homo sapiens) 189 LI:414732.2:2000MAY01 g183232 0 Human beta-glucuronidase mRNA, complete cds. 



  190 LI:900264.2:2000MAY01 g414797 2.00E-81 pyruvate dehydrogenase phosphatase (Bos taurus) 191 LI:335593.1:2000MAY01 g3851553 5.00E-34 RNA-binding protein Nova-2 (Homo sapiens) 192 LI:1189543.1:2000MAY01 g7025507 0 ventral neuron-specific protein 1 NOVA1 (Mus musculus) 193 LG:455450.1:2000FEB18 g4105111 2.10E-20 dehydrin 6 194 LG:1040978.1:2000FEB18 g453189 3.30E-41 acyl carrier protein Human endozepine (putative ligand of benzodiazepine receptor) 195 LG:446649.1:2000FEB18 g181960 2.00E-35 mRNA, complete cds. 



  196 LG:132147.3:2000FEB18 g6446606 0 E3 ub@quitin ligase SMURF1 (Homo sapiens) 197 LI:036034.1:2000FEB01 g9622856 1.00E-33 sorting nexin 15A (Homo sapiens) dJ20B11.1 (ortholog of rat RSEC5 (mammalian exocyst complex subunit)) 198 LG:162161.1:2000MAY19 g5823961 3.00E-87 (Homo sapiens) 199 LG:407214.10:2000MAY19 g9963839 4.00E-54 lipase (Homo sapiens) 200 LG:204626.1:2000MAY19 g3243240 5.10E-41 syntaxin 11 201 LI:007401.1:2000MAY01 g4512103 3.00E-81 rab11 binding protein (Bos @aurus) 202 LI:476342.1:2000MAY01 g790641 2.00E-21 gamma-thionin (Hordeum vulgare) 203 LI:1072759.1:2000MAY01 g2367625 4.00E-21 protein synthesis elongation factor 1-alpha (Rhodotorula mucllaginosa) 204 LG:998857.1:2000FEB18 g2731641 6.10E-13 Fas-ligand associated factor 3 Human genomic DNA of 8p21.3-p22 anti-oncogene of hepatocellular 205 LG:482261.1:

  2000FEB18 94003386 0 colorectal and non-small Cell lung cancer, segment 9/11. 



  206 LG:480328.1:2000FEB18 g246482 0 prohibitin (Human, mRNA, 1043 n@). 



  207 LG:311197.1:2000MAY19 g505033 8.00E-62 mitogen inducible gene mig-2 (Homo sapiens) 208 LG:1054883.1:2000MAY19 g325464 0 Human endogenous retrovirus type C oncovirus sequence. 



  209 LG:399395.1:2000MAY19 g1177607 8.00E-10 pval (Plasmodium vivax) 210 LG:380497.2:2000MAY19 g10504238 3.00E-88 hepatocellular carcinoma-related putative tumor suppressor (Homo 211 LI:272913.22:2000MAY01 g4982485 3.00E-59 apoptosis related protein APR-3 (Homo sapiens)   

 <Desc/Clms Page number 189> 

   TABLE 2 SEQ ID NO:

   Termplate ID Start Stop Frame Pfam Hit Pfam Description E-value 1 LG:1040582.1:2000FEB18 267 539 forward 3 aldo¯ket¯red Aldo/keto reductase family 2.50E-51 2 LG:453570.1:2000FEB18 186 605 forward 3 Glyoxalase Glyoxalase 3.80E-72 3 LG:408751.3:2000FEB18 194 1345 forward 2 Dihydroorafase Dihydroorotase-like 1.40E-19 4 LI:090574.1:2000FEB01 60 776 forward 3 carb¯anhydrase Eukaryotic-type carbonic anhydrase 9.70E-144 6 LI:332176.1:2000FEB01 2 961 forward 2 Glyco¯hydro¯31 Glycosyl hydrolases family 31 4.10E-144 7 LI:403248.2:2000FEB01 191 367 forward 2 Orn¯DAP¯Arg¯deC Pyridoxal-dependent decarboxylase 1.40E-12 8 LG:220992.1:2000MAY19 156 1556 forward 3 Amidase Amidase 1.10E-153 9 LG:1094571.1:2000MAY19 328 720 forward 1 FAD¯Synth Riboflavin kinase / FAD synthetase 2.30E-42     10 LI:350754.4:

  2000MAY01 855 1121 forward 3 Transglut¯core Transglutaminase-like superfamily 2.90E-47 10 LI:350754.4;2000MAY01 1455 2132 forward 3 Transglutamin¯C Transglutaminase family 3.20E-106 10 LI:350754.4:2000MAY01 54 413 forward 3 Transglutamin¯N Transglutaminase family 2.50E-63 11 LI:255828.29:2000MAY01 2 367 forward 2 PK Pyruvate kinase 7.00E-71 11 LI:255828.29:2000MAY01 348 512 forward 3 PK Pyruvate kinase 5.70E-24 12 LI:1190263.1:2000MAY01 281 1750 forward 2 Sulfatase Sulfatase 8.60E-66 14 LG:999414.3:2000FEB18 718 1038 forward 1 7tm¯1 7 transmembrane receptor (rhodopsin family) 3.60E-13 14 LG:999414.3:2000FEB18 1115 1453 forward 2 7tm¯1 7 transmembrane receptor (rhodopsin family) 4.30E-07 18 LG:068682.1:2000FEB18 176 883 forward 2 pkinase Eukaryotic protein kinase domain 1.70E-65 19 LG:242665.1:2000FEB18 190 747 forward 1 ras Ras family 2.30E-34 20 LG:241743.1:

  2000FEB18 199 345 forward 1 PH PH domain 8.00E-06 22 LG:344886.1:2000MAY19 379 957 forward 1 ras Ras family 1.70E-17 25 LG:898771.1:2000MAY19 525 662 forward 3 Fork¯head Fork head domain 1.70E-25 28 LI:1085273.2:2000MAY01 285 1070 forward 3 DSPc Dual specificity phosphatase, catalytic domain 1.30E-39 29 LI:333138.2:2000MAY01 291 1016 forward 3 pkinase Eukaryotic protein kinase domain 2.00E-90 32 LG998283.7:2000FEB18 370 630 forward 1bromodomain Bromodomain 2.60E-29 32 LG998283.7:2000FEB18 4 153 forward 1 PHD PHD-finger 1.90E-12 34 LI:175223.1:2000FEB01 210 431 forward 3 CSD 'Cold-shock' DNA-binding domair 1.40E-18 38 LG:021371.3:2000FEB18 932 1000 forward 2 zf-C2H2 Zinc finger, C2H2 type 2.10E-04 39 LG:475404.1:2000FEB18 176 328 forward 2 KRAB KRAB box 1.10E-15 40 LG:979406.2:2000FEB18 85 273 forward 1 KRAB KRAB box 2.40E-34 41 LG:410726.1:

  2000FEB18 646 834 forward 1 KRAB KRAB box 2.10E-17 41 LG:410726.1:2000FEB18 274 558 forward 1SCAN SCAN domain 8.90E-55 43 LG:1076828.1:2000FEB18 448 516 forward 1 zf-C2H2 Zinc finger, C2H2 type 2.00E-07 44 LG:1076931.1:2000FEB18 173 310 forward 2 KRAB KRAB box 3.40E-21 45 LG:1078121.1:2000FEB18 186 374 forward 3 KRAB KRAB box 2.80E-41   

 <Desc/Clms Page number 190> 

   46 LG:1079203.1:2000FEB18 421 489 forward 1 zf-C2H2 Zinc finger, C2H2 type 6.00E-06 46 LG:1079203.1:2000FEB18 647 715 forward 2 zf-C2H2 Zinc finger, C2H2 type 9.20E-05 47 LG:1082586.1:2000FEB18 414 536 forward 3 KRAB KRAB box 6.80E-12 48 LG:1082774.1:2000FEB18 138 326 forward 3 KRAB KRAB box 1.30E-40 49 LG:1082775.1:2000FEB18 45 230 forward 3 KRAB KRAB box 7.10E-39 49 LG:1082775.1:2000FEB18 840 908 forward 3 zf-C2H2 Zinc finger, C2H2 type 4.40E-05 50 LG:1083120.1:

  2000FEB18 117 266 forward 3 KRAB KRAB box 5.10E-22 51 LG:1087707.1:2000FEB18 162 350 forward 3 KRAB KRAB box 2.80E-40 52 LG:1090915.1:2000FEB18 129 251 forward 3 KRAB KRAB box 7.40E-22 53 LG:1094230.1:2000FEB18 120 308 forward 3 KRAB KRAB box 3.70E-41 54 LG:474848.3:2000FEB18 253 441 forward 1 KRAB KRAB box 2.10E-38 55 LI:251656.1:2000FEB01 242 310 forward 2 zf-C2H2 Zinc finger, C2H2 type 3.90E-08 56 LI:021371.1:2000FEB01 717 785 forward 3 zf-C2H2 Zinc finger, C2H2 type 2.10E-04 57 LI:133095.1:2000FEB01 539 607 forward 2 zf-C2H2 Zinc finger, C2H2 type 4.30E-06 58 LI:236654.2:2000FEB01 805 873 forward 1 zf-C2H2 Zinc finger, C2H2 type 1.40E-04 59 LI:200009.1:2000FEB01 564 632 forward 3 zf-C2H2 Zinc finger, C2H2 type 1.80E-05 60 LI:758502.1:2000FEB01 633 701 forward 3 zf-C2H2 Zinc finger, C2H2 type 2.50E-07 62 LI:789445.1:2000FEB01 71 262 forward 2 KRAB KRAB box 1.60E-27 63 LI:

  789657.1:2000FEB01 542 610 forward 2 zf-C2H2 Zinc finger, C2H2 type 2.60E-06 64 LI:789808.1:2000FEB01 272 340 forward 2 zf-C2H2 Zinc finger, C2H2 type 1.00E-07 64 LI:789808.1:2000FEB01 426 494 forward 3 zf-C2H2 Zinc finger, C2H2 type 3.40E-04 65 LI:792919.1:2000FEB01 31 99 forward 1 zf-C2H2 Zinc finger, C2H2 type 5.30E-06 66 LI:793949.1:2000FEB01 120 308 forward 3 KRAB KRAB box 1.70E-41 67 LI:794389.1:2000FEB01 75 143 forward 3 zf-C2H2 Zinc finger, C2H2 type 8.70E-06 68 LI:796010.1:2000FEB01 276 344 forward 3 zf-C2H2 Zinc finger, C2H2 type 1.00E-07 68 LI:796010.1:2000FEB01 433 501 forward 1 zf-C2H2 Zinc finger, C2H2 type 3.40E-04 69 LI:796324.1:2000FEB01 290 358 forward 2 zf-C2H2 Zinc finger, C2H2 type 1.00E-07 69 LI:796324.1:2000FEB01 450 518 forward 3 zf-C2H2 Zinc finger, C2H2 type 3.40E-04 70 LI:796373.1:2000FEB01 181 249 forward 1 zf-C2H2 Zinc finger, C2H2 type 1.10E-06 71 LI:

  796415.1:2000FEB01 45 230 forward 3 KRAB KRAB box 7.10E-39 72 LI:798636.1:2000FEB01 329 397 forward 2 zf-C2H2 Zinc finger, C2H2 type 2.60E-07 73 LI:800045.1:2000FEB01 364 432 forward 1 zf-C2H2 Zinc finger, C2H2 type 5.30E-07 74 LI:800680.1:2000FEB01 155 319 forward 2 KRAB KRAB box 5.00E-21 75 LI:800894.1:2000FEB01 125 313 forward 2 KRAB KRAB box 6.50E-40 76 LI:801015.1:2000FEB01 22 216 forward 1 KRAB KRAB box 3.00E-24 77 LI:801236.1:2000FEB01 225 293 forward 3 zf-C2H2 Zinc finger, C2H2 type 4.40E-07   

 <Desc/Clms Page number 191> 

   78 LI:803335.1:2000FEB01 220 408 forward 1 KRAB KRAB box 2.10E-38 79 LI:803998.1:2000FEB01 62 130 forward 2 zf-C2H2 Zinc finger, C2H2 type 1.20E-05 80 LI:478757.1:2000FEB01 467 643 forward 2 KRAB KRAB box 2.40E-21 81 LI:808532.1:2000FEB01 53 121 forward 2 zf-C2H2 Zinc finger, C2H2 type 5.70E-05 82 LI:443073.1:

  2000FEB01 176 244 forward 2 zf-C2H2 Zinc finger, C2H2 type 2.50E-05 83 LI:479671.1:2000FEB01 160 312 forward 1 KRAB KRAB box 1.70E-19 84 LI:810078.1:2000FEB01 424 492 forward 1 zf-C2H2 Zinc finger, C2H2 type 1.80E-06 84 LI:810078.1:2000FEB01 587 655 forward 2 zf-C2H2 Zinc finger, C2H2 type 1.20E-05 85 LI:810224.1:2000FEB01 171 239 forward 3 zf-C2H2 Zinc finger, C2H2 type 1.00E-07 86 LI:817052.2:2000FEB01 901 969 forward 1 zf-C2H2 Zinc finger, C2H2 type 8.90E-08 87 LG:892274.1:2000MAY19 96 461 forward 3 dUTPase dUTPase 9.20E-27 87 LG:892274.1:2000MAY19 489 75 forward 3 rvp Retroviral aspartyl protease 5.30E-11 88 LG:1080959.1:2000MAY19 182 322 forward 2 KRAB KRAB box 2.00E-16 89 LG:1054900.1:2000MAY19 78 218 forward 3 KRAB KRAB box 2.30E-17 90 LG:1077357.1:2000MAY19 94 282 forward 1 KRAB KRAB box 4.80E-31 91 LG:1084051.1:

  2000MAY19 195 263 forward 3 zf-C2H2 Zinc finger, C2H2 type 1.80E-06 92 LG:1076853.1:2000MAY19 706 774 forward 1 zf-C2H2 Zinc finger, C2H2 type 1.50E-07 93 LG:481631.10:2000MAY19 96 263 forward 3 KRAB KRAB box 5.70E-25 93 LG:481631.10:2000MAY19 882 950 forward 3 zf-C2H2 Zinc finger, C2H2 type 1.70E-05 94 LG:1088431.2:2000MAY19 175 339 forward 1 KRAB KRAB box 5.00E-21 96 LI:1144007.1:2000MAY01 914 1108 forward 2 KRAB KRAB box 5.90E-05 96 LI:1144007.1:2000MAY01 323 610 forward 2 SCAN SCAN domain 4.10E-60 97 LI:331074.1:2000MAY01 194 262 forward 2 zf-C2H2 Zinc finger, C2H2 type 1.00E-03 98 LI:1170349.1:2000MAY01 185 370 forward 2 KRAB KRAB box 2.50E-29 98 LI:1170349.1:2000MAY01 740 808 forward 2 zf-C2H2 Zinc finger, C2H2 type 5.80E-05 102 LG:101269.1:2000MAY19 556 831 forward 1 IRK Inward rectifier potassium channel 3.50E-65 104 LI:410188.1:

  2000MAY01 3760 4569 forward 1 ion¯trans Ion transport protein 3.70E-97 104 LI:410188.1:2000MAY01 4586 5314 forward 2 ion¯trans Ion transport protein 3.30E-66 105 LI:1188288.1:2000MAY01 751 1215 forward 1 SNF Sodium:neurotroansmitter symporter family 8.50E-113 105 LI:1188288.1:2000MAY01 423 782 forward 3 SNF Sodium:neurotransmitter symporter family 8.60E-74 105 LI:1188288.1:2000MAY01 1187 1438 forward 2 SNF Sodium:neurotransmitter symporter family 5.50E-52 107 LG:451682.1:2000FEB18 117 560 forward 3 proteasome Proteasome A-type and B-type 4.40E-59 108 LG:1077283.1:2000FEB18 110 427 forward 2 pro¯isomerase Cyclophilin type peptidyl-prolyl cis-trans isomerase 1.80E-37 108 LG:1077283.1:2000FEB18 177 278 forward 3 pro¯isomerase Cyclophilin type peptidyl-proly cis-trans isomerase 1.30E-18 109 LG:481436.5:2000FEB18 351 539 forward 3 DnaJ DnaJ domain 2.80E-28 111 LI:373637.1:

  2000FEB01 17 217 forward 2 DnaJ DnaJ domain 6.30E-39   

 <Desc/Clms Page number 192> 

   113 LI:053826.1:2000MAY01 834 1106 forward 3 SCP SCP-like extracelluiar protein 1.10E-17 114 LI:449393.1:2000MAY01 90 788 forward 3 cpn60¯TCP1 TCP-1/cpn60 chaperonin family 9.80E-66 117 LG:345527.1:2000FEB18 667 957 forward 1 helicase¯C Helicases conseved C-terminal domain 7.20E-21 117 LG:345527.1:2000FEB18 8 631 forward 2 SNF2¯N SNF2 and others N-terminal domain 7.20E-44 122 LI:335671.2:2000FEB01 188 475 forward 2 helicase¯C Helicases conserved C-terminal domain 9.10E-13 122 LI:335671.2:2000FEB01 3 95 forward 3 SNF2¯N SNF2 and others N-terminal domain 7.10E-06 128 LG:247388.1:2000MAY19 346 600 forward 1 helicase¯C Helicases conserved C-terminal domain 2.70E-19 128 LG:247388.1:2000MAY19 3 173 forward 3 SNF2¯N SNF2 and others N-terminal domain 1.60E-14 131 LI:331610.2:

  2000MAY01 1415 1699 forward 2 cadherin Cadherin domain 6.00E-20 135 LI:903225.1:2000FEB01 603 764 forward 3 Ribosomal¯L23 Ribosomal protein L23 4.80E-14 138 LI:1169865.1:2000MAY01 593 790 forward 2 ig Immunoglobulin domain 2.30E-08 138 LI:1169865.1:2000MAY01 242 547 forward 2 MHC¯II¯alpha Class II histocompatibillty antigen, aipha domain 1.80E-65 139 LG337818.2:2000FEB18 136 1518 forward 1 p450 Cytochrome P450 1.50E-173 140 LI:337818.1:2000FEB01 654 998 forward 3 p450 Cytochrome P450 3.50E-45 140 LI:337818:1.2000FEB01 136 384 forward 1 p450 Cytochrome P450 4.40E-27 140 LI:337818.1:2000FEB01 359 673 forward 2 p450 Cytochrome P450 5.40E-27 143 LI:414307.1:2000FEB01 590 964 forward 2 C1q C1q domain 2.30E-38 143 LI:414307.1:2000FEB01 365 544 forward 2 Collagen Collagen triple helix repeat (20 copies) 2.50E-10 144 LI:202943.2:

  2000FEB01 36 209 forward 3 sushi Sushi domain (SCR repeat) 1.40E-09 147 LG:120744.1:2000MAY19 301 813 forward 1 vwa von Willebrand factor type A domain 2.00E-51 148 LI:757520.1:2000MAY01 427 1362 forward 1 filament Intermediate filament proteins 7.10E-157 149 LG:160570.1:2000FEB18 260 562 forward 2 Band¯41 FERM domain (Band 4.1 family) 1.60E-22 152 LI:401605.2:2000FEB01 1 129 forward 1 myosin¯head Myosin head (motor domain) 5.90E-07 153 LI:329017.1:2000FEB01 226 336 forward 1 WD40 WD domain, G-beta repeat 5.10E-06 154 LI:401322.1:2000FEB01 156 341 forward 3 tubulin Tubulin/FtsZ family 7.10E-20 154 LI:401322.1:2000FEB01 371 478 forward 2 tubulin Tubulin/FtsZ family 2.50E-06 155 LG:403409.1:2000MAY19 1458 1652 forward 3 FHA FHA domain 3.00E-04 155 LG:403409.1:2000MAY19 78 1193 forward 3 kinesin Kinesin motor domain 6.80E-172 156 LG:233933.5:

  2000MAY19 258 356 forward 3 ank Ank repeat 4.90E-06 157 LI:290344.1:2000MAY01 992 1312 forward 2 spectrin Spectrin repeat 4.10E-07 157 LI:290344.1:2000MAY01 1361 1450 forward 2 WW WW domain 5.40E-08 158 LI:410742.1:2000MAY01 599 889 forward 2 cadherin Cadherin domain 1.80E-21 158 LI:410742.1:2000MAY01 1224 1520 forward 2 cadherin Cadherin domain 9.90E-04 161 LI:347687.113:2000MAY01 214 855 forward 1 Myelin PLP Myelin proteolipid protein (PLP or lipophilin) 7.10E-160 163 LG:45170.1:2000FEB18 130 459 forward 1 Ribosomal¯L32e Ribosomal protein L32 4.80E-57 164 LG:455771.1:200FEB18 69 473 forward 3 Ribosomal¯S12 Ribosomal protein S12 6.60E-78   

 <Desc/Clms Page number 193> 

   165 LG:452089.1:2000FEB18 107 268 forward 2 Ribosomal¯L5 Ribosomal protein L5 2.40E-25 165 LG:452089.1:

  2000FEB18 278 577 forward 2 ribosomal¯L5¯C ribosomal L5P family C-terminus 2.70E-60 166 LG:246415.1:2000FEB18 27 365 forward 3 Ribosomal¯S26e Ribosomal protein S26e 2.40E-59 168 LG:1101445.1:2000FEB18 306 464 forward 3 Ribosomal¯L30 Ribosomal protein L30p/L7e 4.20E-28 171 LI:246422.1:2000FEB01 53 397 forward 2 Ribosomal¯L22e Ribosomal L22e protein family 4.30E-28 171 LI:246422.1:2000FEB01 64 318 forward 1 Ribosomal¯L22e Ribosomal L22e protein family 7.70E-07 172 LG:449404.1:2000MAY19 175 531 forward 1 Ribosomal¯S11 Ribosomal protein S11 6.90E-77 173 LG:449413.1:2000MAY19 99 368 forward 3 Ribosomal¯S12 Ribosomal protein S12 6.10E-47 173 LG:449413.1:2000MAY19 367 504 forward 1 Ribosomal¯S12 Ribosomal protein S12 3.20E-21 174 LG:450105.1:2000MAY19 86 490 forward 2 Ribosomal¯S12 Ribosomal protein S12 6.60E-78 175 LG:460809.1:

  2000MAY19 3 236 forward 3 Ribosomal¯L31e Ribosomal protein L31e 6.00E-17 176 LG:481781.1:2000MAY19 243 671 forward 3 Ribosomal¯S4e Ribosomal family S4e 1.40E-97 177 LG:1101153.1:2000MAY19 89 499 forward 2 Ribosomal¯L22 Ribosomal protein L22p/L17e 5.20E-76 178 LI:257695.20:2000MAY01 110 673 forward 2 Ribosomal¯S9 Ribosomal protein S9/S16 1.60E-40 179 LI:455771.1:2000MAY01 69 473 forward 3 Ribosomal¯S12 Ribosomal protein S12 6.60E-78 181 LI:035973.1:2000MAY01 318 479 forward 3 Ribosomal¯L37e Ribosomal protein L37e 1.60E-13 183 LG:247781.2:2000FEB18 142 426 forward 1 mito¯carr Mitochondrial carrier proteins 1.80E-24 190 LI:900264.2:2000MAY01 1151 1555 forward 2 PP2C Protein phosphatase 2C 2.90E-11 192 LI:1189543.1:2000MAY01 1292 1447 forward 2 KH-domain KH domain 2.50E-13 192 LI:1189543.1:2000MAY01 592 744 forward 1 KH-domain KH domain 5.40E-13 193 LG:455450.1:

  2000FEB18 1 426 forward 1 dehydrin Dehydrins 4.20E-41 194 LG:1040978.1:2000FEB18 278 481 forward 2 pp-binding Phosphopantethein attachment site 3.90E-14 195 LG:446649.1:2000FEB18 80 316 forward 2 ACBP Acyl CoA binding protein 1.60E-44 196 LG:132147.3:2000FEB18 1497 2414 forward 3 HECT HECT-domain (ubiquitin-transferase). 9.90E-138 196 LG:132147.3:2000FEB18 1065 1154 forward 3 WW WW domain 1.50E-12 198 LG:162161.1:2000MAY19 128 385 forward 2 TIG IPT/TIG domain 5.50E-15 200 LG:204626.1:2000MAY19 322 1212 forward 1 Syntaxin Syntaxin 8.60E-44 202 LI:476342.1:200MAY01 159 299 forward 3 Gamma-thionin Gamma-thionins family 1.70E-19 205 LG:482261.1:200FEB18 286 552 forward 1 Gag¯p10 Retroviral GAG p10 protein 6,80E-31 205 LG:482261.1:2000FEB18 1044 1229 forward 3 gag¯p24 gag gene protein p24 (core nucleocapsld 2.00E-15 205 LG:482261.1:

  2000FEB18 1375 1545 forward 1 gag¯p24 gag gene protein p24 (core nucleocapsid 9.50E-15 206 LG:480328.1:2000FEB18 985 1515 forward 1 Band¯7 SPFH domain / Band 7 family 2.60E-39 206 LG:480328.1:2000FEB18 49 117 forward 1 zf-C2H2 Zinc finger, C2H2 type 1.60E-06 210 LG:380497.2:2000MAY19 202 336 forward 1 G-patch G-patch domain 7.00E-17   

 <Desc/Clms Page number 194> 

 
TABLE 3 
Template   ID Start Stop Frame Domain Topology     SEQ ID NO   :

   Type 
1 LG : 1040582. 1 : 2000FEB18 31 117 forward 1 TM N in   1 LG   : 1040582. 1 : 2000FEB18 319 405 forward 1 TM N in 
1 LG :   1040582. 1   :   2000FEB18 108 155 forward 3 TM N out     2 LG   : 453570. 1 :   2000FEB18 361 447 forward 1 TM N in     3 LG   : 408751. 3 :   2000FEB18 1318 1404 forward 1 TM N in     3 LG   : 408751. 3 :   2000FEB18 1025 1099 forward 2 TM N in     3 LG   : 408751. 3 :   2000FEB18 1298 1360 forward 2 TM N in     3 LG   : 408751. 3 : 2000FEB18   1379 1441 forward 2 TM N in     3 LG   : 408751. 3 :   2000FEB18 1463 1537 forward 2 TM N in     3 LG   : 408751. 3 :

     2000FEB18 1047 1133 forward3 TM N in   
3 LG: 408751. 3 :   2000FEB18 1266 1352 forward 3 TM N in     3 LG   : 408751. 3 :   2000FEB18 1419 1469 forward 3 TM N in     4 LI   : 090574. 1 :   2000FEB01 79 144 forward 1 TM N in     4 LI   : 090574. 1 : 2000FEB01 607 678 forward 1 TM N in   4 LI   : 090574.   1   : 2000FEB01 1009 1080 forward 1 TM N in   4 Ll   : 090574. 1 : 2000FEB01 497 583 forward 2 TM N out   4 LI   : 090574. 1 : 2000FEB01 743 829 forward 2 TM N out 
4 LI: 090574. 1 :   2000FEB01 1026 1085 forward 3 TM N out     5 LI   : 229932. 2 :   2000FEB01 76 162 forward 1 TM N out     5 LI   : 229932. 2 :

   2000FEB01 190 276 forward 1 TM N out 
5 LI : 229932. 2 : 2000FEB01 1237 1323 forward 1 TM N out 
5 LI : 229932. 2 : 2000FEB01 68 142 forward 2 TM N in 
5 LI : 229932. 2 : 2000FEB01 335 412 forward 2 TM N in 
5 LI : 229932. 2 :   2000FEB01 758 844 forward 2 TM N in   
5 LI : 229932. 2 : 2000FEB01 1229 1288 forward 2 TM N in 
5 LI : 229932. 2 : 2000FEB01 60 146 forward 3 TM N in 
5 LI : 229932. 2 :   2000FEB01 216 302 forward 3 TM N in   
5 LI : 229932. 2 : 2000FEB01 690 752 forward 3 TM N in 
5 LI : 229932. 2 :   2000FEB01 765 827 forward 3 TM N in   
5 LI : 229932. 2 : 2000FEB01 1209 1289 forward 3 TM N in 
6 LI : 332176. 1 : 2000FEB01 343 399 forward 1 TM N in 
6 LI : 332176. 1 : 2000FEB01 1078 1131 forward 1 TM N in 
6 LI : 332176. 1 :   2000FEB01 1606 1692 forward 1 TM N in   
6 LI : 332176. 1 :

     2000FEB01 2218 2274 forward 1 TM N in   
6 LI : 332176. 1 : 2000FEB01 2383 2433 forward 1 TM N in 
6 LI : 332176. 1 : 2000FEB01 110 196 forward 2 TM N in 
6 LI :   332176.   1 : 2000FEB01 1307 1378 forward 2 TM N in   6 LI   : 332176. 1 : 2000FEB01 1640 1726 forward 2 TM N in 
6 LI : 332176. 1 : 2000FEB01 1946 2005 forward 2 TM N in 
6 LI : 332176. 1 : 2000FEB01 135 200 forward 3 TM N in 
6 LI : 332176. 1 : 2000FEB01 693 752 forward 3 TM N in 
6 LI : 332176. 1 : 2000FEB01 777 839 forward 3 TM N in 
6 LI : 332176. 1 :   2000FEB01 867 929 forward 3 TM N in   
6 LI : 332176. 1 : 2000FEB01 1035 1118 forward 3 TM N in 
6 LI : 332176. 1 : 2000FEB01 1173 1253 forward 3 TM N in 
6 LI : 332176. 1 :   2000FEB01 1572 1658 forward 3 TM N in   
6 LI : 332176. 1 :

     2000FEB01 2121 2180 forward 3 TM N in   
6 LI : 332176. 1 : 2000FEB01 2277 2363 forward 3 TM N in 
6 LI : 332176. 1 :   2000FEB01 2400 2456 forward 3 TM N in     8 LG   : 220992. 1 :   2000MAY19 343 393 forward 1 TM     8 LG   : 220992. 1 :   2000MAY19 646 732 forward 1 TM     8 LG   : 220992. 1 :   2000MAY19 1639 1725 forward 1 TM     8 LG   : 220992. 1 : 2000MAY19 1879 1965 forward 1 TM 

 <Desc/Clms Page number 195> 

   8 LG   : 220992. 1 :   2000MAY19 2005 2088 forward 1 TM     8 LG   : 220992. 1 :   2000MAY19 17 76 forward 2 TM N in     8 LG   : 220992. 1 : 2000MAY19 1646 1732 forward 2 TM N in   8 LG   :

   220992. 1 :   2000MAY19 1850 1933 forward 2 TM N in     8 LG   : 220992. 1 :   2000MAY19 1434 1484 forward 3 TM N out     8 LG   : 220992. 1 :   2000MAY19 1734 1820 forward 3 TM N out     8 LG   : 220992. 1 :   2000MAY19 1974 2036 forward 3 TM N out     8 LG   : 220992. 1 : 2000MAY19 2067 2129 forward 3 TM N out   8 LG   : 220992. 1 : 2000MAY19 2151 2237 forward 3 TM N out   9 LG   :   1094571.   1 :   2000MAY19 781 867 forward 1 TM N in     9 LG   :   1094571.   1 :   2000MAY19 419 505 forward 2 TM N in     9 LG   :   1094571.   1 :

   2000MAY19 767 853 forward 2 TM N in   9 LG   :   1094571.   1 :   2000MAY19 756 842 forward 3 TM N in   10 LI :   350754.     4   : 2000MAY01 277 348 forward 1 TM N in 10 LI : 350754. 4 : 2000MAY01 583 651 forward 1 TM N in 10 LI : 350754. 4 : 2000MAY01 670 747 forward 1 TM N in 10 LI : 350754.   4   : 2000MAY01 381 467 forward 3 TM N in 10 LI : 350754. 4 : 2000MAY01 2469 2555 forward 3 TM N in   12 Ll   :   1190263.   1 : 2000MAY01 664 735 forward 1 TM N in   12 Ll   : 1190263.1 : 2000MAY01 787 861 forward 1 TM N in 12 LI :   1190263.   1 : 2000MAY01 901 954 forward 1 TM N in   12 Ll   :   1190263.   1 : 2000MAY01 188 274 forward 2 TM N in   12 Ll   :

     1190263.   1 : 2000MAY01 455 508 forward 2 TM N in   12 Ll   :   1190263.   1 : 2000MAY01 809 895 forward 2 TM N in   12 Ll   :   1190263.   1 :   2000MAY01 1616 1663 forward 2 TM N in     12 Ll   :   1190263.   1 : 2000MAY01 183 251 forward 3 TM N in 12 LI :   1190263.   1 : 2000MAY01 648 704 forward 3 TM N in   12 Ll   :   1190263.   1 : 2000MAY01 1149 1235 forward 3 TM N in   13 LG   : 270916. 2 : 2000FEB18 173 259 forward 2 TM N out   14 LG   : 999414. 3 : 2000FEB18 109 195 forward 1 TM N out   14 LG   : 999414. 3 :   2000FEB18 358 438 forward 1 TM N out     14 LG   : 999414. 3 :

   2000FEB18 520 591 forward 1 TM N out   14 LG   : 999414. 3 : 2000FEB18 661 744 forward 1 TM N out   14 LG   : 999414. 3 : 2000FEB18 883 969 forward 1 TM N out   14 LG   : 999414. 3 : 2000FEB18 976 1062 forward 1 TM N out   14 LG   : 999414. 3 :   2000FEB18 302 388 forward 2 TM N in     14 LG   : 999414. 3 :   2000FEB18 533 613 forward 2 TM N in     14 LG   : 999414. 3 :   2000FEB18 992 1048 forward 2 TM N in     14 LG   : 999414. 3 :   2000FEB18 1169 1246 forward 2 TM N in     14 LG   : 999414. 3 :   2000FEB18 1307 1366 forward 2 TM N in     14 LG   : 999414. 3 : 2000FEB18 207 284 forward 3 TM N out   14 LG   :

   999414. 3 :   2000FEB18 324 404 forward 3 TM N out   14 LG :   999414.   3 : 2000FEB18 540 599 forward 3 TM N out   14 LG   : 999414. 3 : 2000FEB18 1029 1115 forward 3 TM N out   14 LG   : 999414. 3 :   2000FEB18 1167 1253 forward 3 TM N out     14 LG   : 999414. 3 :   2000FEB18 1314 1373 forward 3 TM N out     15 LG   : 429446. 1 : 2000FEB18 628 699 forward 1 TM N out   15 LG   : 429446. 1 :   2000FEB18 629 682 forward 2 TM N in     15 LG   : 429446. 1 :   2000FEB18 627 713 forward 3 TM N in   16 LI : 057229. 1 :   2000FEB01 10 69 forward 1 TM   16 LI : 057229. 1 : 2000FEB01 118 198 forward 1 TM 16 LI : 057229. 1 :

     2000FEB01 292 360 forward 1 TM   16 LI : 057229. 1 : 2000FEB01 11 67 forward 2 TM 16 LI : 057229. 1 : 2000FEB01 146 226 forward 2 TM 16 LI : 057229. 1 : 2000FEB01 290 355 forward 2 TM 16 LI : 057229. 1 : 2000FEB01 12 71 forward 3 TM N out 

 <Desc/Clms Page number 196> 

   16 LI   : 057229. 1 :   2000FEB01 114 176 forward 3 TM N out   17 LI : 351965. 1 : 2000FEB01 487 573 forward 1 TM 17 LI : 351965. 1 : 2000FEB01 1036 1098 forward 1 TM 17 LI : 351965. 1 : 2000FEB01 492 578 forward 3 TM N in   17 LI   : 351965. 1 : 2000FEB01 969 1055 forward 3 TM N in 17 LI : 351965. 1 : 2000FEB01 1098 1184 forward 3 TM N in   18 LG   : 068682.   1   : 2000FEB18 707 793 forward 2 TM N out   19 LG   : 242665. 1 :

     2000FEB18 10 63 forward 1 TM N out     19 LG   : 242665. 1 :   2000FEB18 12 62 forward 3 TM N out     19 LG   : 242665. 1 :   2000FEB18 333 398 forward 3 TM N out   20 LG : 241743. 1 :   2000FEB18 43 99 forward 1 TM N out   21 LI : 034212. 1 : 2000FEB01 1300 1365 forward 1 TM N in   21 LI   : 034212. 1 : 2000FEB01 1570 1647 forward 1 TM N in   21 LI   : 034212. 1 :   2000FEB01 2386 2472 forward 1 TM N in   21 LI :   034212.   1 :   2000FEB01 2533 2598 forward 1 TM N in   21 LI : 034212. 1 :   2000FEB01 2620 2706 forward 1 TM N in   21 LI : 034212. 1 :   2000FEB01 2740 2826 forward 1 TM N in   21 LI : 034212. 1 : 2000FEB01 719 805 forward 2 TM 21 LI :

   034212. 1 : 2000FEB01 1205 1291 forward 2 TM 21 LI : 034212. 1 : 2000FEB01 1460 1546 forward 2 TM 21 LI :   034212.   1 : 2000FEB01 1685 1768 forward 2 TM 21 LI : 034212. 1 : 2000FEB01 1814 1882 forward 2 TM 21 LI : 034212.   1   :   2000FEB01 2066 2128 forward 2 TM   21 LI : 034212. 1 : 2000FEB01 2156 2218 forward 2 TM 21 LI : 034212. 1 :   2000FEB01 2540 2626 forward 2 TM   21 LI : 034212. 1 : 2000FEB01 2657 2734 forward 2 TM 21 lI : 034212. 1 :   2000FEB01 12 62 forward 3 TM N out   21 LI : 034212. 1 : 2000FEB01 1236 1301 forward 3 TM N out 21 LI : 034212. 1 : 2000FEB01 1590 1646 forward 3 TM N out 21 LI : 034212. 1 : 2000FEB01 1668 1721 forward 3 TM N out 21 LI :   034212.   1 :   2000FEB01 2130 2216 forward 3 TM N out   21 LI : 034212. 1 :

   2000FEB01 2295 2381 forward 3 TM N out 21 LI : 034212. 1 : 2000FEB01 2436 2513 forward 3 TM N out 21 LI : 034212. 1 : 2000FEB01 2538 2624 forward 3 TM N out 21 LI :   034212.   1 : 2000FEB01 2667 2735 forward 3 TM N out 22 LG : 344886. 1 : 2000MAY19 937 1002 forward 1 TM N in 22 LG : 344886. 1 :   2000MAY19 1081 1155 forward 1 TM N in   22 LG : 344886. 1 :   2000MAY19 1696 1782 forward 1 TM N in   22 LG : 344886. 1 :   2000MAY19 413 463 forward 2 TM N in   22 LG : 344886. 1 :   2000MAY19 551 637 forward 2 TM N in   22 LG : 344886. 1 : 2000MAY19 950 1012 forward 2 TM N in 22 LG : 344886. 1 :   2000MAY19 1031 1093 forward 2 TM N in   22 LG : 344886. 1 :   2000MAY19 1112 1183 forward 2 TM N in   22 LG : 344886. 1 :

     2000MAY19 1271 1348 forward 2 TM N in   22 LG :   344886.   1 : 2000MAY19 1634 1720 forward 2 TM N in 22 LG : 344886. 1 :   2000MAY19 567 626 forward 3 TM N in   22 LG : 344886. 1 :   2000MAY1 g 1011 1073 forward 3 TM N in   22 LG : 344886. 1 :   2000MAY19 1089 1151 forward 3 TM N in   22 LG : 344886. 1 :   2000MAY19 1707 1757 forward 3 TM N in   23 LG : 228930. 1 :   2000MAY19 111 167 forward 3 TM N in   24 LG : 338927. 1 :   2000MAY19 934 1020 forward 1 TM N out   24 LG : 338927. 1 : 2000MAY19 1133 1219 forward 2 TM N in 24 LG : 338927. 1 : 2000MAY19 1170 1250 forward 3 TM N in 25 LG : 898771. 1 :   2000MAY19 1261 1314 forward 1 TM N out   25 LG : 898771. 1 :

     2000MAY19 1397 1450 forward 2 TM N out   26 LI : 257664. 67 : 2000MAY01 280 366 forward 1 TM N in 

 <Desc/Clms Page number 197> 

 26 LI : 257664. 67 : 2000MAY01 421 498 forward 1 TM N in   26 LI : 257664.   67 : 2000MAY01 12 71 forward 3 TM N out 27 LI :   001496.   2 :   2000MAY01 399 473 forward 3 TM     28 Ll   :   1085273.   2 : 2000MAY01 2188 2274 forward 1 TM N in 28 LI : 1085273. 2 : 2000MAY01 503 583 forward 2 TM N out 28 LI :   1085273.   2 : 2000MAY01 2126 2194 forward 2 TM N out 28 LI :   1085273.   2 :   2000MAY01 897 968 forward 3 TM N in   29 LI : 333138. 2 :   2000MAY01 1930 2016 forward 1 TM N out   29 LI : 333138. 2 :   2000MAY01 50 103 forward 2 TM   29 LI :

   333138. 2 : 2000MAY01 884 940 forward 2 TM 29 LI : 333138. 2 :   2000MAY01 114 179 forward 3 TM N out   29 LI : 333138. 2 : 2000MAY01 273 356 forward 3 TM N out 29 LI : 333138. 2 : 2000MAY01 819 875 forward 3 TM N out 29 LI : 333138. 2 : 2000MAY01 1581 1667 forward 3 TM N out 30 LI : 338927. 1 : 2000MAY01 1069 1140 forward 1 TM N in 30 LI : 338927. 1 : 2000MAY01 968 1051 forward 2 TM N in 30 LI : 338927.   1   : 2000MAY01 1056 1118 forward 3 TM N out 30 LI : 338927. 1 :   2000MAY01 1155 1217 forward 3 TM N out     31 LG   : 335558. 1 :   2000FEB18 518 604 forward 2 TM N in     31 LG   : 335558. 1 : 2000FEB18 614 682 forward 2 TM N in   31 LG   : 335558. 1 : 2000FEB18 761 829 forward 2 TM N in   31 LG   : 335558. 1 :

     2000FEB18 798 860 forward 3 TM N in     31 LG   : 335558. 1 :   2000FEB18 882 944 forward 3 TM N in     31 LG   : 335558. 1 : 2000FEB18 966 1028 forward 3 TM N in 32 LG : 998283. 7 :   2000FEB18 1066 1146 forward 1 TM N in   32 LG : 998283. 7 :   2000FEB18 23 109 forward 2 TM N in   32 LG : 998283. 7 :   2000FEB18 194 280 forward 2 TM N in   32 LG : 998283. 7 :   2000FEB18 392 478 forward 2 TM N in   32 LG :   998283.   7 : 2000FEB18 527 613 forward 2 TM N in 32 LG : 998283. 7 : 2000FEB18 776 862 forward 2 TM N in 32 LG: 998283. 7 : 2000FEB18 1064 1141 forward 2 TM N in 32 LG:998283. 7 : 2000FEB18 12 65 forward 3 TM N in 32 LG : 998283. 7 :   2000FEB18 147 227 forward 3 TM N in   32 LG : 998283. 7 :

     2000FEB18 684 770 forward 3 TM N in   32 LG : 998283. 7 :   2000FEB18 1011 1097 forward 3 TM N in   33 LI : 402739. 1 : 2000FEB01 415 501 forward 1 TM N in   35 LG   : 981076. 2 : 2000MAY19 388 450 forward 1 TM N in   35 LG   : 981076. 2 : 2000MAY19 20 82 forward 2 TM N out   35 LG   : 981076. 2 :   2000MAY19 389 451 forward 2 TM N out     35 LG   : 981076. 2 :   2000MAY19 464 526 forward 2 TM N out     35 LG   : 981076. 2 : 2000MAY19 539 604 forward 2 TM N out   35 LG   : 981076. 2 :   2000MAY19 438 524 forward 3 TM N in     37 Ll   :   1190250.   1 : 2000MAY01 530 613 forward 2 TM   37 Ll   :   1190250.   1 :

   2000MAY01 558 635 forward 3 TM N out   38 LG   : 021371. 3 : 2000FEB18 122 208 forward 2 TM N in   41 LG   : 410726. 1 :   2000FEB18 22 108 forward 1 TM N in     41 LG   : 410726. 1 : 2000FEB18 385 471 Forward 2 TM N in 42 LG : 200005. 1 : 2000FEB18 166 222 forward 1 TM N out 42 LG : 200005. 1 : 2000FEB18 185 232 forward 2 TM N out 42 LG : 200005. 1 :   2000FEB18 162 248 forward 3 TM N out   46 LG : 1079203.1 : 2000FEB18 11 70 forward 2 TM N in 46 LG :   1079203.   1 :   2000FEB18 125 196 forward 2 TM N in   46 LG :   1079203.   1 :   2000FEB18 965 1051 forward 2 TM N in     47 LG   :   1082586.   1 :   2000FEB18 256 339 forward 1 TM N in     47 LG   :

   1082586. 1 : 2000FEB18 248 316 forward 2 TM N out   49 LG   :   1082775.   1 :   2000FEB18 553 606 forward 1 TM N in   

 <Desc/Clms Page number 198> 

 50 LG :   1083120.   1 :   2000FEB18 214 291 forward 1 TM N out   50 LG :   1083120.   1 :   2000FEB18 233 319 forward 2 TM N out   50 LG : 1083120. 1 :   2000FEB18 252 320 forward 3 TM N in     51 LG   :   1087707.   1 :   2000FEB18 367 453 forward 1 TM N out     51 LG   :   1087707.   1 :   2000FEB18 469 531 forward 1 TM N out     51 LG   : 1087707. 1 :   2000FEB18 667 729 forward 1 TM N out     51 LG   :

     1087707.   1 :   2000FEB18 742 804 forward 1 TM N out     51 LG   :   1087707.   1 :   2000FEB18 407 481 forward 2 TM N in     51 LG   :   1087707.   1 :   2000FEB18 671 739 forward 2 TM N in     51 LG   :   1087707.   1 : 2000FEB18 743 811 forward 2 TM N in   51 LG   :   1087707.   1 :   2000FEB18 570 641 forward 3 TM N out     51 LG   : 1087707. 1 :   2000FEB1 8 747 833 forward 3 TM N out   52 LG :   1090915.   1 : 2000FEB18 11 61 forward 2 TM N in   53 LG   :   1094230.   1 :

     2000FEB18 469 555 forward 1 TM N out     53 LG   :   1094230. 1   :   2000FEB18 449 535 forward 2 TM N out     54 LG   : 474848. 3 :   2000FEB18 445 531 forward 1 TM N out     54 LG   : 474848. 3 :   2000FEB18 456 518 forward 3 TM N out     58 LI   : 236654. 2 : 2000FEB01 221 307 forward 2 TM N out 59 LI : 200009. 1 : 2000FEB01 1045 1131 forward 1 TM N out   59 LI   : 200009. 1 : 2000FEB01 1171 1233 forward 1 TM N out 59 LI : 200009. 1 :   2000FEB01 1076 1162 forward 2 TM N in   59 LI : 200009. 1 : 2000FEB01 1044 1130 forward 3 TM N in   60 LI   : 758502. 1 :   2000FEB01 286 369 forward 1 TM N out   60 Li : 758502. 1 :

   2000FEB01 755 805 forward 2 TM N in   60 LI : 758502.   1 : 2000FEB01 780 833 forward 3 TM N in   62 LI   : 789445. 1 : 2000FEB01 9 80 forward 3 TM N out   63 LI   : 789657. 1 : 2000FEB01 854 937 forward 2 TM N in   64 LI   : 789808. 1 :   2000FEB01 347 400 forward 2 TM N in     65 LI   : 792919. 1 : 2000FEB01 176 256 forward 2 TM   65 LI   : 792919. 1 : 2000FEB01 371 427 forward 2 TM 66 LI : 793949. 1 :   2000FEB01 208 282 forward 1 TM N out   66 LI : 793949. 1 : 2000FEB01 472 558 forward 1 TM N out 66 LI: 793949. 1 : 2000FEB01 455 541 forward 2 TM N out 67 LI : 794389. 1 : 2000FEB01 265 333 forward 1 TM N out 67 LI : 794389. 1 : 2000FEB01 424 477 forward 1 TM N out 67 LI : 794389. 1 : 2000FEB01 384 455 forward 3 TM N in 68 LI :

   796010. 1 : 2000FEB01 351 404 forward 3 TM N in 69 LI : 796324. 1 : 2000FEB01 365 418 forward 2 TM N in 72 LI : 798636. 1 : 2000FEB01 490 543 forward 1 TM N in 73 LI : 800045. 1 :   2000FEB01 627 701 forward 3 TM N in   74 LI : 800680. 1 : 2000FEB01 334 411 forward 1 TM N out 74 LI : 800680. 1 : 2000FEB01 359 421 forward 2 TM N out 75 LI : 800894. 1 : 2000FEB01 536 592 forward 2 TM N in 
75 LI : 800894. 1 : 2000FEB01 300 374 forward 3 TM N out 75 LI : 800894. 1 : 2000FEB01 396 482 forward 3 TM N out 77 LI : 801236. 1 :   2000FEB01 262 318 forward 1 TM N out   78 LI : 803335. 1 : 2000FEB01 412 498 forward 1 TM N out 78 LI : 803335. 1 : 2000FEB01 423 485 forward 3 TM N out   79 Ll   : 803998. 1 :   2000FEB01 221 307 forward 2 TM N out   81 LI : 808532. 1 :

     2000FEB01 472 558 forward 1 TM N in   81 LI : 808532. 1 : 2000FEB01 117 203 forward 3 TM N in 
81 LI :   808532.   1 : 2000FEB01 363 443 forward 3 TM N in   81 LI   : 808532. 1 : 2000FEB01 558 623 forward 3 TM N in 82 LI : 443073. 1 : 2000FEB01 293 379 forward 2 TM N in 
82 LI : 443073. 1 : 2000FEB01 81 152 forward 3 TM N in 
82 LI : 443073. 1 : 2000FEB01 189 260 forward 3 TM N in 

 <Desc/Clms Page number 199> 

 83 LI : 479671. 1 : 2000FEB01 523 579 forward 1 TM N out 85 LI : 810224. 1 : 2000FEB01 246 299 forward 3 TM   87 LG   : 892274. 1 :   2000MAY19 49 105 forward 1 TM N out     87 LG   : 892274. 1 :   2000MAY19 613 681 forward 1 TM N out     87 LG   : 892274. 1 :

     2000MAY19 506 589 forward 2 TM N in     91 LG   :   1084051.   1 :   2000MAY19 301 363 forward 1 TM N in   92 LG :   1076853.   1 :   2000MAY19 964 1050 forward 1 TM N in   92 LG :   1076853.   1 :   2000MAY19 56 130 forward 2 TM N out   92 LG :   1076853.   1 :   2000MAY19 741 818 forward 3 TM N in     93 LG   : 481631. 10 :   2000MAY19 298 357 forward 1 TM N out     93 LG   : 481631. 10 :   2000MAY19 598 654 forward 1 TM N out     94 LG   :   1088431.   2 :   2000MAY19 379 441 forward 1 TM N out     94 LG   :   1088431.   2 :

   2000MAY19 354 431 forward 3 TM N out 95 LI : 401619. 10 : 2000MAY01 157 219 forward 1 TM N out 95 LI : 401619. 10 : 2000MAY01 232 294 forward 1 TM N out   95 LI : 401619.   10 : 2000MAY01 502 576 forward 1 TM N out 95 LI : 401619. 10 : 2000MAY01 146 232 forward 2 TM N in 95 LI : 401619. 10 : 2000MAY01 326 412 forward 2 TM N in 95 LI : 401619. 10 : 2000MAY01 440 490 forward 2 TM N in 95 LI : 401619. 10 : 2000MAY01 512 580 forward 2 TM N in 
95 LI : 401619. 10 : 2000MAY01 186 257 forward 3 TM N in 
95 LI : 401619. 10 : 2000MAY01 528 599 forward 3 TM N in   96 Ll   : 1144007.1 : 2000MAY01 2833 2910 forward 1 TM N in 
96 LI :   1144007.   1 :   2000MAY01 3301 3378 forward 1 TM N in   
96 LI :   1144007.   1 : 2000MAY01 3511 3597 forward 1 TM N in   96 Ll   : 1144007.1 :

   2000MAY01 3634 3696 forward 1 TM N in   96 Ll   :   1144007.   1 : 2000MAY01 3736 3801 forward 1 TM N in   96 Ll   :   1144007.   1 : 2000MAY01 2645 2725 forward 2 TM N out   96 Ll   :   1144007.   1 : 2000MAY01 2879 2965 forward 2 TM N out   96 Ll   :   1144007.   1 : 2000MAY01 3356 3433 forward 2 TM N out   96 Ll   :   1144007.   1 : 2000MAY01 3476 3523 forward 2 TM N out   96 Ll   :   1144007.   1 : 2000MAY01 2772 2858 forward 3 TM N in   96 Ll   :   1144007.   1 : 2000MAY01 3258 3332 forward 3 TM N in   96 Ll   :   1144007.   1 : 2000MAY01 4017 4097 forward 3 TM N in 
97 LI : 331074. 1 :

   2000MAY01 1264 1326 forward 1 TM N in 
97 LI : 331074. 1 : 2000MAY01 1357 1419 forward 1 TM N in 
97 LI : 331074. 1 : 2000MAY01 1450 1512 forward 1 TM N in 
97 LI : 331074. 1 : 2000MAY01 1540 1626 forward 1 TM N in 
97 LI :   331074. 1   :   2000MAY01 1433 1513 forward 2 TM N in   
97 LI : 331074. 1 : 2000MAY01 1574 1660 forward 2 TM N in 
97 LI : 331074. 1 : 2000MAY01 1461 1529 forward 3 TM N in 
97 LI : 331074. 1 : 2000MAY01 1560 1646 forward 3 TM N in   98 Ll   :   1170349.   1 : 2000MAY01 34 102 forward 1 TM N in   99 LG   : 335097. 1 : 2000FEB18 601 672 forward 1 TM N out   99 LG   : 335097. 1 : 2000FEB18 847 909 forward 1 TM N out   99 LG   : 335097. 1 : 2000FEB18 928 981 forward 1 TM N out   99 LG   : 335097. 1 :

     2000FEB18 164 244 forward 2 TM N out     99 LG   : 335097. 1 : 2000FEB18 623 682 forward 2 TM N out   99 LG   : 335097. 1 :   2000FEB18 12 74 forward 3 TM N in     99 LG   : 335097. 1 :   2000FEB18 219 299 forward 3 TM N in     99 LG   : 335097. 1 :   2000FEB18 594 680 forward 3 TM N in   100 LG :   1076451.   1 :   2000FEB18 94 156 forward 1 TM N in   100 LG :   1076451.   1 : 2000FEB18 101 187 forward 2 TM N out 100 LG : 1076451. 1 : 2000FEB18 18 98 forward 3 TM N out 100 LG :   1076451.   1 : 2000FEB18 96 164 forward 3 TM N out 100 LG :   1076451.   1 :

   2000FEB18 216 290 forward 3 TM N out 

 <Desc/Clms Page number 200> 

   101 LI   : 805478. 1 : 2000FEB01 83 136 forward 2 TM N out   101 LI   : 805478. 1 : 2000FEB01 212 298 forward 2 TM N out 102 LG :   101269.   1 :   2000MAY19 655 741 forward 1 TM N in   102 LG : 101269. 1 : 2000MAY19 650 736 forward 2 TM N in 102 LG :   101269. 1 : 2000MAY19 96 182 forward 3 TM N in   102 LG :   101269.   1 :   2000MAY19 249 335 forward 3 TM N in   102 LG : 101269. 1 : 2000MAY19 663 740 forward 3 TM N in   103 LI   : 331087. 1 : 2000MAY01 251 298 forward 2 TM N out 103 LI : 331087. 1 : 2000MAY01 237 311 forward 3 TM 104 LI : 410188. 1 : 2000MAY01 520 591 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 640 711 forward 1 TM N in 104 LI : 410188. 1 :

   2000MAY01 724 810 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 832 879 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 883 969 forward 1 TM N in   104 LI   :   410188.   1 : 2000MAY01 1171 1257 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 1303 1389 forward 1 TM N in 104 LI : 410188. 1 :   2000MAY01 2290 2361 forward 1 TM N in   104 LI : 410188. 1 : 2000MAY01 2389 2460 forward 1 TM N in 104 LI : 410188. 1 :   2000MAY01 2470 2556 forward 1 TM N in   104 LI : 410188. 1 : 2000MAY01 2635 2721 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 2794 2862 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 2878 2964 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 3757 3837 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 3871 3957 forward 1 TM N in   104 LI   : 410188. 1 :

   2000MAY01 3961 4047 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 4111 4194 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 4342 4428 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 4492 4578 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 4714 4794 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 6439 6519 forward 1 TM N in   104 LI : 410188.   1 : 2000MAY01 7492 7575 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 7783 7845 forward 1 TM N in 104 LI :   410188.   1 : 2000MAY01 4673 4735 forward 2 TM N in 104 LI : 410188. 1 : 2000MAY01 4766 4828 forward 2 TM N in   104 Ll   : 410188. 1 : 2000MAY01 4928 5014 forward 2 TM N in 104 LI : 410188. 1 : 2000MAY01 5231 5317 forward 2 TM N in 104 LI : 410188. 1 : 2000MAY01 6341 6409 forward 1 TM N in 104 LI : 410188. 1 : 2000MAY01 7655 7741 forward 2 TM N in 104 LI : 410188. 1 :

   2000MAY01 8060 8146 forward 2 TM N in 104 LI :   410188.   1 : 2000MAY01 4776 4859 forward 3 TM N in 104 LI : 410188. 1 : 2000MAY01 6309 6371 forward 3 TM N in 104 LI : 410188. 1 : 2000MAY01 7704 7775 forward 3 TM N in   105 Ll   :   1188288.   1 : 2000MAY01 457 519 forward 1 TM   105 Ll   :   1188288.   1 : 2000MAY01 841 915 forward 1 TM   105 Ll   :   1188288.   1 : 2000MAY01 958 1038 forward 1 TM   105 Ll   :   1188288.   1 : 2000MAY01 1072 1140 forward 1 TM   105 Ll   :   1188288.   1 : 2000MAY01 1477 1539 forward 1 TM   105 Ll   :   1188288.   1 : 2000MAY01 1564 1626 forward 1 TM   105 Ll   :   1188288.   1 :

   2000MAY01 1810 1896 forward 1 TM   105 Ll   :   1188288.   1 : 2000MAY01 2134 2220 forward 1 TM   105 Ll   :   1188288. 1   :   2000MAY01 2734 2820 forward 1 TM     105 Ll   :   1188288.   1 : 2000MAY01 1067 1147 forward 2 TM N out   105 Ll   :   1188288.   1 : 2000MAY01 1157 1243 forward 2 TM N out   105 Ll   :   1188288.   1 :   2000MAY01 1313 1399 forward 2 TM N out     105 LI   :   1188288.   1 : 2000MAY01 1556 1618 forward 2 TM N out   105 Ll   :   1188288.   1 :

     2000MAY01 2294 2368 forward 2 TM N out   

 <Desc/Clms Page number 201> 

   105   LI:1188288.106 LI 1 :   2000MAY01 435 521 forward 3 TM N in     105 Ll   :   1188288.   1 : 2000MAY01 597 683 forward 3 TM N in   105 Ll   :   1188288.   1 :   2000MAY01 2301 2354 forward 3 TM N in     105 Ll   :   1188288.   1 : 2000MAY01 2700 2753 forward 3 TM N in 106 LI : 427997. 4 : 2000MAY01 148 222 forward 1 TM N in 106 LI : 427997. 4 : 2000MAY01 745 828 forward 1 TM N in 106 LI : 427997. 4 : 2000MAY01 1192 1278 forward 1 TM N in 106 LI : 427997. 4 : 2000MAY01 1351 1434 forward 1 TM N in   106 lui   : 427997. 4 :   2000MAY01 1450 1518 forward 1 TM N in   106 LI : 427997. 4 :

   2000MAY01 1759 1845 forward 1 TM N in 106 LI : 427997. 4 : 2000MAY01 134 220 forward 2 TM N in 106 LI : 427997. 4 : 2000MAY01 749 832 forward 2 TM N in 106 LI : 427997. 4 : 2000MAY01 1031 1087 forward 2 TM N in   106 LI   : 427997. 4 : 2000MAY01 1607 1693 forward 2 TM N in 106 LI : 427997. 4 : 2000MAY01 1730 1816 forward 2 TM N in   106 Ll   : 427997. 4 : 2000MAY01 2111 2191 forward 2 TM N in 106 LI : 427997. 4 : 2000MAY01 150 236 forward 3 TM N in 106 LI : 427997. 4 :   2000MAY01 681 767 forward 3 TM N in   106 LI : 427997. 4 : 2000MAY01 765 851 forward 3 TM N in 106 LI : 427997.   4   : 2000MAY01 1068 1124 forward 3 TM N in 106 LI : 427997. 4 : 2000MAY01 1665 1751 forward 3 TM N in 2000FEB18 1111 1176 forward 1 TM N in : 427997. 4 : 2000MAY01 1782 1856 forward 3 TM N in 107 LG : 451682. 1 :

     2000FEB18 93 155 forward 3 TM   109 LG : 481436. 5 :   2000FEB18 583 669 forward 1 TM N in   109 LG : 481436. 5 :   2000FEB18 769 834 forward 1 TM N in   109 LG : 481436. 5 : 2000FEB18 1164 1241 forward 3 TM N in 109 LG : 481436. 5 :   2000FEB18 575 655 forward 2 TM N out   109 LG : 481436. 5 :   2000FEB18 764 826 forward 2 TM N out   109 LG :   481436.   5 :   2000FEB18 1091 1153 forward 2 TM N out   109 LG :   481436.   5 : 2000FEB18 1187 1249 forward 2 TM N out 109 LG : 481436. 5 :   2000FEB18 84 170 forward 3 TM N in   109 LG : 481436. 5 :   2000FEB18 753 833 forward 3 TM N in   109 LG : 481436. 5 : 110 LI 2000FEB01 866 919 forward 2 TM N out : 793701. 1 :

   2000FEB01 352 405 forward 1 TM N in   110 LI   : 793701. 1 :   2000FEB01 389 475 forward 2 TM N in   111 LI : 373637. 1 : 2000FEB01 412 498 forward 1 TM 111 LI : 373637. 1 : 2000FEB01 434 520 forward 2 TM N out 111 LI : 373637. 1 : 2000FEB01 867 920 forward 3 TM N in   ill LI   : 373637. 1 : 2000FEB01 423 473 forward 3 TM N in 111 LI : 373637. 1 : 113 LI 112 LG : 239368. 2 :   2000MAY19 241 327 forward 1 TM N out   113 : 053826. 1 : 2000MAY01 31 117 forward 1 TM N out   113 LI   : 053826. 1 :   2000MAY01 1102 1188 forward 1 TM N out   LI : 053826. 1 : 2000MAY01 1282 1350 forward 1 TM N out 113 LI : 053826. 1 : 2000MAY01 41 112 forward 2 TM N out 113 LI : 053826. 1 : 2000MAY01 164 238 forward 2 TM N out 113 LI : 053826. 1 : 2000MAY01 461 538 forward 2 TM N out 113 LI :

   053826. 1 : 2000MAY01 1130 1192 forward 2 TM N out 113 LI : 053826. 1 : 2000MAY01 1214 1276 forward 2 TM N out 113 LI : 053826. 1 : 2000MAY01 1307 1378 forward 2 TM N out 113 LI : 053826. 1 :   2000MAY01 126 200 forward 3 TM N in   113 Li : 053826. 1 : 2000MAY01 348 416 forward 3 TM N in 113 LI : 053826. 1 : 2000MAY01 624 683 forward 3 TM N in 113 LI : 053826. 1 : 2000MAY01 1215 1277 forward 3 TM N in 113 LI : 053826. 1 : 2000MAY01 1290 1352 forward 3 TM N in   115   LI:1071427.96:2000MAY01 1072 1140 orward 1 TM 

 <Desc/Clms Page number 202> 

   115 Ll   :   1071427.   96 : 2000MAY01 1297 1383 forward 1 TM 115 LI :   1071427.   96 : 2000MAY01 1459 1536 forward 1 TM   115 LI   :   1071427.   96 :

     2000MAY01 1765 1851 forward 1 TM   115 LI :   1071427.   96 : 2000MAY01 1909 1971 forward 1 TM 115 LI : 1071427.96:2000MAY01 2002 2064 forward 1 TM 115 LI :   1071427.   96 : 2000MAY01 1562 1648 forward 2 TM N out 115 LI :   1071427.   96 : 2000MAY01 1706 1792 forward 2 TM N out 115 LI :   1071427.   96 : 2000MAY01 1823 1885 forward 2 TM N out 115 LI :   1071427.   96 : 2000MAY01 1913 1975 forward 2 TM N out 115 LI :   1071427.   96 : 2000MAY01 2045 2098 forward 2 TM N out 115 LI :   1071427.   96 : 2000MAY01 384 470 forward 3 TM N out 115 LI :   1071427.   96 : 2000MAY01 840 926 forward 3 TM N out 115 LI :   1071427.   96 : 2000MAY01 987 1049 forward 3 TM N out   115 lu   :

     1071427.   96 : 2000MAY01 1092 1154 forward 3 TM N out 115 LI : 1071427. 96 : 2000MAY01 1383 1454 forward 3 TM N out 115 LI :   1071427.   96 : 2000MAY01 1599 1655 forward 3 TM N out 115 LI :   1071427.   96 : 2000MAY01 1767 1844 forward 3 TM N out 115 LI :   1071427.   96 : 2000MAY01 1884 1952 forward 3 TM N out 115 LI :   1071427.   96 : 2000MAY01 2013 2099 forward 3 TM N out 115 LI: 1071427. 96 : 2000MAY01 2127 2189 forward 3 TM N out   116 LI   : 336338. 8 : 2000MAY01 100 186 forward 1 TM N out   116 LI   : 336338. 8 : 2000MAY01 427 513 forward 1 TM N out 116 LI: 336338. 8 : 2000MAY01 110 196 forward 2 TM 116 LI: 336338. 8 : 2000MAY01 281 367 forward 2 TM 116 LI : 336338. 8 :   2000MAY01 422 508 forward 2 TM   116 LI : 336338. 8 :

   2000MAY01 354 416 forward 3 TM N out 116 LI : 336338. 8 : 2000MAY01 432 494 forward 3 TM N out   117 LG   : 345527. 1 :   2000FEB18 46 120 forward 1 TM N out     117 LG   : 345527. 1 :   2000FEB18 917 979 forward 2 TM N out     117 LG   : 345527. 1 :   2000FEB18 1010 1072 forward 2 TM N out     117 LG   : 345527. 1 :   2000FEB18 1112 1198 forward 2 TM N out     117 LG   : 345527. 1 :   2000FEB18 96 182 forward 3 TM N out     117 LG   : 345527. 1 :   2000FEB18 474 536 forward 3 TM N out     117 LG   : 345527. 1 :   2000FEB18 552 614 forward3 TM Nout     118 LG   :   1089383.   1 :

   2000FEB18 43 126 forward 1 TM N out   118 LG   :   1089383.   1 :   2000FEB18 14 100 forward 2 TM     118 LG   :   1089383.   1 :   2000FEB18 140 205 forward 2 TM     118 LG   :   1089383.   1 :   2000FEB18 12 59 forward 3 TM N out   120 LG :   1093216.   1 :   2000FEB18 31 117 forward 1 TM N out   120 LG :   1093216.   1 :   2000FEB18 151 234 forward 1 TM N out   120 LG :   1093216.   1 : 2000FEB18 283 348 forward 1 TM N out 120 LG : 1093216. 1 : 2000FEB18 23 109 forward 2 TM N in 120 LG :   1093216.   1 :   2000FEB18 143 193 forward 2 TM N in   120 LG :   1093216.   1 :

     2000FEB18 48 122 forward 3 TM N out   120 LG : 1093216. 1 : 2000FEB18 180 263 forward 3 TM N out 122 LI : 335671. 2 :   2000FEB01 22 108 forward 1 TM N out   122 LI : 335671. 2 : 2000FEB01 1048 1134 forward 1 TM N out 122 LI : 335671. 2 :   2000FEB01 854 916 forward 2 TM N in   122 LI : 335671. 2 :   2000FEB01 926 988 forward 2 TM N in   122 LI : 335671. 2 : 2000FEB01 998 1072 forward 2 TM N in 122 LI : 335671. 2 : 2000FEB01 399 461 forward 3 TM N out 122 LI : 335671. 2 :   2000FEB01 480 542 forward 3 TM N out     122 LI   :   335671.   2 : 2000FEB01 576 662 forward 3 TM N out 122 LI : 335671. 2 : 2000FEB01 1023 1085 forward 3 TM N out 122 LI : 335671. 2 : 2000FEB01 1098 1160 forward 3 TM N out 122 LI : 335671. 2 :

   2000FEB01 1173 1235 forward 3 TM N out 

 <Desc/Clms Page number 203> 

   123 LI   : 793758. 1 :   2000FEB01 31 117 forward 1 TM N out     123 LI   : 793758. 1 : 2000FEB01 151 234 forward 1 TM N out   123 Li   : 793758. 1 : 2000FEB01 283 348 forward 1 TM N out   123 LI   : 793758. 1 : 2000FEB01 23 109 forward 2 TM N in   123 LI   : 793758. 1 : 2000FEB01 143 193 forward 2 TM N in   123 LI   : 793758. 1 : 2000FEB01 48 122 forward 3 TM N out   123 LI   : 793758. 1 : 2000FEB01 180 263 forward 3 TM N out   124 Li   : 803718. 1 : 2000FEB01 43 126 forward 1 TM N out 124 LI : 803718. 1 : 2000fEB01 14 100 forward 2 TM 124 LI :   803718.   1 : 2000FEB01 140 205 forward 2 TM   124 LI   :

   803718. 1 : 2000FEB01 12 59 forward 3 TM N out   125 LI   : 412179. 1 : 2000FEB01 328 414 forward 1 TM 125 lI : 412179. 1 : 2000FEB01 436 504 forward 1 TM 125 LI : 412179. 1 : 2000FEB01 56 115 forward 2 TM N out 125 LI : 412179. 1 :   2000FEB01 413 475 forward 2 TM N out   125 LI : 412179. 1 :   2000FEB01 512 574 forward 2 TM N out   125 LI : 412179. 1 : 2000FEB01 96 176 forward 3 TM N out 125 LI : 412179. 1 : 2000FEB01 384 446 forward 3 TM N out 125 Li : 412179. 1 :   2000FEB01 462 524 forward 3 TM N out   126 LI : 815679. 1 : 2000FEB01 10 84 forward 1 TM N out 126 Li : 815679. 1 : 2000FEB01 313 399 forward 1 TM N out 126 LI : 815679. 1 : 2000FEB01 946 1032 forward 1 TM N out 126 LI : 815679. 1 : 2000FEB01 1171 1248 forward 1 TM N out   126 LI   : 815679. 1 :

     2000FEB01 323 409 forward 2 TM N in   126 LI : 815679. 1 :   2000FEB01 500 568 forward 2 TM N in   126 LI : 815679. 1 :   2000FEB01 971 1021 forward 2 TM N in   126 LI : 815679. 1 :   2000FEB01 1493 1561 forward2 TM N in   126 LI : 815679. 1 : 2000FEB01 15 92 forward 3 TM N in   126 Li   : 815679. 1 : 2000FEB01 285 356 forward 3 TM N in 126 LI : 815679. 1 :   2000FEB01 690 764 forward 3 TM N in   126 LI : 815679. 1 : 2000FEB01 993 1076 forward 3 TM N in 126 LI : 815679. 1 : 2000FEB01 1626 1712 forward 3 TM N in 127 LI : 481361. 3 : 2000FEB01 199 252 forward 1 TM N out 128 LG : 247388. 1 :   2000MAY19 190 240 forward 1 TM N out   128 LG : 247388. 1 :   2000MAY19 233 319 forward 2 TM N out   128 LG : 247388. 1 :

     2000MAY19 446 532 forward 2 TM N out     130 Ll   : 787618. 1 : 2000MAY01 10 84 forward 1 TM N in   130 LI   : 787618. 1 : 2000MAY01 313 399 forward 1 TM N in 
130 LI : 787618. 1 : 2000MAY01 679 750 forward 1 TM N in   130 LI   : 787618. 1 : 2000MAY01 1018 1098 forward 1 TM N in 
130 LI : 787618. 1 : 2000MAY01 1189 1266 forward 1 TM N in 
130 LI : 787618. 1 : 2000MAY01 323 409 forward 2 TM N out 
130 LI : 787618. 1 :   2000MAY01 500 568 forward 2 TM N out     130 LI   : 787618. 1 : 2000MAY01 944 1030 forward 2 TM N out 
130 LI : 787618. 1 : 2000MAY01 1508 1582 forward 2 TM N out   130 LI : 787618.   1 : 2000MAY01 1616 1702 forward 2 TM N out 
130 LI : 787618. 1 : 2000MAY01 15 92 forward 3 TM N out 
130 LI : 787618. 1 :

   2000MAY01 285 356 forward 3 TM N out 
131 LI : 331610. 2 : 2000MAY01 91 156 forward 1 TM 
131 LI : 331610. 2 : 2000MAY01 277 363 forward 1 TM 
131 LI : 331610. 2 : 2000MAY01 682 744 forward 1 TM 
131 LI : 331610. 2 :   2000MAY01 4126 4212 forward 1 TM   
131 LI : 331610. 2 : 2000MAY01 4951 5001 forward 1 TM 
131 LI : 331610. 2 : 2000MAY01 5023 5109 forward 1 TM 
131 LI : 331610. 2 : 2000MAY01 5128 5190 forward 1 TM 
131 LI : 331610. 2 :   2000MAY01 5407 5469 forward 1 TM   

 <Desc/Clms Page number 204> 

   131 LI   :   331610.   2 : 2000MAY01 5485 5547 forward 1 TM 131 LI :   331610.   2 : 2000MAY01 5563 5625 forward 1 TM   131 LI   : 331610. 2 : 2000MAY01 5728 5805 forward 1 TM   131 LI   : 331610. 2 :

   2000MAY01 5896 5949 forward 1 TM   131 LI   : 331610. 2 :   2000MAY01 6268 6327 forward 1 TM     131 LI   : 331610. 2 : 2000MAY01 6454 6522 forward 1 TM 131 LI : 331610. 2 : 2000MAY01 6559 6645 forward 1 TM   131 LI   : 331610. 2 : 2000MAY01 7477 7539 forward 1 TM   131 LI   : 331610. 2 : 2000MAY01 7552 7614 forward 1 TM   131 LI   : 331610. 2 : 2000MAY01 671 724 forward 2 TM N out   131 LI   : 331610. 2 : 2000MAY01 4127 4213 forward 2 TM N out   131 LI   : 331610. 2 : 2000MAY01 4928 5011 forward 2 TM N out   131 LI   : 331610. 2 : 2000MAY01 5051 5113 forward 2 TM N out   131 LI   : 331610. 2 : 2000MAY01 5135 5197 forward 2 TM N out 131 LI : 331610. 2 :

     2000MAY01 5207 5269 forward 2 TM N out   131 LI : 331610. 2 : 2000MAY01 5537 5611 forward 2 TM N out 131 LI : 331610. 2 : 2000MAY01 5726 5797 forward 2 TM N out 131 LI : 331610. 2 : 2000MAY01 5903 5989 forward 2 TM N out 131 LI : 331610. 2 : 2000MAY01 6392 6478 forward 2 TM N out 131 LI : 331610. 2 : 2000MAY01 6746 6814 forward 2 TM N out 131 LI : 331610. 2 : 2000MAY01 7295 7381 forward 2 TM N out 131 LI : 331610. 2 : 2000MAY01 7586 7633 forward 2 TM N out 131 LI : 331610. 2 : 2000MAY01 2763 2849 forward 3 TM   131 LI   : 331610. 2 : 2000MAY01 4527 4595 forward 3 TM 131 LI : 331610. 2 : 2000MAY01 5079 5165 forward 3 TM 131 LI : 331610. 2 : 2000MAY01 5445 5516 forward 3 TM 131 LI : 331610. 2 : 2000MAY01 5676 5759 forward 3 TM 131 LI : 331610. 2 : 2000MAY01 6255 6341 forward 3 TM   131 LI   : 331610. 2 :

   2000MAY01 6378 6464 forward 3 TM   131 LI   : 331610. 2 :   2000MAY01 6624 6692 forward 3 TM   131 LI : 331610. 2 : 2000MAY01 6705 6779 forward 3 TM 131 LI : 331610. 2 : 2000MAY01 6810 6884 forward 3 TM 131 LI : 331610. 2 : 2000MAY01 7062 7133 forward 3 TM   131 LI : 331610.   2 : 2000MAY01 7677 7748 forward 3 TM 131 LI : 331610. 2 : 2000MAY01 7833 7919 forward 3 TM 132 LG : 982697. 1 :   2000FEB18 355 441 forward 1 TM N in   132 LG : 982697. 1 :   2000FEB18 946 993 forward 1 TM N in   132 LG : 982697. 1 :   2000FEB18 897 983 forward 3 TM N in   132 LG : 982697. 1 :   2000FEB18 1215 1301 forward 3 TM N in     133 LG   :   1080896.   1 :

     2000FEB18 367 426 forward 1 TM N in     133 LG   : 1080896. 1 :   2000FEB18 476 562 forward 2 TM N in     133 LG   : 1080896. 1 : 2000FEB18 815 901 forward 2 TM N in   133 LG   :   1080896.   1 :   2000FEB18 342 395 forward 3 TM N in   134 LI : 811341. 1 : 2000FEB01 562 615 forward 1 TM N out 134 LI : 811341. 1 : 2000FEB01 691 777 forward 1 TM N out 135 LI : 903225. 1 :   2000FEB01 20 100 forward 2 TM N out   135 LI : 903225. 1 : 2000FEB01 12 83 forward 3 TM N out 135 LI : 903225.   1   :   2000FEB01 768 827 forward 3 TM N out     137 LG   : 979580. 1 :   2000MAY19 298 354 forward 1 TM N in     137 LG   : 979580. 1 :

     2000MAY19 826 909 forward 1 TM N in     137 LG   : 979580. 1 :   2000MAY19 934 1020 forward 1 TM N in     137 LG   : 979580. 1 :   2000MAY19 233 289 forward 2 TM N out     137 LG   : 979580. 1 :   2000MAY19 338 418 forward 2 TM N out     137 LG   : 979580. 1 :   2000MAY19 201 272 forward 3 TM N in   138 LI :   1169865.   1 : 2000MAY01 197 283 forward 2 TM N in 138 LI :   1169865.   1 : 2000MAY01 863 949 forward 2 TM N in 

 <Desc/Clms Page number 205> 

   139 LG   : 337818. 2 : 2000FEB18 40 117 forward 1 TM N out   139 LG   : 337818. 2 :   2000FEB18 532 618 forward 1 TM N out     139 LG   : 337818. 2 :

     2000FEB18 907 993 forward 1 TM N out     139 LG   : 337818. 2 :   2000FEB18 1372 1425 forward 1 TM N out   
140 LI : 337818. 1 : 2000FEB01 40 114 forward 1 TM N in 
140 LI : 337818. 1 : 2000FEB18 401 466 forward 2 TM N in 
140 LI : 337818. 1 : 2000FEB01 852 905 forward 3 TM N in   141 LG   : 241577. 4 : 2000MAY19 496 582 forward 1 TM N in 
142 LG : 344786. 4 :   2000MAY19 19 105 forward 1 TM N out   
142 LG : 344786. 4 :   2000MAY19 14 88 forward 2 TM N in   
142 LG : 344786. 4 :   2000MAY19 173 247 forward 2 TM N in   
142 LG : 344786. 4 :   2000MAY19 21 107 forward 3 TM     143 LI : 414307.   1 : 2000FEB01 116 202 forward 2 TM N in 
144 LI : 202943. 2 : 2000FEB01 166 237 forward 1 TM N in   144 LI   :

   202943. 2 :   2000FEB01 263 313 forward 2 TM N out   
144 LI : 202943. 2 :   2000FEB01 276 326 forward 3 TM N in   
146 LI : 815961. 1 : 2000FEB01 232 291 forward 1 TM N out 
146 LI : 815961. 1 : 2000FEB01 81 167 forward 3 TM N out 
146 LI : 815961. 1 : 2000FEB01 243 329 forward 3 TM N out 
146   LI : 815961.   1 : 2000FEB01 354 422 forward 3 TM N out 
146 LI : 815961. 1 : 2000FEB01 573 659 forward 3 TM N out   146 LI   : 815961. 1 : 2000FEB01 741 803 forward 3 TM N out   147 LG   :   120744.   1 :   2000MAY19 181 249 forward 1 TM N out     147 LG   :   120744.   1 :   2000MAY19 188 256 forward 2 TM     147 LG   :   120744.   1 :

     2000MAY19 275 328 forward 2 TM   
148 LI : 757520. 1 :   2000MAY01 2140 2220 forward 1 TM N in   
148 LI : 757520. 1 : 2000MAY01 2293 2379 forward 1 TM N in 
148 Li : 757520. 1 : 2000MAY01 1988 2059 forward 2 TM N in 
148 LI: 757520. 1 : 2000MAY01 2285 2359 forward 2 TM N in 
148 LI : 757520. 1 : 2000MAY01 1677 1763 forward 3 TM 
148 LI : 757520. 1 :   2000MAY01 1995 2066 forward 3 TM     149 LG   : 160570. 1 :   2000FEB18 345 413 forward 3 TM N out     149 LG   :   160570.   1 : 2000FEB18 462 518 forward 3 TM N out 
151 LI : 221285. 1 : 2000FEB01 1375 1452 forward 1 TM N out   152 LI   : 401605. 2 :   2000FEB01 235 321 forward 1 TM N in   
152 LI : 401605. 2 : 2000FEB01 192 263 forward 3 TM N in 
152 LI : 401605. 2 :

   2000FEB01 489 563 forward 3 TM N in 
153 Li : 329017. 1 :   2000FEB01 179 235 forward 2 TM N in   
153 Li : 329017. 1 : 2000FEB01 359 433 forward 2 TM N in 
153 LI : 329017. 1 :   2000FEB01 449 526 forward 2 TM N in     153 Ll   : 329017. 1 :   2000FEB01 617 703 forward 2 TM N in   
153 LI : 329017. 1 : 2000FEB01 920 973 forward 2 TM N in   155 LG   : 403409. 1 : 2000MAY19 136 222 forward 1 TM N out   155 LG   : 403409. 1 :   2000MAY19 973 1029 forward 1 TM N out     155 LG   : 403409. 1 :   2000MAY19 1285 1371 forward 1 TM N out     155 LG   : 403409. 1 : 2000MAY19 182 268 forward 2 TM N in 
156 LG : 233933. 5 :   2000MAY19 148 234 forward 1 TM N out   
156 LG : 233933. 5 :

     2000MAY19 39 125 forward 3 TM N out   
157 LI : 290344. 1 : 2000MAY01 232 312 forward 1 TM N out   157 LI   :   290344. 1   : 2000MAY01 1258 1311 forward 1 TM N out 
157 LI : 290344. 1 : 2000MAY01 3640 3714 forward 1 TM N out 
157 LI:290344.1 : 2000MAY01 4366 4449 forward 1 TM N out 
157 LI : 290344. 1 : 2000MAY01 4468 4548 forward 1 TM N out 
157 LI :   290344. 1   :   2000MAY01 146 226 forward 2.

   TM N out   
157 LI : 290344. 1 :   2000MAY01 3122 3196 forward 2 TM N out   
157 LI :   290344. 1   : 2000MAY01 3833 3919 forward 2 TM N out 

 <Desc/Clms Page number 206> 

 157 LI : 290344. 1 : 2000MAY01 4457 4537 forward 2 TM N out 157 LI :   290344. 1   : 2000MAY01 4760 4846 forward 2 TM N out   157 LI   : 290344. 1 : 2000MAY01 432 503 forward 3 TM N out 157 LI : 290344. 1 : 2000MAY01 1647 1733 forward 3 TM N out 157 LI : 290344. 1 :   2000MAY01 3177 3248 forward 3 TM N out   157 LI : 290344. 1 :   2000MAY01 3594 3680 forward 3 TM N out   157 LI : 290344. 1 : 2000MAY01 3753 3815 forward 3 TM N out 157 LI : 290344. 1 : 2000MAY01 3864 3926 forward 3 TM N out 157 Li : 290344. 1 : 2000MAY01 4443 4526 forward 3 TM N out 158 LI : 410742. 1 :

   2000MAY01   136 210     forward 1 TM N out   158 LI : 410742. 1 :   2000MAY01 2200 2286 forward 1 TM N out   158 LI : 410742. 1 : 2000MAY01 2437 2514 forward 1 TM N out 158 LI : 410742. 1 :   2000MAY01 3149 3229 forward 2 TM N in   158 Li :   410742.   1 : 2000MAY01 3437 3505 forward 2 TM N in 158 LI : 410742. 1 : 2000MAY01 510 578 forward 3 TM N in 158 LI : 410742. 1 : 2000MAY01 1905 1991 forward 3 TM N in 158 LI : 410742. 1 : 2000MAY01 2811 2897 forward 3 TM N in 158 LI : 410742. 1 : 2000MAY01 3168 3254 forward 3 TM N in   159 LG   : 406568. 1 :   2000MAY19 490 549 forward 1 TM N in     159 LG   : 406568. 1 :   2000MAY19 1732 1818 forward 1 TM N in     159 LG   : 406568. 1 :

     2000MAY19 1825 1899 forward 1 TM N in     159 LG   : 406568. 1 :   2000MAY19 1918 2004 forward 1 TM N in     159 LG   : 406568. 1 :   2000MAY19 12 59 forward 3 TM N in     159 LG   : 406568. 1 :   2000MAY19 1935 2018 forward 3 TM N in     159 LG   : 406568. 1 :   2000MAY19 2094 2174 forward 3 TM N in     160 LI   : 283762. 1 : 2000MAY01 1675 1746 forward 1 TM   160 LI   : 283762. 1 : 2000MAY01 2095 2181 forward 1 TM   160 LI   : 283762. 1 : 2000MAY01 2632 2718 forward 1 TM   160 LI   : 283762. 1 :   2000MAY01 2830 2916 forward 1 TM   160 LI :   283762. 1   :

   2000MAY01 2941 3027 forward 1 TM   160 lui   : 283762. 1 : 2000MAY01 3235 3321 forward 1 TM   160 LI   : 283762. 1 : 2000MAY01 3328 3414 forward 1 TM   160 LI   : 283762. 1 : 2000MAY01 3592 3666 forward 1 TM   160 LI   : 283762. 1 : 2000MAY01 3682 3768 forward 1 TM   160 lui   : 283762. 1 :   2000MAY01 4153 4224 forward 1 TM     160 LI   : 283762. 1 : 2000MAY01 4360 4434 forward 1 TM   160 LI   : 283762. 1 : 2000MAY01 4594 4656 forward 1 TM   160 LI   : 283762. 1 : 2000MAY01 4681 4743 forward 1 TM 160 LI : 283762. 1 : 2000MAY01 4885 4962 forward 1 TM 160 LI : 283762. 1 : 2000MAY01 5011 5061 forward 1 TM 
160 LI : 283762. 1 : 2000MAY01 92 178 forward 2 TM N in 160 LI : 283762. 1 :

   2000MAY01 278 364 forward 2 TM N in   160 Li   : 283762. 1 : 2000MAY01 995 1075 forward 2 TM N in 160 LI : 283762. 1 : 2000MAY01 1523 1597 forward 2 TM N in 160 LI : 283762. 1 : 2000MAY01 1817 1903 forward 2 TM N in 160 LI : 283762. 1 : 2000MAY01 2522 2599 forward 2 TM N in 
160 LI : 283762. 1 : 2000MAY01 2666 2752 forward 2 TM N in 160 LI : 283762. 1 : 2000MAY01 2837 2887 forward 2 TM N in 160 LI : 283762. 1 : 2000MAY01 3038 3097 forward 2 TM N in   160 LI   : 283762. 1 : 2000MAY01 3563 3625 forward 2 TM N in 160 LI : 283762. 1 : 2000MAY01 3638 3700 forward 2 TM N in 160 LI : 283762. 1 : 2000MAY01 4067 4144 forward 2 TM N in 160 Li : 283762. 1 : 2000MAY01 4439 4522 forward 2 TM N in 160 LI : 283762. 1 : 2000MAY01 4685 4765 forward 2 TM N in 160 LI : 283762. 1 : 2000MAY01 4784 4843 forward 2 TM N in 160 LI : 283762. 1 :

   2000MAY01 4973 5050 forward 2 TM N in 

 <Desc/Clms Page number 207> 

   160 LI   : 283762. 1 : 2000MAY01 5072 5125 forward 2 TM N in   160 LI   : 283762. 1 : 2000MAY01 693 755 forward 3 TM N out   160 LI   : 283762. 1 :   2000MAY01 765 827 forward 3 TM N out     160 LI   : 283762. 1 : 2000MAY01 840 902 forward 3 TM N out   160 Li   : 283762. 1 : 2000MAY01 1623 1694 forward 3 TM N out   160 LI   : 283762. 1 : 2000MAY01 1800 1880 forward 3 TM N out   160 LI   : 283762. 1 :   2000MAY01 2622 2708 forward 3 TM N out     160 LI   : 283762. 1 :   2000MAY01 2778 2861 forward 3 TM N out     160 LI   : 283762. 1 : 2000MAY01 3144 3230 forward 3 TM N out 160 LI : 283762. 1 :

   2000MAY01 3276 3362 forward 3 TM N out 160 LI : 283762. 1 : 2000MAY01 3441 3527 forward 3 TM N out 160 LI : 283762. 1 : 2000MAY01 3666 3752 forward 3 TM N out 160 LI : 283762. 1 : 2000MAY01 4077 4163 forward 3 TM N out 160 LI : 283762. 1 : 2000MAY01 4245 4331 forward 3 TM N out 10 LI : 283762. 1 : 2000MAY01 4395 4481 forward 3 TM N out 160 LI : 283762. 1 : 2000MAY01 4584 4646 forward 3 TM N out 160 LI : 283762.   1   : 2000MAY01 4662 4724 forward 3 TM N out 160 LI : 283762. 1 : 2000MAY01 4845 4892 forward 3 TM N out 161 LI : 347687. 113 : 2000MAY01 319 405 forward 1 TM N out 161 LI : 347687. 113 : 2000MAY01 463 549 forward 1 TM N out 161 LI :   347687.   113 : 2000MAY01 733 819 forward 1 TM N out   161 LI   : 347687.   113   :   2000MAY01 1240 1293 forward 1 TM N out   161 LI : 347687. 113 :

   2000MAY01 1720 1797 forward 1 TM N out 161 LI : 347687. 113 : 2000MAY01 1861 1908 forward 1 TM N out 161 LI :   347687. 113   :   2000MAY01 1972 2034 forward 1 TM N out   161 LI : 347687. 113 :   2000MAY01 2050 2112 forward 1 TM N out   161 LI : 347687. 113 : 2000MAY01 2308 2394 forward 1 TM N out 161 LI : 347687. 113 : 2000MAY01 977 1057 forward 2 TM N in 161 LI : 347687. 113 : 2000MAY01 1250 1309 forward 2 TM N in 161 LI : 347687. 113 : 2000MAY01 1730 1792 forward 2 TM N in 161 LI : 347687. 113 : 2000MAY01 1808 1870 forward 2 TM N in 161 LI : 347687. 113 : 2000MAY01 1886 1948 forward 2 TM N in 161 LI : 347687. 113 : 2000MAY01 324 398 forward 3 TM N in 161 LI : 347687. 113 : 2000MAY01 948 1034 forward 3 TM N in 161 LI : 347687. 113 : 2000MAY01 1686 1763 forward 3 TM N in 161 LI : 347687. 113 :

   2000MAY01 1791 1874 forward 3 TM N in 161 LI : 347687. 113 :   2000MAY01 2025 2108 forward 3 TM N in   163 LG : 451710. 1 :   2000FEB18 502 588 forward 1 TM N in   163 LG: 451710. 1 :   2000FEB18 453 515 forward 3 TM N in   164 LG : 455771. 1 :   2000FEB18 199 285 forward 1 TM N out   165 LG :   452089.   1 :   2000FEB18 695 772 forward 2 TM N out   165 LG : 452089. 1 :   2000FEB18 708 764 forward 3 TM N out   166 LG : 246415. 1 :   2000FEB18 196 246 forward 1 TM N in   167 LG : 414144. 10 :   2000FEB18 589 672 forward 1 TM N in   167 LG : 414144. 10 :   2000FEB18 615 692 forward 3 TM N out   168 LG :   1101445.   1 : 2000FEB18 787 858 forward 1 TM N out 168 LG :

     1101445.   1 :   2000FEB18 506 592 forward 2 TM N out   169 LG : 452134. 1 :   2000FEB18 276 326 forward 3 TM N out     170 LI   : 903021. 1 :   2000FEB01 109 162 forward 1 TM N out   172 LG : 449404. 1 :   2000MAY19 163 219 forward 1 TM N out   172 LG : 449404. 1 :   2000MAY19 200 280 forward 2 TM N out     173 LG   : 449413. 1 :   2000MAY19 353 439 forward 2 TM N out     177 LG   :   1101153.   1 :   2000MAY19 520 600 forward 1 TM N in     177 LG   :   1101153.   1 :   2000MAY19 585 671 forward 3 TM N in   178 LI : 257695. 20 : 2000MAY01 433 516 forward 1 TM N in 179 LI : 455771. 1 :

     2000MAY01 199 285 forward 1 TM N out   

 <Desc/Clms Page number 208> 

   180 LI   : 274551. 1 : 2000MAY01 81 152 forward 3 TM N out   180 Li   : 274551. 1 : 2000MAY01 216 269 forward 3 TM N out 181 LI : 035973. 1 :   2000MAY01 622 708 forward 1 TM N out   181 LI : 035973. 1 : 2000MAY01 596 682 forward 2 TM N out 181 LI : 035973. 1 : 2000MAY01 588 674 forward 3 TM N out 182 LG : 978427. 5 :   2000FEB18 221 295 forward 2 TM N out   182 LG : 978427. 5 : 2000FEB18 365 433 forward 2 TM N out 182 LG : 978427. 5 :   2000FEB18 198 284 forward 3 TM N out     183 LG   :   247781.   2 :   2000FEB18 22 108 forward 1 TM N in     183 LG   : 247781. 2 :

     2000FEB18 1114 1200 forward 1 TM N in     183 LG   : 247781. 2 : 2000FEB18 1149 1235 forward 3 TM N in 185 LI : 333307. 2 : 2000FEB01 24 98 forward 3 TM N out   187 LG   : 414732. 1 :   2000MAY19 40 93 forward 1 TM N out     187 LG   :   414732.   1 :   2000MAY19 156 233 forward 3 TM N out     188 LG   : 413910. 6 :   2000MAY19 385 441 forward 1 TM N out     188 LG   : 413910. 6 :   2000MAY19 886 948 forward 1 TM N out     188 LG   : 413910. 6 :   2000MAY19 104 190 forward 2 TM N out     188 LG   : 413910. 6 :   2000MAY19 387 461 forward 3 TM N out     188 LG   : 413910. 6 :

     2000MAY19 921 1007 forward 3 TM N out     189 LI   : 414732. 2 :   2000MAY01 34 93 forward 1 TM N out   189 LI : 414732. 2 : 2000MAY01 24 110 forward 3 TM N out 189 LI : 414732. 2 : 2000MAY01 159 236 forward 3 TM N out   190 LI   : 900264. 2 : 2000MAY01 730 807 forward 1 TM N in   190 LI   : 900264. 2 : 2000MAY01 1018 1092 forward 1 TM N in   190 LI   : 900264. 2 : 2000MAY01 1294 1350 forward 1 TM N in   190 LI   : 900264. 2 : 2000MAY01 1519 1578 forward 1 TM N in   190 LI   : 900264. 2 : 2000MAY01 2311 2397 forward 1 TM N in   190 LI   : 900264. 2 :   2000MAY01 2509 2562 forward 1 TM N in   190 LI: 900264. 2 :   2000MAY01 2752 2808 forward 1 TM N in   190 LI : 900264. 2 :

   2000MAY01 3103 3165 forward 1 TM N in 190 LI : 900264. 2 : 2000MAY01 3178 3240 forward 1 TM N in 190 LI : 900264. 2 : 2000MAY01 3253 3315 forward 1 TM N in 190 LI : 900264. 2 : 2000MAY01 3424 3510 forward 1 TM N in   190 LI   : 900264. 2 :   2000MAY01 3520 3603 forward 1 TM N in   190 LI : 900264. 2 : 2000MAY01 3883 3945 forward 1 TM N in 190 LI : 900264. 2 :   2000MAY01 3982 4044 forward 1 TM N in     190 LI   : 900264. 2 : 2000MAY01 68 154 forward 2 TM 190 LI : 900264. 2 : 2000MAY01 188 274 forward 2 TM 190 LI : 900264. 2 : 2000MAY01 1079 1165 forward 2 TM 190 LI : 900264. 2 :   2000MAY01 2285 2359 forward 2 TM   190 LI : 900264. 2 : 2000MAY01 2732 2812 forward 2 TM 190 LI : 900264. 2 : 2000MAY01 3095 3172 forward 2 TM 190 LI : 900264. 2 : 2000MAY01 3260 3319 forward 2 TM 190 LI :

   900264. 2 : 2000MAY01 3434 3505 forward 2 TM 190 LI : 900264. 2 : 2000MAY01 3515 3601 forward 2 TM 190 LI : 900264. 2 : 2000MAY01 3662 3748 forward 2 TM 190 LI : 900264. 2 : 2000MAY01 3842 3913 forward 2 TM 190 LI : 900264. 2 : 2000MAY01 3992 4063 forward 2 TM 190 LI : 900264. 2 :   2000MAY01 198 248 forward 3 TM N in     190 LI   : 900264. 2 : 2000MAY01 1080 1133 forward 3 TM N in 190 LI :   900264.   2 : 2000MAY01 1431 1517 forward 3 TM N in 190 LI : 900264. 2 : 2000MAY01 1518 1571 forward 3 TM N in 190 LI : 900264. 2 : 2000MAY01 1740 1814 forward 3 TM N in 190 LI : 900264. 2 : 2000MAY01 2409 2480 forward 3 TM N in 190 LI : 900264. 2 : 2000MAY01 2928 2993 forward 3 TM N in 190 LI : 900264. 2 : 2000MAY01 3096 3161 forward 3 TM N in 

 <Desc/Clms Page number 209> 

 
190 LI : 900264. 2 :

     2000MAY01 3342 3404 forward 3 TM N in   
190 LI :   900264. 2   : 2000MAY01 3447 3509 forward 3 TM N in 
190 LI : 900264. 2 : 2000MAY01 3531 3614 forward 3 TM N in 
190 LI : 900264. 2 : 2000MAY01 3987 4064 forward 3 TM N in   191 LI   : 335593. 1 : 2000MAY01 685 771 forward 1 TM N in 
191 LI : 335593. 1 : 2000MAY01 1273 1335 forward 1 TM N in 
191 LI : 335593. 1 : 2000MAY01 1366 1428 forward 1 TM N in   191 Ll   : 335593. 1 :   2000MAY01 710 757 forward 2 TM N in   
191 LI : 335593. 1 : 2000MAY01 1250 1336 forward 2 TM N in 
191 LI : 335593. 1 : 2000MAY01 1358 1408 forward 2 TM N in 
191 LI : 335593. 1 : 2000MAY01 1448 1525 forward 2 TM N in   191 LI   : 335593. 1 :   2000MAY01 1604 1690 forward 2 TM N in   
191 LI : 335593. 1 :

   2000MAY01 81 128 forward 3 TM N in 
191 LI : 335593. 1 : 2000MAY01 246 296 forward 3 TM N in 
191 LI : 335593. 1 :   2000MAY01 807 866 forward 3 TM N in   
191 LI : 335593. 1 : 2000MAY01 876 947 forward 3 TM N in 
191 LI : 335593. 1 : 2000MAY01 1155 1217 forward 3 TM N in 
191 LI : 335593. 1 : 2000MAY01 1233 1295 forward 3 TM N in 
191 LI : 335593. 1 : 2000MAY01 1359 1445 forward 3 TM N in   192 Ll   :   1189543.   1 : 2000MAY01 1765 1842 forward 1 TM   192 Ll   :   1189543.   1 : 2000MAY01 1861 1935 forward 1 TM 
192 LI :   1189543.   1 :   2000MAY01 2236 2307 forward 1 TM     192 Ll   :   1189543.   1 :   2000MAY01 2356 2442 forward 1 TM     192 Ll   :

     1189543.   1 :   2000MAY01 2476 2544 forward 1 TM     192 Ll   :   1189543.   1 : 2000MAY01 2659 2712 forward 1 TM 
192 LI : 1189543.1:2000MAY01 3097 3174 forward 1 TM   192 Ll   :   1189543.   1 : 2000MAY01 3217 3288 forward 1 TM   192 Ll   :   1189543.   1 : 2000MAY01 3439 3492 forward 1 TM   192 Ll   :   1189543.   1 : 2000MAY01 860 946 forward 2 TM   192 Ll   :   1189543.   1 : 2000MAY01 1016 1099 forward 2 TM   192 Ll   :   1189543.   1 : 2000MAY01 1145 1216 forward 2 TM 
192 LI :   1189543.   1 :   2000MAY01 1601 1672 forward 2 TM     192 Ll   :

     1189543.   1 : 2000MAY01 1691 1768 forward 2 TM   192 Ll   :   1189543.   1 :   2000MAY01 2411 2485 forward 2 TM     192 Ll   :   1189543.   1 : 2000MAY01 2831 2917 forward 2 TM   192 Ll   :   1189543.   1 :   2000MAY01 3080 3166 forward 2 TM     192 Ll   :   1189543.   1 : 2000MAY01 3227 3310 forward 2 TM   192   LI:1189543. 1 :   2000MAY01 1155 1229 forward 3 TM N out   
192 LI :   1189543.   1 : 2000MAY01 1683 1766 forward 3 TM N out   192 Ll   :   1189543.   1 : 2000MAY01 1770 1838 forward 3 TM N out   192 Ll   :   1189543.   1 :

     2000MAY01 2019 2069 forward 3 TM N out     192 Ll   :   1189543.   1 : 2000MAY01 2352 2438 forward 3 TM N out 
192 LI:1189543. 1 : 2000MAY01 2508 2594 forward 3 TM N out 
192 LI :   1189543.   1 :   2000MAY01 3030 3101 forward 3 TM N out   
192 LI :   1189543.   1 : 2000MAY01 3183 3263 forward 3 TM N out   192 Ll   :   1189543.   1 : 2000MAY01 3360 3446 forward 3 TM N out   193 LG   : 455450. 1 :   2000FEB18 422 490 forward 2 TM N out     194 LG   :   1040978.   1 : 2000FEB18 500 586 forward 2 TM N out   194 LG   : 1040978.1 :   2000FEB18 276 332 forward 3 TM N out   
196 LG : 132147. 3 :

   2000FEB18 259 345 forward 1 TM N out 
196 LG :   132147.   3 : 2000FEB18 418 504 forward 1 TM N out 
196 LG : 132147. 3 : 2000FEB18 718 780 forward 1 TM N out 
196 LG : 132147. 3 : 2000FEB18 1477 1548 forward 1 TM N out 
196 LG : 132147. 3 : 2000FEB18 1585 1647 forward 1 TM N out 
196 LG :   132147.   3 : 2000FEB18 1690 1752 forward 1 TM N out 
196 LG :   132147.   3 : 2000FEB18 2560 2637 forward 1 TM N out 

 <Desc/Clms Page number 210> 

 196 LG :   132147. 3   :   2000FEB18 2731 2790 forward 1 TM N out   196 LG : 132147. 3 : 2000FEB18 2908 2976 forward 1 TM N out 196 LG :   132147.   3 :   2000FEB18 3082 3168 forward 1 TM N out   196 LG : 132147. 3 : 2000FEB18 3184 3243 forward 1 TM N out 196 LG : 132147. 3 :

     2000FEB18 3376 3462 forward 1 TM N out   196 LG : 132147. 3 : 2000FEB18 1451 1531 forward 2 TM N out 196 LG : 132147. 3 : 2000FEB18 1538 1615 forward 2 TM N out 196 LG :   132147.   3 :   2000FEB18 2741 2827 forward 2 TM N out   196 LG : 132147. 3 : 2000FEB18 2960 3031 forward 2 TM N out 196 LG : 132147. 3 : 2000FEB18 3050 3112 forward 2 TM N out 196 LG :   132147.   3 : 2000FEB18 1626 1703 forward 3 TM N in 196 LG: 132147. 3 : 2000FEB18 2508 2594 forward 3 TM N in 196 LG :   132147.   3 : 2000FEB18 2919 2987 forward 3 TM N in 196 LG :   132147.   3 :   2000FEB18 3177 3263 forward 3 TM N in   196 LG :   132147.   3 :   2000FEB18 3372 3422 forward 3 TM N in   197 LI : 036034. 1 :

   2000FEB01 157 219 forward 1 TM N out 197 LI :   036034.   1 : 2000FEB01 395 457 forward 2 TM N in 197 LI : 036034. 1 : 2000FEB01 479 541 forward 2 TM N in 197 LI : 036034. 1 :   2000FEB01 563 625 forward 2 TM N in   197 LI : 036034. 1 : 2000FEB01 647 709 forward 2 TM N in   198 LG   :   162161.   1 :   2000MAY19 372 458 forward 3 TM N in   199 LG : 407214. 10 :   2000MAY19 34 120 forward 1 TM N out     199 LG   : 407214. 10 :   2000MAY19 44 124 forward 2 TM N out     199 LG   : 407214. 10 :   2000MAY19 203 289 forward 2 TM N out   200 LG : 204626. 1 :   2000MAY19 19 99 forward 1 TM N out   202 LI : 476342. 1 : 2000MAY01 39 122 forward 3 TM N out 203 LI : 1072759. 1 :

   2000MAY01 409 495 forward 1 TM N in   203 Ll   :   1072759.   1 : 2000MAY01 889 951 forward 1 TM N in   203 LI   :   1072759.   1 :   2000MAY01 1387 1458 forward 1 TM N in     203 Ll   :   1072759.   1 : 2000MAY01 1687 1770 forward 1 TM N in   203 Ll   :   1072759.   1 :   2000MAY01 392 478 forward 2 TM N out     203 Ll   :   1072759.   1 : 2000MAY01 1055 1132 forward 2 TM N out 203 LI :   1072759.   1 :   2000MAY01 1424 1507 forward 2 TM N out     203 Ll   :   1072759.   1 : 2000MAY01 1694 1768 forward 2 TM N out 203 LI : 1072759. 1 :

   2000MAY01 1191 1277 forward 3 TM N out   203 Ll   :   1072759.   1 : 2000MAY01 1677 1760 forward 3 TM N out 204 LG : 998857. 1 :   2000FEB18 1195 1281 forward 1 TM N in   204 LG : 998857. 1 : 2000FEB18 164 226 forward 2 TM N out 204 LG : 998857. 1 : 2000FEB18 344 400 forward 2 TM N out 204 LG : 998857. 1 :   2000FEB18 398 460 forward 2 TM N out   204 LG : 998857. 1 :   2000FEB18 1478 1561 forward 2 TM N out   205 LG : 482261. 1 :   2000FEB18 19 93 forward 1 TM N out   205 LG : 482261. 1 : 2000FEB18 890 961 forward 2 TM N out 205 LG : 482261. 1 : 2000FEB18 1070 1123 forward 2 TM N out 205 LG : 482261. 1 :   2000FEB18 21 89 forward 3 TM N out   205 LG : 482261. 1 : 2000FEB18 1242 1292 forward 3 TM N out 206 LG : 480328. 1 :

   2000FEB18 436 522 forward 1 TM N out 206 LG : 480328. 1 : 2000FEB18 568 642 forward 1 TM N out 206 LG : 480328. 1 : 2000FEB18 769 849 forward 1 TM N out 206 LG :   480328. 1   :   2000FEB18 967 1029 forward 1 TM N out   206 LG : 480328. 1 : 2000FEB18 56 130 forward 2 TM N in 206 LG : 480328. 1 :   2000FEB18 194 280 forward 2 TM N in   206 LG : 480328. 1 : 2000FEB18 396 482 forward 3 TM N out 206 LG : 480328. 1 :   2000FEB18 747 818 forward 3 TM N out   207 LG :   311197.   1 :   2000MAY19 241 315 forward 1 TM N in   207 LG :   311197.   1 : 2000MAY19 527 613 forward 2 TM N out 

 <Desc/Clms Page number 211> 

 208 LG :   1054883.   1 :   2000MAY19 76 129 forward 1 TM N out   208 LG : 1054883. 1 :

   2000MAY19 83 145 forward 2 TM N out 209 LG : 399395. 1 :   2000MAY19 163 216 forward 1 TM N out   211 LI : 272913. 22 : 2000MAY01 37 123 forward 1 TM N in 

 <Desc/Clms Page number 212> 

   TABLE 4 SEQ SEQ SEQ ID Component ID Component ID Component NO: ID Start Stop NO: ID Start Stop NO:

   ID Start Stop 4 70513533D1 496 1069 5 60123152B1 714 1379 1 g1260446 2 316 4 70317606D1 522 961 5 1602161H1 603 798 1 6791379H1 1 397 4 1296898H1 262 495 5 6217784H1 857 1351 1 g1614819 215 655 4 6338333H1 640 1159 5 6217935H1 859 1356 1 g1647514 244 543 4 g4332126 1 454 5 1473544R6 214 653 2 5911492F8 1 467 4 2659966F6 1 192 5 1473544T1 249 653 2 5911492H1 1 271 4 2659966T6 1 194 5 1473544H1 408 653 2 5911492T8 303 633 4 70513545V1 31 585 5 60123139B1 948 1362 3 5311056H1 591 753 4 70514030D1 31 577 5 60124858B2 982 1362 3 6866213H1 784 1388 4 70514030V1 31 589 5 g4457589 222 601 3 5659105H1 1261 1340 4 70513041V1 31 366 5 1473544T6 247 569 3 5498383R6 1291 1674 4 2659966H1 1 247 5 g715576 191 567 3 g5553287 1 315 4 70512591V1 31 171 5 g993694 255 560 3 6989857H1 1 436 4 70512591D1 31 169 5 2866203H1 459 557 3 6955370H1 22 540 4 70318172D1 57 447 5 g682774 196 472 3 g4534562 24 

  504 4 70513426V1 61 680 5 g3213293 1 454 3 g4390046 24 500 4 70320498D1 247 638 5 g3148379 141 356 3 g1192915 25 170 4 034207H1 1 290 6 4835189H1 679 932 3 g2003054 31 344 4 70515532V1 24 12 6 g1920265 798 1061 3 6818987J1 33 250 4 70317785D1 295 738 6 4541025H1 804 1057 3 6818431J1 33 570 4 70516168V1 24 391 6 2330975H1 952 1172 3 g2003419 45 421 4 70321122D1 370 783 6 2330975R6 952 1311 3 g1551472 61 213 4 70514482D1 24 278 6 2430651T6 1141 1667 3 6147606H1 71 625 4 70514482V1 24 278 6 2571536T6 1144 1680 3 6990907H1 383 885 4 70318245D1 25 395 6 3878850T6 1244 1662 3 6866026H1 381 973 4 70318040D1 29 409 6 g2930491 1302 1714 3 7067123H1 525 1069 4 70514959V1 1 300 6 g3844521 1455 1711 3 5498383F6 573 1055 5 g2958900 175 294 6 6623593H1 104 689 3 5498383H1 573 811 5 167606H1 1 173 6 6623593J1 634 1236 4 1709025H1 198 445 5 60123139D2 1 307 6 4724029H1 1 133 4 70319971D1 397 784 5 

  60123152D1 1 368 6 5577111H1 14 200 4 70320592d1 412 733 5 60123139D1 11 156 6 g2183982 84 445 4 70320769D1 462 809 5 6127857H1 1 475 6 3878850F6 119 524 4 70317687D1 464 962 5 g706800 1 340 6 3878850H1 120 399 4 6483492H1 184 713 5 g690740 11 385 6 g2783388 239 721 4 5006925H1 88 362 5 4454268H1 377 645 6 g2785249 239 710   

 <Desc/Clms Page number 213> 

   6 g1920471 284 439 8 2346879T6 167 442 8 3881903H1 1460 1700 6 g1784782 388 828 8 2313878H1 229 495 8 3014303T6 1504 1714 6 6551102H1 429 951 8 6081177H1 264 720 8 490059H1 1515 1763 6 6550902H1 429 1042 8 3773491H1 306 476 8 1803962T6 1575 1714 6 6551002H1 429 957 8 6413047H1 401 935 8 692274H1 1583 1765 6 g3645387 447 909 8 4212712H1 407 675 8 2313878T6 1587 1714 6 2430651H1 448 703 8 6918705H1 413 837 8 g2436491 1623 1714 6 2430651R6 448 680 8 3014942H1 416 684 8 g3277091 1654 1714 6 3712318H1 471 7590 8 

  1807684F6 483 905 8 3319247H1 1718 1969 6 g981405 485 787 8 18039762F6 483 841 8 4917483H1 1916 2145 6 g1975879 1 219 8 1803962H1 483 760 8 6826179J1 1971 2309 6 g1784993 585 909 8 1807684H1 483 746 8 6826179H1 1971 2309 6 1467511T7 1 438 8 3876768H1 529 819 9 3088820H1 537 807 6 6326238H1 8 306 8 6024789H1 553 695 9 2012535H1 561 787 6 526662H1 25 208 8 4062033H1 555 779 9 3232447H1 618 863 6 2856544H1 34 146 8 4634711H1 615 877 9 2536817H2 670 926 7 1980062R6 2 491 8 3014303H1 667 964 9 g2209764 832 1178 7 1980059H1 1 280 8 3014303F6 667 1096 9 7260050H1 1 537 7 5573392H1 30 283 8 1633402H1 672 885 9 2457623F6 101 601 8 2642346H1 1 239 8 5865873H1 725 997 9 6987326H1 110 654 8 2662640H1 2 95 8 6552378H1 881 1430 9 7032756H1 151 720 8 2523793H1 11 212 8 2892794H1 907 1089 9 6457158H1 350 878 8 3155877H1 12 278 8 2892794F6 907 1389 9 2890632F6 351 531 8 3539049H1 13 237 8 6406936H1 

  935 1476 9 2893095F6 351 909 8 3179390H1 13 320 8 4902732H1 962 1225 9 2893095H1 351 614 8 5543678H1 15 210 8 1492959H1 970 1171 9 7166124H1 401 930 8 6834974H1 15 623 8 g1686447 974 1299 9 4760777H1 427 714 8 257055R6 16 423 8 5929258H1 974 1266 9 3160745H1 437 710 8 133343H1 16 157 8 3509940H1 974 1230 9 g2006716 479 776 8 484198H1 16 163 8 4588644H1 983 1230 9 g2209659 481 940 8 257055H1 17 224 8 6979634H1 991 1412 9 3362481H1 514 763 8 2346879H1 18 247 8 946914H1 1010 1310 9 1751755H1 520 735 8 2346879F6 18 442 8 g5854943 1083 1460 9 6141843H1 536 793 8 g1985882 19 366 8 5544986H1 1095 1303 10 6584158H1 14 592 8 3156918H1 21 155 8 3860122H1 1133 1400 10 3401851H1 4 241 8 4934879H1 22 297 8 g4260489 1228 1701 10 3403550H1 1 259 8 g1751206 41 388 8 1724521H1 1385 1493 10 3404251H1 3 258 8 4024568H1 42 189 8 1724521F6 1385 1714 10 g1137215 2430 2639 8 258140H1 58 433 8 3026692H1 

  1429 1687 10 3378612H1 2451 2638   

 <Desc/Clms Page number 214> 

   10 6584049H1 6 562 12 70557043V1 221 595 14 6420632H1 79 637 10 3402034H1 10 229 12 70562198V1 227 819 14 2195346F6 1 450 10 g307503 16 2638 12 70562312V1 245 894 14 2195346H1 344 450 10 70779637V1 2077 2632 12 70561005V1 249 895 14 3933140F6 11 351 10 7258175H1 2121 2603 12 70559925V1 254 840 14 3933140H1 96 351 10 3961980H1 2130 2252 12 70556220V1 274 841 14 6020638H1 1 263 10 6584189T1 2152 2553 12 70561793V1 282 939 14 g5880310 52 255 10 6584158T1 2159 2659 12 70559002V1 293 888 14 g2156573 11 254 10 g3785069 2394 2641 12 6127888H1 351 824 14 g2264198 30 253 10 981592T6 2170 2701 12 70557039V1 359 611 14 6936789H1 11 173 10 3385383T6 2171 2601 12 70559636V1 391 1033 14 5043956H1 11 159 10 1221889T6 2202 2700 12 70561656V1 409 852 15 1897573H1 1 259 10 70779908V1 2348 2654 12 70560742V1 507 

  990 15 4337619T6 137 645 10 70776665V1 1884 2562 12 70561641V1 594 1066 15 5436383H1 598 849 10 70775642V1 1981 2519 12 g873095 657 1032 16 1671029F6 1 443 10 70776673V1 2022 2640 12 70449484V1 674 894 16 1671029H1 72 144 10 6584049T1 2035 2569 12 6848717H1 732 1253 16 1671029T6 388 443 10 3401765H1 2050 2287 12 70558944V1 1495 2173 16 g2238932 388 443 10 3401379H1 15 260 12 70450051V1 1550 1731 16 g2243675 388 443 10 3404170H1 18 267 12 70561063V1 1607 2025 17 g2219785 236 531 11 6905943H1 1 534 12 g5361507 1719 2172 17 5099781H1 378 604 12 70562343V1 1 628 12 g4735148 1778 2172 17 3254347R6 527 1132 12 70560334V1 1 538 12 g4875543 1779 2195 17 5668261H1 661 889 12 70559056V1 2 275 12 758040T6 1784 2147 17 4289028H1 781 1061 12 70560532V1 12 269 12 g872996 1824 2188 17 g3931955 822 1267 12 70559641V1 12 383 12 g3840560 1830 2175 17 g3401348 823 1271 12 70559213V1 12 511 12 

  g4333943 2114 2199 17 g2825326 825 1144 12 70561550V1 12 575 13 2734453F6 1 435 17 2750359R6 824 1242 12 70561738V1 12 582 13 2734453H1 1 252 17 2750359T6 847 1241 12 70559422V1 13 631 14 4860612T7 1184 1574 17 2351445H1 151 367 12 70559480V1 12 687 14 g902319 1020 1492 17 2750359H1 968 1241 12 758040H1 12 272 14 g778351 1137 1386 17 2203345H1 1018 1169 12 70559415V1 13 605 14 g1638619 1140 1268 17 3254347H1 979 1089 12 758040R6 12 310 14 6929341H1 724 1222 17 4822216H1 1202 1393 12 70561876V1 13 585 14 1675892T6 754 1203 17 3184753F6 1 472 12 70559130V1 35 384 14 1720010F6 696 1079 17 5108214T6 60 258 12 g2576304 119 2175 14 1720010H1 696 922 17 3184753T6 60 312 12 70561182V1 150 812 14 4711951H1 675 833 17 g4287070 152 617 12 70560479V1 217 834 14 5496406H1 546 795 17 g2524411 160 493   

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  427 21 2670050H1 722 974 17 g2788109 236 513 20 4923878H1 211 494 21 4118973H1 762 1013 18 6829315H1 314 884 20 6847871H1 274 451 21 2744119H1 784 968 18 g3109791 492 811 20 g5151844 373 585 21 g1081212 787 1107 18 g5452473 492 650 21 g4281322 2017 2463 21 5576610H1 879 1134 18 g4564783 26 423 21 3934096H1 2040 2211 21 6418368H1 926 1226 18 G5438746 1 423 21 3934274H1 2042 2187 21 5864111H1 950 1233 18 g3805312 34 423 21 1648485T6 2045 2159 21 5033538H1 997 1264 18 g4372490 78 423 21 1648242H1 2052 2198 21 3511167H1 1010 1288 18 g2954208 76 423 21 1648485H1 2052 2198 21 5805062H1 1021 1225 18 g2954218 142 422 21 1648485F6 2052 2198 21 4873652H1 1038 1293 18 g3307490 142 344 21 4676772H1 2056 2317 21 6614566H1 1064 1565 18 2011384H1 190 263 21 1004843T6 2092 2693 21 2645969F6 1085 1588 19 3106785H1 747 1023 21 g4533314 2101 2207 21 2645969H1 1085 1330 19 g565430 783 1150 21 

  3416826H1 2134 2365 21 g2112831 1112 1336 19 g1505888 812 1132 21 5304471H1 2184 2431 21 3742279H1 1144 1422 19 g892901 859 1159 21 1310089F6 2250 2718 21 6334985H1 1249 1754 19 g892900 1 179 21 1310089H1 2250 2448 21 5292787H2 1287 1357 19 3436153H1 1 269 21 2461918H1 2266 2461 21 g853299 1365 1679 19 g4737696 1 452 21 g4330296 2658 2953 21 5742830H1 1399 1713 19 839090H1 73 301 21 g3178479 2713 2950 21 777713H1 1400 1625 19 6938671H1 206 685 21 g856243 2748 3020 21 777713R6 1401 1702 19 6939071H1 207 731 21 3951063F6 1 417 21 2291319H1 1424 1651 19 g646973 427 675 21 3951063H1 1 285 21 3241291H1 1436 1673 19 g1277657 484 934 21 g2114724 215 609 21 1832630H1 1481 1750 19 2755039H1 596 767 21 1804812F6 311 871 21 1804812T6 1571 2172 19 838332T6 615 1116 21 1804812H1 311 582 21 1481768H1 1572 1793 19 4800144H1 651 916 21 1804772H1 311 530 21 3730896H1 1583 1874 19 2084151H1 664 945 

  21 2662164H1 346 588 21 4442950H1 1631 1773 20 6199407H1 1 558 21 2011927H1 365 608 21 2794894H1 1648 1896 20 2519336F6 37 448 21 4863613H1 380 645 21 4161422H1 1664 1913 20 2519336H1 37 198 21 3934325H1 386 672 21 4163675H1 1665 1779 20 6793417H1 61 331 21 3763239H1 402 671 21 g2787436 1672 1875 20 1639314H1 71 248 21 1004843R6 502 864 21 1898917T6 1693 2159 20 6818283H1 75 652 21 1004843H1 502 706 21 5026603H1 1695 1945 20 1984661R6 94 490 21 g846375 547 889 21 3726809H1 1753 2047 20 1984662H1 94 368 21 5810205H1 585 885 21 1891483T6 1803 2160 20 1984661T6 127 543 21 384851H1 652 926 21 2293309H1 1836 2077 20 4021069H1 177 388 21 g1861137 688 1180 21 1214225H1 1850 2020   

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   21 g856337 1855 2128 22 5681165H1 629 889 22 g3147585 1357 1758 21 g3733909 1913 2198 22 5277977H1 637 892 22 g2053942 1361 1759 21 g3890798 1917 2386 22 

  4645753H1 69 921 22 g1332315 1371 1839 21 5696574H1 1925 2048 22 6193452H1 705 1000 22 3918780h1 1373 1665 21 g1081162 1942 2201 22 6193441H1 705 963 22 g3884754 179 1765 21 2645969T6 1964 2222 22 746066H1 713 961 22 5207656H1 1382 1616 21 g1860963 1970 2387 22 4771093H1 754 1022 22 2292948H1 1388 1552 21 777713T6 1983 2420 22 g2054100 771 1123 22 22 1373715H1 1388 1603 21 g4149122 1984 2203 22 g4762720 796 1252 22 4742391H1 1388 1647 21 4875545H1 1994 2292 22 g2321843 801 1217 22 2749392H1 1400 1627 22 487652H1 1 242 22 g3017276 804 1256 22 g1241849 1408 1559 22 487652R6 1 232 22 g483457 850 1222 22 g6046888 1408 1785 22 g2003010 47 328 22 g4762705 865 1252 22 2294209T6 1428 1945 22 6442754H1 49 581 22 4338534H1 870 1155 22 g1471742 1441 1766 22 6152037H1 50 234 22 628211H1 887 1051 22 22 g4332659 1471 1772 22 7072795H1 75 624 22 5563275H1 922 1144 22 3842415 1473 1785 22 

  3126080H1 75 234 22 5286207H1 952 1210 22 g2841585 1474 1756 22 491193H1 83 234 22 6160626H1 953 1244 22 g2003011 1479 1772 22 4751976H1 90 232 22 6838871H1 1013 1491 22 2501320H1 1513 1738 22 7100707H1 100 609 22 2400686H1 1068 1153 22 g3961438 1525 1988 22 2292643H1 102 232 22 g866503 1107 1440 22 g2022791 1559 1757 22 6140970H1 105 232 22 6747627H1 1122 1593 22 g6132921 1559 1977 22 4066069H1 110 234 22 g920433 1143 1481 22 5690247H1 1568 1843 22 5007390H1 113 231 22 2294209R6 1179 1688 22 g1496515 1580 1977 22 4773161H1 130 415 22 2294209H1 1179 1444 22 g2252260 1620 1984 22 6993149H1 145 680 22 g1471741 1213 1600 22 g3739177 1650 1831 22 572575H1 150 232 22 g1332314 1213 1712 22 5338636H1 1671 1915 22 5756677H1 180 483 22 6021757H1 1214 1822 22 5710854H2 1686 2004 22 6064882H1 346 644 22 2189963H1 1229 1505 22 3223930H1 1686 1772 22 5372689H1 367 514 22 g5766946 1310 1760 22 

  4255692H1 1701 1756 22 3123257H1 367 493 22 g2524669 1309 1776 22 g1210253 1725 1980 22 g2022792 381 648 22 g2178309 1317 1448 22 g920434 1730 1946 22 5068149H1 503 782 22 g4762067 1326 1779 22 g1210208 1763 1980 22 5894157H1 560 759 22 g5361693 1328 1778 22 4320678H1 1841 1973 22 5898812H1 560 853 22 g4074013 1332 1773 22 g884068 1924 2233 22 2621801H1 561 824 22 g5849550 1331 1762 23 6991066H1 1 190 22 6401738H1 574 844 22 6736883H1 1355 1730 23 4753733H1 121 279 22 2122453H1 622 902 22 g2183535 1356 1766 23 4753733F8 122 613 22 2122453F6 622 764 22 g4332913 1357 1772 23 7065882H1 183 679   

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   3 g5676063 251 722 25 6862186H1 519 806 27 2966432H1 1421 1627 23 1576986F6 270 661 25 g5543132 541 939 27 g3419012 1500 1961 23 1576986H1 270 349 25 g4196555 541 821 27 g2178116 1505 1630 23 1576986T6 275 683 25 g5636120 541 982 27 

  g2715860 1512 1962 23 g3843718 364 724 25 g3246528 541 923 27 3891858T6 1559 1901 23 g3750491 408 722 25 g5636142 541 1018 27 3891858F6 1566 1961 23 3604635H1 409 584 25 g2568732 577 1024 27 3891858H1 1566 1796 23 g793096 600 881 25 3747228H1 589 887 27 925628T6 1574 1943 24 6756043H1 101 504 25 6600395H1 587 999 27 925628H1 1574 1797 24 3845553H1 368 574 25 6978931H1 605 954 27 g5113339 1766 2184 24 5057288F9 382 1021 25 2240520H1 629 730 27 7286802H1 1 500 24 5057288H1 382 660 25 2240520F6 641 967 27 3490274H1 26 319 24 5065461F8 404 839 25 5626829H1 663 931 27 6817649H1 55 308 24 2359950H1 625 748 25 g2162199 663 863 27 6779822H1 68 617 24 6112349H1 792 1081 25 6862047H1 699 978 27 1343838H1 166 433 24 5444375H1 912 1131 25 7281807H1 809 1041 28 6827746J1 1 328 24 g4264503 989 1390 25 g2209650 815 1308 28 g4372436 2 487 24 3385663T6 1011 1248 25 6216076H1 841 1322 28 7277307H1 1 

  320 24 3845553T6 1009 1248 25 7238641H1 862 978 28 g1109781 68 2457 24 3385663F6 1 315 25 6210267H1 868 970 28 g2987914 238 414 24 3385663H1 1 257 25 7039186H1 940 1508 28 3242379H1 460 594 24 5468404H1 3 271 25 g2209760 1092 1569 28 5843682H1 675 814 24 5468404F8 3 587 26 7281748H1 1 439 28 6244385H1 750 960 24 5072501H1 11 292 26 7281705H1 1 574 28 3560794H1 878 978 24 6917202H1 10 259 27 925628R6 1574 1961 28 6146228H1 1389 1559 24 4148204T6 1176 1391 27 6466550H1 228 746 28 g5454470 1759 2219 24 4148204H1 1183 1435 27 035057H1 483 638 28 g5634013 1818 2245 24 4148204F6 1183 1390 27 2173285F6 571 968 28 g3933775 1835 2188 24 g4532148 1256 1361 27 2173285H1 571 762 28 6824121H1 1864 2239 24 213841R6 1325 1390 27 1743077R6 577 1069 28 6824121J1 1864 2239 24 213841T6 1325 1393 27 1743077H1 577 892 28 6789580H1 1898 2361 24 213841H1 1325 1390 27 3344276H1 598 856 28 g5596185 1915 

  2221 24 213833H1 1325 1390 27 6579246H1 697 1025 28 g1137662 1994 2365 25 7204172H1 1 536 27 5637939F8 825 1101 28 g3917233 2020 2409 25 6819816H1 368 566 27 5637939H1 825 1082 28 3222719H1 2019 2299 25 6836688H1 383 960 27 5624530R8 836 1190 28 g4487317 2029 2459 25 6349781H2 434 616 27 5637939R8 1016 1380 28 g2878126 2045 2386 25 7280863H1 477 880 27 3483518H1 1163 1470 28 925319R6 2061 2458 25 g4081510 513 860 27 6817649J1 1402 1969 28 925319T6 2061 2419   

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   28 925319H1 2061 2370 29 70254664V1 444 930 29 70248162V1 1486 1604 28 g6463796 2103 2467 29 70254648V1 463 958 29 70254647V1 612 1111 28 2175458H1 2139 2397 29 70257273V1 531 636 29 7077379H1 627 1163 28 g1153903 2165 2458 29 g3778732 559 1002 29 7237267H1 1907 2197 28 g1114183 2165 2444 29 4023624H1 1 286 29 70660270V1 1926 2390 28 g3844190 2203 2458 29 70249613V1 1 198 

  29 70255193V1 1976 2586 28 6193937H1 2265 2534 29 3269702H1 8 254 29 70656301V1 1979 2231 28 g5364673 2276 2476 29 70249634V1 142 374 29 71273576V1 1979 2197 29 6450124H1 992 1374 29 1286494H1 165 402 29 6620779H1 2006 2603 29 70254601V1 1036 1539 29 7191139H2 175 755 29 70655675V1 2023 2430 29 70250522V1 1050 1299 29 5841654H1 175 446 29 70255204V1 2038 2632 29 70255407V1 1073 1570 29 7120789H1 398 702 29 1741694T6 2052 2625 29 g2004277 1088 1409 29 70255670V1 413 949 29 6121206F8 2130 2669 29 70247748V1 1116 1378 29 70255767V1 1672 2119 29 5267406H1 2070 2344 29 70249044V1 1117 1269 29 3272240F6 1165 1617 29 6120592H1 2110 2682 29 70256246V1 1130 1541 29 3272240H1 1166 1413 29 6128080H1 2110 2689 29 70248757V1 1132 1311 29 70257558V1 1171 1601 29 6120292H1 2110 2671 29 7331424H1 1149 1638 29 70248599V1 1202 1420 29 6127177H1 2110 2671 29 70248822V1 1157 1333 29 70250795V1 1247 

  1524 29 3269702T6 2111 2623 29 70656623V1 1723 2219 29 70249059V1 1304 1679 29 70254909V1 2152 2688 29 661089H1 1723 1986 29 70250085V1 1310 1485 29 4001246R6 2158 2517 29 661089R6 1723 2224 29 70251290V1 1314 1573 29 4001246H1 2158 2458 29 6444789H1 1742 2329 29 70255060V1 1326 1720 29 70659041V1 2163 2394 29 70248827V1 1769 2103 29 70254762V1 1330 1860 29 g5864796 2171 2685 29 6551207H1 1771 2379 29 70257824V1 1339 1525 29 g2913171 2184 2622 29 6745795H1 1775 1933 29 70255199V1 1341 1753 29 6752303H1 2193 2671 29 6977014H1 1787 2347 29 70250692V1 1345 1576 29 70658326V1 2194 2559 29 70258294V1 1791 2007 29 70257468V1 1347 1462 29 70256172V1 2209 2671 29 70254633V1 1805 2279 29 70251556V1 1351 1435 29 2287809R6 2232 2681 29 70255157V1 1838 2268 29 70255729V1 1361 1869 29 2287809T6 2232 2629 29 6746363H1 1854 2506 29 70255867V1 1392 1903 29 2287809H1 2232 2458 29 70655542V1 1865 

  1948 29 70255422V1 1366 1870 29 6834934H1 2233 2671 29 6561163H1 1872 2335 29 70254725V1 1690 2218 29 2255790H1 2260 2543 29 7239061H1 1879 2417 29 70254417V1 1671 1916 29 70657495V1 2264 2391 29 70247905V1 1879 2117 29 7033280H1 1691 2365 29 6835034H1 2288 2570 29 70255827V1 764 1239 29 1741694R6 1410 1866 29 70254456V1 2316 2671 29 70255250V1 842 1401 29 1741694H1 1410 1587 29 661089T6 2355 2639 29 70248754V1 866 1233 29 70256209V1 1414 1915 29 70249168V1 2452 2682 29 70256314V1 917 1512 29 7079231H1 1418 1892 29 6059187H1 2608 2671   

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   29 6059196H1 2606 2682 30 3385663T6 1033 1284 30 6917202H1 10 263 29 70255727V1 977 1493 30 3845553T6 1031 1284 30 3385663H1 1 261 29 70257455V1 1692 1951 30 70776533V1 1086 1284 30 5468404F8 3 596 29 70257816V1 1397 1604 30 70776877V1 1087 1284 30 3385663F6 319 29 70257088V2 1659 1980 30 

  4148204T6 1174 1389 30 70775648V1 4 416 29 70255058V1 1663 2172 30 4148204F6 1181 1388 30 70778292V1 4 577 29 5779331H1 1663 1928 30 4148204H1 1181 1433 30 70779496V1 4 568 29 70248161V1 1487 1604 30 g4532148 1254 1359 30 70775549V1 4 538 29 6536779H1 1490 2108 30 5072501H1 11 296 30 70776773V1 4 576 29 70254435V1 1509 2113 30 70770035V1 207 641 31 g3254719 1 445 29 70254449V1 1538 1906 30 70769523V1 245 581 31 g5745388 1 462 29 70257853V1 1570 1900 30 70779544V1 337 548 31 g3959110 1 353 29 70254760V1 1571 1970 30 70776851V1 339 867 31 g897505 1 215 29 70255434V1 1577 2080 30 70779695V1 4 418 31 g5233046 1 295 29 5191169H1 1609 1875 30 5468404H1 3 275 31 G2929868 1 406 29 70257088V1 1659 1980 30 6756043H1 102 509 31 g3016800 1 358 29 70255260V1 1694 2245 30 7190732H1 163 722 31 6202689H1 1 548 29 70255174V1 1695 2113 30 71087882V1 4 560 31 g2435161 9 456 29 g756281 1698 2066 30 

  70777729V1 4 492 31 4707801H1 1197 1296 29 70247381V1 1704 2145 30 70781241V1 4 467 31 806768H1 1211 1421 29 70659718V1 1723 2028 30 70780840V1 4 442 31 6915008H1 1320 1761 29 70254418V1 675 1188 30 70779497V1 342 547 31 3036834H1 1578 1846 29 70258031V1 699 905 30 3845553H1 372 583 31 3036834F6 1578 2016 29 7178470H1 651 1108 30 5057288F9 386 1043 31 5387714H1 1632 1886 29 70256308V1 672 1122 30 5057288H1 386 669 31 7122625H1 443 835 30 70780965V1 4 464 30 70776801V1 399 933 31 691516J1 622 1129 30 2359950H1 634 758 30 5065461F8 408 858 31 5092947H1 899 1177 30 70777278V1 648 1196 30 70778496V1 429 737 31 5370334H1 939 1050 30 70780989V1 653 1111 30 70777098V1 456 752 31 5092947F56 899 1330 30 70779952V1 697 1217 30 70780976V1 476 1081 31 5017752H1 959 1229 30 70777355V1 715 1195 30 70777266V1 503 1128 31 4707801F6 1197 1714 30 70771142V1 716 1207 30 70776726V1 513 1093 31 

  5505129H1 17 282 30 70775784V1 751 1132 30 70772943V1 541 740 31 5514010H1 17 254 30 70769785V1 764 1127 30 70777725V1 576 1166 31 5505122H1 17 228 30 6112349H1 809 1108 30 70779028V1 613 766 31 1225071H1 52 262 30 70776223V1 839 1239 30 213833H1 1323 1388 31 g2929769 56 178 30 5444375H1 933 1164 30 213841T6 1323 1391 31 6314660H1 60 613 30 5057288T8 997 1251 30 213841R6 1323 1388 31 2682720F6 173 606 30 g4264503 1011 1388 30 213841H1 1323 1388 31 6516656H1 275 76   

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   31 6201718H1 312 599 32 042027H1 310 561 36 6769153J1 1 529 31 2682720H1 324 606 32 g1291521 316 662 36 6764824J1 3 589 32 3665294H1 706 823 32 030668H1 319 524 36 g6048166 3 423 32 g1139437 903 977 32 g897225 340 705 37 653157H1 82 310 32 3663680H1 706 950 32 g921054 340 660 37 509350H1 107 307 32 5040414H1 731 947 32 g897232 340 676 37 3386816H1 1 183 32 g1139431 

  904 977 32 5018641T6 368 996 37 3888124H1 4 268 32 1772915R6 911 1308 32 g2933440 374 696 37 6779840H1 9 549 32 1772915H1 911 966 32 1876772T6 379 940 37 3159561H1 58 220 32 4849209H1 1000 1259 32 g2703395 564 979 37 6250084H1 67 593 32 1772915T6 1024 1540 32 2652012T6 566 930 37 2809974H1 67 324 32 g4691031 1318 1588 32 G185336 575 1000 37 3386816F6 1 622 32 6298418H1 1 314 32 3124683H1 622 881 37 3672749H1 81 364 32 3403590H1 1 232 32 g1313829 631 867 37 4005442H1 2 285 32 6327716H1 98 415 32 4200739H1 671 956 37 70821167V1 627 1066 32 g1068995 238 588 32 2244549H1 671 882 37 70822737V1 643 949 32 69210109H1 255 457 32 2684460H1 696 909 37 70832705V1 715 823 32 4307678H1 254 563 33 g3232386 1 362 37 412478T6 835 1013 32 g1858272 267 664 33 g3181700 7 419 37 6779840J1 894 1315 32 2812311H1 503 816 33 6109493H1 322 613 37 g2631213 986 1049 32 2812294H1 505 826 34 3081155F6 1 417 37 

  5501816H1 1053 1261 32 4976432H1 536 792 34 3081155H1 2 315 37 5501816F6 1053 1293 32 g5037963 518 976 34 3081155T6 31 463 37 4005442T6 487 1008 32 4976496H1 536 801 34 g1648354 58 455 37 70818940V1 506 1068 32 g3736768 525 978 34 5800009H1 103 522 37 059074H1 601 765 32 g4986041 540 979 35 3517533R6 332 653 37 2862291H1 375 652 32 4124067H1 552 792 35 3517533H1 332 621 37 g3934153 376 841 32 g4073888 527 978 35 g5178468 190 667 37 g3900180 404 840 32 g5526438 564 978 35 3504115H1 162 477 37 391612F1 432 1051 32 5209523H1 413 705 35 3082646H1 123 415 37 g895347 1 84 32 4109819H1 416 696 35 259700H1 1 278 37 4005442F6 1 326 32 3726063H1 426 688 35 7169509H1 1 477 37 391612R1 353 898 32 4671035H1 421 693 35 6768733H1 58 292 37 412478R6 373 699 32 2311003H1 454 685 35 3176620H1 66 320 37 412478H1 373 600 32 5206768H1 478 723 35 3176620F6 66 551 37 3386816T6 337 818 32 679406H1 480 737 

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  71266530V1 732 1246 106 71118652V1 1408 2023 106 71120385V1 1121 1448 106 2681128F6 695 1180 106 7126623V1 1448 1751 106 g2198304 1133 1220 106 g2541182 2106 2424 106 71265047V1 731 1261 106 71119740V1 1134 1424 106 g3039868 2147 2225 106 71119759V1 731 1234 106 4174878H1 1137 1420 106 3931985H1 896 1182 106 71266124V1 732 1366 106 71117317V1 1153 1724 106 3934538H1 895 1200 106 71118641V1 731 1311 106 71119085V1 1197 1825 106 3934758H1 895 1195 106 71118731V1 731 1292 106 3792653H1 1224 1433 106 3934357H1 895 1200 106 71265792V1 731 1260 106 6329373H1 1235 1780 106 3662984H1 1006 1254 106 1915736R6 731 1137106 71119545V1 1267 1954 106 70529227V1 1009 1601 106 2681128H1 696 984 106 71120838V1 1275 1741 106 71265619V1 1031 1689 106 70531848V1 706 1260 106 4717156H1 1277 1501 106 5946829H1 1044 1325 106 71264971V1 725 1481 106 5391806H1 1282 1481 106 71266345V1 1097 1749 106 

  71266413V1 922 1608 106 71118747V1 1295 1960 106 71120487V1 1102 1594 106 71117117V1 921 1580 106 70527304V1 1301 1969 106 71266010V1 1097 1765 106 3672409H1 937 1168 106 71120713V1 1306 1885 106 5947885H1 1100 1363 106 71118560V1 968 1537 106 71118117V1 1311 1871 106 3173959H1 1322 1589 106 71119741V1 973 1452 106 5290693H1 1314 1610 106 70059533V1 1322 1585 106 3964084H1 1006 1294 106 5287845H1 1314 1441 106 g1981676 1322 1654 106 3628624H1 1936 2251 106 70530468V1 1320 1851 106 70062034V1 1326 1794 106 g2969311 1938 2222 106 3173959F6 1322 1886 107 5913661H1 1 283   

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   107 5913661F8 1 569 111 g3040122 248 503 115 g990246 1645 1912 107 5913661F9 1 575 111 g3051904 251 501 115 957795H1 1653 1903 107 5913661T6 213 821 111 g3804542 368 750 115 472488H1 1653 1882 108 6796436H1 1 435 111 5282615F6 635 1106 115 472488R1 1654 2125 108 

  g5837313 111 559 111 5282615H1 884 1106 115 302216H1 1655 1877 109 2536867F6 918 1430 112 g2563121 1 166 115 1876833H1 1654 1913 109 2536867H2 918 1187 112 2792728F6 1 440 115 2697195H1 1656 1898 109 g1926046 932 1379 112 2792728T6 1 417 115 5377444H1 1666 1918 109 g1925836 933 1201 112 g3040744 1 338 115 2431879H1 1668 1914 109 3244923F6 993 1220 112 g2714186 1 401 115 777024H1 1669 1901 109 3244923H1 993 1213 112 7157205H1 1 461 115 3236114H1 1675 1928 109 2878889H1 1010 1304 112 g4371924 71 205 115 1641892H1 1677 1880 109 2878889F6 1010 1349 112 1394888T6 90 304 115 5569350H1 1678 1920 109 g858504 1080 1411 112 1624877H1 94 288 115 3857806H1 1686 1976 109 5567006H1 1081 1257 112 2792728H1 147 439 115 g2751499 1694 2029 109 1896278H1 1106 1341 113 g2943715 1 1450 115 g876527 1697 2020 109 g784668 1209 1286 113 6487571H1 657 1161 115 4069649H1 1708 1834 109 3364252F6 1 432 113 

  6487571F9 657 1207 115 4307481H1 1724 1900 109 3364252H1 1 226 113 70681361V1 692 1244 115 031299H1 1750 1920 109 096666H1 66 233 113 70681601V1 692 1178 115 3623362H1 1779 2032 109 096675H1 67 240 113 1544823R6 692 1181 115 g5036497 1858 2136 109 7132087H1 255 624 113 70681277V1 692 1164 115 3624213H1 1872 2081 109 4030547F8 461 1001 113 1544823H1 692 898 115 g2411009 1907 2079 109 4030547H1 462 715 113 g4686743 879 1327 115 4721623H1 1918 2011 109 5077091H1 800 1072 113 70680264V1 950 1079 115 2839772H2 1922 2209 110 2717953H1 1 259 113 6476403H1 998 1525 115 6056374H1 1933 2132 110 2806157F6 27 606 114 6272292H2 1 507 115 6056674H1 1933 2136 110 2806157H1 26 323 114 5910821T8 365 636 115 3802178H1 1933 2135 110 2724233T6 367 954 114 5910821T9 365 662 115 4371036H1 1940 2136 111 166942F1 1 624 114 5910821F8 365 793 115 412662H1 1939 2136 111 g3755789 134 505 114 5910821H1 365 676 

  115 1907478H1 1950 2136 111 g3109437 134 208 115 1551035H1 1625 1845 115 5050475H1 1966 2136 111 g3037830 135 509 115 2716914H1 1625 1873 115 6447704H1 1966 2106 111 g3180013 139 583 115 4876737H1 1631 1915 115 g3888474 1988 2321 111 g4270829 142 429 115 7091270H1 1631 1955 115 5379172H1 1989 2238 111 g3594985 142 562 115 674945H1 1636 1903 115 5015647H1 1994 2136 111 5282615T6 168 751 115 670546H1 1636 1756 115 3327673H1 1995 2251 111 g2942533 248 563 115 2119143H1 1637 1891 115 3567634H1 2021 2137 111 g2953832 248 509 115 2815231H1 1643 1910 115 1739210H1 2025 2215   

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   115 880124H1 2032 2132 115 4696530H1 245 433 115 2886241H1 1493 1744 115 2354054H1 2037 2132 115 928643H1 289 554 115 1222871H1 1495 1739 115 1235532H1 2060 2132 115 4529248H1 370 618 115 4193225H1 1498 1707 115 2350867H1 2062 2132 115 3942806H1 418 694 115 

  7065251H1 1500 2097 115 g983285 2072 2421 115 7280921H1 466 677 115 2152140H1 1501 1772 115 4190021H1 2084 2132 115 g2013891 480 698 115 3763167H1 1502 1559 115 g2017399 2116 2329 115 g2013455 480 709 115 3571390H1 1504 1790 115 6550613H1 2153 2494 115 5422141H1 481 726 115 942026H1 1509 1756 115 5195955H1 2155 2420 115 3110608H1 525 776 115 4533306T1 1513 2067 115 3907635H1 2185 2464 115 5659913H1 620 896 115 2124615H1 1517 1816 115 g3166884 2193 2356 115 5545664F8 647 982 115 3684042H1 1522 1808 115 g994523 2232 2476 115 5545664F6 647 1087 115 6715567H1 1523 2096 115 g1099949 2244 2491 115 3163564H1 751 1011 115 3322645H1 1523 1782 115 g757333 2245 2513 115 761584H1 768 982 115 g1873672 1524 2006 115 4534796H1 2255 2463 115 6887167J1 837 1454 115 3590863H1 1530 1809 115 1543657H1 2262 2468 115 1852303H1 1011 1084 115 60123902B1 1538 2104 115 4754806H1 2295 2496 115 6948478H1 1087 

  1504 115 1002539H1 1538 1639 115 1947635H1 2336 2483 115 4158605H1 1206 1469 115 6077425H1 1541 1858 115 3930538H1 2360 2496 115 5270074H1 1238 1440 115 6513731H1 1548 2079 115 2760689H1 2377 2496 115 2365846H1 1239 1469 115 3785763H1 1548 1837 115 5563236H1 2404 2496 115 2444605H1 1244 1464 115 538349H1 1550 1775 115 4310883H1 2408 2496 115 3115706H1 1255 1482 115 1832140H1 1551 1753 115 2778679H1 2439 2496 115 6728257H1 1349 1938 115 1669989H1 1553 1767 115 725638H1 1 243 115 5717548H1 1401 1922 115 450252H1 1553 1773 115 6733284H1 96 640 115 5796003H1 1427 1849 115 737082H1 1556 1778 115 3096672H1 98 410 115 779492H1 1436 1699 115 7065802H1 1560 2096 115 7034622H1 100 606 115 2279382H1 1436 1702 115 6741001H1 1563 2068 115 g777461 120 197 115 5370473H1 1439 1656 115 83532H1 1566 1869 115 5198834H1 146 385 115 4152676H1 1450 1712 115 1599938T6 1569 2099 115 2394482H2 157 372 115 

  60123909B1 1458 2086 115 4226362H1 1573 1848 115 4970513H1 161 431 115 6960268H1 1466 1902 115 529930H1 1578 1721 115 4969579H1 160 361 115 3449619H1 1461 1706 115 4771124H1 1577 1846 115 377654H1 163 374 115 7039654H1 1461 1970 115 4715169H1 1583 1859 115 305879H1 187 444 115 3890456H1 1461 1758 115 4533768T1 1584 2101 115 307158H1 189 433 115 6966324H1 1466 2051 115 4895425H1 1591 1861 115 115 3319063H1 215 496 115 765511H1 1469 1812 115 4348930H1 1593 1852 115 5504343H1 219 449 115 4940482T9 1481 2031 115 4348733H1 1594 1854 115 3513751H1 230 473 115 6409578H1 1486 1978 115 g2616416 1596 1833 115 4775727H1 230 504 115 6734637H1 1495 1912 115 5698825H1 1596 1847   

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   115 2288814H1 1597 1844 119 3437984H1 274 530 122 60110854B2 1164 1287 115 5762293H1 1598 2136 119 2544176H1 332 520 122 g2884782 1 452 115 3082229T6 1601 1987 120 

  2807456F6 1 508 122 g2220423 1 398 115 3737781H1 1598 1897 120 2807456H1 1 249 122 g1267721 1 282 115 883098H1 1600 1835 120 2807456T6 122 671 122 g3918260 1 411 115 5336847H1 1601 1837 121 g3595066 1 357 123 2807456F6 194 701 115 2741220H1 1601 1860 121 4665764H1 1 257 123 2807456H1 1 249 115 880546H1 1600 1841 122 70151773V1 587 917 123 2807456T6 31 580 115 6398288H1 1601 1744 122 6020347701 266 822 123 270567H1 78 161 115 3252954H1 1608 1863 122 522228H1 955 1199 123 269931H1 374 499 115 3779658H1 1618 1921 122 522135H1 955 1090 123 269626H1 1824 2053 116 g1891130 669 957 122 026879H1 768 1022 123 269080H1 128 352 116 7333578H1 1 523 122 60203621U1 1 548 123 270403H1 85 349 116 6545439H1 141 676 122 60203622U1 118 507 123 270910R1 1824 2022 116 93805536 534 966 122 4401727H1 579 846 124 587588R6 220 555 116 93322110 534 772 122 g2540618 526 801 124 587588T6 44 555 116 5610773H1 

  537 788 122 2807905H1 484 793 124 587588H1 1 165 117 6929774H1 1 513 122 70152547V1 797 1219 125 3321035F6 317 786 117 6052078J1 72 520 122 3812508H1 140 438 125 3321035H1 330 595 117 6052078H1 72 520 122 g1265991 198 338 125 g1319620 414 927 117 4970577H1 120 381 122 3860472H1 256 546 125 g2741801 414 556 117 4970577F6 120 483 122 3520754H1 334 621 125 g2841030 1354 1422 117 6292129H1 423 637 122 70152228V1 391 1018 125 g2933104 421 906 117 6294687H1 423 647 122 4350225H1 364 638 126 5259815H1 1 206 117 2807905H1 555 863 122 70155823V1 419 986 126 3568526H1 71 366 117 g2540618 597 871 122 g3919706 1 424 126 1289824F6 181 734 117 4401727H1 650 916 122 2512390F6 1 316 126 g928730 693 889 117 5729803H1 731 1236 122 g1792877 1 365 126 764159H1 693 849 117 g1301257 787 1245 122 g4187765 4 446 126 6620992H1 718 1297 117 026879H1 838 1092 122 g2905531 4 102 126 839936R1 825 1369 117 

  g1303063 897 1111 122 70156040V1 960 1356 126 1289824H1 181 349 117 22135H1 1025 1160 122 70151954V1 828 1353 126 839936H1 825 1066 117 52228H1 1025 1269 122 2512390H1 14 316 126 3869224H1 996 1286 118 587588R6 1 336 122 60202389B1 858 1335 126 1685280F6 522 955 118 587588T6 1 512 122 60202388B1 870 1329 126 3223525H1 1137 1457 118 g1069975 229 539 122 60202388B2 924 1329 126 3843717H1 1 293 119 g2809760 1 443 122 999391H1 37 270 126 g1395923 1198 1531 119 g2934256 68 387 122 2512390T6 38 315 126 1685280H1 522 754 119 2785236H2 220 481 122 4970577T6 45 618 126 5028090H1 1333 1598   

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   126 4216695H1 1404 1656 130 4032140H1 1443 1700 131 6881955J1 2681 3266 126 1289824T6 249 852 130 1597096H1 1442 1628 131 2825680F6 7992 8265 126 g2540596 600 897 130 3438058T6 1483 1957 131 t5036098 8059 8265 126 5724304H1 664 1233 130 4032240T9 1491 

  1900 131 4027974T6 6903 7418 126 1947742T6 688 858 130 g3804916 1564 1997 131 5717756H1 6914 7382 126 g1225270 688 889 130 g3432508 1574 2002 131 2717848T6 6942 7421 126 3438058F6 269 562 130 g3554781 1621 2002 131 4722802H1 6965 7069 126 3438058H1 318 562 130 1947742T6 689 861 131 g3594812 7015 7468 126 3438058T6 45 516 130 g928730 693 892 131 6403421H1 7053 7317 126 g3804916 6 436 130 3223525H1 1152 1474 131 93163456 7067 7469 126 g3254781 1 379 130 5259815H1 1 206 131 2649733F6 7123 7465 127 3504571H1 910 1216 130 3568526H1 71 366 131 2649733H1 7123 7369 127 g2055741 1038 1358 130 1289824F6 181 737 131 7003170H1 7124 7465 127 g1270278 1025 1374 130 1289824H1 181 349 131 g683322 7127 7465 127 2733544H1 1137 1405 130 g2540596 600 900 131 2649733T6 7128 7424 127 g1162686 1144 1490 130 4216695H1 1421 1676 131 2484677H1 7142 7369 127 g1109059 1197 1484 130 3438058F6 1437 1733 131 

  g6075627 7147 7469 127 g1774715 1212 1519 130 3438058H1 1437 1682 131 g3048962 7149 768 127 g3897241 1316 1718 130 1597096F6 1442 2036 131 212391H1 7200 7437 127 5854467H1 1401 1557 130 1685280H1 1243 1477 131 g6506945 7080 7465 127 g1898243 1492 1691 130 5028090H1 1348 1616 131 g6073337 7082 7469 128 g2358498 616 997 130 7213258H1 1225 1803 131 g2630574 7111 7470 128 183176H1 747 971 130 g1395923 1213 1549 131 5207126H2 2813 2981 128 183176R6 507 971 130 3928775H1 696 891 11 2825680H1 7994 8265 128 183176R1 359 971 130 1289824T6 249 855 131 g560960 8006 8268 128 2733388H1 659 888 130 3843717H1 1153 1448 131 g796025 8011 8277 128 5616358H1 602 878 130 764159H1 693 852 131 1415181H1 8034 8265 128 g2824012 433 792 130 5724304H1 664 1213 131 3336518H1 3434 3669 128 g4762579 167 600 130 6040888H1 665 891 131 4723427H1 3458 3606 128 7104793H1 1 520 130 g1225270 689 892 131 70390598D1

  3509 4085 128 4004284H1 207 474 130 7153412H1 656 1189 131 264846H1 2524 2856 129 3928775H1 1 192 130 3640283T9 1701 1932 131 6357277H1 2531 2839 129 2562126T6 40 182 130 3640283T8 1701 1908 131 5544294H1 2708 2828 130 94902006 709 895 130 g3538751 1690 2005 131 2101112T6 7755 8220 130 2562126T6 708 852 130 3640283F8 1701 1949 131 7162079H1 218 671 130 839936R1 828 1385 130 6620992H1 717 1277 131 g2599501 1 4447 130 839936H1 828 1073 131 3404480H1 1921 2103 131 6044517J1 7709 8237 130 1685280F6 1040 1477 131 5665320H1 2177 2358 131 810518H1 8140 8262 130 3869224H1 1001 1301 131 872922H1 7624 7881 131 2101112H1 7763 8008   

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   131 2101112R6 7764 8137 131 g5675561 3974 4452 131 5036970H1 7696 7961 131 6559424H1 7774 8284 131 6618788H1 3991 4472 131 g2212423 7200 7465 131 g2140984 7774 8165 131 g3144228 4048 4452 131 32820246 7255 7409 

  131 3781662H1 7815 8126 131 g2751511 4080 4441 131 3323350H1 7271 7524 131 744157H1 7818 8046 131 1863153F6 4273 4666 131 g2768029 7301 7465 131 4774224T9 7821 8181 131 1863161F6 4273 4526 131 3322677H1 7313 7591 131 g2324704 7831 8266 131 1863153H1 4273 4520 131 g1515911 7317 7464 131 g3796940 7834 8265 131 385245H1 4275 4498 131 3865622H1 7330 7526 131 95839127 7843 8265 131 g4970402 4364 4819 131 g317850 7332 7592 131 g3932489 7870 8265 131 5857654H1 4368 4636 131 g3174898 7357 7469 131 g683058 7964 8268 131 g1624652 4395 4466 131 4253321H1 7391 7470 131 g1225252 7984 8268 131 g1023422 4479 4773 131 4244366H1 7390 7465 131 2825680T6 7985 8226 131 g1023318 4511 4801 131 3334656H1 7394 7606 131 3278368H1 7719 7959 131 3341807F6 4527 4888 131 6355568H1 3040 3236 131 4726587H1 7619 7844 131 3341807H1 4527 4766 131 g1881143 3040 3347 131 2108075H1 7405 7654 131 4027974F6 4592 4881 

  131 1372008H1 7499 7667 131 g1241115 7445 7723 131 4027926H1 4592 4822 131 2292865H1 7499 7721 131 4632120H1 7488 7765 131 6024929H1 4601 4923 131 g915845 7561 7784 131 4632247H1 7488 7765 131 2203255H1 4755 5006 131 2289132H1 7571 7802 131 7154510H1 7499 7643 131 4184071H1 4839 5084 131 5832896H1 7593 7874 131 6162751H1 7497 8049 131 3322055F6 4994 5518 131 4774272H1 7612 7881 131 1593082F6 3583 4007 131 3322055H1 4994 5271 131 1393295H1 7499 7692 131 1593043H1 3583 3803 131 1863161T6 5057 5459 131 1772773H1 7522 7784 131 1593082H1 3583 3803 131 811828H1 5105 5352 131 6269862H1 6136 6658 131 7236349H1 3664 4161 131 789137H1 5167 5226 131 g1975380 6238 6568 131 4004354H1 3665 3798 131 g3077295 5179 5635 131 3389337H1 6336 6619 131 439020H1 3680 3902 131 3242918H1 5346 5610 131 6706684H1 6370 6913 131 3480603H1 3698 3865 131 4578433H1 5545 5787 131 3282024F6 6416 6946 131 g1678362 

  3734 3890 131 5694130H1 5702 5886 131 3282024H1 6416 6662 131 g4691014 3746 4204 131 4255728H1 5783 6052 131 3854583H1 6428 6705 131 g6132554 3779 4207 131 5673677H1 5815 5974 131 2509302H1 6442 6670 131 g3756265 3780 4206 131 g1880292 5855 6068 131 7225908H1 6547 7110 131 g1199039 3835 4151 131 5768386H1 5903 6448 131 3147875H1 6581 6852 131 g819518 3859 4212 131 g1442274 5974 6178 131 3797563H1 6583 6873 131 2155889F6 3867 4265 131 g1678263 6029 6205 131 g2398342 1058 1351 131 2155889H1 3867 4106 131 3607889H1 6044 6335 131 6881955H1 1921 2185 131 3513325H1 3880 4124 131 g709099 6063 6392 131 2717848F6 6584 7049 131 70391721D1 3896 4321 131 g769480 6063 6274 131 2717848H1 6584 6832 131 2155889T6 3964 4405 131 g692094 6065 6419 131 4058694H1 6629 6895   

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   131 025240H1 6665 6932 133 g5109774 443 863 137 3960046F8 333 926 131 

  4270965H1 6782 6955 133 g5526440 420 841 137 g4114677 609 1055 131 3341807T6 6814 7417 133 g4599202 303 725 137 g3674688 744 1053 131 3322055T6 6820 7418 133 6306770H1 1 450 137 2705604T6 744 1011 131 4027974T9 6822 7361 134 4186114H1 683 1024 137 g3429159 744 1052 131 g691854 684 7235 134 4186114F6 683 1091 137 1871586H1 744 869 131 4029613H1 6873 7102 134 4186114T6 921 1587 137 1998855H1 744 926 131 g708808 6874 7171 134 4165023H1 674 955 137 2135293H1 763 1036 131 g565754 6874 7098 135 2268189H1 1 231 137 2135293F6 763 1227 131 124772H1 6888 7030 135 2268189R6 1 379 137 2100630H1 865 1055 132 1630826T6 1136 1410 135 1470335H1 37 230 137 292419T6 874 1001 132 g5813217 1172 1255 135 g2505442 142 353 137 3960046T8 887 1009 132 1631555T6 1194 1410 135 g2505398 157 521 138 6100950H1 1 213 132 3878121H1 1210 1271 135 g2458763 157 579 138 63025444H1 151 468 132 1986029H1 1210 1281 135 

  1445774H1 262 507 138 6144369H1 166 755 132 2956806H1 1210 1284 135 2268189T6 334 885 138 6144369F8 166 749 132 1372584H1 1214 1417 135 368103H1 520 763 138 6099148H1 208 488 132 854216H1 1 247 135 g2328398 608 929 138 6281896H1 383 528 132 854312R6 1 445 135 2322571H1 690 923 138 6111359H1 570 864 132 854312H1 1 151 136 6114201H1 1 292 138 6028709H1 650 932 132 2465049H1 150 358 136 3287273H1 260 505 138 6144369T8 846 1259 132 2465049F6 150 697 136 g79441 375 522 139 5056523H1 2466 2604 132 2271395H1 192 455 136 g879517 78 440 139 2271032H1 893 1152 132 2271395R6 192 704 136 g870148 135 450 139 4243388H1 919 1251 132 g2184123 1896 582 136 g883117 77 465 139 4552488H1 958 1120 132 g1692736 348 740 136 2120815H1 450 565 139 g1953334 885 1086 132 2465049T6 519 1047 136 1631555H1 450 565 139 g1953371 885 986 132 1601994H1 535 677 136 1631555F6 450 565 139 g713204 1029 1191 132 

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   139 g5887689 2244 2601 139 2061567H1 1698 1970 140 2271032R6 402 880 139 911966H1 2249 2503 139 6158833H1 

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  2340 139 6829715H1 860 1357 140 5946104H1 1420 1655 139 5291302H1 2152 2398 140 1272843F1 1 331 140 2700155F6 1432 1943 139 g5038163 2168 2608 140 1272843F6 1 587 140 2700155H1 1432 1631 139 g2355286 2200 2589 140 1272843H1 1 245 140 2061567T6 1461 2064 139 g3146568 2200 2604 140 1274319H1 21 218 140 3365965H1 1493 1618 139 5307668H1 1567 1691 1409 2598183H1 24 263 140 1749882T6 1549 2081 139 g1968949 1345 1812 140 3256051H1 25 268 140 70366789D1 1569 1883 139 5840503H2 1578 1835 140 3857549H1 306 615 140 1752020H1 1599 1768 139 g2229641 1360 1792 140 1749882F6 320 680 140 2700155T6 1573 2073 139 6725031H1 1675 2299 140 1749882H1 320 594 140 1889107T6 1609 2072 139 476160H1 1389 1655 140 g1953334 394 595 140 3236592H2 1635 1850 139 2061567R6 1698 2177 140 g1953371 394 495 140 5291302H1 1662 1908   

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  3050 3634 158 70478359V1 3235 3666   

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   158 6515587H1 3242 3671 158 70478180V1 2901 3570 159 3028782F6 505 841 158 70478211V1 3217 3682 158 70465864V1 2900 3534 159 3028782H1 505 807 158 4558722H1 1289 1534 158 70479279V1 2923 3570 159 1005592H1 599 889 158 3618747F6 1904 2401 158 70476782V1 2927 3466 159 4646393H1 801 1061 158 g190751 50 3497 158 70469166V1 2935 3628 159 5278286H1 912 1125 158 g1736195 979 1234 158 70476816V1 2992 3529 159 2521969F6 950 1354 158 2624295R6 1016 1412 158 7048392V1 2746 3208 159 983403H1 1063 1335 158 2624295H1 1016 1225 158 70478255V1 2751 3368 159 983403R6 1064 1315 158 70466944V1 3104 3692 158 70478182V1 3467 4041 159 188949H1 1106 1306 158 70480113V1 3129 3556 158 70498607V1 3496 3697 159 983403T6 1123 1347 158 70468718V1 3142 3695 158 70465130V1 3440 3697 159 988925H1 1126 1349 158 70477732V1 

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  158 70466101V1 3337 3682 159 4852614H1 1363 1587 158 70466253V1 2697 3264 158 70468914V1 3348 3697 159 3016113F6 1376 1647 158 70469171V1 2699 3374 158 70479545V1 3359 3667 159 3011865H1 1376 1459 158 70469220V1 2765 3324 159 3874759H1 1 296 159 058529H1 1376 1522 158 70481022V1 2759 3486 159 3685395H1 3 271 159 3958442H1 1377 1474 158 2624295T6 2762 3351 159 3692640H1 4 270 159 5276870H1 1376 1535 158 70469934V1 2769 3329 159 3692640F6 4 342 159 3578346H1 1382 1634 158 70480048V1 2644 3023 159 g3116688 4 443 159 3016113H1 1383 1669 158 70478224V1 2650 3136 159 3445829H2 6 264 159 6337413H1 1390 1507 158 70467878V1 2660 3107 159 306745H1 13 386 159 6338013H1 1390 1881 158 70481284V1 2587 3175 159 945423H1 11 293 159 6335720H1 1390 1883 158 70467582V1 2607 3072 159 988248R1 179 383 159 3688195H1 1398 1695 158 70465805V1 2630 3318 159 988248H1 179 291 159 4151882H1 1401 1641 158 

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  g1225697 3410 3783 161 6441810H1 2085 2530 160 3595466H1 2452 2678 160 g985312 3414 3790 161 7076574H1 2068 2555 160 3120055H1 2600 2875 160 g944631 3420 3511 161 70346502D1 2070 2384 160 4028493H1 2623 2861 160 2471283H1 3421 3645 161 477287H1 2073 2352 160 6918376H1 2649 3021 160 3561909H1 3421 3714 161 80523H1 2073 2269 160 833106R1 2689 3287 160 g965314 3442 3674 161 6763871H1 2078 2569 160 833106H1 2689 2941 160 2748973H1 3446 3719 161 7079082H1 2081 2565 160 g1068214 2708 3062 160 5621772H1 3458 3598 161 6996593H1 2081 2575 160 g1068219 2719 3068 160 g1064141 3477 3785 161 5693746H1 2096 2319 160 386166H1 2777 3061 160 7033911H1 3483 4011 161 4760746H1 2081 2371 160 3163260H1 2798 3086 160 517085H1 3502 3727 161 4760754H1 2082 2371 160 6490409H1 2830 3338 160 517085R6 3503 3982 161 3466673H1 2085 2180 160 2122144H1 2851 3109 160 4655472H1 3524 3710 161 3955495H1 2106 2385 160 

  g1484539 2902 3404 160 3521721H1 3525 3865 161 3945275H1 2213 2490 160 3120466H1 2924 3191 160 2668464H1 3540 3800 161 6891449H1 2214 2316 160 g1062289 2966 3116 160 2285765H1 3578 3795 161 4195061H1 2215 2518 160 4178517H1 3003 3265 160 2796870H1 3597 3846 161 3781801H1 2096 2379 160 2505067H1 3016 3259 160 2761118H1 3598 3859 161 2570710H1 2219 2467 160 2505367H1 3016 3245 160 5544423H1 3615 3699 161 4690839H1 2097 2352 160 7034903H1 3023 3630 160 338142H1 3622 3850 161 g683009 2100 2389 160 g1259693 3042 3222 160 4545187H1 3627 3871 161 5682533H1 2099 2378 160 7315867H1 3070 3471 160 2243019H1 3628 3885 161 5575001H1 2106 2253 160 5370265H1 3126 3359 160 2772059H1 3629 3889 161 1505114H1 2127 2344 160 g2022547 3176 3463 160 2960117H1 3636 3937 161 4091605H1 2114 2422 160 2707316H1 3197 3502 160 g1969950 3641 4014 161 5832027H1 2123 2377 160 2894887H1 3197 3446 160 g1947603 3654 

  3817 161 5401121H1 2219 2429 160 g796865 3229 3473 160 5002748H1 3687 3844 161 5388353H1 2219 2494 160 3523324H1 3238 3589 160 4062722H1 3721 4010 161 2704568H1 2127 2350 160 g1479499 3257 3560 160 7107841H1 3740 4101 161 5834524H1 2132 2405 160 g1548639 3257 3626 160 g104914 3749 4082 161 7103141H1 2155 2265 160 g943258 3257 3559 160 5546516H1 3750 3928 161 4315855H1 2139 2389 160 g1067965 3257 3571 161 g1219848 2061 2389 161 5834443H1 2139 2343 160 5371320H1 3264 3396 161 5406570H1 2061 2269 161 664952H1 2142 2367 160 4441134H1 3378 3656 161 5451646H1 2063 2243 161 1992383H1 2143 2344 160 g682946E 3380 3626 161 g20009331 2063 2316 161 3468549H1 2219 2487   

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  7071563H1 2208 2569 161 g1062711 1172 1485 161 5015603H1 1648 1813 161 6891449J1 2214 2315 161 g875401 1174 1475 161 2963466H1 1724 2028 161 554022H1 2202 2438 161 161704H1 1178 1392 161 4316745H1 1724 2020 161 453253H1 2273 2435 161 5781411H1 1181 1459 161 4315891H1 1744 2022 161 6739828H1 2219 2805 161 532473H1 11931433 161 4091142H1 1749 2023 161 4691246H1 2208 2424 161 3945042H1 1194 1480 161 4753020H1 1751 2007 161 3470773H1 2209 2454 161 4758039H1 1193 1440 161 6518022H1 1757 1838 161 5967249H1 2224 2744 161 3466710H1 1203 1464 161 4316639H1 1772 2046 161 6765073J1 2275 2692 161 5860513H1 1221 1490 161 2705655H1 1800 2083 161 666644H1 2212 2384 161 6770719H1 1238 1355 161 5961924H1 1790 2298 161 5579450H1 2212 2365 161 5656957H1 1238 1487 161 4695643H1 1820 2036 161 1618034H1 2212 2418 161 5204013H1 1238 1505 161 2707689H1 1896 2192 161 7088449H1 1 552 161 4254309H1 1257 1459 

  161 4342587H1 1979 2088 161 7073146H1 170 726 161 5578708H1 1259 1489 161 6770516H1 1988 2569 161 6784888H1 439 960 161 5013751HY1 1264 1491 161 4342620H1 1992 2246 161 6882093H1 479 990 161 4019484H1 1278 1543 161 6882093J1 1996 2568 161 6882161H1 479 992 161 6063870H1 1281 1564 161 6886296J1 2001 2481 161 6766193H1 480 961 161 3471004H1 1298 1570 161 7253724H1 2024 2531 161 6977268H1 504 925 161 5206590H1 1304 1546 161 2423349H1 2027 2272 161 7230035H1 585 1180 161 6746950H1 1304 1831 161 4316734H1 2032 2342 161 7075220H1 629 1180 161 5857309H1 1303 1576 161 4256579H1 2036 2265 161 1485846F6 646 910 161 5968610H1 1308 1851 161 6763709H1 2023 2568 161 g197576 664 884 161 g2012398 1308 1663 161 6771870J1 2031 2578 161 1486578H1 676 912 161 3469688H1 1317 1580 161 4772777H1 2046 2149   

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  167 5587272H1 18 282 161 4691683H1 2058 2293 164 5911540H1 1 250 167 4997069H1 18 284 161 5205527H1 2058 2293 164 5911540T8 78 570 167 1551008H1 18 211 161 5575884H1 2060 2251 165 5905252F8 1 497 167 5347226H1 19 275 161 366389H1 2061 2306 165 5905252F6 35 576 167 6819761H1 19 616 161 5955966H1 2315 2492 165 5905252H1 35 313 167 5117110H1 20 289 161 5205250H2 2316 2479 165 5905252T6 376 821 167 3447366H2 19 275 161 4776444H1 2311 2566 166 4020439F8 1 391 167 2445594H1 19 249 161 6517485H1 2347 2825 166 2773907F6 1 173 167 4622780H1 19 288 161 2292420H1 2408 2647 166 2773907H1 1 146 167 2111981H1 20 282 161 6552809H1 2494 2887 166 4020439H1 1 115 167 552401H1 20 258 161 6552988H1 2494 2887 166 4020439T8 6 503 167 2461616H1 19 197 161 6552963H1 2494 2887 166 2773907T6 129 435 167 5379341H1 19 275 161 4619412H1 2571 2791 166 g820143 313 435 167 3820792H1 20 310 161 4775811H1 2566 2801 

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   167 g889083 366 638 172 5908301H1 1 311 180 4290049F6 1 353 167 6819761J1 396 1013 172 6271267H2 24 492 180 4290049H1 1 124 167 g916666 408 513 172 

  590831T9 248 586 180 5493752H1 172 444 167 4249123H1 452 539 173 5907939T9 1 517 181 6729842H1 1 412 167 6481276H1 584 800 173 5907939F8 1 551 181 5401350H1 1 105 167 4191747H1 624 709 173 5907939H1 1 310 181 6057617H1 56 643 167 638004H1 648 753 173 6271008H2 10 483 181 5401350T9 82 666 167 1452178H1 676U 753 174 5912415F8 1 376 181 g3214092 406 782 167 g2615681 692 753 174 5912415H1 1 299 181 3524102H1 479 779 167 g1665347 697 753 174 5912415F6 12 565 182 7030475H1 1 524 167 g2558364 697 753 174 5912415T9 66 535 182 703027H1 3 376 168 6795278H1 184 698 175 4119207F6 1 336 183I 5271230H1 1485 1748 168 6796542H1 1 546 175 4119207T6 1 336 183 240641H1 1485 1657 168 6796380H1 8 563 175 4119207H1 1 175 183 2948213H1 1486 1768 168 6795463H1 8 523 176 5905477F6 1 564 183 g1291432 1492 1896 168 3941984H1 26 315 176 5905477H1 1 271 183 353018H1 1496 1708 168 g1260435 112 287 176 5905477T9 

  411 932 183 349497H1 1496 1680 168 6798249H1 184 635 177 5907791F8 1 360 183 g2276981 1533 1859 168 6791366H1 238 804 177 5907791H1 1 280 183 6804414J1 1537 2080 168 6790685H1 239 699 177 5907791F6 1 305 183 3499213H1 1537 1841 168 1242854H1 698 885 177 5907791T9 155 682 183 g2537462 1542 1981 169 2904954T6 1 522 177 5907791T6 248 733 183 1551014H1 1549 1763 169 4739603H1 11 303 178 4770137H1 1 144 183 808076H1 1554 1769 169 5614905H1 213 492 178 5564253H1 2 235 183 2307944H1 1557 1762 169 g2006850 277 557 178 606101H1 1 169 183 G2222952 1559 1851 169 2008385H1 464 562 178 4650476H1 1 271 183 2804178H1 1565 1852 169 2014576T6 464 523 178 592893H1 2 130 183 6801505J1 1566 1881 169 2014576H1 464 586 178 781453H1 17 276 183 g888315 1569 1927 169 3294227T6 223 557 178 2793564H1 28 317 183 g2056786 1570 2035 169 2014576R6 464 553 178 888943H1 151 287 183 6901949H1 1 518 169 1716729H1 

  501 556 178 6589872H1 194 698 183 4183549H1 91 259 170 3966795F6 1 365 178 2190828T6 207 699 183 6980581H1 104 452 170 3966795H1 1 267 178 1503654H1 225 491 183 5839778H1 107 359 170 3274864H1 14 268 178 4200384H1 388 662 183 808102H1 172 406 170 3966795T6 23 659 179 5911540F8 1 460 183 4323252H1 224 469 171 3033193F6 1 272 179 5911540H1 1 250 183 g2003585 258 517 171 3033193H1 1 216 179 5911540T9 27 568 183 g900863 361 447 171 3033193T6 129 445 179 5911540T8 78 569 183 6979927H1 437 785 172 5908301F8 1 519 180 g4325750 1 103 183 582110H1 487 739   

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   183 3323870H1 499 778 183 3697260H1 962 1179 183 5483319H1 1402 1501 183 2115479R6 515 913 183 1331860H1 981 1179 183 5322481H1 1403 1641 183 4779981H1 538 793 183 g1644929 1018 1179 183 5948083H1 1409 1714 183 1818066H1 552 808 183 g1880762 1046 1163 183 1996549H1 1415 1554 183 

  1818066F6 552 893 183 6804414H1 1065 1601 183 1996549R6 1415 1751 183 g4438745 584 797 183 1722938H1 1074 1327 183 5405753H1 1434 1586 183 5450643H1 614 851 183 5451478H1 1104 1348 183 5293687H2 1434 1680 183 7066292H1 636 1078 183 1500938H1 1108 1304 183 935623R1 1465 1989 183 3538459H1 648 855 183 5950551H1 1120 1413 183 936611H1 1465 1767 183 2115479H1 655 913 183 3376840H1 1121 1367 183 935623H1 1465 1708 183 4090518H1 669 864 183 5950519H1 1121 1320 183 3409340H1 1473 1629 183 6077621H1 686 797 183 4193013H1 1121 1438 183 1819126H1 1480 1748 183 4947628H1 702 832 183 4556920H1 1124 1261 183 1819126F6 1480 1979 183 4759655H1 713 993 183 g1046489 1143 1470 183 g2053565 1480 1888 183 1699317H1 715 931 183 g1046497 1143 1455 183 g2209615 1481 1929 183 4303659H1 740 991 183 1283213H1 1202 1390 183 5879274H1 1482 1754 183 591324R6 752 1180 183 1283213F6 1202 1658 183 4570795H1 2032 

  2292 183 591324H1 752 975 183 4225341H1 1206 1487 183 g1046490 2044 2349 183 591174H1 752 955 183 6714888H1 1214 1778 183 1416514H1 2046 2291 183 5998787H1 772 1179 183 1346996H1 1259 1485 183 4193606H1 2048 2324 183 1672160H1 778 991 183 1348304H1 1259 1481 183 g1858344 2051 2338 183 1672152H1 778 991 183 1348303H1 1259 1481 183 5021880T1 2055 2300 183 4694794H1 784 1045 183 6954001H1 1261 1764 183 1281333H1 2058 2294 183 2972664H2 782 1078 183 g2107139 1290 1761 183 2222539H1 2060 2298 183 1718469H1 795 1006 183 4720048H1 1308 1583 183 2222539F6 2060 2338 183 1718480H1 795 998 183 1720725H1 1328 1538 183 2222539T6 2061 2300 183 3270027H1 802 1059 183 1722139H1 1328 1553 183 5341229H1 2066 2265 183 927251R1 806 1419 183 5970873H1 1334 1887 183 2659133H1 2067 2280 183 927251H1 806 1058 183 4693221H1 1344 1601 183 g3871000 2069 2338 183 g1986696809 992 183 2571078H1 1343 1591 183 

  g4524292 2077 2340 183 5102583H1 821 1068 183 6121921H1 1351 1904 183 g1858279 2081 2342 183 7094153H1 822 1103 183 4215895H1 1363 1625 183 1301182H1 2082 2343 183 5948962H1 843 1137 183 g2188904 1372 1857 183 2256984H1 2105 2338 183 1571253H1 845 1013 183 g1646485 1374 1468 183 6498361H1 2106 2338 183 5371617H1 860 1101 183 004844H1 1386 1669 183 2574115H1 2106 2337 183 3784982H1 895 1195 183 6313521H1 1386 1950 183 2273027H1 2107 2310 183 6980693H1 893 1257 183 2427675H1 1392 1622 183 g4899838 2119 2346 183 4770011H1 901 1166 183 5478565H1 1401 1608 183 g3051313 2128 2338 183 1695218H1 903 1119 183 5480821H1 1402 1635 183 3142955H1 2158 2340   

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  2036 183 953800H1 2012 2296 183 2247168H1 2197 2344 183 5928305H1 1801 2111 183 953800R1 2012 2338 183 2246357H1 2225 2339 183 683816H1 1805 2040 183 4977128H1 2022 2281 183 2247256H1 2254 2339 183 5451469H1 1808 2048 183 g1046498 2026 2338 183 3812939H1 2274 2338 183 2402750H1 1811 1919 184 3497339T6 1 492 183 5103254H1 2282 2339 183 1851976H1 1816 2034 184 g1060678 211 436 183 7064872H1 1575 2105 183 3702341H1 1825 2066 184 6258709H1 213 444 183 6818666H1 1579 1827 183 2963989H1 1819 2096 184 2570554H1 239 484 183 4726069H1 1581 1854 183 g1441785 1826 2091 184 2570554R6 239 634 183 g4662834 1586 2036 183 5777701H1 1828 2105 184 6200064H1 459 921 183 g1182379 1589 1804 183 g3898324 1832 2042 184 g734035 473 792 183 g4874675 1588 2033 183 1819126T6 1843 2298 184 1400373F6 474 1007 183 5661656H1 1589 1849 183 1484988H1 1847 2095 184 1400373H1 474 733 183 6868292H1 1591 2060 183 

  2106229T6 1851 2298 184 g2167556 488 897 183 3706916H1 1605 1904 183 g4762523 1859 2033 184 6389638H1 542 823 183 g2208265 1633 2033 183 g46205071860 2039 184 6344656H1 598 885 183 2424009H1 1649 1907 183 1964264H1 1872 2033 184 6344688H1 606 888 183 1470490H1 1663 1849 83 1964264R6 1872 2040 184 6328470H1 664 1018 183 797359H1 1664 1903 183 1964264T6 1872 2002 184 g4148622 723 1072 183 3245252H1 1669 1936 183 g2945963 1876 2340 184 6338470H1 793 1018 183 4781187H1 1671 1939 183I 4696529H1 1885 2095 184 3784456H1 876 120 183 g1688385 1690 2024 183 g4564438 1886 2339 184 3781165H1 943 1049 183 1413737H1 1712 1978 183 g3741810 1886 2341 184 g733951 1031 1403 183 g2279273 1737 2033 183 2913361H1 1889 1977 184 g734304 1149 1333 183 g1146486 1741 1926 183 6862628H1 1894 2033 184 3554877H1 1 294 183 g1644873 1745 2038 183 g3742173 1905 2340 185 4111213H1 1 243 183 g888316 1749 2033 183 

  4083420 1910 2338 185 5624259R8 1 350 183 099594H1 1754 1972 183 g3678703 1912 2342 185 3139315H1 126 427 183 3996904H1 1754 1913 183 6327536H1 1912 2340 185 5545642H1 130 363 183 3916961H1 1759 2048 183 g2910125 1916 2341 185 3141553H1 139 412 183 3917411H1 1759 2036 183 g3308355 1919 2342 185 g564381 139 369 183 6801505H1 1759 2125 183 2556534H1 1933 2178 185 987690H1 154 424 183 910557H1 1785 1850 183 g3922126 1935 2338 185 4833664H1 169 319 183 2921975H1 1788 2061 183 g4083267 1936 2338 185 116132H1 214 403 183 5003193H1 1790 2060 183 g4083582 1951 2338 185 3691576H1 244 399 183 g1442965 1791 2033 183 g1646486 1958 2340 185 1667502F6 265 644 183 5018209H1 1797 1957 183 g2902884 1998 2339 185 3283023H1 267 528   

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   185 3959293H2 290 564 189 70395867D1 1 542 190 g816766 4165 4460 185 2675859H1 347 596 189 2525961F6 4 452 190 

  3041690H1 4157 4451 185 2676108H1 347 594 189 2525961H1 5 246 190 977648R1 4063 4431 18 52676122H1 347 591 189 70395557D1 40 642 190 g1193159 4182 4458 185 5301231H1 387 569 189 70397658D1 59 589 190 977648T1 4063 4415 185 1598950H1 387 567 189 6560586H1 67 616 190 977648H1 4063 4386 185 2352332H1 465 626 189 70396453D1 72 478 190 978306H1 4063 4319 185 5038795H1 459 689 189 70396167D1 119 654 190 g2051455 4184 4460 185 2534205H1 459 705 189 70396459D1 118 585 190 5280171H1 4187 4442 185 3252993H1 464 711 189 70396121D1 192 481 190 g3886427 4204 4347 185 1742040H1 490 721 189 g1357976 238 526 190 1381539H1 4210 4457 185 4592168H1 498 648 189 2525961T6 256 680 190 552969H1 4210 4458 185 g29244 1 255 189 70396053D1 260 679 190 2760603H1 4210 4453 185 2635196H1 1 249 189 70396654D1 278 769 190 g3647431 4212 4454 186 6428735H1 867 1184 189 70395799D1 279 680 190 1917113H1 4245 4457 187 

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  2402420H1 4083 4349 190 2819748T6 3815 4417 188 5479637T1 23 159 190 1340154H1 4091 4319 190 3445036H1 3816 4085 188 7289635H1 196 762 190 2791792R6 4108 4475 190 6735779H1 3822 4433 188 6559813H1 603 1192 190 2079255T6 4053 4414 190 2279717T6 382 4418 188 6858544H1 732 1210 190 g2884988 4111 4451 190 3720714H1 3832 4020 188 5929387H1 845 1124 190 g1189027 4060 4458 190 4828820H1 3844 4119 188 5778630H1 845 1114 190 2412005H1 4120 4352 190 1369089H1 3847 4077 188 4017917H1 851 1115 190 g2354871 4123 4450 190 1833781H1 3850 4130 188 5206602H1 905 1185 190 g2350487 4133 4457 190 1965941T6 3865 4422 188 4776841H1 1078 1295 190 g5662773 4156 4466 190 2559627H1 3868 3984 188 6314480H1 1093 1213 190 g5639034 4061 4453 190 2285422H1 3881 4083   

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  3000   

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  7290084H1 3269 3683 190 1305496F6 798 1267 190 71226687V1 1457 2020 190 7011256H1 3273 3657 190 70145686V1 798 1046 190 70864162V1 1463 2044   

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  1498 1770 196 g2207390 2175 2518 198 6764201J1 1 585 196 g4524899 1511 1896 196 908344R2 2180 2516 198 986476R6 5 479 196 g768901 1513 1820 196 908344H1 2180 2285 198 986476H1 5 303 196 g571029 1512 1840 196 4748325H1 2199 2467 198 4180212H1 8 260 196 g880779 1513 1841 196 4746557H1 2200 2439 198 g3933038 75 566 196 g832097 1513 1884 196 g1859648 2231 2520 198 4029248H1 122 377 196 4623266H1 1566 1825 196 5565332H1 2244 2501 198 4029227H1 122 370 196 3519427H1 1590 1955 196 g778446 2320 2632 199 g4196260 1 320 196 3680478H1 1608 1840 196 g831074 2319 2636 199 g2218495 1 313 196 6332293H1 1668 2034 196 g2053046 2330 2794 199 3685061H1 1 293 196 4832174H1 1735 1965 196 3031124H1 2353 2644 199 6821713J1 1 518 196 g1329683 1739 2321 196 2277831H1 2391 2660 199 5372078H1 25 226 196 g787502 1779 2009 196 2277831R6 2391 2516 199 4205860H1 169 437 196 g864605 1779 2053 196 g2552923 2437 

  2906 199 4205654H1 169 416 196 g787457 1779 2024 196 g832055 2523 2923 200 6868907H1 1 542 196 g841915 1778 2047 196 g1329627 2545 2922 200 1543453R6 24 440 196 5589418H1 1784 2030 196 g499052 2547 2834 200 1543453H1 24 217 196 5071250H1 1803 2062 196 g4311136 2547 2993 200 5055563H1 38 307 196 3865563H1 1843 2217 196 g4988520 2547 3007 200 5055563F9 38 607 196 1955962H1 1894 2151 196 g4740301 2547 2833 200 4778273F8 126 752 196 4750673H2 1945 2009 196 g3422370 2548 2923 200 4778273H1 126 397 196 442154H1 1966 2272 196 g4289096 2548 2904 200 2724784H1 137 372 196 261197H1 1965 2281 196 g3431513 2549 2876 200 2925738H1 339 605 196 444335H1 966 2225 196 000140H1 2555 2911 200 4778273T9 559 1136 196 4515301H1 2028 2284 196 2543809H1 2555 2770 200 1543453T6 610 1221 196 3780534H1 2055 2353 196 2435619H1 2555 2713 200 1921258H1 832 1046 196 2191782F6 2063 2373 196 g5113593 2555 2917 200 

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   203 6080872H1 1264 1862 203 6150959H1 647 898 203 6110012H1 754 1069 203 5824738H1 1269 1837 203 6162572H1 648 1211 203 6135068H1 757 1061 203 3390106T6 1274 1662 203 5803707H1 648 950 203 6035941H1 759 1259 203 60221621V1 1274 1677 203 6111936H1 653 955 203 6163478H1 764 1336 203 7004345H1 1284 1909 203 6111235H1 656 969 203 5968127H1 760 1255 203 60109238B1 1286 1912 203 

  5953419H1 659 1020 203 6178490H1 763 1018 203 7186303H1 1290 1881 203 5995735H1 659 974 203 60204862U1 771 1320 203 7025213H1 1297 1900 203 6002288H1 659 917 203 6108986H1 773 1083 203 7182261H1 1299 1807 203 6144013H1 664 1176 203 6097994H1 787 1118 203 7193486H2 1306 1929 203 6166779H1 664 1176 203 6165174H1 793 1081 203 6736012H1 1319 1907 203 6052294H1 666 889 203 6115201H1 792 1129 203 5970654H1 1322 1912 203 6053478J1 664 1250 203 5798369H1 797 1419 203 5770017H1 1327 1919 203 6052294J1 666 1098 203 5801102H1 797 1105 203 5802957H1 1327 1561 203 5995222H1 671 1001 203 6112951H1 797 997 203 5800770H1 1328 1601 203 5990146H1 671 994 203 6109295H1 803 1129 203 5804521H1 1331 1550 203 6079085H1 671 956 203 6110881H1 805 1081 203 5995788H1 1330 1639 203 5798802H1 672 1277 203 6179893H1 804 1105 203 7331927H1 1332 1935 203 5975323H1 681 1238 203 5955376H1 806 1170 203 5769517H1 

  1333 1918 203 7327981H2 684 1173 203 7359669H1 815 1090 203 5967562H1 1333 1856 203 6155086H1 691 1046 203 6176408H1 813 1095 203 6163438H1 1337 1896 203 6183751H1 699 1001 203 7004683H1 820 1427 203 5953896H1 1338 1640 203 6183830H1 699 983 203 7004582H1 820 1396 203 5789044H1 1338 1633 203 6215696H1 701 1298 203 71033163V1 820 1364 203 5975734H1 1339 1916 203 6109481H1 701 1023 203 5974746H1 827 1459 203 6078714H1 1338 1668 203 6030242H2 702 1001 203 7325468H1 829 1480 203 5786342H1 1338 1646 203 6163760H1 706 1074 203 6035163H1 839 1457 203 6175953H1 1340 1636 203 6153438H1 710 1236 203 6112093H1 835 1148 203 5805592H1 1340 1639 203 6111179H1 710 982 203 6177785H1 836 1124 203 5952736H1 1351 1684 203 6116630H1 723 1044 203 5798782H1 836 917 203 3693154F6 1354 1935 203 6151576H1 726 998 203 6150235H1 848 1459 203 6217933H1 1356 1756 203 6111753H1 732 1001 103 5952964H1 849 1130 

  203 7160128H1 628 1197 203 5824855H1 736 1199 203 6218804H1 851 1196 203 6117709H1 630 956 203 5991422H1 740 1085 203 5804744H1 853 1162 203 6180232H1 632 859 203 6180759H1 740 1069 203 5771556H1 858 1477 203 6173095H1 645 974 203 6135676H1 741 1031 203 5994894H1 856 1166 203 6132472H1 645 956 203 5954296H1 741 983 203 71036246V1 861 1368 203 6150854H1 647 996 203 6154930H1 741 1095 203 5915476H1 858 1096 203 5804474H1 647 944 203 6145090H1 747 1336 203 6163363H1 863 1499 203 6043158H1 647 922 203 7215869H1 750 1359 203 5796628H1 870 1046   

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  4571391F8 75 628 203 7325279H1 201 812 203 6172790H1 890 1176 203 5771842H1 79 676 203 6116494H1 204 493 203 5801236H1 902 1168 203 6108158H1 79 418 203 5953527H1 210 543 203 6113354H1 906 1275 203 6176812H1 79 395 203 5892842H1 214 483 203 6032732H1 906 1459 203 6177958H1 79 393 203 5993811H1 218 546 203 6130142H1 908 1245 203 6179512H1 79 393 203 6109818H1 226 448 203 6168522H1 913 1264 203 6181431H1 79 384 203 6044864H1 228 751 203 6037518H1 915 1525 203 7008218H1 79 694 203 6182368H1 242 578 203 6032546H1 914 1542 203 6215727H1 79 590 203 7326560H2 250 893 203 60104786D4 912 1222 203 6151732H1 84 317 203 6033805H1 253 837 203 6176164H1 924 1214 203 5973312H1 84 615 203 6101869H1 257 545 203 6133149H1 913 1026 203 60126922U1 86 662 203 6170505H1 265 596 203 7320972H1 920 1515 203 5974449H1 88 675 203 6152125H1 265 596 203 6033149H1 923 1523 203 6111184H1 90 170 203 6137458H1 265 

  568 203 5970892H1 924 1548 203 6113115H1 90 429 203 5958352H1 265 856 203 6036731H1 925 1529 203 6000095H1 91 474 203 5975525H1 294 899 203 5970884H1 924 1510 203 6182051H1 91 394 203 5838053H1 296 585 203 6113366H1 923 1026 203 7272693H1 91 677 203 5796171H1 304 858 203 7185073H1 932 1524 203 7385648H1 96 287 203 6112187H1 310 639 203 5827058H1 935 988 203 6135054H1 99 403 203 6165284H1 313 858 203 5770524H1 940 1589 203 6182449H1 104 467 203 5826529H1 320 764 203 6114346H1 936 1210 203 5775693H1 104 719 203 6172048H1 336 644 203 5990721H1 942 1263 203 6175825H1 106 381 203 6035764H1 326 932 203 5975145H1 947 1489 203 5968167H1 118 728 203 6966778H1 329 425 203 6044290H1 941 1226 203 60120598D1 108 278 203 6172080H1 328 576 203 5768644H1 957 1532 203 6147691H1 122 616 203 7182401H1 354 954 203 5971326H1 948 1554 203 6115311H1 133 425 203 7269240H1 357 994 203 6110988H1 946 1173 

  203 5975411H1 137 630 203 6029089H1 360 667 203 6099321H1 948 1236 203 5915392H1 144 416 203 6100553H1 367 674 203 6173091H1 62 390 203 6130193H1 148 465 203 5825841H1 387 1002 203 6036155H1 65 613 203 7328558H1 147 830 203 7214023H1 396 923 203 6180780H1 65 387 203 5996192H1 166 467 203 6109604H1 393 729 203 5770305H1 66 615 203 5977403H1 168 658 203 6143986H1 411 1031 203 7215805H1 67 610 203 5989334H1 177 480 203 6117765H1 421 543 203 7024381H1 67 430 203 6109928H1 179 509 203 6136677H1 423 744   

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  7180879H1 457 1063 203 60264017D1 32 569 203 5914782H1 57 278 203 5797850H1 461 930 203 7079629H2 44 358 203 7359725H1 39 685 203 6115841H1 461 756 203 5972573H1 41 686 203 6084122H1 40 673 203 6137438H1 468 772 203 7160586H1 34 619 203 7083662H1 39 685 203 6731970H1 474 1055 203 6784013H1 35 645 203 7358734H1 39 682 203 6108268H1 478 793 203 7083178H1 36 637 203 7181284H1 39 557 203 6115462H1 491 718 203 7182216H1 36 592 203 60264004D1 39 552 203 5891369H1 517 775 203 7184701H1 36 578 203 6080769H1 50 645 203 6001442H1 524 1096 203 6170753H1 36 341 203 7212965H1 39 444 203 7360854H1 525 694 203 7261837H1 36 644 203 6174304H1 39 387 203 5974453H1 539 1087 203 6180976H1 37 372 203 6117564H1 40 363 203 6109032H1 534 897 203 6170740H1 37 340 203 6132067H1 39 350 203 5892176H1 534 868 203 7262607H1 39 648 203 6099630H1 39 322 203 5953990H1 534 804 203 7220667H1 38 636 203 6134055H1 62 

  379 203 5831262H1 534 726 203 7182859H1 39 616 203 5994380H1 42 384 203 5955474H1 543 886 203 6099372H1 39 324 203 6098224H1 42 362 203 5955458H1 543 888 203 7260783H1 39 675 203 6132993H1 42 337 203 6111439H1 544 769 203 7316234H1 39 709 203 7213881H1 43 644 203 6111316H1 546 881 203 4625563F9 39 673 203 6081567H1 43 636 203 5797137H1 577 1094 203 7160372H1 39 643 203 6132164H1 43 385 203 6133207H1 586 897 203 7160758H1 39 615 203 6101109H1 43 377 203 7328606H1 589 1133 203 7216608H1 39 623 203 5992072H1 43 372 203 7261854H1 590 1225 203 7219614H1 39 613 203 6078760H1 43 365 203 6100514H1 596 898 203 7213493H1 39 643 203 5995191H1 43 374 203 6040074H1 599 1199 203 7160235H1 39 555 203 6171974H1 43 359 203 5824550H1 606 1060 203 7317368H1 39 420 203 5989890H1 43 350 203 2814920F7 612 1092 203 6097675H1 39 380 203 6181359H1 44 353 203 6181437H1 617 905 203 6098127H1 39 375 203 

  6110331H1 43 347 203 5771704H1 626 1260 203 6171834H1 39 358 203 6111434H1 43 339 203 6131361H1 624 937 203 6169216H1 39 372 203 6168435H1 43 284 203 71216557V1 628 1188 203 6131659h1 39 358 203 7268992h1 45 675 203 6141011h1 1 156 203 6177913h1 39 350 203 6081557h1 44 614 203 6108212H1 1 324 203 6109871H1 39 347 203 6110567H1 43 283 203 6177861H1 2 301 203 6132882H1 39 346 203 7272829H1 45 685   

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  1364 1676 203 6152884H1 1564 1869 203 6170754H1 47 353 203 6042367H1 1364 1661 203 g6471957 1565 1928 203 6107830H1 47 235 203 6035204H1 1366 1929 203 5955189H1 1566 1854 203 7275183H1 48 644 203 6170972H1 1365 1682 203 6030104H2 1566 1861 203 7182041H1 49 616 203 6181601H1 1365 1667 203 5805947H1 1565 1840 203 7182971H1 49 616 203 5802968H1 1367 1676 203 5771690H1 1566 1928 203 5991415H1 48 388 203 4362784F8 1372 1927 203 g6030549 1568 1928 203 6112705H1 48 373 203 5996082H1 1377 1699 203 g6198562 1567 1928 203 6098861H1 48 347 203 5996003H1 1375 1666 203 6097965H1 1568 1889 203 6099983H1 48 243 203 5995203H1 1382 1721 203 6151677H1 1571 1866 203 7283631H1 49 718 203 6133437H1 1383 1700 203 5974518H1 1571 1926 203 6039521H1 49 650 203 6178414H1 1393 1699 203 g6229210 1574 1933 203 5992224H1 49 375 203 6179001H1 1401 1548 203 6028702H1 1579 1813 203 6170022H1 49 387 203 5769429H1 

  1403 1918 203 g6470581 1580 1920 203 5992273H1 49 363 203 5785086H1 1404 1722 203 g6198908 1587 1928 203 6114538H1 48 348 203 5801902H1 1405 1725 203 g6044672 1588 1928 203 7272102H1 50 635 203 5792833H1 1404 1477 103 g6470803 1511 1930 203 6136863H1 50 389 203 5949501H1 1415 1784 203 g6027906 1513 1934 203 6085163H1 68 331 203 g6228962 1428 1937 203 g6074740 1524 1928 203 6115314H1 50 306 203 g6228709 1432 1938 203 g6026164 1523 1928 203 7270954H1 51 687 203 g6300732 1431 1938 203 g6133768 1528 1932 203 7190289H2 51 619 203 60126922B1 1434 1887 203 6179968H1 1526 1784 203 4624253F8 54 733 203 6173439H1 1434 1747 203 5991468H1 1528 1863 203 6037863H1 54 590 203 5769804H1 1434 1920 203 5786634H1 1530 1832 203 5973394H1 54 630 203 g6301637 1436 1938 203 5786701H1 1530 1715 203 6168342H1 54 412 203 g6302015 1437 1936 203 5786734H1 1532 1652 203 6733426H1 73 419 203 5953730H1 1440 1530 

  203 g6047869 1534 1921 203 5770643H1 55 644 203 5770049H1 1440 1530 203 6176458H1 1547 1825 203 6733166H1 54 647 203 7082145H1 1440 1907 103 5816770H1 1550 1883 203 7321688H1 58 593 203 g6301063 1443 1936 203 5817968H1 1550 1746 203 6038935H1 58 635 203 6133907H1 1444 1778 203 g6471065 1550 1928 203 6134077H1 56 399 203 g6039672 1445 1932 203 5817174H1 1550 1832 203 7185189H1 56 643 203 g6301561 1453 1938 203 g6471718 1552 1937   

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   203 g6464005 1552 1937 203 g6131743 1480 1935 203 5946071H1 1668 1917 203 6108818H1 1554 1787 203 g6044247 1480 1928 203 5950118H1 1668 1905 203 7181625H1 1553 1920 203 6034847H1 1482 1911 203 6157445H1 1668 1850 203 g6043984 1488 1938 203 g6117455 1481 1933 203 5946973H1 1668 1920 203 g6476041 1488 1930 203 g6464482 1481 1928 203 5767935H1 1670 1920 203 g6075390 1489 1937 23 g6036141 1481 1928 203 

  1767973H1 1670 1912 203 g6028910 1490 1937 203 g6047637 1481 1928 203 5947508H1 1672 1934 203 g6073495 1490 1930 203 g6117632 1482 1930 203 5790266H1 1674 1920 203 g6401184 1490 1930 203 g6463078 1482 1936 203 g6400408 1676 1938 203 g6073672 1490 1937 203 g6035703 1480 1936 203 7338126H1 1680 1932 203 g6036791 1489 1928 203 g6076131 1482 1932 203 7350525H1 1686 1936 203 g6026147 1492 1935 203 g6196780 1482 1932 203 g6398110 1686 1938 203 g6036640 1492 1920 203 g6031028 1483 1930 203 g6402278 1686 1938 203 6157091H1 1492 1813 203 g6464375 1484 1928 203 6157246H1 1686 1827 203 g6033899 1493 1928 203 g6043231 1484 1929 203 g6197883 1687 1928 203 g6198865 1493 1938 203 g6117300 1485 1933 203 g6046641 1691 1928 203 6219812H2 1500 1886 203 g6200216 1484 1938 203 g6400764 1692 1928 203 6192668H1 1501 1814 203 g6040103 1485 1932 203 g6076220 1693 1928 203 5768793H1 1505 1920 203 g6399002 

  1485 1926 203 5794559H1 1695 1926 203 6191666H1 1501 1804 203 g6029144 1486 1934 203 g6033633 1695 1926 203 g6473184 1502 1934 203 g6043655 1487 1937 203 5787563H1 1695 1925 203 g6746087 1502 1928 203 g6196472 1489 1934 203 g6117328 1708 1937 203 5993364H1 1503 1813 203 g6034277 1815 1920 203 g6044253 1710 1929 203 g6438645 1503 1928 203 g6117025 1815 1920 203 g6398246 1721 1928 203 g6076043 1504 1928 203 g6199037 1815 1920 203 g6131945 1465 1929 203 6043152H1 1519 1787 203 g6198912 1823 1928 203 6181714H1 1466 1806 203 g6472040 1509 1937 203 g6473327 1833 1928 203 g6048081 1466 1928 203 6179110H1 1511 1806 203 g6463164 1841 1930 203 g6047077 1466 1932 203 g6400409 1476 1929 203 g920960H1 1844 1925 203 g6132102 1468 1929 203 g6036079 1476 1928 203 g6198784 18546 1926 203 g6398647 1468 1929 203 g6086332 1477 1934 203 5787770H1 1722 1981 203 g6400444 1468 1929 203 g6199417 1477 1933 

  203 5789227H1 1722 1927 203 g6086266 1468 1928 203 g6044232 1478 1936 203 g6074506 1728 1938 203 g6132321 1469 1928 203 g6029466 1478 1935 203 6166065H1 1741 1956 203 g6132239 1470 1930 203 g6439518 1479 1934 203 g6029762 1746 1876 203 g6074116 1468 1928 203 g6462956 1479 1934 203 5786021H1 1754 1926 203 5990574H1 1470 1804 203 g6439604 1480 1937 203 g6438899 1763 1928 203 g6400620 1470 1930 203 g6196631 1480 1938 203 5992585H1 1769 1920 203 g6074889 1470 1928 203 g6439647 1479 1930 203 6158235H1 1865 1940 203 g6473566 1469 1928   

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  6828967J1 813 1145 204 g1616031 448 783 203 g6047234 1474 1930 204 6825030H1 823 1116 204 6824349J1 147 796 203 7180844H1 1459 1928 204 6825030J1 822 1115 204 4516155H1 541 775 203 g6035938 1464 1916 204 1470306H1 921 1114 204 4672305H1 94 298 203 g6439666 1475 1928 204 6827717J1 434 1076 204 6829925H1 1303 1619 203 g6036991 1475 1928 204 1945447H1 808 1051 204 1426502H1 1438 1679 203 g6047968 1475 1930 204 5870352H1 729 1007 204 6829925J1 1303 1618 203 g6036801 1476 1937 204 6826536J1 1157 1756 204 6824269J1 875 1484 203 g6196212 1600 1928 204 6822128J1 829 984 204 6824288J1 805 1385 203 6177877H1 1603 1888 204 6826536H1 1143 1742 204 6825511J1 786 1392 203 g6039199 1605 1928 204 g3056033 1065 1305 204 g5856181 886 1309 203 g6438679 1604 1920 204 g5837846 816 1289 204 g824615 1112 1275 203 g6400557 1611 1928 204 g2986193 1027 1297 204 6802929J1 833 1289 203 5817262H1 1615 1904 204 

  6831310H1 845 1278 204 g5127144 929 1299 203 5817362H1 1615 1906 204 1631532H1 1227 1297 204 g2344128 1075 1315 203 5820474H1 1615 1915 204 g3150672 914 1287 204 6458046H1 929 1285 203 6102129H1 1619 1928 204 92236974 899 1287 204 g2397910 1064 1302 203 g607467 1653 1932 204 g4889379 1021 1302 204 g2278427 1157 1301 203 g6132060 1656 1935 204 2255054R6 393 777 204 g3058045 823 1284 203 3775108F9 1656 1912 204 6498130H1 312 777 204 6820991H1 747 1284 203 g6198405 1665 1929 204 2255054H1 549 777 204 2255054T6 705 1267 203 6178153H1 1665 1928 204 6826447H1 357 752 204 6822128H1 514 951 203 g6045038 1666 1911 204 6826447J1 357 752 204 6802929H1 535 920 203 5770026H1 1668 1946 204 1579339H1 502 713 204 6825384H1 424 928 203 5792284H1 1668 1920 204 4443559H1 474 613 204 6824288H1 498 991 203 g6028253 1588 1930 204 2042892H1 315 568 204 6824091J1 335 879 203 g6475978 1593 1932 204

  3358238H1 329 566 204 3512547H1 1455 1695 203 6175948H1 1593 1891 204 4750087H1 185 453 204 6804257H1 1157 1691 203 g6132639 1594 1928 204 6824269H1 381 880 204 3700061H1 1404 1683 203 g6463340 1597 1928 204 6824349H1 350 851 204 1426502F6 1335 1679 203 g6046756 1596 1928 204 2184748H1 179 463 304 755545H1 1439 1679 204 6844571H1 664 1221 204 6756704J1 235 808 204 6498381H1 1158 1746 204 1299074T6 715 1264 204 2184748F6 1 463 205 185853856 1354 1583 204 1751671T6 860 1258 204 6756496H1 234 432 205 181099356 1360 1586 204 6941680H1 769 1245 204 6756704H1 255 432 205 1810792H1 1281 1506   

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  1129 207 g4299176 248 676 205 2689550F6 1 511 206 3873170H1 876 2028 207 g5630519 273 675 205 2689550H1 1 265 206 4575663H1 912 1190 207 5743674T7 354 545 205 6476804H1 3 563 206 6155913H1 953 1276 207 5743674H1 1 297 205 1545750H1 17 163 206 4543658H1 1005 1249 207 4127943H1 1 246 205 g2244465 68 341 206 5064775H1 1104 1345 207 5743674R7 1 183 205 1494653H1 351 402 206 g1014062 1144 1418 208 1952854T6 1 565 205 1858538H1 400 682 206 6249620H1 1173 1668 209 2917244T6 1 567 205 2218749F6 474 916 206 2881132F6 1223 1489 209 2917244H1 1 156 205 2228636F6 474 908 206 2881132H1 1223 1496 209 2917244F6 1 567 205 2228636H1 474 714 206 g1949025 1228 1546 210 5507875H1 1 219 205 1724288H1 642 852 206 4552262H1 1238 1500 210 5507875F6 1 393 205 1724417H1 642 852 206 950155H1 1278 1523 210 3510289H1 1 276 205 1724417F6 642 998 206 1705026H1 1316 1527 210 6985373H1 20 382 205 1724064H1 642 841 

  206 4398180H1 1331 1571 210 6985355H1 20 540 205 1724064F6 642 1096 206 3179825T6 1418 1909 210 4715879H1 20 238 205 6549336H1 1002 1419 206 2487001T6 1441 2006 210 645341H1 21 220 205 4822845H1 1024 1261 206 6097383H1 1482 1798 210 645341R6 21 432 205 1810993F6 1281 1558 206 g2251371 1596 2047 210 4551228H1 20 212 205 1810993H1 1281 1518 206 g4072810 1631 2050 210 3182009H1 24 326 206 6045641H1 1 361 206 g3889774 1700 2050 210 3182022H1 24 321 206 6045641J1 1 364 206 g4087488 1749 2056 210 5156573H1 27 272 206 3179825H1 120 424 206 g1014063 1798 2030 210 4800844H1 28 277 206 3179825F6 120 672 206 2881132T6 1809 2005 210 5864355H1 31 324 206 4758644H1 141 409 206 g1195443 1885 2047 210 6219781H1 32 251 206 2966833H1 222 484 206 g2155148 1942 2053 210 3375196H1 35 280 206 5372448H1 327 481 207 6803514H1 503 984 210 6306192H1 38 512 206 2686267H1 359 619 207 6803514J1 242 823 210 

  g575091 38 316 206 3722865H1 425 558 207 6286909H2 316 799 210 2924014F6 38 385 206 5036810H1 463 731 207 g4568721 260 685 210 2924014H1 38 247 206 4164428H1 519 787 207 g3742689 209 684 210 2825826H1 56 355 206 3799675H1 563 731 207 g5365250 586 679 210 3695750H1 62 182 206 4708839H1 568 851 207 g5676607 451 679 210 941644R6 66 275 206 2952970H1 602 865 207 g4111591 263 679 210 5310424H1 66 282 206 3561988H1 633 928 207 g2539496 299 678 210 3580982H1 36 340 206 856538H1 642 846 207 g5769156 224 677 210 941644R1 66 365   

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TABLE 5 SEQ ID NO :

   Template ID Tissue Distribution   Liver-41   %, Pancreas-34%, Cardiovascular System 
1 LG :1040582. 1 : 2000FEB18 - 14% 
2 LG : 453570. 1 :   2000FEB18 Nervous System-100%   
3 LG : 408751. 3 :   2000FEB18   Sense Organs-63%, Nervous   System-22%   
4 LI : 090574. 1 : 2000FEB01 Nervous System - 46%, Unclassified/Mixed - 36% 
5 LI : 229932. 2 : 2000FEB01 Musculoskeletal System-80% 
6 LI : 332176. 1 : 2000FEB01 Urinary   Tract-95%   
Respiratory System-60%, Hemic and Immune 
7 LI : 403248. 2 :   2000FEB01     System-40%   
8 LG : 220992. 1 :   2000MAY1 9 Embryonic Structures-17%, Male Genitalia-12%   
Liver-19%, Embryonic Structures-16%, 
9 LG :   1094571.   1 :

     2000MAY19   Cardiovascular System-14%   Skin-47%,   Stomatognathic System-27%, Sense 
10 LI : 350754. 4 : 2000MAY01 Organs    14%   
11 LI : 255828. 29 : 2000MAY01   MuscuWoskeletal   System-100% 
12 LI :   1190263.   1 : 2000MAY01 Urinary   Tract-80%,   Urinary Tract-15% 
13 LG : 270916. 2 : 2000FEB18 Female Genitalia - 100% 
Embryonic Structures-30%, Urinary Tract-13%, 
Digestive System-11%, Musculoskeletal System- 
14 LG : 999414. 3 :   2000FEB18 11 %   
Urinary Tract-80%, Hemic and Immune System- 
15 LG : 429446. 1 : 2000FEB18 2 
Male Genitalia-71%, Hemic and Immune System- 
16 LI : 057229. 1 : 2000FEB01   29%   
17 LI : 351965. 1 : 2000FEB01 Unclassified/Mixed-53%, Male Genitalia-12% 
18 LG : 068682. 1 :

     2000FEB18 Unclassified/Mixed-49%, Male Genitalia-27%   
Germ Cells-47%, Female Genitalia-13%, Male 
19 LG : 242665. 1 :   2000FEB18 Genitalia-12%     Liver-27%,   Urinary Tract-27%, Respiratory System- 
20 LG : 241743. 1 : 2000FEB18 14% 
Digestive System-24%, Musculoskeletal System- 
21 LI : 034212. 1 : 2000FEB01 22%, Nervous System-11% 
22 LG : 344886. 1 :   2000MAY19   Germ Cells-24%, Nervous System-12% 
Embryonic Structures-43%, Nervous System-29%, 
Respiratory System-14%, Hemic and Immune 
23 LG :   228930.   1 :   2000MAY19 System-14%   
Digestive System-23%, Unclassified/Mixed-21%, 
Embryonic Structures-19%, Hemic and immune 
24 LG : 338927. 1 :

   2000MAY19 Svstem - 19% 
Pancreas-13%, Embryonic Structures-11 %, 
Female Genitalia-10%, Urinary Tract-10%, Hemic and Immune System-10%, Cardiovascular System- 
25 LG : 898771. 1 :   2000MAY19 10%   
26 LI : 257664. 67 : 2000MAY01 Hemic and Immune System-100% 
Endocrine System-27%, Female   Genitalia-25%,   
27 LI :   001496.   2 : 2000MAY01 Embryonic Structures-25% 
Digestive System-29%,   Skin-24%,   Endocrine 
28 LI :   1085273.   2 : 2000MAY01   System-16%   
Exocrine   Glands-61%, Nervous   System-13%, 
29 LI : 333138. 2 :   2000MAY01   Nervous System-11 % 
Embryonic Structures-51 %, Digestive System-17% 
30 LI : 338927. 1 : 2000MAY01 
Endocrine System-45%, Nervous System-18%, 
31 LG : 335558. 1 :

     2000FEB18 ExocrineGlands-11%   

 <Desc/Clms Page number 265> 

 32 LG : 998283. 7 :   2000FEB18   Sense Organs-33%, Germ Cells-18%    Unclassified/Mixed-78%, Male Genitalia-11%,   33 LI :   402739.   1 : 2000FEB01 Hemic and Immune   System-11   % 34 LI :   175223.   1 :   2000FEB01   Embryonic Structures-99% 
Endocrine System-28%, Nervous System-22%, 
Respiratory System-17%, Female Genitalia-17%, 35 LG : 981076. 2 :   2000MAY19   Hemic and Immune Svtem-17% 36 Ll :   1008973.   1 : 2000MAY01 Nervous System-57%, Digestive System-41 % 37 LI :   1190250.   1 :

   2000MAY01 Female   Genitalia-48%,   Respiratory System-25%   Liver-23%,   Endocrine System-17%, Hemic and 38 LG : 021371. 3 :   2000FEB18 immune System-17%   39 LG : 475404. 1 :   2000FEB18   Skin-82% 
Liver-46%, Connective Tissue-31%, Nervous 40 LG : 979406. 2 : 2000FEB18 System-15% 
Embryonic Structures-52%, Endocrine System- 41 LG : 410726. 1 :   2000FEB18 26%     Unclassified/Mixed-26%,   Cardiovascular System- 42 LG : 200005. 1 :   2000FEB18   14%, Female Genitalia-13% 43 LG :   1076828.   1 :   2000FEB18 Unclassified/Mixed-69%,   Urinary Tract-25%   Unclassified/Mixed-63%, Musculoskeletal   System- 44 LG :   1076931.   1 : 2000FEB18 20%, urinary Tract-11% 45 LG :

     1078121.   1 :   2000FEB18   Female Genitalia-75%, Nervous System-25% 
Female   Genitalia-42%,   Cardiovascular System- 46 LG : 1079203. 1 :   2000FEB18 33%,   Hemic and Immune System-17% 47 LG :   1082586.   1 :   2000FEB18 Respiratory   System-100% 48 LG :   1082774.   1 :   2000FEB18 Respiratory System-50%,   Female Genitalia-50% 49 LG :   1082775.   1 :   2000FEB18 Female Genitalia-75%, Nervous System-25%   50 LG :   1083120.   1 : 2000FEB18 Nervous System-100% 51 LG :   1087707.   1 :   2000FEB18 Stomatognathic   System-98% 
Embryonic Structures-44%, Connective Tissue- 52 LG :   1090915.   1 :

   2000FEB18   l 9%   53 LG :   1094230.   1 : 2000FEB18 Female Genitalia-100% 
Connective Tissue-44%, Exocrine   Glands-44%,   54 LG : 474848. 3 :   2000FEB18   Hemic and Immune System-11% 
Nervous System-38%, Digestive System-38%, Male 55 LI : 251656. 1 : 2000FEB01   Genitalia-25%   
Hemic and Immune System-69%, Endocrine 56 LI : 021371. 1 :   2000FEB01   System-14% 57 LI :   133095.   1 : 2000FEB01 Respiratory System-67%, Nervous System-13%   Unclassified/Mixed-30%,   Respiratory System-19%, 
Nervous System-13%, Digestive System-13% 58 LI : 236654. 2 : 2000FEB01   Unclassified/Mixed-37%,   Urinary   Tract-16%,   59 LI : 200009. 1 :

   2000FEB01 Cardiovascular System-15%   Unclassified/Mixed-78%,   Musculoskeletal System- 60 LI : 758502. 1 : 2000FEB01 22% 
Nervous System-56%,   Skin-27%,   Connective Tissue 61 LI : 344772. 1 :   2000FEB01-13%   62 LI : 789445. 1 : 2000FEB01 Endocrine System-100% 
Urinary   Tract-31%, Female Genitalia-19%,   
Digestive System-19%, Hemic and Immune System 63 LI : 789657. 1 : 2000FEB01 - 
Exocrine   Glands-44%,   Female   Genitalia-33%,   64 LI : 789808. 1 : 2000FEB01 Nervous System-22% 65 LI : 792919. 1 :   2000FEB01   Respiratory System-100% 

 <Desc/Clms Page number 266> 

 
Female Genitalia-42%, Endocrine System-19%, 66 LI : 793949. 1 : 2000FEB01 Exocrine Glands-13% 67 LI : 794389. 1 :   2000FEB01   Endocrine System-100% 68 LI :

   796010. 1 : 2000FEB01 Exocrine Glands-100% 69 LI : 796324. 1 : 2000FEB01 Female Genitalia-100% 70 LI : 796373. 1 : 2000FEB01 Respiratory System-100% 71 LI : 796415. 1 : 2000FEB01 Nervous System-100% 72 LI : 798636. 1 : 2000FEB01 Hemic and Immune System-100% 73 LI : 800045. 1 : 2000FEB01   Female Genitalia-60%, Male Genitalia-40%   74 LI : 800680. 1 : 2000FEB01 Cardiovascular System-100% 75 LI : 800894. 1 : 2000FEB01 Respiratory System-50%, Digestive System-50% 76 LI : 801015. 1 : 2000FEB01 Male Genitalia-100% 77 LI :   801236.   1 : 2000FEB01 Endocrine System-100% 78 LI : 803335. 1 : 2000FEB01 Connective Tissue-100% 
Nervous System-38%, Digestive System-38%, Male 79 LI : 803998. 1 : 2000FEB01   Genitalia-25%   80 LI : 478757. 1 : 2000FEB01 Digestive System-100% 81 LI : 808532. 1 :

   2000FEB01 Hemic and   Immune System-100%   82 LI : 443073. 1 : 2000FEB01 Digestive System-100% 
Exocrine   Glands-80%,   Hemic and Immune System 83 LI : 479671. 1 : 2000FEB01-20% 84 LI : 810078. 1 : 2000FEB01 Digestive System-100% 85 LI : 810224. 1 : 2000FEB01 Digestive System-100% 
Nervous System-24%,   Unclassified/Mixed-18%,   86 LI : 817052. 2 : 2000FEB01 Exocrine Glands-14% 
Embryonic Structures-63%, Digestive   System-30%   87 LG : 892274. 1 :   2000MAY19   
Digestive System-40%, Respiratory System-30%, 88 LG :   1080959.   1 :   2000MAY19   Hemic and Immune   System-30%   89 LG :   1054900.   1 :

     2000MAY19   Digestive System-100% 
Nervous System-38%, Female   Genitalia-38%,   90 LG :   1077357.   1 :   2000MAY19 Male Genitalia-25%     Pancreas-31 %,   Digestive System-22%, Hemic and 91 LG : 1084051. 1 :   2000MAY19 immune System-16%   
Female   Genitalia-23%,     Unclassified/Mixed-23%,   
Cardiovascular System-18%, Exocrine Glands- 92 LG :   1076853.   1 :   2000MAY19     18%   
Female Genitalia-22%, Nervous   System-17%,   93 LG : 481631. 10 :   2000MAY19   Exocrine   Glands-17%,   Urinary Tract-17% 
Exocrine Glands-67%, Cardiovascular System- 94 LG :   1088431.   2 :

     2000MAY19   33% 
Endocrine System-18%, Embryonic Structures- 95 LI : 401619. 10 : 2000MAY01   16%,   Pancreas-15% 
Hemic and Immune System-27%, Female 96   Ll   :   1144007.   1 : 2000MAY01 Genitalia-13% 
Endocrine System-28%, Sense Organs-22%, 97 LI : 331074. 1 : 2000MAY01 Connective Tissue-10% 98 LI :   1170349.   1 : 2000MAY01 Endocrine System-91 % 
Embryonic   Structures-24%, Musculoskeletal   System 99 LG : 335097.   1   :   2000FEB18-19%, Nervous System-16%   100 LG :   1076451.   1 :   2000FEB18   Nervous System-100% 101 LI : 805478. 1 : 2000FEB01   Skin-100%   
Endocrine System-33%, Embryonic Structures- 102 LG :   101269.   1 :

     2000MAY19 33%, Urinary Tract-30%   103 LI : 331087. 1 : 2000MAY01   Liver-82%,   Hemic and Immune System-13% 

 <Desc/Clms Page number 267> 

 
Cardiovascular System-81 %, Cardiovascular 104 LI : 410188. 1 : 2000MAY01 System-12% 105 LI :   1188288.   1 : 2000MAY01 Nervous System-73%   Liver-16%, Male Genitalia-13%,   Embryonic 106 LI : 427997. 4 : 2000MAY01   Structures-11 %   107 LG : 451682. 1 :   2000FEB18   Nervous System-100% 108 LG :   1077283.   1 :   2000FEB18 Liver-86%,   Hemic and Immune   System-14%   
Embryonic Structures-41 %, Endocrine System- 109 LG : 481436. 5 :   2000FEB18 20%,   Hemic and immune System-13% 
Endocrine System-43%, Urinary Tract-36%, 110 LI : 793701. 1 :

   2000FEB01 Respiratory System-21 % 111 LI : 373637. 1 : 2000FEB01 Germ Cells-74%, Unclassified/Mixed-16% 
Digestive System-43%, Male   Genitalia-24%,   112 LG : 239368. 2 :   2000MAY19   Endocrine System-24% 
Germ   Cells-66%,     Unclassified/Mixed-22%,   Male 113 LI : 053826. 1 : 2000MAY01 Genitalia-12% 114 LI : 449393. 1 : 2000MAY01 Nervous System-100% 115 Ll :   1071427.   96 : 2000MAY01   Stomatognathic   System-13% 116 LI : 336338. 8 : 2000MAY01   Unclassified/Mixed-55%,   Connective Tissue-26% 
Urinary Tract-24%, Hemic and Immune System- 117 LG : 345527. 1 :   2000FEB18 24%, Respiratory System-18%   118 LG :   1089383.   1 :

     2000FEB18   Connective Tissue-73%, Female Genitalia-27% 
Female   Genitalia-38%,   Exocrine Glands-31 %, 
Male   Genitalia-15%,   Hemic and immune System-   119   LG : 1092522. 1 :   2000FEB18 15%   120 LG :   1093216.   1 :   2000FEB18 Urinary Tract-100%   
Embryonic Structures-86%, Hemic and Immune 121 LI : 270318. 3 : 2000FEB01   System-14%   
Unclassified/Mixed-34%, Hemic and Immune 122 LI : 335671. 2 :   2000FEB01   System-20%, Urinary Tract-17% 123 LI : 793758. 1 : 2000FEB01 Nervous   System-62%,   Urinary Tract-38% 124 LI : 803718. 1 : 2000FEB01 Female   Genitalia-100%   125 LI : 412179. 1 : 2000FEB01 Endocrine System-100% 126 LI : 815679. 1 :

   2000FEB01 Digestive System-75% 
Embryonic Structures-28%, Skin-20%, 127 LI :   481361.   3 : 2000FEB01   Unclassified/Mixed-16%   
Cardiovascular System-33%, Endocrine System- 128 LG :   247388.   1 :   2000MAY19 21%, MaleGenitalia-21%   129 LG : 255789. 10 :   2000MAY19   Endocrine System-56%, Urinary Tract-44% 
Endocrine   System-22%,   Digestive   System-13%,   130 LI : 787618. 1 : 2000MAY01 Endocrine System-12% 
Sense Oraans-18% Nervous   System-11%.   

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   Digestive System-34%, Liver-17%, Female 139 LG : 337818. 2 :   2000FEB18 Genitalia-11 %   
Digestive   System-27%,   Liver-19%, Female 140 LI : 337818. 1 : 2000FEB01 Genitalia-15% 
Pancreas-48%, Endocrine System-24%, 141 LG :   241577.   4 : 2000MAY19 Respiratory System-14% 
Respiratory System-67%, Digestive System-22%, 142 LG : 344786. 4 :   2000MAY19   Nervous   System-11     %   
Endocrine System-44%,   Unclassified/Mixed-17%,   143 LI :   414307.   1 : 2000FEB01 Nervous System-11 % 144 LI : 202943. 2 : 2000FEB01 Embryonic Structures-100% 145 LI : 246194. 2 : 2000FEB01 Germ Cells-75%, Pancreas-13% 146 LI : 815961. 1 : 2000FEB01 Digestive System-99% 
Skin-33%, Embryonic   Structures-21 %,   Digestive 147 LG :

     120744.   1 :   2000MAY19 System-21 %   
Musculoskeletal System-45%, Cardiovascular 148 LI : 757520. 1 : 2000MAY01 System-26%,   Skin-24%     149   LG :   160570.   1 :   2000FEB18   Skin-84%, Female Genitalia-16% 
Male   Genitalia-50%,   Hemic and Immune System- 150 LI : 350398. 3 :   2000FEB01   50% 151 LI : 221285. 1 : 2000FEB01 Endocrine System-42%, Nervous   System-21%   153 LI : 329017. 1 : 2000FEB01 Endocrine System-62%,   Unclassified/Mixed-24%   154 LI : 401322. 1 : 2000FEB01 Sense   Organs-44%,     Liver-22%,   Skin-14% 
Respiratory   System-18%, Female Genitalia-16%,   155 LG : 403409. 1 :

     2000MAY19   Cardiovascular System-13% 156 LG : 233933. 5 :   2000MAY19   Digestive System-100% 
Connective Tissue-40%, Nervous   System-19%,   157 LI : 290344. 1 : 2000MAY01   Embryonic Structures-12%   158 LI :   410742.   1 : 2000MAY01 Respiratory System-47%, Skin-42%   Stomatognathic   System-57%, Musculoskeletal 159 LG : 406568. 1 :   2000MAY19   System-21   %,   Cardiovascular System-16% 160 LI : 283762. 1 : 2000MAY01 Sense Organs-25% 161 LI : 347687. 113 : 2000MAY01 Nervous System-45%, Nervous System-38% 162 Ll :   1146510.   1 : 2000MAY01 Skin-94% 163 LG : 451710. 1 : 2000FEB18 Connective Tissue-89%, Nervous System-11% 164 LG :   455771.     1   :

   2000FEB18 Nervous System-100% 165 LG : 452089. 1 :   2000FEB18   Nervous System-100% 166 LG : 246415. 1 :   2000FEB18   Pancreas-83%, Nervous System-17% 
Cardiovascular System-17%, Connective Tissue- 167 LG : 414144. 10 : 2000FEB18 12% 168 LG :   1101445.   1 :   2000FEB18 Liver-91 %   
Hemic and Immune System-64%, Male Genitalia- 169 LG :   452134.   1 : 2000FEB18 36% 170 LI : 903021. 1 : 2000FEB01 Male Genitalia-100% 171 LI : 246422. 1 : 2000FEB01 Hemic and immune System-100% 172 LG : 449404. 1 :   2000MAY19 Nervous System-100%   173 LG : 449413. 1 :   2000MAY19 Nervous System-100%   174 LG : 450105. 1 :   2000MAY19   Nervous System-100% 175 LG : 460809. 1 :   2000MAY19 Exocrine Glands-100%   176 LG :

     481781.   1 :   2000MAY19 Nervous System-100%   177 LG :   1101153.   1 :   2000MAY19   Nervous System-100% 
Exocrine   Glands-28%,   Endocrine System-19%, 178 LI : 257695. 20 : 2000MAY01 Nervous System-16%, Digestive System-16% 179 LI : 455771. 1 : 2000MAY01   Nervous System-100%   
Nervous System-60%, Hemic and Immune System- 180 LI : 274551. 1 : 2000MAY01 40% 

 <Desc/Clms Page number 269> 

 
Embryonic Structures-58%, Digestive System-26%, 181 LI: 035973. 1 : 2000MAY01 Nervous System-16% 182 LG : 978427. 5 :   2000FEB18   Nervous System-100% 183 LG : 247781. 2 :   2000FEB18   Nervous System-11% 184 LI : 034583. 1 :

   2000FEB01 Nervous System-35%, Endocrine   System-35%   
Cardiovascular System-28%, Urinary   Tract-27%,   185 LI : 333307. 2 : 2000FEB01   Musculoskeletal System-17%   186 LI : 814710. 2 : 2000FEB01 Respiratory System-100% 187 LG : 414732. 1 :   2000MAY19   Endocrine System-82%, Nervous System-18% 
Connective Tissue-55%, Nervous System-15%, 188 LG : 413910. 6 : 2000MAY19 Embryonic Structures-13% 189 LI : 414732. 2 : 2000MAY01 Endocrine System-80%, Nervous System-20% 190 LI : 900264. 2 :   2000MAY01   Urinary   Tract-15%,   Male Genitalia-12% 
Urinary Tract-46%, Endocrine System-17%, Germ 191 LI : 335593. 1 : 2000MAY01 Cells-14% 
Stomatognathic System-35%, Digestive System- 192   Ll   :   1189543.   1 : 2000MAY01 14% 193 LG : 455450. 1 :

     2000FEB18   Nervous System-100% 194 LG :   1040978.   1 :   2000FEB18   Nervous System-100% 195 LG : 446649. 1 :   2000FEB18 Liver-80%, Hemic and Immune System-13%     Unclassified/Mixed-17'0,   Sense Organs-16%, 196 LG :   132147.   3 :   2000FEB18   Embryonic Structures-10% 197 LI : 036034. 1 : 2000FEB01 Nervous System-80% 
Unclassified/Mixed-53%, Cardiovascular System- 198 LG :   162161.   1 :   2000MAY19 21%, NervousSYstem-16%     Unclassified/Mixed-40%,   Respiratory System-24%, 199 LG : 407214. 10 :   2000MAY19   Cardiovascular   System-16%   
Digestive System-41 %, Exocrine   Glands-24%,   200 LG : 204626. 1 :

   2000MAVY19 Female Genitalia-18%   Unclassified/Mixed-31 %,   Nervous System-25%, 201 LI : 007401. 1 : 2000MAY01   Urinary Tract-11 %   202 LI : 476342. 1 : 2000MAY01 Connective Tissue-77%, Nervous System-23% 
Hemic and Immune System-27%, Musculoskeletal 203   U : 1072759.   1 : 2000MAY01 System-19%, Endocrine   System-11   % 204 LG : 998857. 1 : 2000FEB18 Digestive System-58%, Pancreas- 12% 205 LG : 482261. 1 :   2000FEB18 Male Genitalia-85%, Respirstory System-15%   
Skin-20%, Germ Cells - 18%, Female Genitalia- 206 LG :   480328. 1   : 2000FEB18 10% 
Germ Cells-44%, Digestive System-15%, Male 207 LG : 311197. 1 :   2000MAY19 Genitalia-11%   208 LG :   1054883.   1 :   2000MAY19   Endocrine System-100% 209 LG :

   399395. 1 :   2000MAY19 Hemic   and immune System-100% 
Germ   Cells-23%,   Exocrine Glands-14%, 210 LG : 380497. 2 :   2000MAY19   Connective Tissue-13% 211 LI : 272913. 22 : 2000MAY01   Female Genitalia-100%   

 <Desc/Clms Page number 270> 

   TABLE 6 SEQ ID NO:

   Frame Length Strat Stop GI Number Probability Score Annotation 212 3 115 198 542 g399660 3.00E-51 aldehyde reductase [Rattus norvegicus] 212 3 115 198 542 g7677318 7.00E-51 aldehyde reductase [Mus musculus] 212 3 115 198 542 g6013149 2.00E-48 aldehyde reductase [Homo sapiens] 213 3 161 3 485 g2909424 2.00E-60 Glyoxalase I [Cicer arietinum] 213 3 161 3 485 g2113825 2.00E-58 Glyoxalase I [Brassica juncea] 213 3 161 3 485 g1177314 4.00E-57 glyoxalase-I [Lycopersicon esculentum] 214 3 332 2 997 g8671168 0 hypothetical protein [Homo sapiens] 214 2 332 2 997 g8886025 0 collapsin response mediator protein-5 [Homo sapiens] 214 2 332 2 997 g8671360 1.00E-179 Ulip-like protein [Rattus norvegicus] 215 3 274 12 833 g29600 2.00E-86 carbonic anhdrase I (AA 1-261) [Homo sapiens] 215 3 274 12 833 g179793 2.00E-86 carbonic anhydrase I (EC 4.2.1.)

   [Homo sapiens] 215 3 274 12 833 g29587 4.00E-84 carbonic anhydrase II (AA 1-260) [Homo sapiens] 216 1 182 742 1287 91043188 1.00E-102 unnamed protein product [Homo sapiens] 216 1 182 742 1287 g9949721 3.00E-49 probable acetyl-coa synthetase [Pseudomonas aeruginosa] 216 1 182 742 1287 g9655831 7.00E-46 prpE protein [vibrio cholerae] 217 2 359 2 1078 g2104689 1.00E-111 alpha glucosidase II, alpha subunit [Mus musculus] 217 2 359 2 1078 g7672977 1.00E-111 glucosidase II alpha subunit [Homo sapiens] The ha1225 gene product is related to human alpha-glucosidase.

   [Homo 217 2 359 2 1078 g577295 1.00E-110 sapiens] 218 2 110 161 490 g9653274 1.00E-26 ornithine decarboxylase-2 [Xenopus laevis] 218 2 110 161 490 g200124 5.00E-18 ornithine decarboxylase [Mus pahari] 218 2 110 161 490 g53518 1.00E-17 ornithine decarboxylase [Mus musculus] 219 3 549 36 1682 g10435462 0 unnamed protein product [Homo sapiens] 219 3 549 36 1682 g7023375 0 unnamed protein product [Homo sapiens] 219 3 549 36 1682 g10433608 1.00E-164 unnamed protein product [Homo sapiens] 220 1 264 1 792 g7023634 3.00E-92 unnamed protein product [Homo sapiens] similar to C. elegans r10H10.6 and S. cerevisiae YD8419.03c [Drosophila 220 1 264 1 792 g3213202 3.00E-49 melanogaster] 220 1 264 1 792 g7298960 3.00E049 CG2846 gene product [Drosophila melanogaster] 221 3 701 33 2135 g307504 0 transglutaminase E3 [Homo sapiens] TGM3 (PROTEIN-GLUTAMINE GLUTAMYLTRANSFERASE E3 PRECURSOR (EC 2.3.2.

   13) (TGASE E3) (TRANSGLUTAMINASE 3).) [Homo 221 3 701 33 2135 g4467804 0 sapiensl 221 3 701 33 2135 g309521 0 transgiutaminase E3 [Mus musculus]   

 <Desc/Clms Page number 271> 

   222 2 150 2 451 g35505 7.00E-65 pyruvate kinase [Homo sapiens] 222 2 150 2 451 g189998 7.00E-65 M2-type pyruvate kinase [Homo sapiens] pyruvate kinase;

   ATP:pyruvate 2-o-phosphotransferase [Oryctolagus 222 2 150 2 451 g2623945 3.00E-64 cuniculus] 223 2 234 866 1567 g2576305 1.00E-128 arylsulphatase [Homo sapiens] 223 2 234 866 1567 g791002 3.00E-82 ARSD [Homo sapiens] 223 2 234 866 1567 g791004 4.00E-75 ARSE [Homo sapiens] 224 2 86 2 259 225 2 173 1049 1567 g4092820 8.00E-62 BC319430¯7 [Homo sapiens] 225 2 173 1049 1567 g2792016 2.00E-54 olfactory receptor [Homo sapiens] 225 2 173 1049 1567 g4092819 2.00E-54 BC319430¯5 [Homo sapiens] 226 2 68 86 289 g8272468 4.00E-15 envelope protein [Homo sapiens] 226 2 68 86 289 g4773880 4.00E-15 envelope protein precursor [Homo sapiens] 226 2 68 86 289 g4262296 4.00E-15 envelope protein [Homo sapiens] 227 1 70 79 288 g11231093 1.00E-11 hypothetical protein [Macaca fascicularis] 227 1 70 79 288 g10435559 3.00E-10 unnamed protein product [Homo sapiens] 227 1 70 79 288 g7020625 2.00E-09 unnamed 

  protein product [Homo sapiens] 228 2 117 836 1186 g5726235 3.00E-18 unknown protein U5/2 [multiple sclerosis associated retrovirus element] 229 2 294 2 883 g404634 1.00E-59 serine/threonine kinase [Mus musculus] 229 2 294 2 883 g2738898 3.00E-59 protein kinase [Mus musculus] 229 2 294 2 883 g8101585 2.00E-54 testis specific serine kinase-3 [Mus musculus] 230 1 326 1 978 g2117166 1.00E-160 Ras like GTPase [Homo sapiens] 230 1 326 1 978 g466271 1.00E-140 Rar protein [Homo sapiens] 230 1 326 1 978 g3036779 1.00E-102 match: multiple proteins;

   RAR (RAS like GTPASE) like [Homo sapiens] 231 1 182 40 585 g5763838 1.00E-66 dJ593C16.1 (ras GTPase activating protein) [Homo sapiens] 231 1 182 40 585 g4417207 1.00E-66 synGAP-d [Rattus norvegicus] 231 1 182 40 585 g4105589 1.00E-66 nGAP [Homo sapiens] 232 1 358 58 1131 g1469876 1.00E-103 The KIAA0147 gene product is related to adenylyl cyclase.

   [Homo sapiens] 232 1 358 58 1131 g6850952 1.00E-86 vartul-2 protein [Drosophila melanogaster] 232 1 358 58 1131 g6782322 1.00E-86 Vartul-1 protein [Drosophila melanogaster] 233 1 194 370 951 g7008402 1.00E-107 kappa B-ras 1 [Homo sapiens] 233 1 194 370 951 g7239257 1.00E-103 kappaB-Ras1 [Mus musculus] 233 1 194 370 951 g7008404 8.00E-75 kappa B-ras 2 [Homo sapiens] 234 2 222 17 682 g9368448 1.00E-111 phospholipase C-beta-1a [Homo sapiens] 234 2 222 17 682 g9368450 1.00E-111 phospholipase C-beta-1b [Homo sapiens] 234 2 222 17 682 g206218 1.00E-110 phospholipase C-1 [Rattus sp.]   

 <Desc/Clms Page number 272> 

   235 3 185 126 680 g3599940 1.00E-57 faciogenital dysplasia protein 2 [Mus musculus] 235 3 185 126 680 g10440426 8.00E-42 FLJ00048 protein [Homo sapiens] 235 3 185 126 680 g595425 4.00E-20 FGD1 [Homo sapiens] 236 2 192 707 1282 237 3 61 204 386 238 2 335 

  17 1021 g3005085 2.00E-92 hook1 protein [Homo sapiens] 238 2 335 17 1021 g5706448 2.00E-92 dJ782L23.1 (HOOK1) [Homo sapiens] 238 2 335 17 1021 g3005087 2.00E-70 hook2 protein [Homo sapiens] 239 1 346 1261 2298 g1109782 1.00E-105 protein-tyrosine phosphatase [Homo sapiens] 239 1 346 1261 2298 g1781037 1.00E-76 neuronal tyrosine threonine phosphatase 1 [Mus musculus] 239 1 346 1261 2298 g10241798 5.00E-11 hypothetical protein SCE41.24c [Streptomyces coelicolor] 240 3 298 147 1040 g4678722 1.00E-156 hypothetical protein [Homo sapiens] dJ272L16.1 (Rat Ca2+/Calmodulin dependent Protein Kinase LIKE protein)

   240 3 298 147 1040 g4007153 1.00E-153 [Homo sapiens] 240 3 298 147 1040 g2077934 1.00E-152 Protein Kinase [Rattus norvegicus] 241 1 133 133 531 g10440426 1.00E-34 FLJ00048 protein [Homo sapiens] 241 1 133 133 531 g3599940 2.00E-16 faciogenital dysplasia protein 2 [Mus musculus] 242 2 354 821 1882 g11907572 1.00E-143 TSC22-related inducible leucine zipper 1b [Mus musculus] 242 2 354 821 1882 g1181619 1.00E-106 a variant of TSC-22 [Gallus gallus] 242 2 354 821 1882 g3327152 9.00E-16 KIAA0669 protein [Homo sapiens] 243 1 237 1 711 g6683492 1.00E-105 bromodomain PHD finger transcription factor [Homo sapiens] contains similarity to Pfam domain: PF00439 (Bromodomain), Score=125.5, Evalue=1.5e-35, N=1; PF00628 (PHD-finger), Score=102.0, E-value=3.8e-27, 243 1 237 1 711 g3876452 9.00E-53 N=2 [Caenorhabditis elegans] predicted using Genefinder¯contains similarity to Pfam domain:

   PF00439 (Bromodomain), Score=125.5, E-value=1.5e-35, N=1; PF00628 (PHD-finger), 243 1 237 1 711 g3876449 9.00E-53 Score=102.0, E-value=3.8e-27, N=2 [Caenorhabditis elegans] 244 1 161 1 483 g6330736 1.00E-42 KIAA1234 protein [Homo sapiens] 244 1 161 1 483 g11244871 1.00E-40 dioxin receptor repressor [Homo sapiens] 244 1 161 1 483 g4164151 4.00E-35 AhR repressor [Mus musculus] 245 3 151 54 506 g10433955 9.00E-44 unnamed protein product [Homo sapiens] 245 3 151 54 506 g7295442 1.00E-16 CG17334 gene product [Drosophila melanogaster] 245 3 151 54 506 g2745892 1.00E-12 Y box transcription factor [Mus musculus] supported by Genscan and several ESTs;

   C83049 (NID:g3062006), AA823760 (NID:g2893628), AA215791 (NID:g1815572), Al095488 246 2 160 173 652 g3924670 4.00E-68 (NID:g3434464), and AA969095 (NID:g3144275) [Homo sapiens]   

 <Desc/Clms Page number 273> 

   246 2 160 173 652 g5640105 2.00E-59 homoeobox protein LSX [Homo sapiens] 246 2 160 173 652 g6523391 6.00E-59 phtf protein [Mus musculus] 247 3 160 108 587 g6939732 1.00E-52 transcription factor Eiongin A2 [Homo sapiens] dJ886K2.1 (elongin A; RNA polymerase; RNA polymerase II;

   RNA polymerase 247 3 160 108 587 g4581412 1.00E-29 II elongation factor.) [Homo sapiens] 247 3 160 108 587 g992563 1.00E-29 eiongin A [Homo sapiens] 248 1 171 25 537 g11907923 4.00E-29 enhancer of polycomb [Homo sapiens] 248 1 171 25 537 g3757890 3.00E-18 enhancer of polycomb [Drosophila melanogaster] 248 1 171 25 537 g7303589 3.00E-18 E(Pc) gene product [Drosophila melanogaster] bA465L10.2 (novel C2H2 type zinc finger protein similar to chicken FZF-1)

   249 2 449 266 1612 g10443047 0 [Homo sapiens] 249 2 449 266 1612 g10438918 0 unnamed protein product [Homo sapiens] 249 2 449 266 1612 g984814 8.00E-98 zinc finger protein [Gallus gallus] 250 2 127 140 520 g10434195 2.00E-64 unnamed protein product [Homo sapiens] 250 2 127 140 520 g6467206 3.00E-36 gonadotropin inducible transcription repressor-4 [Homo sapiens] 250 2 127 140 520 g6330394 4.00E-34 KIAA1198 protein [Homo sapiens] 251 1 157 1 471 9340446 2.00E-17 zinc finger protein 7 (ZFP7)

   [Homo sapiens] 251 1 157 1 471 g4325310 2.00E-17 zinc-finger protein 7 [Homo sapiens] 251 1 157 1 471 g6007771 5.00e-17 KID2 [Mus musculus] 252 1 305 145 1059 g6002480 3.00E-49 BWSCR2 associated zinc-finger protein BAZX2 [Homo sapiens] 252 1 305 145 1059 g9963806 3.00E-47 zinc finger protein ZNF287 [Homo sapiens] 252 1 305 145 1059 g11527849 8.00E-43 zinc finger protein SKAT2 [Mus musculus] 253 2 717 305 2455 g10047335 0 KIAA1629 protein [Homo sapiens] 253 2 717 305 2455 g1504006 1.00E-96 similarto human ZFY protein. [Homo sapiens] 253 2 717 305 2455 g7243280 4.00E-66 KIAA1441 protein [Homo sapiens] 254 1 211 1 633 g10047183 3.00E-49 KIAA1559 protein [Homo sapiens] 254 1 211 1 633 g5080758 2.00E-45 BC331191¯1 [Homo sapiens] 254 1 211 1 633 g498721 3.00E-44 zinc finger protein [Homo sapiens] 255 2 103 2 310 g498152 2.00E-20 ha0946 protein is Kruppel-related.

   [Homo sapiens] 255 2 103 2 310 g7576272 2.00E-20 bA393J16.1 (zinc finger protein 33a (KOX 31)) [Homo sapiens] 255 2 103 2 310 g10440081 2.00E-19 unnamed protein product [Homo sapiens] 256 3 84 135 386 g347906 2.00E-26 zinc finger protein [Homo sapiens] 256 3 84 135 386 g3342002 1.00E-25 hematopoietic cell derived zinc finger protein [Homo sapiens] 256 3 84 135 386 g8163824 5.00E-25 krueppel-like zinc finger protein HZF2 [Homo sapiens] 257 1 194 103 684 g10435738 4.00E-74 unnamed protein product [Homo sapiens] 257 1 194 103 684 g1017722 8.00E-73 repressor transcriptional factor [Homo sapiens]   

 <Desc/Clms Page number 274> 

   257 1 194 103 684 g7959207 3.00E-71 KIAA1473 protein [Homo sapiens] 258 1 129 28 414 g2072955 6.00E-07 p40 [Homo sapiens] 258 1 129 28 414 g483915 8.00E-07 ORF1,

   encodes a 40 kDa product [Homo sapiens] 258 1 129 28 414 g339776 8.00E-07 ORF1 codes for a 40 kDa product [Homo sapiens] 259 3 93 75 353 g3329372 4.00E-36 DNA-binding protein [Homo sapiens] 259 3 93 75 353 g7959207 1.00E-33 KIAA1473 protein [Homo sapiens] 259 3 93 75 353 g184452 3.00E-33 Krueppel-related DNA-binding protein [Homo sapiens] 260 3 193 369 947 g8099348 1.00E-38 zinc finger protein [Homo sapiens] 260 3 193 369 947 g5730196 2.00E-38 Kruppel-type zinc finger [Homo sapiens] 260 3 193 369 947 g8050899 4.00E-38 ZNF180 [Homo sapiens] 261 3 111 3 335 g7023216 1.00E-14 unnamed protein product [Homo sapiens] 261 3 111 3 335 g3406676 6.00E-14 zinc finger protein 54 [Mus musculus] 261 3 111 3 335 g9802037 3.00E-13 zinc finger protein SBZF3 [Homo sapiens] 262 3 137 75 485 g186774 1.00E-26 zinc finger protein [Homo sapiens] 262 3 137 75 485 g2384653 6.00E-26 Krueppel family zinc 

  finger protein [Homo sapiens] 262 3 137 75 485 g8163824 6.00E-26 krueppel-like zinc finger protein HZF2 [Homo sapiens] 263 3 68 51 254 g7239109 2.00E-15 HSPC059 [Homo sapiens] 263 3 68 51 254 g347906 4.00E-15 zinc finger protein [Homo sapiens] 263 3 68 51 254 g7023216 2.00E-14 unnamed protein product [Homo sapiens] 264 3 101 90 392 g3329372 8.00E-35 DNA-binding protein [Homo sapiens] 264 3 101 90 392 g4559318 7.00E-32 BC273239¯1 [Homo sapiens] 264 3 101 90 392 g184452 9.00e-32 Krueppel-related DNA-binding protein [Homo sapiens] 265 1 96 184 471 g4589588 5.00E-22 KIAA0972 protein [Homo sapiens] 265 1 96 184 471 g4514561 6.00E-22 KRAB-containing zinc-finger potein KRAZ2 [Mus musculus] 265 1 96 184 471 g7576272 2.00E-21 bA393J16.1 (zinc finger protein 33a (KOX 31))

   [Homo sapiens] 266 2 251 2 754 g55471 1.00E-134 Zfp-29 [Mus musculus] 266 2 251 2 754 g1020145 3.00E-73 DNA binding protein [Homo sapiens] 266 2 251 2 754 g6002478 3.00E-72 BWSCR2 associated zinc-finger protein BAZ1 [Homo sapiens] bA465L10.2 (novel C2H2 type zinc finger protein similar to chicken FZF-1)

   267 3 522 36 1601 g10443047 0 [Homo sapiens] 267 3 522 36 1601 g10438918 0 unnamed protein product [Homo sapiens] 267 3 522 36 1601 g984814 2.00E-97 zinc finger protein [Gallus gallus] 268 2 267 2 802 g9886891 4.00E-45 zinc finger protein 276 C2H2 type [Mus musculus] 268 2 267 2 802 g11611571 3.00E-43 hypothetical protein [Macaca fascicularis] 268 2 267 2 802 g453376 4.00E-43 zinc finger protein PZF [Mus musculus] 269 2 286 2 859 g2754696 9.00E-08 high molecular nuclear antigen [Gallus gallus]   

 <Desc/Clms Page number 275> 

   269 2 286 2 859 g2078483 9.00E-06 antifreeze glycopeptide AFGP polyprotein precursor [Boreogadus saida] PR-domain zinc finger protein 6 isoform A; PR-domain family protein 3 isoform 270 3 194 270 851 g8575782 1.00E-112 A; PRDM6A;

   PFM3A [Homo sapiens] 270 3 194 270 851 g10437767 1.00E-26 unnamed protein product [Homo sapiens] 270 3 194 270 851 g7295698 9.00E-26 CG15436 gene product [Drosophila melanogaster] 271 3 263 3 791 g6409345 1.00E-107 zinc finger protein ZNF180 [Homo sapiens] 271 3 263 3 791 g80500899 1.00E-107 ZNF180 [Homo sapiens] 271 3 263 3 791 g200407 1.00E-101 pMLZ-4 [Mus musculus] 272 2 142 290 715 g4062983 5.00E-65 Eos protein [Mus musculus] 272 2 142 290 715 g9408382 4.00E-46 eos [Raja eglanteria] 272 2 142 290 715 g11612390 3.00E-42 zinc finger transcription factor Eos [Homo sapiens] KRAB zinc finger protein;

   Method: conceptual translation supplied by author 273 2 164 2 493 g1049301 3.00E-25 [Homo sapiens] 273 2 164 2 493 g10047251 9.00E-25 KIAA1588 protein [Homo sapiens] 273 2 164 2 493 g8809810 1.00E-19 KRAB zinc finger protein [Mus musculus] 274 2 107 509 829 g1237278 2.00E-36 zinc finger protein [Cavia porcellus] 274 2 107 509 829 g7023417 4.00E-36 unnamed protein product [Homo sapiens] 274 2 107 509 829 g11917507 5.00E-36 HPF1 protein [Homo sapiens] 275 3 105 336 650 g9801232 2.00E-51 bA508N22.2 (zinc finger protein 37a (KOX 21))

   [Homo sapiens] 275 3 105 336 650 g829151 2.00E-51 ZNF37A [Homo sapiens] 275 3 105 336 650 g5730196 4.00E-36 Kruppel-type zinc finger [Homo sapiens] 276 1 149 1 447 g7656698 3.00E-91 Zinc finger protein 222 [Homo sapiens] 276 1 149 1 447 g6118381 3.00E-91 zinc finger protein ZNF222 [Homo sapiens] 276 1 149 1 447 g6118383 1.00E-81 zinc finger protein ZNF223 [Homo sapiens] 277 3 101 90 392 g3329372 1.00E-30 DNA-binding protein [Homo sapiens] 277 3 101 90 392 g4559318 3.00E-29 BC273239¯1 [Homo sapiens] 277 3 101 90 392 g1124876 5.00E-29 Krueppel-related DNA-binding protein [Homo sapiens] 278 3 137 6 416 g11062533 2.00E-46 bA245E14.1 (novel zinc finger protein similar to ZFP47)

   [Homo sapiens] 278 3 137 6 416 g5640017 2.00E-46 zinc finger protein ZFP113 [Mus musculus] 278 3 137 6 416 g186774 5.00E-46 zinc finger protein [Homo sapiens] 279 3 97 165 455 g829151 2.00E-27 ZNF37A [Homo sapiens] 279 3 97 165 455 g9801232 2.00E-27 bA508N22.2 (zinc finger protein 37a (KOX 21)) [Homo sapiens] 279 3 97 165 455 g3702137 9.00E-20 dJ733D15.1 (Zinc-finger protein) [Homo sapiens] 280 2 97 182 472 g9801232 4.00E-29 bA508N22.2 (zinc finger protein 37a (KOX21))

   [Homo sapiens] 280 2 97 182 472 g829151 4.00E-29 ZNF37A [Homo sapiens] 280 2 97 182 472 g200407 4.00E-21 pMLZ-4 [Mus musculus]   

 <Desc/Clms Page number 276> 

   281 1 179 31 567 g10442700 3.00E-61 zinc-finger prtein ZB RK1 [Homo sapiens] 281 1 179 31 567 g10435411 3.00E-61 unnamed protein product [Homo sapiens] 281 1 179 31 567 g10954044 3.00E-61 KRAB zinc finger protein ZFQR [Homo sapiens] 282 3 87 369 629 g8099348 2.00E-14 zic finger protein [Homo sapiens] 282 3 87 369 629 g498725 2.00E-14 zinc finger protein [Homo sapiens] 282 3 87 369 629 g495568 2.00E-13 zinc finger protein [Homo sapiens] 283 2 1723 2 517 g6007771 4.00E-97 KID2 [Mus musculus] 283 2 172 2 517 g2970038 2.00E-93 HKL1 [Homo sapiens] 283 2 172 2 517 g205067 2.00E-93 zinc finger protein [Rattus norvegcus] 284 1 151 1 453 g1806134 5.00E-57 zinc finger protein [Mus musculus] 284 1 151 1 453 

  g538413 5.00E-57 zinc finger protein [Mus musculus] 284 1 151 1 453 g186774 3.00E-55 zinc finger protein [Homo sapiens] 285 2 89 83 349 g97023216 2.00E-18 unnamed protein product [Homo sapiens] 285 2 89 83 349 g9802037 4.00E-16 zinc finger protein SBZF3 [Homo sapiens] 285 2 89 83 349 g7239109 7.00E-15 HSPC059 [Homo sapiens] 286 2 146 62 499 g2739353 7.00E-56 ZNF91L [Homo sapiens] 286 2 146 62 499 g7959207 5.00E-50 KIAA1473 protein [Homo sapiens] 286 2 146 62 499 g3342002 7.00E-50 hematopoietic cell derived zinc finger protein [Homo sapiens] 287 1 78 1 234 g487785 4.00E-16 zin finger protein ZNF163 [Homo sapiens] 287 1 78 1 234 g5262560 7.00E-15 hypothetical protein [Homo sapiens] 287 1 78 1 234 g10434856 9.00E-15 unnamed protien product [Homo sapiens] 288 3 126 78 455 g9963804 4.00E-47 zincfinger protein ZNF286 [Homo sapiens] 288 3 126 78 455 g5640017 2.00E-46 zin finger protein 

  ZFP113 [Mus musculus] 288 3 126 78 455 g7020166 4.00E-46 unnamed proein product [Homo sapiens] 289 1 96 151 438 g4589588 5.00E-22 KRAB-containing zinc-finger protein KRAZ2 [Mus musculus] 289 1 96 151 438 g4514561 6.00E-22 KRAB-containing zinc-finger protein KRAZ2 [Mus Musculus] 289 1 96 151 438 g7576272 2.00E-21 bA393J16.1 (zinc finger protein 33a (KOX 31)) [Homo sapiens] 290 1 149 118 564 g7959207 1.00E-26 KIAA1473 protein [Homo sapiens] 290 1 149 118 564 g498736 3.00E-26 zinc finger protein [Homo sapiens] 290 1 149 118 564 g4454678 4.00E-23 zinc finger protein 4 [Homo sapiens] 291 2 134 152 553 g498152 1.00e-06 ha0946 protein is Kruppel-related.

   [Homo sapiens] 291 2 134 152 553 g10440081 1.00E-06 unnamed protein product [Homo sapiens] 291 2 134 152 553 g7576272 1.00E-06 bA393J16.1 (zinc finger protein 33a (KOX 31)) [Homo sapiens] 292 2 212 2 637 g7656698 1.00E-133 Zinc finger protein 222 [Homo sapiens] 292 2 212 2 637 g6118381 1.00E-133 zinc finger protein ZNF222 [Homo sapiens] 292 2 212 2 637 g6118383 1.00E-122 zinc finger protein ZNF223 [Homo sapiens]   

 <Desc/Clms Page number 277> 

   293 2 108 2 325 g4567179 2.00E-33 BC37295¯1 [Homo sapiens] 293 2 108 2 325 g10434142 9.00E-31 unnamed protein product [Homo sapiens] 293 2 108 2 325 g51817149 9.00E-31 hypothetical protein [Homo sapiens] 294 1 83 97 345 g930123 9.00E-24 Zinc inger protein (583 AA)

   [Homo sapiens] 294 1 83 97 345 g487785 8.00E-23 zinc finger protein ZNR136 [Homo sapiens] 294 1 83 97 345 g5262560 1.00E-22 hypothetical protein [Homo sapiens] 295 1 180 1 540 g498719 2.00E-83 zinc finger protein [Homo sapiens] 295 1 180 1 540 g3953593 3.00E-69 Zinc finger protein s11-6 [Mus musculus] 295 1 180 1 540 g6467206 4.00E-68 gonadotropin inducible transcription repressor-4 [Homo sapiens] 296 3 97 57 347 g9801232 3.00E-28 bA508N22.2 (zinc finger protein 37a (KOX 21))

   [Homo sapiens] 296 3 97 57 347 g829151 3.00E-28 ZNF37A [Homo sapiens] 296 3 97 57 347 g881564 4.00E-20 ZNF157 [Homo sapiens] 297 1 217 421 1071 g6331377 1.00E-131 KIAA1285 protein [Homo sapiens] 297 1 217 421 1071 g1020145 6.00E-53 DNA binding protein [Homo sapiens] 297 1 217 421 1071 g2224593 1.00E-52 KIAA0326 [Homo sapiens] 298 3 137 15 425 g4456989 4.00E-20 protease [Homo sapiens] 298 3 137 15 425 g9558703 4.00E-20 protease [Homo sapiens] 298 3 137 15 425 g1780976 4.00E-20 protease [Human endogenous retrovirus K] 299 2 169 59 565 g10434856 2.00E-40 unnamed protein product [Homo sapiens] 299 2 169 59 565 g5262560 2.00E-40 hypothtical protein [Homo sapiens] 299 2 169 59 565 g930123 1.00E-31 zinc finger protein (583 AA)

   [Homo sapiens] 300 3 135 3 407 g10434856 3.00E-35 unnamed protein product [Homo sapiens] 300 3 135 3 407 g5262560 3.00E-35 hypothetical protein [Homo sapiens] 300 3 135 3 407 g10434195 2.00E-27 unnamed protein product [Homo sapiens] 301 1 170 22 531 g10047297 2.00E-23 KIAA1611 protein [Homo sapiens] 301 1 170 22 531 g7023216 2.00E-22 unnamd protein product [Homo sapiens] 301 1 170 22 531 g347906 5.00E-16 zinc finger protein [Homo sapiens] 302 3 181 3 545 g5931821 8.00E-79 dJ228H13.3 (zinc fingerprotein)

   [Homo sapiens] 302 3 181 3 545 g6807587 8.00E-79 hypothetical protein [Homo sapeins] 302 3 181 3 545 g488555 2.00E-63 zinc finger protein ZNF135 [Homo sapeins] 303 1 263 1 789 g506502 1.00E-141 NK10 [Mus musculus] 303 1 263 1 789 g488555 1.00E-92 zinc finger protein ZNF135 [Homo sapiens] 303 1 263 1 789 g8453103 7.00E-88 zinc finger protein [Homo sapiens] 304 3 340 18 1037 g7023216 1.00E-142 unnamed protein product [Homo sapiens] 304 3 340 18 1037 g7023703 2.00E-89 unnamed protein product [Homo sapiens] 304 3 340 18 1037 g10436789 7.00E-54 unnamed protein product [Homo sapeins]   

 <Desc/Clms Page number 278> 

   305 1 89 103 369 g7023216 2.00E-18 unnamed protein product [Homo sapiens] 305 1 89 103 369 g9802037 4.00E-16 zinc finger protein sBZF3 [Homo sapiens] 305 1 89 103 369 g7239109 7.00E-15 HSPC059 [Homo sapiens] 306 1 80 1 240 g7959865 9.00E-20 PRO2032 

  [Homo sapines] 306 1 80 1 240 g8099520 6.00E-11 muscleblind [Mus musculus] 306 1 80 1 240 98515711 2.00E-10 Exp35 [Homo sapiens] 307 2 385 176 1333 g3869259 0 ZNF202 beta [Homo sapiens] 307 2 386 176 1333 g7328045 0 hypothetical protein [Homo sapiens] putative kruppel-related zinc finger protein NY-REN-23 antigen [Homo 307 2 386 176 1333 G59360097 1.00E-123 sapiens] 308 2 368 71 1174 g3882241 0 KIAA0760 protein [Homo sapiens] 308 2 368 71 1174 g6760445 0 Smad- and Olf-interacting zinc finger protein [Homo sapiens] 308 2 368 71 1174 g2149792 0 Roaz [Rattus norvegicus] 309 2 175 191 715 g487787 8.00E-15 zine finger protein ZNF14 [Homo sapiens] 309 2 175 191 715 g10047183 9.00E-31 KIAA1559 protein [Homo sapiens] 309 2 175 191 715 g4567179 2.00e-29 BC37295¯1 [Homo sapiens] 310 2 78 521 754 311 1 61 394 576 312 1 73 172 390 g2587027 4.00E-13 HERV-E envelope glycoprotein [Homo sapiens] 

  312 1 73 172 390 g2587024 4.00E-13 HERV-E envelope glycoprotein [Homo sapiens] 312 1 73 172 390 g1049232 2.00E-10 HERV-E envelope protein [Human endogenous retrovirus] 313 1 184 304 855 g8132311 2.00E-74 inwardly-rectifying potassium channel Kir5.1 [Homo sapiens] 313 1 184 304 855 g8132295 2.00E-74 inwardly-rectifying potassium channel Kir5.1 [Homo sapiens] 313 1 184 304 855 g8132293 2.00E-74 inwardly-rectifying potassium channel kir5.1 [Homo sapiens] 314 2 219 164 820 g7105926 2.00E-22 calcium channel alpha2-delta3 subunit [Homo sapiens] 314 2 219 164 820 g4186073 2.00E-22 calcium channel alpha2-delta-C subunit [Mus musculus] 314 2 219 164 820 g9929977 2.00e-22 hypothetical protein [Macaca fascicularis] 315 1 1603 1 4809 g184039 0 sodium channel alpha subunit [Homo sapiens] 315 1 1603 1 4809 g6782382 0 voltage-gated sodium channel [Mus musculus] 315 1 1603 1 4809 g206858 0 sodium 

  channel alpha-subunit [Rattus norvegicus] gamma-aminobutyric acid transporter type 3, GABA transporter type 3, GAT-3 316 3 200 240 839 g913242 5.00E-71 [human, fetal brain, Petide, 632 aa] [Homo sapiens] 316 3 200 240 839 g204220 2.00E-69 beta-alanine-sensitive neuronal GABA transporter [Rattus norvegicus] 316 3 200 240 839 g202535 2.00E-69 GABA transporter [Rattus norvegicus] 317 3 329 3 989 g6996442 4.00E-61 CTL1 protein [Homo sapiens] 317 3 329 3 989 g6996589 1.00E-59 CTL1 protein [Rattus norvegicus]   

 <Desc/Clms Page number 279> 

   317 3 329 3 989 g6996587 2.00E-51 CTL1 protein [Torpedo marmorata] ESTs AU058081(E30812),AU058365(E50679), AU030138(E50679) correspond to a region of the predicted gene.;

   Similar to Spinacia oleracea 318 3 256 3 770 g5091520 1.00e-134 mRNA for proteasome 37kD subunit.(X96974) [Oryza sativa] ESTs AU058081(E3082),AU075427(E30384) correspond to a region of the predicted gene.#Similar to Spinacia oleracea proteasome 27 kD subunit 318 3 256 3 770 g8096329 1.00E-134 (P52427) [Oryza Sativa] ESTs AU058081(E3082),AU075427(E30384) correspond to a region of the predicted gene.#Similar to Spinacia oleracea proteasome 27 kD subunit 318 3 256 3 770 98096319 1.00E-134 (P52427) [Oryza sativa] 319 2 76 2 229 g951425 2.00E-07 housekeeping protein [Rattus norvegicus] 319 2 76 2 229 g5759144 2.00E-07 cyclophilin A [Mus musculus] 319 2 76 2 229 g50621 2.00e-07 Cyclophilin (AA 1 - 164)

   [Mus musculus] 320 3 276 354 1181 g7019854 1.00E-84 unnamed protein product [Homo sapiens] 320 3 276 354 1181 g6567172 7.00E-84 mDj10 [Mus musculus] 320 3 276 354 1181 g10436329 5.00E-81 unnamed protein product [Homo sapiens] 321 1 115 328 672 g1049232 3.00E-24 HERV-E envelope protein [Human endogenous retrovirus] 321 1 115 328 672 g2587024 2.00E-23 HERV-E envelope glycoprotein [Homo sapiens] 321 1 115 328 672 g2587027 2.00E-23 HERV-E envelope glycoprotein [Homo sapiens] 322 3 227 3 683 g2286123 6.00E-33 testis specific DNAj-homolog [Mus musculus] 322 3 227 3 683 g6681592 1.00E-32 DnaJ homolog [Homo sapiens] 322 3 227 3 683 g6648623 1.00E-32 DNAi homolog [Homo sapiens] 323 3 100 153 452 324 3 142 840 1265 g2943716 5.00E-81 25 kDa trypsin inhibitor [Homo sapiens] 324 3 142 840 1265 g9885193 5.00E-54 dJ881L22.3 (novel protein similar to a trypsin inhibitor)

   [Homo sapiens] 324 3 142 840 1265 g432682 2.00E-52 late gestation lung protein 1 [Rattus noregicus] 325 3 263 3 791 96957716 1.00E-135 putative chaperonin [Arabidopsis thaliana] 325 3 263 3 791 g9755653 1.00E-132 TCP-1 chaperonin-like protein [Arabidopsis thaliana] 325 3 263 3 791 95295933 2.00E-93 chaperonin containing TCP-1 zeta-1 subunit [Mus musculus] 326 2 357 23 1093 g3882167 1.00E-171 KIAA0723 protein [Homo sapiens] similar to Homo sapiens mRNA for KIAA0723 protein with GenBank 326 2 357 23 1093 g9956070 1.00E-171 Accession Number AB018266.1 # 326 2 357 23 1093 g6563246 1.00E-170 matrin 3 [Homo sapiens] 327 2 100 656 955 328 2 303 2 910 98980660 1.00E-158 proliferation-associated SNF2-like protein [Homo sapiens] 328 2 303 2 910 g805296 1.00E-149 lymphocyte specific helicase [Mus musculus]   

 <Desc/Clms Page number 280> 

   similar to Mus musculus 

  lymphocyte specfic helicase mRNA with GenBank 328 2 303 2 910 g9956001 8.00E-86 Accession Number U25691.1 [Homo sapiens] 329 2 72 167 382 330 2 76 80 307 ORF derived from D1 leader region and integrase coding region [Homo 331 2 74 446 667 g2104910 1.00E-29 sapiens] 331 2 74 446 667 g2104914 5.00E-21 ORF derived from protease and integrase coding regions [Homo sapiens] 331 2 74 446 667 g4959374 5.00E-21 pol protein [Homo sapeins] 332 3 67 57 257 333 2 192 302 877 98980660 8.00E-92 proliferation-Associated SNF2-like protein [Homo sapiens] similar to mus musculus lymphocyte specific helicase mRNA with GenBank 333 2 192 302 877 g9956001 8.00E-92 Accession Number U25691.1 [Homo sapiens] 333 2 192 302 877 g7022306 1.00E-89 unnamed protein product [Homo sapiens] ORF derived from D1 leader egion and integrase coding region [Homo 334 2 74 446 667 g2104910 1.00E-30 sapiens] 334 2 74 446 667 

  g2104914 5.00E-21 ORF derived from protease and integase coding regions [Homo sapiens] 334 2 74 446 667 g4959374 5.00e-21 pol protein [Homo sapiens] 335 2 72 167 382 orf; encodes putative chimeric protein with SET domain in N-terminus with similarity to several other human, Drosopila, nematode and yeast proteins 336 2 55 557 721 g2231380 8.00E-12 [Homo sapiens] 336 2 55 557 721 g3005702 8.00E-12 unknown [Homo sapiens] 336 2 55 557 721 g1263081 1.00E-11 mariner transposase [Homo sapiens] 337 3 107 1614 1934 338 3 147 63 503 g10047265 7.00E-81 KIAA1595 protein [Homo sapiens] 338 3 147 63 503 g10176757 3.00E-26 ATP-dependent RNA helicase-like protein [Arabidopsis thaliana] 338 3 147 63 503 g3776011 3.00E-26 RNA helicase [Arabidopsis thaliana] 339 1 257 199 969 g10434055 1.00E-128 unnamed protein product [Homo sapiens] 339 1 257 199 969 g7243213 1.00E-126 KIAA1416 protein [Homo

  sapiens] dJ620E11.1 (novel Helicase C-terminal domain and SNF2 N-terminal domains 339 1 257 199 969 g11345539 1.00E-120 containing protein, similar to KIAA0308) [Homo sapiens] 340 3 63 3 191 341 1 112 1639 1974 342 3 427 2097 3377 g2599502 0 protocadherin 68 [Homo sapiens] 342 3 427 2097 3377 g7243181 4.00E-49 KIAA1400 protein [Homo sapiens] 342 3 427 2097 3377 g4099551 5.00E-48 OL-protocadherin [Mus musculus]   

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   343 2 144 635 1066 g10436424 1.00E-10 unnamed protein product [Homo sapiens] 344 2 97 557 847 345 3 75 675 899 g2587027 4.00E-13 HERV-E envelope glycoprotein [Homo sapiens] 345 3 75 675 899 g2587024 4.00E-13 HERV-E envelope glycoprotein [Homo sapiens] 345 3 75 675 899 g1049232 2.00E-10 HERV-E envelope protein [Human endogenous retrovirus] 346 3 135 399 803 g9368839 2.00E-71 hypothetical protein [Homo sapiens] 346 3 135 

  399 803 g2739452 6.00E-58 ribosomal protein L23A [Homo sapiens] 346 3 135 399 803 g1399086 6.00E-58 ribosomal protein L23a [Homo sapiens] 347 2 55 179 343 348 2 129 425 811 gt11493463 2.00E-22 PRO2852 [Homo sapiens] 348 2 129 425 811 g9280152 5.00E-22 unnamed portein product [Macaca fasicularis] 348 2 129 425 811 g10437485 5.00E-21 unnamed protein product [Homo sapiens] 349 2 291 122 994 g673417 1.00E-152 class II antigen [Homo sapiens] 349 2 291 122 994 9703089 1.00E-152 MHC class II DP3-alpha [Homo sapiens] 349 2 291 122 994 g758100 1.00E-137 SB classll histocompatibility antigen alpha- chain [Homo sapiens] cytochrome P450 2B-Bx=phenobarbital-inducible [rabbits, kidney, Peptide,

   491 350 1 517 1 1551 g402843 1.00E-144 aa] [Oryctolagus cuniculus] 350 1 517 1 1551 g404777 1.00E-144 cytochrome P-450 2B-Bx [Oryctolagus cuniculus] 350 1 517 1 1551 g164959 1.00E-142 cytochrome P-450 [Oryctolagus cuniculus] 351 1 232 1300 1995 dJ857M17.2 (novel protein similar to cytochrome c oxidase subunit IV 352 1 220 67 726 g11863734 2.00E-80 (COX4)) [Homo sapiens] 352 1 220 67 726 g8809758 9.00E-42 cytochrome c oxidase subunit IV isoform 2 precursor [Thunnus obesus] 352 1 220 67 726 g2809498 3.00E-41 cytochrome c oxidase subunit IV [Gorilla gorilla] 353 1 95 1 285 354 2 331 2 994 g11229985 1.00E-176 unnamed protein product [Homo sapiens] 354 2 331 2 994 g11229992 6.00E-57 unnamed protein product [Mus musculus] 354 2 331 2 994 g30095 6.00E-49 collagen subunit (alpha-1 (X))

   3 [Homo sapiens] 355 3 93 54 332 g11177164 4.00E-12 polydom protein [Mus musculus] 355 3 93 54 332 g391669 4.00E0-07 Hikaru genki type4 product precursor [Dorsophila melangogaster] 355 3 93 54 332 g391667 4.00E-07 hikaru genki type3 product precursor [Drosophila melanogaster] 356 1 112 1 336 357 3 73 192 410 358 1 239 181 897 g4582324 1.00E-129 dJ708F5.1 (PUTATIVE novel Collagen alpha 1 LIKE protein) [Homo sapiens] 358 1 239 181 897 g1732121 4.00E-36 cartilage matrix protein [Homo sapiens]   

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   358 1 239 181 897 g180654 2.00E-35 cartilage matrix protein [Homo sapiens] 359 1 528 4 1587 g1903218 0 type I intermediate filament of hair keratin [Homo sapiens] 359 1 528 4 1587 g7161771 0 keratin [Homo sapiens] 359 1 528 4 1587 g4103156 0 hair keratin basic 5;

   keratin Hb5 [Mus musculus] 360 2 157 161 631 g11034725 2.00E-64 hNBL4 [Homo sapiens] 360 2 157 161 631 g466548 3.00E-63 NBL4 [Mus musculus] 360 2 157 161 631 g2822458 5.00E-54 band 4.1-like protein 4 [Danio rerio] 361 3 65 54 248 g3724141 6.00E-08 myosin I [Rattus norvegicus] 361 3 65 54 248 g3882175 6.00E-08 KIAA0727 protein [Homo sapiens] dJ111C20.1 (similar to Chlamydomonas radial spoke protein 3)

   [Homo 362 3 517 3 1553 g6855339 1.00E-120 sapiens] 362 3 517 3 1553 g18218 1.00E-75 spoke protein [Chlamydomonas reinhardtii] 362 3 517 3 1553 g7295323 9.00E-47 CG10099 gene product [Drosophila melanogaster] 363 2 60 314 493 364 1 239 127 843 g1813638 9.00E-53 PF20 [Chlamydomonas reinhardtii] 364 1 239 127 843 g3983133 2.00E-47 pf20 homolog [Trypanosoma brucei] 364 1 239 127 843 g607003 1.00E-37 beta transducin-like protein [Podospora anserina] 365 1 160 1 480 366 3 757 3 2273 g8896164 0 kinesin-like protein GAKIN [Homo sapiens] 366 3 757 3 2273 g10697238 0 KIF13A [Mus musculus] 366 3 757 3 2273 g117616134 0 kinesin-like protein RBKIN2 [Homo sapiens] 367 3 162 3 488 g11231085 1.00E-56 hypothetical protein [Macaca fascicularis] 367 3 162 3 488 g7385113 2.00E-18 ankyrin 1 [Bos taurus] 367 3 162 3 488 g747710 2.00E-18 alt.

   ankyrin (variant 2.2) [Homo sapiens] 368 2 635 308 2212 g1353782 0 dystrophin-related protein 2 [Homo sapiens] 368 2 635 308 2212 g11066165 0 dystrophin-related protein 2 A-form splice variant [Rattus norevgicus] 368 2 635 308 2212 g11066167 0 dystrophin-related protein 2 B-form splice variant [Rattus norvegicus] 369 3 433 999 2297 g190752 0 pemphigus vulgaris antigen [Homo sapiens] 369 3 433 999 2297 g2290200 1.00E-176 desmoglein 3 [Mus musculus] 369 3 433 999 2297 g416178 2.00E-58 desmoglein 2 [Homo sapiens] 370 3 531 3 1595 g28969 7.00E-71 64 Kd autoantigen [Homo sapiens] 370 3 531 3 1595 g6934240 8.00E-61 tropomodulin 2 [Homo sapiens] 370 3 531 3 1595 g7288857 3.00E-60 neural tropomodulin N-Tmod [Mus musculus] The KIAA0143 gene product is related to a putative C.elegans gene encoded 371 2 257 383 1153 g1469868 1.00E-124 on cosmid C32D5.

   [Homo sapiens] 371 2 257 383 1153 g4589550 4.00E-82 KIAA0953 protein [Homo sapiens]   

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   371 2 257 383 1153 g7304005 1.00E-55 cmp44E gene product [alt 1] [Drosophila melanogaster] 372 1 242 139 864 g387514 1.00E-123 DM-20 protein [Mus musculus] 372 1 242 139 864 g190088 1.00E-123 DM-20 [Homo sapiens] 372 1 242 139 864 g200409 1.00E-122 proteolipid protein variant Dm-20 [Mus musculus] 373 2 60 380 559 374 1 157 22 492 g7268562 1.00E-59 ribosomal protein L32-like protein [Arabidopsis thaliana] 374 1 157 22 492 g5816996 1.00E-59 ribosomal protein L32-like protein [Arabidopsis thaliana] 374 1 157 22 492 g10177580 7.00E-59 ribosomal protein L32 [Arabidopsis thaliana] 375 3 158 3 476 g643074 4.00E-76 putative 40S ribosomal protein s12 [Fragaria x ananassa] 375 3 158 3 476 g6716785 1.00E-75 40s ribosomal protein S23 [Euphorbia esula] 375 

  3 158 3 476 g7413571 6.00E-75 putative protein [Arabidopsis thaliana] 376 2 238 14 727 g10799832 1.00E-93 ribosomal protein L11-like [Nicotiana tabacum] 376 2 238 14 727 g7630065 4.00E-93 ribosomal protein L11-like [Arabidopsis thaliana] 376 2 238 14 727 g11908058 4.00E-93 ribosomal protein L11, cytosolic [Arabidopsis thaliana] 377 3 102 3 308 g57131 7.00E-41 ribosomal protein S26 [Rattus norvegicus] 377 3 102 3 308 g296452 7.00E-41 ribosomal protein S26 [Homo sapiens] 377 3 102 3 308 g3335024 7.00E-41 ribosomal protein S26 [Homo sapiens] dJ475N16.3 (novel protein similar to RPL7A (60S ribosomal protein L7A))

   378 1 102 316 621 g6969165 6.00E-53 [Homo sapiens] 378 1 102 316 621 g6687301 2.00E-21 60S ribosomal protein L7 [Cyanophora paradoxa] 378 1 102 316 621 g200785 1.00E-20 ribosomal protein L7 [Mus musculus] 379 3 177 3 533 g206736 1.00E-82 ribosomal protein L7 [Rattus norvegicus] 379 3 177 3 533 g200785 2.00E-80 ribosomal protein L7 [Mus musculus] 379 3 177 3 533 g554269 2.00E-80 ribosomal protein L7 [Mus musculus] 380 2 86 257 514 g550025 2.00E-31 ribosomal protein S10 [Homo sapiens] 380 2 86 257 514 g57127 3.00E-30 ribosomal protein S10 (AA 1-165) [Rattus norvegicus] 380 2 86 257 514 g9581772 3.00E-29 bA371L19.2 (similar to ribosomal protein S10)

   [Homo sapiens] 381 1 97 286 576 g36140 2.00E-31 ribosomal protein L7 [Homo sapiens] 381 1 97 286 576 g307388 2.00E-31 ribosomal protein L7 [Homo sapiens] 381 1 97 286 576 g1335288 2.00E-31 ribosomal protein L7 [Homo sapiens] 382 1 82 70 315 g409074 2.00E-19 HBp15/L22 [Sus scrofa] 382 1 82 70 315 g409072 2.00E-19 HBp15/L22 [Mus musculus] 382 1 82 70 315 g409070 2.00E-19 HBp15/L22 [Homo sapiens] 383 1 180 46 585 g4886269 2.00E-75 putative ribosomal protein S14 [Arabidopsis thaliana] 383 1 180 46 585 96006890 6.00E-75 putative 40S ribosomal protein s14;

   67401-66292 [Arabidopsis thaliana] 383 1 180 46 585 g4678226 3.00E-74 40S ribosomal protein S14 [Arabidopsis thaliana]   

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   384 3 118 21 374 g643074 2.00E-49 putative 40S ribosomal protein s12 [Fragaria x ananassa] 384 3 118 21 374 g6716785 6.00E-49 40s ribosomal protein S23 [Euphoriba esula] 384 3 118 21 374 g7413571 3.00E-48 putative protein [Arabidopsis thaliana] 385 2 164 2 483 g643074 4.00E-76 putative 40S ribosomal protein s12 [Fragaria x ananassa] 385 2 164 2 493 g6716785 1.00E-75 40s ribosomal protein S23 [Euphoriba esula] 385 2 164 2 493 g7413571 6.00E-75 putative protein [Arabidopsis thaliana] 386 3 101 3 305 g36130 1.00E-22 ribosomal protein L31 (AA 1-125) [Homo sapiens] 386 3 101 3 305 g1655596 1.00E-22 ribosomal protein L31 [Homo sapiens] 386 3 101 3 305 g57115 1.00E-22 ribosomal protein L31 (AA 1-125)

   [Rattus norvegicus] 387 3 259 3 779 g2331301 1.00E-122 ribosomal protein S4 type 1 [Zea mays] 387 3 259 3 779 g2345154 1.00E-120 ribsomal protein S4 [Zea mays] 387 3 259 3 779 g746687 1.00E-116 ribosomal protein S4 [Arabidopsis thaliana] 388 2 184 2 553 g2668748 1.00E-95 ribosomal protein L17 [Zea mays] 388 2 184 2 553 g19104 8.00E-85 ribosomal protein L17-2 [Hordeum vulgare] 388 2 184 2 553 g19102 1.00E-82 ribosomal protein L17-1 [Hordeum vulgare] 389 2 152 2 457 g338447 5.00E-28 RPS16 [Homo sapiens] 389 2 152 2 457 g57714 5.00E-28 ribosomal protein S16 (AA 1-146)

   [Rattus rattus] 389 2 152 2 457 g200796 2.00E-27 16S ribosomal protein [Mus musculus] 390 3 158 3 476 g643074 4.00E-76 putative 40S ribosomal protein s12 [Fragria x ananassa] 390 3 158 3 476 g6716785 1.00E-75 40s ribosomal protein S23 [Europhorbia esula] 390 3 158 3 476 g7413571 6.00E-75 putative protein [Arabidopsis thaliana] 391 1 94 34 315 392 3 83 303 551 g57121 3.00E-18 ribosomal protein L37 [Rattus norvegicus] 392 3 83 303 551 g292441 3.00E-18 ribosomal protein L37 [Homo sapiens] ribosomal protein L37 {C2-C2 zinc-finger-like} [human, HeLa cells, Peptide,

   97 392 3 83 303 551 g1839334 3.00E-18 aa] [Homo sapiens] 393 2 174 2 523 g10433651 3.00E-80 unnamed protein product [Homo sapiens] 393 2 174 2 523 g10434617 3.00E-80 unnamed protein product [Homo sapiens] 393 2 174 2 523 g545998 6.00E-79 tricarboxylate carrier [Rattus sp.] 394 3 183 3 551 395 1 399 1 1197 g11907599 0 protein kinase HIPK2 [Homo sapiens] 395 1 399 1 1197 g5815141 0 nuclear body associated kinase 1b [Mus musculus] 395 1 399 1 1197 g5815139 0 nuclear body associated kinase 1a [Mus musculus] 396 1 301 109 1011 g7688667 1.00E-161 PC326 protein [Homo sapiens] 396 1 301 109 1011 g2734854 1.00E-08 Mus musculus Dentin Matrix Protein 1 [] 396 1 301 109 1011 g6137020 1.00E-08 dentin matrix protein-1 [Mus musculus]   

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   397 2 105 2 316 g178281 1.00E-47 AHNAK nucleoprotein [Homo sapiens] 397 2 105 2 316 g50675 2.00E-47 

  desmoyokin [Mus musculus] 397 2 105 2 316 g897824 5.00E-47 AHNAK gene product [Homo sapiens] 398 1 153 202 660 g183233 1.00E-34 beta-glucuronidase precursor (EC 3.2.1.31) [Homo sapiens] 398 1 153 202 660 g3549609 2.00E-33 beta-glucuronidase [Cercopithecus aethiops] 398 1 53 202 660 g4102553 3.00E-29 mutant beta-glucuronidase [Fells catus] 399 1 161 106 588 g7022046 1.00E-36 unnamed protein product [Homo sapiens] 399 1 161 106 588 g7670456 5.00E-34 unnamed protein product [Mus musculus] 399 1 161 106 588 g8671586 1.00E-29 ataxin 2-binding protein [Homo sapiens] 400 1 153 205 663 g183233 1.00E-34 beta-glucuronidase precursor (EC 3.2.1.31)

   [Homo sapiens] 400 1 153 205 663 g3549609 2.00E-33 beta-glucuronidase [Cercopithecus aethiops] 400 1 153 205 663 g4102553 3.00E-29 mutant beta-glucuronidase [Felis catus] 401 3 135 651 1055 g414797 9.00E-58 pyruvate dehydrogenase phosphatase [Bos taurus] 401 3 135 651 1055 g3298607 3.00E-56 pyruvate dehydrogenase phosphatase isoenzyme 1 [Rattus norvegicus] 401 3 135 651 1055 g7688679 3.00E-53 pyruvate dehydrogenase [Homo sapiens] 402 3 129 30 416 403 1 299 1 897 g440878 1.00E-149 onconeural ventral antigen-1 [Homo sapiens] 403 1 299 1 897 g7025507 1.00E-137 ventral neuron-specific protein 1 NOVA1 [Mus musculus] 403 1 299 1 897 g2673961 9.00E-99 astrocytic NOVA-like RNA-binding protein [Homo sapiens] 404 1 142 1 426 g4105111 1.00E-43 dehydrin 6 [Hordeum vulgare] 404 1 142 1 426 g6017938 4.00E-43 dehydrin;

   DHN6 [Hordeum vulgare] 404 1 142 1 426 g5738195 1.00E-28 abscisic acid response protein [Prunus dulcis] 405 2 168 2 505 g453189 9.00E-59 acyl carrier protein [Zea mays] 405 2 168 2 505 g166971 4.00E-49 acyl carrier protein III [Hordeum vulgare] 405 2 168 2 505 g166969 6.00E-41 acyl carrier protein II [Hordeum vulgare] 406 2 117 2 352 g203923 1.00E-40 diazepam binding inhibitor [Rattus norvegicus] 406 2 117 2 352 g1228089 1.00E-40 multifunctional acyl-CoA-binding protein [Rattus norvegicus] 406 2 117 2 352 g203925 1.00E-40 diazepam binding inhibitor [Rattus norvegicus] 407 3 804 3 2414 g10953883 0 ubiquitin E3 ligase SMURF2 [Homo sapiens] 407 3 804 3 2414 g10047327 0 KIAA1625 protein [Homo sapiens] 407 3 804 3 2414 g6446606 0 E3 ubiquitin ligase SMURF1 [Homo sapiens] 408 1 220 244 903 g9622856 9.00E-24 sorting nexin 15A [Homo sapiens] 408 1 220 244 903 g2529709 1.00E-23 unknown 

  [Homo sapiens] 408 1 20 244 903 g9622854 1.00E-23 sorting nexin 15 [Homo sapiens] dJ20B11.1 (ortholog of rat RSEC5 (mammalian exocyst complex subunit)) 409 2 168 80 583 g5823961 2.00E-87 [Homo sapiens]   

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   409 2 168 80 583 g2827158 2.00E-84 resc5 [Rattus norvegicus] 409 2 168 80 583 g7295804 8.00E-29 CG8843 gene product [Drosophila melanogaster] 410 2 108 194 517 g9963839 1.00E-50 lipase [Homo sapiens] 411 1 314 277 1218 g3243240 4.00E-56 syntaxin 11 [Homo sapiens] 411 1 314 277 1218 g4104685 1.00E-53 syntaxin 11 [Homo sapiens] 411 1 314 277 1218 g3248918 8.00E-46 syntaxin 11 [Homo sapiens] 412 2 143 212 640 g4512103 3.00E-57 rab11 binding protein [Bos taurus] 412 2 143 212 640 g6049150 8.00E-43 WD-containing protein [Rattus norveglcus] 413 1 122 1 366 414 2 86 623 880 415 3 213 183 821 416 1 263 40 828 g9558701 3.00E-31 gag [Homo 

  sapiens] 416 1 263 40 828 g5802824 3.00E-31 Gag-Pro-Pol protein [Homo sapiens] 416 1 263 40 828 g5802821 3.00E-31 Gag-Pro-Pol protein [Homo sapiens] 417 1 175 940 1464 g246483 1.00E-63 prohibitin [human, Peptide, 272 aa] [Homo sapiens] 417 1 175 940 1464 g206384 2.00E-63 prohibitin [Rattus norvegicus] 417 1 175 940 1464 g541732 2.00E-63 prohibitin or B-cell receptor associarted protein (BAP)

   32 [Mus musculus] 418 2 272 167 982 g505033 6.00E-75 mitogen inducible gene mig-2 [Homo sapiens] 418 2 272 167 982 g10727293 5.00E-33 CG14991 gene product [alt 2] [Drosophila melanogaster] 418 2 272 167 982 g7292434 5.00E-33 CG14991 gene product [alt 1] [Drosophila melanogaster] 419 1 167 16 516 g2587027 3.00E-34 HERV-E envelope glycoprotein [Homo sapiens] 419 1 167 16 516 g2587024 3.00E-34 HERV-E envelope glycoprotein [Homo sapiens] 419 1 167 16 516 g1049232 2.00E-31 HeRV-E envelope protein [Human endogenous retrovirus] 420 2 59 227 403 421 1 216 1 648 g10504238 1.00E-101 hepatocellular carcinoma-related putative tumor suppressor [Homo sapiens] 421 1 216 1 648 g7020759 7.00E-75 unnamed protein product [Homo sapiens] contains similarity to Pfam domain;

   PF01585 (G-patch domain), Score=67.0, E 421 1 216 1 648 g3880143 1.00E-28 value-1.3e-16, N=1 [Caenorhabditis elegans] 422 1 162 1 486 g4982485 7.00E-55 apoptosis related protein APR-3 [Homo sapiens] 422 1 162 1 486 g4689122 3.00E-49 HSPC013 [Homo sapiens]   

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   Table 7 Program Description Reference Parameter Threshold ABI FACTURA A program that remove vector sequences and Applied Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences. 



  ABI/PARACEL FDF A Fast Data Finder useful in comparing and Applied Biosystems, Foster City, CA; Mismatch <50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA. 



  ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA. 



  BLAST A Basic Local Alignment Search Tool useful in Altschul, S.F. et al. (1990) J. Mol. Biol. ESTs: Probability value= 1.0E-8 sequence similarity search for amino acid and 215:403-410; Altschul, S.F. et al. (1997) or less nucleic acid sequences. BLAST includes five Nucleic Acids Res. 25:3389-3402. Full Lenght sequences: Probability functions: blastp, blastn, blastx, tblastn, and tblastx. value= 1.0E-10 or less FASTA A Pearson and Lipman algorithm that searches for Pearson, W.R. and D.J. Lipman (1988) Proc. ESTs: fasts E value=1.06E-6 similarity between a query sequence and a group of Natl, Acad Sci. USA 85:2444-2448; Pearson, Assembled ESTs: fasta Identity= sequences of the same type. FASTA comprises as W.R. (1990) Methods Enzymol. 183:63-98; 95% or greater and least five functions: fasta, tfasta, fastx, tfastx, and Smith, T.F. and M.S.

   Waterman (1981) Match length=200 bases or greater; ssearch. Adv. Appl. Math. 2:482-489. fastx E value=1.0E-8 or less Full Length sequences: fastx score=100 or greater BLIMPS A BLocks IMProved Searcher that matches a Henikoff, S. and J.G. Hnikoff (1991) Nucleic Probability value= 1.0E-3 or less sequence against those in BLOCKS, PRINTS, Acids Res. 19:6565-6572; Henikoff, J.G. and DOMO, PRODOM, and PFAM databases to search S. henikoff (1996) Methods Enzymol. for gene families, sequence homology, and structural 266:88-105; and Attwood, T.K. et al. (1997) J. fingerprint regions. Chem. Inf. Comput. Sci. 37:417-424. 



  HMMER An algorithm for searching a query sequence against Krogh, A. et al. (1994) J. Mol. Biol. PFAM hits: Probability value= hidden Markov model (HMM)-based databases of 235:1501-1531; Sonnhammer, E.L.L. et al. 1.0E-3 or less protein family consensus sequences, such as PFAM. (1988) Nucleic Acids Res. 26:320-322; Signal peptide hits: Score= 0 or Durbin, R. et al. (1998) Our World View, in a greater Nutshell, Cambridge Univ. Press, pp. 1-350.   

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    Table 7 (cont.) Program Description Reference Parameter Threshold ProfileScan An algorithm that searches for structural and sequence Gribskov, M. et al. (1988) CABIOS 4:61-66; Normalized quality score# GCGmotifs in protein sequences that match sequence patterns Gribskov, M. et al. (1989) Methods Enzymol. specified "HIGH" value for that defined in Prosite. 183:146-159; Bairoch, A. et al. (1997) particular Prosite motif. 



  Nucleic Acids Res. 25:217-221. Generally, score=1.4-2.1. 



  Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8:175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194. 



  Phrap A Phils Revised Assembly Program including SWAT and Smith, T.F. and M.S. Waterman (1981) Adv. Score= 120 or greater; CrossMatch, programs based on efficient implementation Appl. Math. 2:482-489; Smith, T.F. and M.S. Match length= 56 or greater of the Smith-Waterman algorithm, useful in searching Waterman (1981) J. Mol. Biol. 147:195-197; sequence homology and assembling DNA sequences. and Green, P., University of Washington, Seattle, WA. 



  Consede A graphical tool for viewing and editing Phrap assemblies. Gordon, D. et al. 91998) Genome Res. 8;195-202, SPScan A weight matrix analysis program that cans protein Nielson, H. et al. (1997) Protein Engineering Score=3.5 or greater sequences for the presence of secretory signal peptides. 10:1-6; Claverie, J.M. and S. Audic (1997) CABIOS 12:431-439. 



  TMAP A program that uses weight matrices to delineate Persson, B. and P. Argos (1994) J. Mol. Biol. transmembrane segments on protein sequences and 237:182-192; Persson, B. and P. Argos (1996) determine orientation. Protein Sci. 5:363-371. 



  TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182. 



  Motifs A program that searches amino acid sequences for patterns Bairoch, A. et al. (1997) Nucleic Acids Res. 25:217-221; that matched those defined in Prosite. Wisconsin Package Program Manual, version 9, page M51-59, Genetics Computer Group, Madison, WI.  

Claims

CLAIMS What is claimed is : 1. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of : a) a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d).

2. An isolated polynucleotide of claim 1, comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO : 1-211.

3. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 1.

4. A composition for the detection of expression of diagnostic and therapeutic polynucleotides comprising at least one of the polynucleotides of claim 1 and a detectable label.

5. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 1, the method comprising : a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.

6. A method for detecting a target polynucleotide in a sample, said target polynucleotide comprising a sequence of a polynucleotide of claim 1, the method comprising : a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and <Desc/Clms Page number 290> b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.

7. A method of claim 5, wherein the probe comprises at least 30 contiguous nucleotides.

8. A method of claim 5, wherein the probe comprises at least 60 contiguous nucleotides.

9. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 1.

10. A cell transformed with a recombinant polynucleotide of claim 9.

11. A transgenic organism comprising a recombinant polynucleotide of claim 9.

12. A method for producing a diagnostic and therapeutic polypeptide, the method comprising : a) culturing a cell under conditions suitable for expression of the diagnostic and therapeutic polypeptide, wherein said cell is transformed with a recombinant polynucleotide of claim 9, and b) recovering the diagnostic and therapeutic polypeptide so expressed.

13. A purified diagnostic and therapeutic polypeptide (DITHP) encoded by at least one of the polynucleotides of claim 2.

14. An isolated antibody which specifically binds to a diagnostic and therapeutic polypeptide of claim 13.

15. A method of identifying a test compound which specifically binds to the diagnostic and therapeutic polypeptide of claim 13, the method comprising the steps of : a) providing a test compound ; b) combining the diagnostic and therapeutic polypeptide with the test compound for a sufficient time and under suitable conditions for binding ; and c) detecting binding of the diagnostic and therapeutic polypeptide to the test compound, thereby identifying the test compound which specifically binds the diagnostic and therapeutic polypeptide. <Desc/Clms Page number 291>

16. A microarray wherein at least one element of the microarray is a polynucleotide of claim 3.

17. A method for generating a transcript image of a sample which contains polynucleotides, the method comprising the steps of : a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray of claim 16 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.

18. A method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence of claim 1, the method comprising : a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.

19. A method for assessing toxicity of a test compound, said method comprising : a) treating a biological sample containing nucleic acids with the test compound ; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 1 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 1 or fragment thereof ; c) quantifying the amount of hybridization complex ;

and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.

20. An array comprising different nucleotide molecules affixed in distinct physical locations on a solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target <Desc/Clms Page number 292> polynucleotide, said target polynucleotide having a sequence of claim 1.

21. An array of claim 20, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.

22. An array of claim 20, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide 23. An array of claim 20, which is a microarray.

24. An array of claim 20, further comprising said target polynucleotide hybridized to said first oligonucleotide or polynucleotide.

25. An array of claim 20, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.

26. An array of claim 20, wherein each distinct physical location on the substrate contains multiple nucleotide molecules having the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another physical location on the substrate.

27. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of : a) an amino acid sequence selected from the group consisting of SEQ ID NO : 212-422, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO : 212-422, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO : 212-422, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO : 212-422.