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WO2003016493A2 - Transporteurs et canaux ioniques - Google Patents

Transporteurs et canaux ioniques Download PDF

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Publication number
WO2003016493A2
WO2003016493A2 PCT/US2002/026323 US0226323W WO03016493A2 WO 2003016493 A2 WO2003016493 A2 WO 2003016493A2 US 0226323 W US0226323 W US 0226323W WO 03016493 A2 WO03016493 A2 WO 03016493A2
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polynucleotide
seq
polypeptide
amino acid
sequence
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PCT/US2002/026323
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English (en)
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WO2003016493A3 (fr
Inventor
Y. Tom Tang
Preeti G. Lal
Henry Yue
Mariah R. Baughn
Danniel B. Nguyen
Monique G. Yao
Barrie D. Bell
Mark L. Borowsky
Sally Lee
Brooke M. Emerling
Yuming Xu
Shanya D. Becha
Ann E. Gorvad
Yalda Azimzai
Huibin Yue
Vicki S. Elliott
Ernestine A. Lee
Junming Yang
Patricia M. Lehr-Mason
Jayalaxmi Ramkumar
Soo Yeun Lee
Mary Faris
Christopher Turner
Michael Furness
Jenny L. Buchbinder
Narinder K. Chawla
Joana X. Li
Ian J. Forsythe
Jennifer A. Griffin
Kimberly J. Gietzen
Anita Swarnakar
April J.A. Hafalia
Erika A. Lindquist
Xin Jiang
Alan A. Jackson
Amy D. Wilson
Pei Jin
Reena Khare
Joseph P. Marquis
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Incyte Genomics, Inc.
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Priority to AU2002332573A priority Critical patent/AU2002332573A1/en
Priority to CA002458625A priority patent/CA2458625A1/fr
Priority to JP2003521802A priority patent/JP2005503790A/ja
Publication of WO2003016493A2 publication Critical patent/WO2003016493A2/fr
Publication of WO2003016493A3 publication Critical patent/WO2003016493A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to novel nucleic acids, transporters and ion channels encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of transport, neurological, muscle, irnmunological and cell proliferative disorders.
  • the invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and transporters and ion channels.
  • Eukaryotic cells are surrounded and subdivided into functionally distinct organelles by hydrophobic lipid bilayer membranes which are highly impermeable to most polar molecules.
  • Cells and organeiles require transport proteins to import and export essential nutrients and metal ions including K + , NH , P ⁇ > SO 4 % sugars, and vitamins, as well as various metabolic waste products.
  • Transport proteins also play roles in antibiotic resistance, toxin secretion, ion balance, synaptic neurotiansmission, kidney function, intestinal absorption, tumor growth, and other diverse cell functions (Griffith, J. and C. Sansom (1998) The Transporter Facts Book, Academic Press, San Diego CA, pp. 3-29).
  • Transport can occur by a passive concentration-dependent mechanism, or can be linked to an energy source such as ATP hydrolysis or an ion gradient.
  • Proteins that function in transport include carrier proteins, which bind to a specific solute and undergo a conformational change that translocates the bound solute across the membrane, and channel proteins, which form hydrophilic pores that allow specific solutes to diffuse through the membrane down an electrochemical solute gradient.
  • Carrier proteins which transport a single solute from one side of the membrane to the other are called uniporters.
  • 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).
  • 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 system.
  • Sodium-coupled transporters include the mammalian glucose transporter (SGLT1), iodide transporter (NIS), and multivitarnin 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).
  • MFS major facilitator superfamily
  • MFS transporters are single polypeptide carriers that transport small solutes in response to ion gradients.
  • Members of the MFS are found in all classes of living organisms, and include transporters for sugars, oligosaccharides, phosphates, nitrates, nucleosides, monocarboxylates, and drugs.
  • MFS transporters found in eukaryotes all have a structure comprising 12 transmembrane segments (Pao, S.S. et al. (1998) Microbiol. Molec. Biol. Rev. 62:1-34).
  • the largest family of MFS transporters is the sugar transporter family, which includes the seven glucose transporters (GLUT1-GLUT7) found in humans that are required for the transport of glucose and other hexose sugars. These glucose transport proteins have unique tissue distributions and physiological functions.
  • GLUTl provides many cell types with their basal glucose requirements and transports glucose across epithelial and endothelial barrier tissues;
  • GLUT2 facilitates glucose uptake or efflux from the liver;
  • GLUT3 regulates glucose supply to neurons;
  • GLUT4 is responsible for insulin- regulated glucose disposal; and
  • GLUT5 regulates fructose uptake into skeletal muscle.
  • 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.
  • TM transmembrane
  • 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 ir bitor selectivities.
  • cardiac muscle and tumor cells have transporters that differ in their K ⁇ values for certain substrates, including stereoselectivity for L- over D-lactate, and in their sensitivity to inhibitors.
  • 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.
  • ATP-binding cassette (ABC) transporters are members of a superfamily of membrane proteins that transport substances ranging from small molecules such as ions, sugars, amino acids, peptides, and phospholipids, to lipopeptides, large proteins, and complex hydrophobic drugs.
  • ABC transporters 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 maybe encoded by a single gene, as is the case for the cystic fibrosis transmembrane regulator (CFTR), or by separate genes.
  • NBD nucleotide-binding domains
  • MSD membrane-spanning domains
  • each gene product When encoded by separate genes, each gene product contains a single NBD and MSD. These "half- molecules" form homo- andheterodimers, such as Tapl and Tap2, the endoplasmic reticulum-based major Hstocompatibility (MHC) peptide transport system.
  • MHC major Hstocompatibility
  • CFTR cystic fibrosis
  • ALDP adrenoleukodystrophy protein
  • Zellweger syndrome peroxisomal membrane protein-70, PMP70
  • hyperinsulinemic hypoglycemia sulfonylurea receptor, SUR.
  • MDR multidrug resistance
  • a number of metal ions such as iron, zinc, copper, cobalt, manganese, molybdenum, selenium, nickel, and chromium are important as cofactors for a number of enzymes.
  • copper is involved in hemoglobin synthesis, connective tissue metabolism, and bone development, by acting as a cofactor in oxidoreductases such as superoxide dismutase, ferroxidase (ceraloplasmin), and lysyl oxidase.
  • Copper and other metal ions must be provided in the diet, and are absorbed by transporters in the gastrointestinal tract. Plasma proteins transport the metal ions to the liver and other target organs, where specific transporters move the ions into cells and cellular organelles as needed. Imbalances in metal ion metabolism have been associated with a number of disease states (Danks, D.M. (1986) J. Med. Genet. 23:99-106).
  • Fatty acid transport protein 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, TN. et al. (1998) J. Biol. Chem. 273:27420-27429).
  • the lipocalin superfamily constitutes a phylogenetically conserved group of more than forty proteins that function as extracellular ligand-binding proteins which bind and transport small hydrophobic molecules. Members of this family function as carriers of retinoids, odorants, chromophores, pheromones, allergens, and sterols, and in a variety of processes including nutrient transport, cell growth regulation, immune response, and prostaglandin synthesis. A subset of these proteins maybe multifunctional, serving as either abiosynthetic enzyme or as a specific enzyme inhibitor. (Tanaka, T. et al. (1997) J. Biol. Chem. 272:15789-15795; and van't Hof, W. et al. (1997) J. Biol. Chem. 272:1837-1841.)
  • Hpocalin family display unusually low levels of overall sequence conservation. Pairwise sequence identity often falls below 20%. Sequence similarity between family members is limited to conserved cysteines which form disulfide bonds and three motifs which form a juxtaposed cluster that functions as a target cell recognition site.
  • the lipocalins share an eight stranded, anti- parallel beta-sheet which folds back on itself to form a continuously hydrogen-bonded beta-barrel.
  • the pocket formed by the barrel functions as an internal ligand binding site. Seven loops (LI to L7) form short beta-hairpins, except loop LI which is a large omega loop that forms a lid to partially close the internal ligand-binding site (Flower (1996) Biochem. J. 318:1-14).
  • Lipocalins are important transport molecules. Each Hpocalin associates with a particular ligand and delivers that ligand to appropriate target sites within the organism.
  • Retinol-binding protein (RBP), one of the best characterized Hpocalins, transports retinol from stores within the liver to target tissues.
  • Apolipoprotein D (apo D), a component of high density lipoproteins (HDLs) and low density lipoproteins (LDLs), functions in the targeted collection and delivery of cholesterol throughout the body. Lipocalins are also involved in cell regulatory processes.
  • Apo D which is identical to gross- cystic-disease-fluid protein (GCDFP)-24, is a progesterone/pregnenolone-binding protein expressed at high levels in breast cyst fluid. Secretion of apo D in certain human breast cancer cell lines is accompanied by reduced cell proliferation and progression of cells to a more differentiated phenotype. Similarly, apo D and another Hpocalin, ⁇ r acid glycoprotein (AGP), are involved in nerve cell regeneration. AGP is also involved in anti-inflammatory and immunosuppressive activities. AGP is one of the positive acute-phase proteins (APP); circulating levels of AGP increase in response to stress and inflammatory stimulation.
  • APP positive acute-phase proteins
  • AGP accumulates at sites of inflammation where it inhibits platelet and neutiophil activation and inhibits phagocytosis.
  • the immunomodulatory properties of AGP are due to glycosylation.
  • AGP is 40% carbohydrate, making it unusuany acidic and soluble.
  • the glycosylation pattern of AGP changes during acute-phase response, and deglycosylated AGP has no immunosuppressive activity (Flower (1994) FEBS Lett. 354:7-11; Flower (1996) supra).
  • the Hpocalin superfamily also includes several animal allergens, including the mouse major urinary protein (mMUP), the rat ⁇ -2-microgloobuHn (rA2U), the bovine ⁇ -lactoglobuHn ( ⁇ lg), the cockroach allergen (Bla g4), bovine dander allergen (Bos d2), and the major horse allergen, designated Equus caballus allergen 1 (Equ cl).
  • Equ cl is a powerful allergen responsible for about 80% of anti- horse IgE antibody response in patients who are chronically exposed to horse allergens. It appears that Hpocalins may contain a common structure that is able to induce the IgE response (Gregoire, C. et al., (1996) J. Biol. Chem. 271:32951-32959).
  • Lipocalins are used as diagnostic and prognostic markers in a variety of disease states.
  • the plasma level of AGP is monitored during pregnancy and in diagnosis and prognosis of conditions including cancer chemotherapy, renal disfunction, myocardial infarction, arthritis, and multiple sclerosis.
  • RBP is used clinically as a marker of tubular reabsorption in the kidney
  • apo D is a marker in gross cystic breast disease (Flower (1996) supj'a).
  • Hpocalin animal allergens may help in the diagnosis of allergic reactions to horses (Gregoire supra), pigs, cockroaches, mice and rats.
  • Mitochondrial carrier proteins are transmembrane-spanning proteins which transport ions and charged metaboHtes between the cytosol and the mitochondrial matrix. Examples include the ADP, ATP carrier protein; the 2-oxoglutarate/malate carrier; the phosphate carrier protein; the pyruvate carrier; the dicarboxylate carrier which transports malate, succinate, fumarate, and phosphate; the tricarboxylate carrier which transports citrate and malate; and the Grave's disease carrier protein, a protein recognized by IgG in patients with active Grave's disease, an autoirnmune disorder resulting in hyperthyroidism.
  • the Grave's disease carrier protein a protein recognized by IgG in patients with active Grave's disease, an autoirnmune disorder resulting in hyperthyroidism.
  • Proteins in this family consist of three tandem repeats of an approximately 100 amino acid domain, each of which contains two transmembrane regions (Stryer, L. (1995) Biochemistry. W.H. Freeman and Company, New York NY, p. 551; PROSITE PDOC00189 Mitochondrial energy transfer proteins signature; Online MendeHan Inheritance in Man (OMLM) *275000 Graves Disease).
  • This class of transporters also includes the mitochondrial uncoupling proteins, which create proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from ATP synthesis. The result is energy dissipation in the form of heat.
  • Mitochondrial uncoupling proteins have been impHcated as modulators of thermoregulation and metaboHc rate, and have been proposed as potential targets for drugs against metaboHc diseases such as obesity (Ricquier, D. et al. (1999) J. Int. Med. 245:637-642). Ion Channels
  • the electrical potential of a cell is generated and maintained by contiolling the movement of ions across the plasma membrane.
  • the movement of ions requires ion channels, which form ion- selective pores within the membrane.
  • ion channels There are two basic types of ion channels, ion transporters and gated ion channels.
  • Ion transporters utiHze 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.
  • 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, Ca 2+ -ATPase, and H + -ATPase, are activated by a phosphorylation event.
  • P-class ion transporters are responsible for maintaining resting potential distributions such that cytosoHc concentrations of Na + and Ca 2+ are low and cytosoHc 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 utiHze a proton gradient to generate ATP from ADP and inorganic phosphate (P j ).
  • the P- ATPases are hexamers of a 100 kD subunit with ten transmembrane domains and several large cytoplasmic regions that may play a role in ion binding (Scarborough, G.A. (1999) Curr. Opin. CeH Biol. 11:517-522).
  • the V- ATPases are composed of two functional domains: the Y x domain, a peripheral complex responsible for ATP hydrolysis; and the V 0 domain, an integral complex responsible for proton translocation across the membrane.
  • the F- ATPases are structuraHy and evolutionarily related to the V- ATPases.
  • the F-ATPase F 0 domain contains 12 copies of the c subunit, a highly hydrophobic protein composed of two transmembrane domains and containing a single buried carboxyl group in TM2 that is essential for proton transport.
  • the V-ATPase V 0 domain contains three types of homologous c subunits with four or five transmembrane domains and the essential carboxyl group in TM4 or TM3. Both types of complex also contain a single a subunit that maybe involved in regulating the pH dependence of activity (Forgac, M. (1999) J. Biol. Chem. 274:12951-12954).
  • the resting potential of the cell is utiHzed in many processes involving carrier proteins and gated ion channels.
  • Carrier proteins utiHze the resting potential to transport molecules into and out of the ceH.
  • A-mino acid and glucose transport into many ceHs is linked to sodium ion co-transport
  • Gated Ion Channels control ion flow by regulating the opening and closing of pores.
  • the abiHty to control ion flux through various gating mechanisms aUows ion channels to mediate such diverse signaling and homeostatic functions as neuronal and endocrine signaling, muscle contraction, fertilization, and regulation of ion and pH balance. Gated ion channels are categorized according to the manner of regulating the gating function.
  • MechanicaHy-gated channels open their pores in response to mechanical stress; voltage-gated channels (e.g., Na + , K + , Ca 2+ , and Cl " channels) open their pores in response to changes in membrane potential; and Hgand-gated channels (e.g., acetylchoHne-, serotonin-, and glutamate-gated cation channels, and GABA- and glycine-gated chloride channels) open their pores in the presence of a specific ion, nucleotide, or neurotransmitter.
  • the gating properties of a particular ion channel i.e., its threshold for and duration of opening and closing
  • auxiliary channel proteins and/or post translational modifications such as phosphorylation.
  • MechanicaHy-gated or mechanosensitive ion channels act as transducers for the senses of touch, hearing, and balance, and also play important roles in cell volume regulation, smooth muscle contraction, and cardiac rhythm generation.
  • a stretch-inactivated channel (SIC) was recently cloned from rat kidney.
  • the SIC channel belongs to a group of channels which are activated by pressure or stress on the cell membrane and conduct both Ca 2+ and Na + (Suzuki, M. et al. (1999) J. Biol. Chem. 274:6330-6335).
  • the pore-forming subunits of the voltage-gated cation channels form a superfamily of ion channel proteins.
  • the characteristic domain of these channel proteins comprises six transmembrane domains (S1-S6), a pore-forming region (P) located between S5 and S6, and intraceUular amino and carboxy termini.
  • S1-S6 transmembrane domains
  • P pore-forming region
  • the P region contains information specifying the ion selectivity for the channel.
  • a GYG tripeptide is involved in this selectivity (Isl i, T.M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11651-11656).
  • Voltage-gated Na + and K + channels are necessary for the function of electricaUy excitable ceHs, such as nerve and muscle ceUs.
  • Action potentials which lead to neurotransmitter release and muscle contraction, arise from large, transient changes in the permeabiHty 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 ceH, further depolarizing the membrane and opening more voltage-gated Na + channels, which propagates the depolarization down the length of the ceH.
  • Depolarization also opens voltage-gated potassium channels. Consequently, potassium ions flow outward, which leads to repolarization of the membrane.
  • Voltage-gated channels utiHze 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 ⁇ subunit that associates with two smaHer auxiHary subunits, ⁇ l and ⁇ 2.
  • the ⁇ 2 subunit is a integral membrane glycoprotein that contains an extraceHular lg domain, and its association with ⁇ and ⁇ l subunits correlates with increased functional expression of the channel, a change in its gating properties, as weH as an increase in whole ceH capacitance due to an increase in membrane surface area (Isom, L.L. et al. (1995) CeU 83:433-442).
  • Non voltage-gated Na + channels include the members of the amiloride-sensitive Na + channel/degenerin (NaC/DEG) family. Channel subunits of this family are thought to consist of two transmembrane domains flanking a long extraceHular loop, with the amino and carboxyl termini located within the ceH.
  • the NaC/DEG family includes the epithelial Na + channel (ENaC) involved in Na + reabsorption in epifheHa including the airway, distal colon, cortical coHecting duct of the kidney, and exocrine duct glands. Mutations in ENaC result in pseudohypoaldosteronism type 1 and Liddle's syndrome (pseudohyperaldosteronism).
  • the NaC/DEG family also includes the recently characterized H + -gated cation channels or acid-sensing ion channels (ASIC).
  • ASIC subunits are expressed in the brain and formheteromultimeric Na + -permeable channels. These channels require acid pH fluctuations for activation.
  • ASIC subunits show homology to the degenerins, a family of mechanicaUy- gated channels originaUy isolated from C. elegans. Mutations in the degenerins cause neurodegeneration. ASIC subunits may also have a role in neuronal function, or in pain perception, since tissue acidosis causes pain (Waldmann, R. and M. Lazdunski (1998) Curr. Opin. Neurobiol. 8:418-424; Eglen, R.M. et al. (1999) Trends Pharmacol. Sci. 20:337-342).
  • K + channels are located in aU ceH types, and may be regulated by voltage, ATP concentration, or second messengers such as Ca 2+ and cAMP.
  • K + channels are involved in protein synthesis, control of endocrine secretions, and the maintenance of osmotic equiHbrium across membranes.
  • K + channels are responsible for setting the resting membrane potential.
  • the cytosol contains non-diffusible anions and, to balance this net negative charge, the ceH contains a Na + - K + pump and ion channels that provide the redistribution of Na + , K + , and Cl " .
  • the pump actively transports Na + out of the ceH and K + into the ceH in a 3 :2 ratio.
  • Ion channels in the plasma membrane aHow K + and Cl " to flow by passive diffusion. Because of the high negative charge within the cytosol, - Cl" flows out of the ceH.
  • the flow of K + is balanced by an electromotive force pulling K + into the ceH, and a K + concentration gradient pushing + out of the ceH.
  • the resting membrane potential is primarily regulated by K ⁇ flow (Salkoff, L. and T. Jegla (1995) Neuron 15:489-492).
  • Potassium channel subunits of the Shaker-Hks superfamily aU have the characteristic six transmembrane/1 pore domain structure.
  • the Shaker-like channel family includes the voltage- gated K + channels as weH as the delayed rectifier type channels such as the human ether-a-go-go related gene (HERG) associated with long QT, a cardiac dysrythmia syndrome (Curran, M.E. (1998) Curr. Opin. Biotechnol. 9:565-572; Kaczorowski, G.J. and M.L. Garcia (1999) Curr. Opin. Chem. Biol. 3:448-458).
  • HERG human ether-a-go-go related gene
  • Kir channels have the property of preferentiaHy conducting K + currents in the inward direction. These proteins consist of a single potassium selective pore domain and two transmembrane domains, which correspond to the fifth and sixth transmembrane domains of voltage-gated K + channels. Kir subunits also associate as tetramers.
  • the Kir family includes ROMK1, mutations in which lead to Bartter syndrome, a renal tubular disorder. Kir channels are also involved in regulation of cardiac pacemaker activity, seizures and epilepsy, and insulin regulation (Doupnik, CA. et al. (1995) Curr. Opin. Neurobiol. 5:268-277; Curran, supra).
  • TWTK K + channel family includes the mammaHan TWIK-1, TREK-1 and TASK proteins.
  • the voltage-gated Ca 2+ channels have been classified into several subtypes based upon their electrophysiological and pharmacological characteristics.
  • L-type Ca 2+ channels are predominantly expressed in heart and skeletal muscle where they play an essential role in excitation-contraction coupling.
  • T-type channels are important for cardiac pacemaker activity, while N-type and P/Q-type channels are involved in the control of neurotransmitter release in the central and peripheral nervous system.
  • the L-type and N-type voltage-gated Ca 2+ channels have been purified and, though their functions differ dramaticaUy, they have similar subumt compositions.
  • the channels are composed of three subunits.
  • the ⁇ x subunit forms the membrane pore and voltage sensor, while the o ⁇ and ⁇ subunits modulate the voltage-dependence, gating properties, and the current ampHtude of the channel.
  • These subunits are encoded by at least six ⁇ 1? one o ⁇ , and four ⁇ genes.
  • a fourth subumt, ⁇ has been identified in skeletal muscle (Walker, D. et al. (1998) J. Biol. Chem. 273:2361-2367; McCleskey, E.W. (1994) Curr. Opin. Neurobiol. 4:304-312).
  • the high-voltage-activated Ca 2+ channels that have been characterized biochemicaUy include complexes of a pore-forming alphal subunit of approximately 190-250 kDa; a transmembrane complex of alpha2 and delta subumts; an intraceHular beta subunit; and in some cases a transmembrane gamma subumt.
  • a variety of alphal subunits, alpha2delta complexes, beta subunits, and gamma subumts are known.
  • the Cavl family of alphal subunits conduct L-type Ca 2+ currents, which initiate muscle contraction, endocrine secretion, and gene transcription, and are regulated primarily by second messenger-activated protein phosphorylation pathways.
  • the Cav2 family of alphal subunits conduct N-type, P/Q-type, and R-type Ca 2+ currents, which initiate rapid synaptic transmission and are regulated primarily by direct interaction with G proteins and SNARE proteins and secondarily by protein phosphorylation.
  • the Cav3 family of alphal subunits conduct T-type Ca 2+ currents, which are activated and inactivated more rapidly and at more negative membrane potentials than other Ca 2+ current types.
  • the distinct structures and patterns of regulation of these three famiHes of Ca 2+ channels provide an array of Ca 2+ entry pathways in response to changes in membrane potential and a range of possibiHties for regulation of Ca 2+ entry by second messenger pathways and interacting proteins (CatteraH, W.A. (2000) Annu. Rev.
  • the alpha-2 subunit of the voltage-gated Ca 2+ -channel may include one or more Cache domains.
  • An extraceHular Cache domain may be fused to an intraceUular catalytic domain, such as the histidine kinase, PP2C phosphatase, GGDEF (a predicted diguanylate cyclase), HD-GYP (a predicted phosphodiesterase) or adenylyl cyclase domain, or to a noncatalytic domain, like the methyl-accepting, DNA-binding winged heHx-turn-heHx, GAF, PAS or HAMP (a domain found in istidine kinases, denylyl cyclases, ethyl-binding proteins and phosphatases).
  • SmaU molecules are bound via the Cache domain and this signal is converted into diverse outputs depending on the intraceUular domains (Anantharaman, V. and Aravind,
  • Trp The transient receptor family (Trp) of calcium ion channels are thought to mediate capacitative calcium entry (CCE).
  • CCE is the Ca 2+ influx into ceUs to resupply Ca 2+ stores depleted by the action of inositol triphosphate (IP3) and other agents in response to numerous hormones and growth factors.
  • IP3 inositol triphosphate
  • Trp and Trp-Hke were first cloned from Drosophila and have similarity to voltage gated Ca 2+ channels in the S3 through S6 regions. This suggests that Trp and/or related proteins may form ammaHan CCE channels (Zhu, X. et al. (1996) CeU 85:661-671; Boulay, G. et al. (1997) J. Biol. Chem.
  • Melastatin is a gene isolated in both the mouse and human, whose expression in melanoma ceUs is inversely correlated with melanoma aggressiveness in vivo.
  • the human cDNA transcript corresponds to a 1533-amino acid protein having homology to members of the Trp family. It has been proposed that the combined use of malastatin mRNA expression status and tumor thickness might aUow for the determination of subgroups of patients at both low and high risk for developing metastatic disease (Duncan, L.M. et al (2001) J. Clin. Oncol. 19:568-576).
  • Chloride channels are necessary in endocrine secretion and in regulation of cytosoHc and organeUe pH.
  • Cl' enters the ceU across a basolateral membrane through an Na + , R7C1" cotransporter, accumulating in the ceU above its electrochemical equiHbrium concentration.
  • 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.
  • CFTR is a member of the ABC transporter family, and is composed of two domains each consisting of six transmembrane domains foUowed by a nucleotide-binding site. Loss of CFTR function decreases transepitheHal 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 obstructive pulmonary disease” (Al-Awqati, Q. et al. (1992) J. Exp. Biol. 172:245-266).
  • the voltage-gated chloride channels are characterized by 10-12 transmembrane domains, as weU as two smaU globular domains known as CBS domains.
  • the CLC subumts probably function as homotetramers.
  • CLC proteins are involved in regulation of ceU volume, membrane potential stabilization, signal transduction, and transepitheHal transport. Mutations in CLC-1, expressed predominantly in skeletal muscle, are responsible for autosomal recessive generalized myotonia and autosomal dominant myotonia congenita, while mutations in the kidney channel CLC-5 lead to kidney stones (Jentsch, TJ. (1996) Curr. Opin. Neurobiol. 3:13-310).
  • Ligand-gated channels open their pores when an extraceHular or intraceUular mediator binds to the channel.
  • Neurotransmitter-gated channels are channels that open when a neurotransmitter binds to their extraceHular domain. These channels exist in the postsynaptic membrane of nerve or muscle ceUs.
  • Chloride channels open in response to inhibitory neurotransmitters, such as ⁇ -aminobutyric acid (GABA) and glycine, leading to hyperpolarization of the membrane and the subsequent generation of an action potential.
  • GABA ⁇ -aminobutyric acid
  • Neurotransmitter-gated ion channels have four transmembrane domains and probably function as pentamers (Jentsch, supra). Amino acids in the second transmembrane domain appear to be important in determirdng channel permeation and selectivity (Sather, W.A. et al. (1994) Curr. Opin. Neurobiol. 4:313-323).
  • Ligand-gated channels can be regulated by intraceUular second messengers.
  • calcium-activated K + channels are gated by internal calcium ions.
  • an influx of calcium during depolarization opens K + channels to modulate the magnitude of the action potential (Ishi et al, supra).
  • the large conductance (BK) channel has been purified from brain and its subunit composition determined.
  • the ⁇ subunit of the BK channel has seven rather than six transmembrane domains in contrast to voltage-gated K + channels.
  • the extra transmembrane domain is located at the subunit N- terminus.
  • a 28-arnino-acid stretch in the C-terminal region of the subunit contains many negatively charged residues and is thought to be the region responsible for calcium binding.
  • the ⁇ subunit consists of two transmembrane domains connected by a glycosylated extraceHular loop, with intraceUular N- and C-termini (Kaczorowski, supra; Vergara, C. et al. (1998) Curr. Opin. Neurobiol. 8:321-329). CycHc nucleotide-gated (CNG) channels are gated by cytosoHc cycHc nucleotides.
  • CNG channels also represent a major pathway for Ca 2+ entry into neurons, and play roles in neuronal development and plasticity.
  • CNG channels are tetramers containing at least two types of subunits, an subunit which can form functional homomeric channels, and a ⁇ subunit, which modulates the channel properties.
  • AU CNG subunits have six transmembrane domains and a pore forming region between the fifth and sixth transmembrane domains, similar to voltage-gated K + channels.
  • a large C-terminal domain contains a cycHc nucleotide binding domain, while the N-terminal domain confers variation among channel subtypes (ZufaU, F. et al. (1997) Curr. Opin. Neurobiol. 7:404-412).
  • ion channel proteins may also be modulated by a variety of intraceUular signaling proteins.
  • Many channels have sites for phosphorylation by one or more protein kinases including protein kinase A, protein kinase C, tyrosine kinase, and casein kinase II, aH of which regulate ion channel activity in ceHs.
  • Kir channels are activated by the binding of the G ⁇ subunits of heterotrimeric G-proteins (Reimann, F. and F.M. Ashcroft (1999) Curr. Opin. CeH. Biol. 11:503-508).
  • Other proteins are involved in the locaHzation of ion channels to specific sites in the ceU membrane.
  • Such proteins include the PDZ domain proteins known as MAGUKs (membrane-associated guanylate kinases) which regulate the clustering of ion channels at neuronal synapses (Craven, S.E. and D.S. Bredt (1998) CeU 93:495-498).
  • MAGUKs membrane-associated guanylate kinases
  • Human diseases caused by mutations in ion channel genes include disorders of skeletal muscle, cardiac muscle, and the central nervous system. Mutations in the pore-forming subunits of sodium and chloride channels cause myotonia, a muscle disorder in which relaxation after voluntary contraction is delayed. Sodium channel myotonias have been treated with channel blockers. Mutations in muscle sodium and calcium channels cause forms of periodic paralysis, while mutations in the sarcoplasmic calcium release channel, T-tubule calcium channel, and muscle sodium channel cause maHgnant hyperthermia. Cardiac arrythmia disorders such as the long QT syndromes and idiopathic ventricular fibrillation are caused by mutations in potassium and sodium channels (Cooper, E.G. and L.Y.
  • AU four known human idiopathic epilepsy genes code for ion channel proteins (Berkovic, S.F. and I.E. Scheffer (1999) Curr. Opin. Neurology 12:177-182).
  • Other neurological disorders such as ataxias, hemiplegic migraine and hereditary deafness can also result from mutations in ion channel genes (Jen, J. (1999) Curr. Opin. Neurobiol. 9:274-280; Cooper, supra).
  • Ion channels have been the target for many drug therapies. Neurotransmitter-gated channels have been targeted in therapies for treatment of msomnia, 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). Various classes of ion channels also play an important role in the perception of pain, and thus are potential targets for new analgesics. These include the vaniUoid-gated ion channels, which are activated by the vaniUoid capsaicin, as weU as by noxious heat.
  • T-ceU activation depends upon calcium signaling, and a diverse set of T-ceU specific ion channels has been characterized that affect this signaling process.
  • Channel blocking agents can inhibit secretion of lymphokines, ceH proHferation, and lling of target ceUs.
  • T-ceU potassium channel Kvl.3 A peptide antagonist of the T-ceU potassium channel Kvl.3 was found to suppress delayed-type hypersensitivity and aUogenic responses in pigs, vaHdating the idea of channel blockers as safe and efficacious immunosuppressants (Cahalan, M.D. and K.G. Chandy (1997) Curr. Opin. Biotechnol. 8:749-756).
  • Microarrays are analytical tools used in bioanalysis.
  • a microarray has a pluraHty of molecules spatiaHy distributed over, and stably associated with, the surface of a soHd support.
  • Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of appHcations, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
  • array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specificaUy related to a particular genetic predisposition, condition, disease, or disorder.
  • the potential appHcation of gene expression profiling is relevant to improving the diagnosis, prognosis, and treatment of cancers, such as breast cancer, lung cancer, prostate cancer, ovarian cancer, and bone cancer, as weU as the treatment of vascular iru ⁇ ammation and immune responses, Hver toxicity, and neurological disorders.
  • cancers such as breast cancer, lung cancer, prostate cancer, ovarian cancer, and bone cancer, as weU as the treatment of vascular iru ⁇ ammation and immune responses, Hver toxicity, and neurological disorders.
  • BRCA1 and BRCA2 are known to greatly predispose a woman to breast cancer and maybe passed on from parents to children (Gish, supra).
  • this type of hereditary breast cancer accounts for only about 5% to 9% of breast cancers, while the vast majority of breast cancer is due to non-inherited mutations that occur in breast epitheHal ceHs.
  • epidermal growth factor EGF
  • EGFR epidermal growth factor
  • EGFR expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis.
  • EGF has effects on ceH functions related to metastatic potential, such as ceU motiHty, chemotaxis, secretion and differentiation.
  • the abundance of erbB receptors, such as HER- 2/neu, HER-3, and HER-4, and their Hgands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S.S. et al. (1994) Am. J. Clin. Pathol. 102:S13-S24).
  • CeU lines derived from human mammary epitheHal ceHs at various stages of breast cancer provide a useful model to study the process of maHgnant transformation and tumor progression as it has been shown that these ceU lines retain many of the properties of their parental tumors for lengthy culture periods (Wistuba, LI. et al. (1998) Clin. Cancer Res. 4:2931-2938). Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epitheHal ceHs at various stages of maHgnant transformation.
  • Lung cancer is the leading cause of cancer death in the United States, affecting more than 100,000 men and 50,000 women each year. Nearly 90% of the patients diagnosed with lung cancer are cigarette smokers. Tobacco smoke contains thousands of noxious substances that induce carcinogen metaboHzing enzymes and covalent DNA adduct formation in the exposed bronchial epitheHum. In nearly 80% of patients diagnosed with lung cancer, metastasis has already occurred. Most commonly lung cancers metastasize to pleura, brain, bone, pericardium, and Hver. The decision to treat with surgery, radiation therapy, or chemotherapy is made on the basis of tumor histology, response to growth factors or hormones, and sensitivity to inhibitors or drugs. With current treatments, most patients die within one year of diagnosis. EarHer diagnosis and a systematic approach to identification, staging, and treatment of lung cancer could positively affect patient outcome.
  • Non SmaU CeU Lung Carcinoma (NSCLC) group includes squamous ceH carcinomas, adenocarcinomas, and large ceU carcinomas and accounts for about 70% of aU lung cancer cases.
  • Adenocarcinomas typicaUy arise in the peripheral airways and often form mucin secreting glands.
  • Squamous ceH carcinomas typicaUy arise in proximal airways.
  • SCLC SmaU CeU Lung Carcinoma
  • Lung cancer ceUs accumulate numerous genetic lesions, many of which are associated with cytologicaUy visible chromosomal aberrations.
  • the high frequency of chromosomal deletions associated with lung cancer may reflect the role of multiple tumor suppressor loci in the etiology of this disease. Deletion of the short arm of chromosome 3 is found in over 90% of cases and represents one of the earHest genetic lesions leading to lung cancer. Deletions at chromosome arms 9p and 17p are also common.
  • Other frequently observed genetic lesions include overexpression of telomerase, activation of oncogenes such as K-ras and c-myc, and inactivation of tumor suppressor genes such as RB, p53 and CDKN2.
  • throhibospondin- 1, fibronectin, interceUular adhesion molecule 1, and cytokeratins 6 and 18 were previously observed to be differentiaUy expressed in lung cancers.
  • Wang et al. 2000; Oncogene 19:1519-1528) used a combination of microarray analysis and subtractive hybridization to identify 17 genes differentiaUy overexpresssed in squamous ceU carcinoma compared with normal lung epitheHum.
  • the known genes they identified were keratin isoform 6, KOC, SPRC, IGFb2, connexin 26, plakofiUin 1 and cytokeratin 13.
  • Prostate cancer is a common maHgnancy in men over the age of 50, and the incidence increases with age. In the US, there are approximately 132,000 newly diagnosed cases of prostate cancer and more than 33,000 deaths from the disorder each year.
  • cancer ceHs arise in the prostate, they are stimulated by testosterone to a more rapid growth. Thus, removal of the testes can indirectly reduce both rapid growth and metastasis of the cancer.
  • prostatic cancers Over 95 percent of prostatic cancers are adenocarcinomas which originate in the prostatic acini. The remaining 5 percent are divided between squamous ceU and transitional ceU carcinomas, both of which arise in the prostatic ducts or other parts of the prostate gland.
  • prostate cancer develops through a multistage progression ultimately resulting in an aggressive tumor phenotype.
  • the initial step in tumor progression involves the hyperproHferation of normal luminal and/or basal epitheHal ceUs.
  • Androgen responsive ceUs become hyperplastic and evolve into early-stage tumors.
  • early-stage tumors are often androgen sensitive and respond to androgen ablation, a population of androgen independent ceUs evolve from the hyperplastic population.
  • ceUs represent a more advanced form of prostate tumor that may become invasive and potentiaUy become metastatic to the bone, brain, or lung.
  • a variety of genes maybe differentiaUy expressed during rumor progression.
  • LOV loss of heterozygosity
  • FISH Fluorescence in situ hybridization
  • PSA prostate specific antigen
  • PSA is a tissue-specific serine protease almost exclusively produced by prostatic epitheHal ceHs.
  • the quantity of PSA correlates with the number and volume of the prostatic epitheHal ceUs, and consequently, the levels of PSA are an exceUent indicator of abnormal prostate growth.
  • Men with prostate cancer exhibit an early linear increase in PSA levels foHowed by an exponential increase prior to diagnosis.
  • PSA levels are also influenced by factors such as inflammation, androgen and other growth factors, some scientists maintain that changes in PSA levels are not useful in detecting individual cases of prostate cancer.
  • Current areas of cancer research provide additional prospects for markers as weU as potential therapeutic targets for prostate cancer.
  • EGF Epidermal Growth Factor
  • FGF Fibroblast Growth Factor
  • TGF ⁇ Tumor Growth Factor alpha
  • TGF- ⁇ family of growth factors are generaHy expressed at increased levels in human cancers and the high expression levels in many cases correlates with advanced stages of maHgnancy and poor survival (Gold, L.I. (1999) Grit. Rev. Oncog. 10:303-360).
  • FinaHy there are human ceH lines representing both the androgen-dependent stage of prostate cancer (LNCap) as weU as the androgen-independent, hormone refractory stage of the disease (PC3 and DU-145) that have proved useful in studying gene expression patterns associated with the progression of prostate cancer, and the effects of ceU treatments on these expressed genes (Chung, T.D. (1999) Prostate 15:199-207).
  • Ovarian cancer is the leading cause of death from a gynecologic cancer.
  • the majority of ovarian cancers are derived from epitheHal ceHs, and 70% of patients with epitheHal ovarian cancers present with late-stage disease. As a result, the long-term survival rate for this disease is very low. Identification of early-stage markers for ovarian cancer would significantly increase the survival rate.
  • Genetic variations involved in ovarian cancer development include mutation of p53 and microsateUite instabiHty. Gene expression patterns likely vary when normal ovary is compared to ovarian tumors. Bone cancer
  • Osteosarcoma is the most common maHgnant bone tumor in children. Approximately 80% of patients present with non-metastatic disease. After the diagnosis is made by an initial biopsy, treatment involves the use of 3-4 courses of neoadjuvant chemotherapy before definitive surgery, foHowed by post-operative chemotherapy. With currently available treatment regimens, approximately 30-40% of patients with non-metastatic disease relapse after therapy. Currently, there is no prognostic factor that can be used at the time of initial diagnosis to predict which patients wiU have a high risk of relapse. The only significant prognostic factor predicting the outcome in a patient with non-metastatic osteosarcoma is the histopathologic response of the primary tumor resected at the time of definitive surgery.
  • the degree of necrosis in the primary tumor is a reflection of the tumor response to neoadjuvant chemotherapy.
  • a higher degree of necrosis (good or favorable response) is associated with a lower risk of relapse and a better outcome.
  • Patients with a lower degree of necrosis (poor or unfavorable response) have a much higher risk of relapse and poor outcome even after complete resection of the primary tumor.
  • poor outcome cannot be altered despite modification of post-operative chemotherapy to account for the resistance of the primary tumor to neoadjuvant chemotherapy.
  • Atherosclerosis is a pathological condition characterized by a chronic local inflammatory response within the vessel waH of major arteries. Disease progression results in the formation of atherosclerotic lesions, unstable plaques which occasionaUy rupture, precipitating a catastrophic thrombotic occlusion of the vessel lumen. Atherosclerosis and the associated coronary artery disease and cerebral stroke represent the most common causes of death in bonHzed nations. Although certain key risk factors have been identified, a fuH molecular characterization that elucidates the causes and identifies aU potential therapeutic targets for this complex disease has not been achieved. Molecular characterization of atherosclerosis requires identification of the genes that contribute to lesion growth, stabiHty, dissolution, rupture and induction of occlusive vessel thrombi.
  • Blood vessel waHs are composed of two tissue layers: an endotheHal ceH (EC) layer which comprises the lumenal surface of the vessel, and an underlying vascular smooth muscle ceU (VSMC) layer.
  • EC endotheHal ceH
  • VSMC vascular smooth muscle ceU
  • the inflammatory response is a complex vascular reaction mediated by numerous cytokines, chemokines, growth factors, and other signaling molecules expressed by activated ECs, VSMCs and leukocytes. Ihflarrrmation protects the organism during trauma and infection, but can also lead to pathological conditions such as atherosclerosis. Activation of vascular endotheHum is a central event in a wide range of physiological and disease processes such as vascular tone regulation, coagulation and thrombosis, atherosclerosis, inflammation and some infectious diseases.
  • pro-iriflammatory cytokines mterleukin (IL)-l and tumor necrosis factor (TNF)
  • IL mterleukin
  • TNF tumor necrosis factor
  • leukocytes particularly neutrophils and monocytes/macrophages, accumulate in the extravascular space, where they remove injurious agents by phagocytosis and oxidative kiUing, a process accompanied by release of toxic factors, such as proteases and reactive oxygen species.
  • IL-1 and TNF induce pro-inflammatory, thrombotic, and anti-apoptotic changes in gene expression by signaling through receptors on the surface of ECs and VSMCs; these receptors activate transcription factors such as NF&B as weU as AP-1, IRF-1, and NF-GMa, leading to alterations in gene expression.
  • Genes known to be differentiaUy regulated in EC by IL-1 and TNF include E selectin, VCAM-1, ICAM-1, PAF, IEB ⁇ , IAP-1, MCP-1, eotaxin, ENA-78, G-CSF, A20, ICE, and complement C3 component.
  • tissue inhibitor of metaUoproteinase 1, ferritin Hght chain, and manganese superoxide dismutase were found to be differentiaUy expressed in rheumatoid arthritis (RA) relative to inflammatory bowel disease (IBD). Further, IL-3, chemokine Gro ⁇ , and metaUoproteinase matrix metaUo-elastase were expressed in both RA and IBD.
  • RA rheumatoid arthritis
  • IBD inflammatory bowel disease
  • IL-3, chemokine Gro ⁇ , and metaUoproteinase matrix metaUo-elastase were expressed in both RA and IBD.
  • Haley et al. found a 20-fold increase in eotaxin, an eosinophil chemotactic factor. The overexpression of eotaxin and its receptor CCR3 in atherosclerotic lesions was confirmed by northern analysis.
  • HCAECs Human coronary artery endotheHal ceHs
  • HCAECs Human coronary artery endotheHal ceHs
  • UAAECs Human umbiHcal artery endotheHal ceUs
  • UtMVECs Human uterine myometrium microvascular endotheHal ceUs
  • HAAECs Human Iliac Artery EndotheHal CeHs
  • HAVECs Human umbiHcal vein endotheHal ceHs
  • ECV304 is a human endotheHal line.
  • AD Alzheimer's disease
  • AD A progressive and irreversible brain disorder, AD is characterized by three major pathogenic episodes involving (a) an aberrant processing and deposition of beta-amyloid precursor protein (betaAPP) to form neurotoxic beta-amyloid (betaA) peptides and an aggregated insoluble polymer of betaA that forms the senile plaque, (b) the estabHshment of intraneuronal neuritic tau pathology yielding widespread deposits of agyrophiHc neurofibriHary tangles (NFT) and (c) the initiation and proHferation of a brain-specific inflammatory response.
  • betaAPP beta-amyloid precursor protein
  • betaA neurotoxic beta-amyloid
  • NFT neurofibriHary tangles
  • cPLA2 inducible oxidoreductase cyclooxygenase-2 and cytosoHc phosphoHpase A2
  • Tumor necrosis factor-alpha is a pleiotropic cytokine that plays a central role in mediation of the inflammatory response through activation of multiple signal transduction pathways.
  • TNF- ⁇ is produced by activated lymphocytes, macrophages, and other white blood ceUs, and activates endotheHal ceHs.
  • terferon-gamma also known as Type ⁇ interferon or immune interferon, is a cytokine produced primarily by T-lymphocytes and natural killer ceUs. Mature IFN ⁇ exists as noncovalently- Hnked homodimers. IFN ⁇ displays antiviral, antiproHferative, immunoregulatory, and proinflarnmatory activities and is important in host defense mechanisms.
  • JJFN- ⁇ induces the production of cytokines; upregulates the expression of class I and II MHC antigens, Fc receptor, and leukocyte adhesion molecule; modulates macrophage effector functions; influences isotype switching; potentiates the secretion of immunoglobuHns by B ceHs; augments TH1 ceU expansion; and maybe required for TH1 ceU differentiation.
  • IFN ⁇ exerts its biological activities by binding to specific ceU surface receptors, which display high affinity binding sites. The IFN ⁇ receptor is present on almost aU ceU types except mature erythrocytes. Upon binding to its receptor, IFN ⁇ triggers the activation of JAK-1 and JAK-2 kinases resulting in the phosphorylation of STATl.
  • Both IFN ⁇ and TNF- ⁇ are considered proinflammatory cytokines.
  • Cross-talk can exist between the signal transduction pathways of two cytokines; for example, signal transduction cascades initiated by two different cytokines lead to the activation of NfkB.
  • Liver toxicity The human C3 A ceU line is a clonal derivative of HepG2/C3 (hepatoma ceU line, isolated from a 15-year-old male with Hver tumor), which was selected for strong contact irj-hibition of growth. The use of a clonal population enhances the reproducibiHty of the ceHs.
  • C3 A cells have many characteristics of primary human hepatocytes in culture: i) expression of insulin receptor and insulin- like growth factor II receptor; n) secretion of a high ratio of serum albumin compared with ⁇ - fetoprotein; in) conversion of ammonia to urea and glutamine; iv) metaboHsm of aromatic amino acids; and v) proHferation in glucose-free and insuHn-free medium.
  • the C3A ceU line is now weU established as an in vitro model of the mature human Hver (Mickelson et al. (1995) Hepatology 22:866-875; Nagendra et al. (1997) Am. J. Physiol. 272:G408-G416).
  • the potential appHcation of gene expression profiling is relevant to measuring the toxic response to potential therapeutic compounds and of the metaboHc response to therapeutic agents.
  • diseases treated with steroids and disorders caused by the metaboHc response to treatment with steroids include adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatoceHular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease. It is desirable to measure the toxic response to potential therapeutic compounds and of the metaboHc response to therapeutic agents.
  • Steroids are a class of Hpid-soluble molecules, including cholesterol, bile acids, vitamin D, and hormones, that share a common four-ring structure based on cyclopentanoperhydrophenanthrene and that carrry out a wide variety of functions.
  • Steroid hormones produced by the adrenal cortex, ovaries, and testes, include glucocorticoids, mineralocorticoids, androgens, and estrogens.
  • Steroid hormones are widely used for fertility control and in anti-inflammatory treatments for physical injuries and diseases such as arthritis, asthma, and auto-immune disorders.
  • Progesterone a naturaUy occurring progestin, is primarily used to treat amenorrhea, abnormal uterine bleeding, or as a contraceptive.
  • MAH Medroxyprogesterone
  • MAH is a synthetic progestin with a pharmacological activity about 15 times greater than progesterone.
  • MAH is usuaUy used for the treatment of renal and endometrial carcinomas, amenorrhea, abnormal uterine bleeding, and endometriosis associated with hormonal imbalance.
  • the primary contraceptive effect of exogenous progestins involves the suppression of the midcycle surge of LH. The exact mechanism of action, however, is unknown.
  • progestins diffuse freely into target ceUs and bind to the progesterone receptor.
  • Target ceUs include the female reproductive tract, mammary gland, hypothalamus, and pituitary. Once bound to the receptor, progestins slow the frequency of release of gonadotropin releasing hormone (GnRH) from the hypothalamus and blunt the pre-ovulatory LH surge, thereby preventing foUicular maturation and ovulation.
  • GnRH gonadotropin releasing hormone
  • MAH stimulatory effect on the respiratory centers has been used clinicaUy to treat low blood oxygenation due to sleep apnea, chronic obstructive pulmonary disease, or hypercapnia (excess of CO 2 in blood).
  • Beclomethasone is a synthetic glucocorticoid that is used for treating steroid-dependent asthma, reHeving symptoms associated with aUergic or nonaUergic (vasomotor) rhinitis, or for preventing recurrent nasal polyps foHowing surgical removal.
  • the anti-inflammatory and vasoconstrictive effects of intranasal beclomethasone are 5,000 times greater than those produced by hydrocortisone.
  • Budesonide is a corticosteroid used to control symptoms associated with aUergic rhinitis or asthma.
  • Dexamethasone is a synthetic glucocorticoid used in anti-inflammatory or immunosuppressive compositions.
  • Prednisone is metaboHzed in the Hver to its active form, prednisolone, a glucocorticoid with anti-inflammatory properties.
  • Betamethasone is a synthetic glucocorticoid with antiinflammatory and immunosuppressive activity and is used to treat psoriasis and fungal infections, such as athlete's foot and ringworm. By comparing both the levels and sequences expressed in tissues from subjects exposed to or treated with steroid compounds with the levels and sequences expressed in normal untreated tissue it is possible to determine tissue responses to steroids.
  • Budesonide (Bude) is a corticosteroid used to control symptoms associated with aUergic rhinitis or asthma.
  • Prednisone is a corticosteroid that is metaboHzed in the Hver to its active form, prednisolone. Prednisone is roughly four times more potent as a glucocorticoid than hydrocortisone. Prednisone is intermediate between hydrocortisone and dexamethasone in duration of action. Prednisone is used in conditions such as aUograft rejection, asthma, systemic lupus erythematosus, and many other inflammatory states.
  • Glucocorticoids are naturaUy occurring hormones that prevent or suppress inflammation and immune responses when administered at pharmacological doses. At the molecular level, unbound glucocorticoids readily cross ceU membranes and bind with high affinity to specific cytoplasmic receptors. Subsequent to binding, transcription and, ultimately, protein synthesis are affected. The result can include inhibition of leukocyte infiltration at the site of inflammation, interference in the function of mediators of inflammatory response, and suppression of humoral immune responses. The anti-inflammatory actions of corticosteroids are thought to involve phosphoHpase A2 inhibitory proteins, coUectively caHed Hpocortins. Lipocortins, in turn, control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic.
  • compositions including nucleic acids and proteins, for the diagnosis, prevention, and treatment of transport, neurological, muscle, immunological and ceU proHferative disorders.
  • Various embodiments of the invention provide purified polypeptides, transporters and ion channels, referred to coUectively as 'TRICH' and individuaUy as 'TRICH-1,' 'TRICH-2,' 'TRICH-3,' 'TRICH-4,' 'TRICH-5,' 'TRICH-6,' 'TRICH-7,' 'TRICH-8,' 'TRICH-9,' 'TRICH-10,' 'TRICH- 11,' 'TRICH-12,' 'TRICH-13,' 'TRICH-14,' 'TRICH-15,' 'TRICH-16,' 'TRICH-17,' 'TRICH-18,' 'TRICH-19,' 'TRICH-20,' 'TRICH-21,' 'TRICH-22,' 'TRICH-23,' 'TRICH-24,' 'TRICH-25,' and 'TRICH-26' and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment
  • Embodiments also provide methods. for utiHzing the purified transporters and ion channels and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology.
  • Related embodiments provide methods for utiHzing the purified transporters and ion channels and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
  • An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l- 26, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-26.
  • Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-26.
  • StiU another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-26.
  • polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:l-26. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:27-52.
  • StiU another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably Hnked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ LD NO: 1-26, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26.
  • Another embodiment provides a ceH transformed with the recombinant polynucleotide.
  • Yet another embodiment provides a transgenic organism comprising the recombinant polyn
  • Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-26.
  • the method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said ceU is transformed with a recombinant polynucleotide comprising a promoter sequence operably Hnked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • Yet another embodiment provides an isolated antibody which specificaHy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, and d) an immunogenic fragment of a polypeptide having an a ino acid sequence selected from the group consisting of SEQ ID NO: 1-26.
  • StiU yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of .a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-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 specificaUy hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex.
  • the method can include detecting the amount of the hybridization complex.
  • the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • StiU yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a poly
  • the method comprises a) ampHfying said target polynucleotide or fragment thereof using polymerase chain reaction ampHfication, and b) detecting the presence or absence of said ampHfied target polynucleotide or fragment thereof.
  • the method can include detecting the amount of the ampHfied target polynucleotide or fragment thereof.
  • compositions comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, and d) an immunogemc fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, and a pharmaceuticaUy acceptable excipient.
  • the composition can comprise an amino acid sequence selected from the group consisting of SEQ JD NO:l-26.
  • Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional TRICH, comprising administering to a patient in need of such treatment the composition.
  • Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
  • Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional TRICH, comprising administering to a patient in need of such treatment the composition.
  • StiU yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-26.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
  • Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional TRICH, comprising administering to a patient in need of such treatment the composition.
  • Another embodiment provides a method of screening for a compound that specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, b) a polypeptide comprising a naturaUy occurring -imino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26.
  • the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specificaUy binds to the polypeptide.
  • Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-26, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO: 1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-26.
  • the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
  • StiU yet another embodiment 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 SEQ ID NO:27-52, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, 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.
  • Another embodiment provides 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 selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52, n) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52, in) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)
  • 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 selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52, ii) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:27-52, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of u), and v) an RNA equivalent of i)-iv).
  • the target polynucleotide can comprise a fragment of a polynucleotide 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 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.
  • Table 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.
  • Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
  • Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 Hsts the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
  • Table 5 shows representative cDNA Hbraries for polynucleotide embodiments.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA Hbraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with appHcable descriptions, references, and threshold parameters.
  • Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with aHele frequencies in different human populations.
  • a host ceH includes a pluraHty of such host ceUs
  • an antibody is a reference to one or more antibodies and equivalents thereof known to those skiUed in the art, and so forth.
  • TRICH refers to the amino acid sequences of substantiaUy purified TRICH obtained from any species, particularly a mammaHan species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which intensifies or mimics the biological activity of
  • AUeHc variant is an alternative form of the gene encoding TRICH.
  • AHeHc variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered.
  • a gene may have none, one, or many aHeHc variants of its naturaUy occurring form.
  • Common mutational changes which give rise to aUeHc variants are generaUy ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding TRICH include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as TRICH or a polypeptide with at least one functional characteristic of TRICH. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oHgonucleotide probe of the polynucleotide encoding TRICH, and improper or unexpected hybridization to aUeHc variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding TRICH.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionaHy equivalent TRICH.
  • DeHberate amino acid substitutions maybe made on the basis of one or more similarities in polarity, charge, solubiHty, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of TRICH is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and argirjdne.
  • Amino acids with uncharged polar side chains having similar hydrophiHcity values may include: asparagine and glutamine; and serine and threonine.
  • Airjino acids with uncharged side chains having similar hydrophiHcity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence can refer to an oHgopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturaUy occurring or synthetic molecules.
  • amino acid sequence is recited to refer to a sequence of a naturaUy occurring protein molecule
  • amino acid sequence and Hke terms are not meant to Hmit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • AmpHfication relates to the production of additional copies of a nucleic acid. AmpHfication may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid ampHfication technologies weU known in the art.
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of TRICH. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of TRICH either by directly interacting with TRICH or by acting on components of the biological pathway in which TRICH participates.
  • antibody refers to intact immunoglobulin molecules as weU as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind TRICH polypeptides can be prepared using intact polypeptides or using fragments containing smaU peptides of interest as the immunizing antigen.
  • the polypeptide or oHgopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • chemicaHy Commonly used carriers that are chemicaHy coupled to peptides include bovine serum albumin, thyroglobuHn, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specificaUy to antigenic determinants (particular regions or three-dimensional structures on the protein).
  • An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to eHcit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oHgonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial Hbraries.
  • Aptamer compositions maybe double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-Hke molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide maybe replaced by 2'-F or -NH ⁇ , which may improve a desired property, e.g., resistance to nucleases or longer Hfetime in blood.
  • Aptamers maybe conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • Aptamers maybe specificaUy cross-linked to their cognate Hgands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
  • introduction refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturaUy occurring enzymes, which normaHy act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense"
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oHgonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, orbenzylphosphonates; oHgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oHgonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'- deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription.
  • the complementary antisense molecule base-pairs with a naturaUy occurring nucleic acid sequence produced by the ceU to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologicalcaUy active refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule.
  • immunologicalaUy active or ''immunogenic refers to the capabiHty of the natural, recombinant, or synthetic TRICH, or of any oHgopeptide thereof, to induce a specific immune response in appropriate a mals or ceHs and to bind with specific antibodies.
  • Complementary describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
  • composition comprising a given polynucleotide and a “composition comprising a given polypeptide” can refer to any composition containing the given polynucleotide or polypeptide.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotides encoding TRICH or fragments of TRICH may be employed as hybridization probes. The probes maybe stored in freeze-dried form and maybe associated with a stabiHzing agent such as a carbohydrate.
  • the probe In hybridizations, the probe maybe deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncaUed bases, extended using the XL-PCR kit (AppHed Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVJEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especiaUy the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitutions generaHy maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha heHcal conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemicaUy modified polynucleotide or polypeptide.
  • Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • “Exon shuffling” refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus aUowrng acceleration of the evolution of new protein functions.
  • a “fragment” is a unique portion of TRICH or a polynucleotide encoding TRICH which can be 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.
  • a fragment may comprise from about 5 to about 1000 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes maybe at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments maybe preferentiaUy selected from certain regions of a molecule.
  • 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.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
  • a fragment of SEQ DD NO:27-52 can comprise a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:27-52, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO:27-52 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and ampHfication technologies and in analogous methods that distinguish SEQ ID NO:27-52 from related polynucleotides.
  • the precise length of a fragment of SEQ DD NO:27-52 and the region of SEQ DD NO:27-52 to which the fragment corresponds are routinely determinable by one of ordinary skfll in the art based on the intended purpose for the fragment.
  • a fragment of SEQ DD NO: 1-26 is encoded by a fragment of SEQ DD NO:27-52.
  • a fragment of SEQ DD NO: 1-26 can comprise a region of unique arnino acid sequence that specificaUy identifies SEQ DD NO:l-26.
  • a fragment of SEQ DD NO:l-26 can be used as an iinmunogenic peptide for the development of antibodies that specificaUy recognize SEQ DD NO:l-26.
  • the precise length of a fragment of SEQ DD NO:l-26 and the region of SEQ DD NO:l-26 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
  • a “fuU length” polynucleotide is one containing at least a translation initiation codon (e.g., methionine) foUowed by an open reading frame and a translation termination codon.
  • a “fuU length” polynucleotide sequence encodes a "fuU length” polypeptide sequence.
  • “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • the terms “percent identity” and “% identity,” as appHed to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aHgned 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 aHgnment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AHgnment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AHgnment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to aHgn a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • BLAST 2 Sequences 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 2.0.12 (April-21-2000) set at default parameters. Such default parameters maybe, for example:
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ DD number, or maybe 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 shown herein, in the tables, figures, or Sequence Listing, 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 a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that aU encode substantiaUy the same protein.
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aHgned using a standardized algorithm.
  • Methods of polypeptide sequence aHgnment are weU-known. Some aHgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generaUy preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • NCBI BLAST software suite may be used.
  • BLAST 2 Sequences Version 2.0.12 (April-21-2000) with blastp set at default parameters.
  • Such default parameters may be, for example:
  • Percent identity maybe measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ DD number, or maybe 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 supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • HACs Human artificial chromosomes
  • chromosomes are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain aU of the elements required for chromosome repHcation, segregation and maintenance.
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and stiU retains its original binding abiHty.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s).
  • the washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions aUowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for anneaHng of nucleic acid sequences are routinely determinable by one of ordinary skiU in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68 °C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • GeneraUy stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point (TJ for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • 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, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Useful variations on these wash conditions wiU be readily apparent to those of ordinary skiU 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 encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a soHd support (e.g., paper, membranes, filters, chips, pins or glass sHdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
  • soHd support e.g., paper, membranes, filters, chips, pins or glass sHdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed.
  • insertion and “addition” refer to changes in an arnino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect ceUular and systemic defense systems.
  • an “immunogenic fragment” is a polypeptide or oHgopeptide fragment of TRICH which is capable of eHciting an immune response when introduced into a Hving organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or oHgopeptide fragment of TRICH which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a pluraHty of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • array element refers to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of TRICH. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of TRICH.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oHgonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which maybe single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • “Operably Hnked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a 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.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oHgonucleotide of at least about 5 nucleotides in length Hnked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubiHty to the composition.
  • PNAs preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their Hfespan in the ceU.
  • Post-translational modification of an TRICH may involve Hpidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic miHeu of TRICH.
  • Probe refers to nucleic acids encoding TRICH, their complements, or fragments thereof, which are used to detect identical, aUeHc or related nucleic acids.
  • Probes are isolated oHgonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, Hgands, chen ⁇ luminescent agents, and enzymes.
  • Primarymers are short nucleic acids, usuaHy DNA oHgonucleotides, which maybe 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 ampHfication (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
  • Probes and primers as used in the present invention typicaUy 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, 25, 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 tables, figures, and Sequence Listing, may be used.
  • 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).
  • OHgonucleotides 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 oHgonucleotides 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 pubHc from the Genome Center at University of Texas South West Medical Center, DaHas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
  • Primer3 primer selection program (available to the pubHc from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows the user to input a "misprirning Hbrary," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oHgonucleotides 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 pubHc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence aHgnments, thereby aHowing selection of primers that hybridize to either the most conserved or least conserved regions of aHgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oHgonucleotides and polynucleotide fragments.
  • oHgonucleotides 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 partiaUy complementary polynucleotides in a sample of nucleic acids. Methods of oHgonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a nucleic acid that is not naturaUy 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 accompHshed 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.
  • a recombinant nucleic acid may include a nucleic acid sequence operably Hnked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a ceU.
  • 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.
  • a "regulatory element” refers to a nucleic acid sequence usuaUy derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionucHdes; enzymes; fluorescent, chemilummescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • An "RNA equivalent,” in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that aH occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • sample is used in its broadest sense.
  • a sample suspected of containing TRICH, nucleic acids encoding TRICH, or fragments thereof may comprise a bodily fluid; an extract from a ceH, chromosome, organeUe, or membrane isolated from a ceU; a ceH; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specificaUy binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition. 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 comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody wiH reduce the amount of labeled A that binds to the antibody.
  • substantiallyUy purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturaUy associated.
  • substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, sHdes, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the coUective pattern of gene expression by a particular ceH type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient ceU. Transformation may occur under natural or artificial conditions according to various methods weU known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host ceU. The method for transformation is selected based on the type of host ceU being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, Hpofection, and particle bombardment.
  • transformed ceHs includes stably transformed ceUs in which the inserted DNA is capable of repHcation either as an autonomously repHcating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the ceUs of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques weU known in the art.
  • the nucleic acid is introduced into the ceU, directly or indirectly by introduction into a precursor of the ceU, by way of deHberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872).
  • 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, 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 40% 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 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an
  • a spHce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate spHcing 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 polynucleotides that vary from one species to another.
  • the resulting polypeptides wiH generaUy 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 nucleotide base.
  • SNPs single nucleotide polymorphisms
  • the presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • 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 85%, at least 90%, at least 91%, at least 92%, at least 10 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
  • TRICH the polynucleotides encoding TRICH, and the use of these compositions for the diagnosis, treatment, or prevention of transport, neurological, muscle, immunological and ceU proHferative disorders.
  • Table 1 summarizes the nomenclature for the fuU length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to 0 a single Incyte project identification number (Incyte Project DD). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ DD NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide DD) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ DD NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide DD) as shown.
  • Column 6 shows the Incyte DD numbers of physical, fuU length clones corresponding to the polypeptide and polynucleotide sequences of the invention.
  • the fuU length clones encode polypeptides which have at least 95% sequence identity to the polypeptide sequences shown in column 3.
  • Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ DD NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide DD) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank DD NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME DD NO:) of the nearest PROTEOME database homologs.
  • Column 4 shows the probabiHty scores for the matches between each polypeptide and its homolog(s).
  • Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where appHcable, aU of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2
  • Table 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIF'S program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
  • Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
  • Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were appHed.
  • Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties estabHsh that the claimed polypeptides are transporters and ion channels.
  • SEQ DD NO:l is 49% identical, from residue Sll to residue K626, to human CTLl protein (GenBank DD g6996442) as determined by the Basic Local AHgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiHty score is 9.0e-168, which indicates the probabiHty of obtaining the observed polypeptide sequence aHgnment by chance. SEQ DD NO:l also contains an eight transmembrane heHces regions as determined by using a hidden Markov model for the prediction of transmembrane heHces.
  • SEQ DD NO:3 is 57% identical, from residue E10 to residue V115, to human SLC11A3 iron transporter (GenBank DD g8895485) as determined by the Basic Local AHgnment Search Tool (BLAST).
  • BLAST Basic Local AHgnment Search Tool
  • the BLAST probabiHty score is 4.7e- 25, which indicates the probabiHty of obtaining the observed polypeptide sequence aHgnment by chance.
  • SEQ DD NO:6 is 88% identical, from residue Ml to residue S944, to rat potassium channel (GenBank DD g2745729) as determined by the Basic Local AHgnment Search Tool (BLAST).
  • SEQ DD NO:6 also contains a PAC motif, a PAS domain, a cycHc nucleotide-binding domain, and an ion transport protein domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)- based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ DD NO:10 is 99% identical, from residue Ml to residue 1418, 95% identical, from residue S420 to residue S680, and 94% identical, from residue P665 to residue H894, to human Eag-related gene member 2 (GenBank DD gl 1878259) as determined by the Basic Local AHgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiHty score is 0.0, which indicates the probabiHty of obtaining the observed polypeptide sequence aHgnment by chance.
  • SEQ DD NO:10 is locaHzed to the plasma membrane, has transporter and channel activity and is a voltage-gated potassium channel, as determined by BLAST analysis using the PROTEOME database.
  • SEQ DD NO:10 also contains a PAC domain, cycHc nucleotide binding domain and ion transport domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.)
  • SEQ DD NO: 10 contains five tiansmembrane-spanning regions as determined by TMHMMR analysis. Data from further BLAST analyses of the PRODOM and DOMO databases provide additional corroborative evidence that SEQ DD NO: 10 is a potassium channel.
  • SEQ DD NO:l 1 is 86% identical, from residue A94 to residue S785, to rat potassium channel (GenBank DD g2745729) as determined by the Basic Local AHgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiHty score is 0.0, which indicates the probabiHty of obtaining the observed polypeptide sequence aHgnment by chance.
  • BLAST Basic Local AHgnment Search Tool
  • SEQ DD NO: 11 is locaHzed to the plasma membrane, is homologous to rat ether-a-go-go related 2, which is a slowly activating delayed rectifier potassium channel, and may faciHtate the differentiation of pre- vertebral neurons (PROTEOME DD 331276
  • SEQ DD NO:ll also contains a cycHc nucleotide-binding domain and an ion transport protein domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ DD NO: 14 is 38% identical, from residue Q13 to residue S1049, to Schizosaccharomyces pombe membrane ATPase (GenBank DD g3451312) as determined by the Basic Local AHgnment Search Tool (BLAST).
  • SEQ DD NO:14 is locaHzed to the membrane, and is a member of the P-type, Ca2+-type, ATPase subfamily, as determined by BLAST analysis using the PROTEOME database. SEQ DD NO: 14 also contains an E1-E2 ATPase domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ DD NO:14 is a membrane ATPase.
  • SEQ DD NO:19 is 98% identical, from residue Ml to residue L602, to human sodium-dependent Hgh-affinity dicarboxylate transporter (GenBank DD g8132324) as determined by the Basic Local AHgnment Search Tool (BLAST).
  • BLAST Basic Local AHgnment Search Tool
  • the BLAST probabiHty score is 0.0, which indicates the probabiHty of obtaining the observed polypeptide sequence aHgnment by chance.
  • SEQ DD NO: 19 also has homology to proteins that have transporter gene function and are sodium-dependent dicarboxylate transporters, as determined by BLAST analysis using the PROTEOME database.
  • SEQ DD NO:19 also contains a sodium-dependent dicarboxylate transporter domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and BLAST analyses provide further corroborative evidence that SEQ DD NO:19 is a sodium-dependent dicarboxylate transporter.
  • HMM hidden Markov model
  • SEQ DD NO:2 SEQ DD NO:4,-5, SEQ DD NO:7-9, SEQ DD NO.12-13, SEQ DD NO:15-18, and SEQ DD NO:20-26 were analyzed and annotated in a simUar manner.
  • the algorithms and parameters for the analysis of SEQ DD NO:l-26 are described in Table 7. As shown in Table 4, the full length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the fuU length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or ampHfication technologies that identify SEQ DD NO:8-14 or that distinguish between SEQ DD NO:8-14 and related polynucleotides.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte cDNAs derived from tissue-specific cDNA Hbraries or from pooled cDNA
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the fuU length polynucleotides.
  • the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database i.e., those sequences including the designation "ENST”).
  • the polynucleotide fragments described in column 2 maybe derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM” or "NT") or the NCBI RefSeq Protein Sequence Records (i.e.
  • polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
  • a polynucleotide sequence identified as VLJXXXX_N 1 _N 2 _YYYW_N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was appHed, and rTOTis the number of the prediction generated by the algorithm, and N 2 ⁇ 3 ...
  • polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stietching" algorithm.
  • a polynucleotide sequence identified as mXXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stietching" algorithm was appHed, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
  • RefSeq identifier (denoted by " ⁇ M,” “ ⁇ P,” or “NT”) maybe used in place of the GenBank identifier (ie., gBBBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
  • the foHowing Table Hst examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
  • Genomic location and EST composition data are combined to predict the exons and resulting transcript.
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA Hbraries for those fuU length polynucleotides which were assembled using Incyte cDNA sequences.
  • the representative cDNA Hbrary is the Incyte cDNA Hbrary which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides.
  • the tissues and vectors which were used to construct the cDNA Hbraries shown in Table 5 are described in Table 6.
  • Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations.
  • Columns 1 and 2 show the polynucleotide sequence identification number (SEQ DD NO:) and the corresponding Incyte project identification number (PDD) for polynucleotides of the invention.
  • Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST DD), and column 4 shows the identification number for the SNP (SNP DD).
  • Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the fuU- length polynucleotide sequence (CB1 SNP).
  • Column 7 shows the aUele found in the EST sequence.
  • Columns 8 and 9 show the two aHeles found at the SNP site.
  • Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the aUele found in the EST.
  • Columns 11-14 show the frequency of aUele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of aUele 1 in the population was too low to be detected, while n/a (not available) indicates that the aUele frequency was not determined for the population.
  • TRICH variants are one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the TRICH amino acid sequence, and which contains at least one functional or structural characteristic of TRICH.
  • Various embodiments also encompass polynucleotides which encode TRICH.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ DD NO:27-52, which encodes TRICH.
  • the polynucleotide sequences of SEQ DD NO:27-52 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • the invention also encompasses variants of a polynucleotide encoding TRICH.
  • a variant polynucleotide wiU have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding TRICH.
  • a particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ DD NO:27-52 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ DD NO:27-52.
  • Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of TRICH.
  • a polynucleotide variant of the invention is a spHce variant of a polynucleotide encoding TRICH.
  • a spHce variant may have portions which have significant sequence identity to a polynucleotide encoding TRICH, but wiU generaUy have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate spHcing of exons during mRNA processing.
  • a spHce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding TRICH over its entire length; however, portions of the spHce variant wiH have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding TRICH.
  • a polynucleotide comprising a sequence of SEQ DD NO:34 and a polynucleotide comprising a sequence of SEQ DD NO:43 are spHce variants of each other;
  • a polynucleotide comprising a sequence of SEQ DD NO:46 and a polynucleotide comprising a sequence of SEQ DD NO:52 are spHce variants of each other;
  • a polynucleotide comprising a sequence of SEQ DD NO:39 and a polynucleotide comprising a sequence of SEQ DD NO:50 are spHce variants of each other;
  • a polynucleotide comprising a sequence of SEQ DD NO:32, a polynucleotide comprising a sequence of SEQ D NO:36, and a polynucleotide comprising a sequence of SEQ DD NO:37 are spHce variants of
  • polynucleotides which encode TRICH and its variants are generaUy capable of hybridizing to polynucleotides encoding naturaUy occurring TRICH under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding TRICH or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non-naturaUy occurring codons. Codons maybe selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utiHzed by the host.
  • RNA transcripts having more desirable properties such as a greater half-Hfe, than transcripts produced from the naturaUy occurring sequence.
  • the invention also encompasses production of polynucleotides which encode TRICH and TRICH derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic polynucleotide may be inserted into any of the many available expression vectors and ceU systems using reagents weU known in the art.
  • synthetic chemistry may be used to introduce mutations into a polynucleotide encoding TRICH or any fragment thereof.
  • Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ DD NO:27-52 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511). Hybridization conditions, including annealing and wash conditions, are described in "Definitions.''
  • Methods for DNA sequencing are weU known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (AppHed Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE ampHfication system (Invitrogen, Carlsbad CA).
  • sequence preparation is automated with machines such as the MICROLAB 2200 Hquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (AppHed Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (AppHed Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are weU known in the art (Ausubel et al., swpra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853).
  • the nucleic acids encoding TRICH may be extended utiHzing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to ampHfy unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods AppHc. 2:318-322).
  • Another method, inverse PCR uses primers that extend in divergent directions to ampHfy unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (TrigHa, T. et al.
  • a third method involves PCR ampHfication of DNA fragments adjacent to known sequences inhuman and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods AppHc. 1:111-119).
  • multiple restriction enzyme digestions and Hgations maybe used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
  • primers maybe designed using commerciaUy available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate 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 template at temperatures of about 68°C to 72°C
  • Hbraries that have been size-selected to include larger cDNAs.
  • random-primed Hbraries which often include sequences containing the 5' regions of genes, are preferable for situations in which an oHgo d(T)
  • Genomic Hbraries does not yield a fuU-length cDNA. Genomic Hbraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • CapiHary electrophoresis systems which are commerciaUy available maybe used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capiUary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/Hght intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppHed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controUed.
  • CapiUary electrophoresis is especiaHy preferable for sequencing smaU DNA fragments which may be present in limited amounts in a particular sample.
  • TRICH may be cloned in recombinant DNA molecules that direct expression of TRICH, or fragments or functional equivalents thereof, in appropriate host ceHs. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantiaUy the same or a functionaUy equivalent polypeptides maybe produced and used to express TRICH.
  • the polynucleotides of the invention can be engineered using methods generaUy known in the art in order to alter TRICH-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oHgonucleotides may be used to engineer the nucleotide sequences.
  • oHgonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce spHce variants, and so forth.
  • the nucleotides of the present invention maybe subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C et al. (1999) Nat. Biotechnol. 17:793-797; Christians, EC et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of TRICH, such as its biological or enzymatic activity or its abiHty to bind to other molecules or compounds.
  • MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C et al. (1999) Nat. Biotechnol. 17:793-797; Christians, EC et al
  • DNA shuffling is a process by which a Hbrary of gene variants is produced using PCR-mediated recombination of gene fragments. The Hbrary 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.
  • 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.
  • fragments of a given gene maybe 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 naturaUy occurring genes in a directed and controHable manner.
  • polynucleotides encoding TRICH may be synthesized, in whole or in part, using one or more chemical methods weU known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).
  • TRICH itself or a fragment thereof may be synthesized using chemical methods known in the art.
  • peptide synthesis can be performed using various solution-phase or soHd-phase techniques (Creighton, T. (1984) Proteins. Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60; Roberge, J.Y. et al. (1995) Science 269:202-204). Automated synthesis maybe achieved using the ABI 431 A peptide synthesizer (AppHed Biosystems). AdditionaHy, the amino acid sequence of TRICH, or any part thereof, maybe altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturaUy occurring polypeptide.
  • the peptide may be substantiaUy purified by preparative high performance Hquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421).
  • the composition of the synthetic peptides may be corrfirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
  • an appropriate expression vector i.e., a vector which contains the necessary elements for transcriptional and translational contiol of the inserted coding sequence in a suitable host.
  • These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3 'untranslated regions in the vector and in polynucleotides encoding TRICH. Such elements may vary in their sitesngth and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding TRICH. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where a polynucleotide sequence encoding TRICH and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • regulatory sequences such as enhancers, constitutive and inducible promoters, and 5' and 3 'untranslated regions in the vector and in polynucleotides encoding TRICH. Such elements may vary in their sitesngth and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides
  • exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
  • Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host ceU system used (Scharf, D. et al. (1994) Results Probl. CeU Differ. 20:125-162).
  • Methods which are weU known to those skiHed in the art may be used to construct expression vectors containing polynucleotides encoding TRICH and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel et al., supra, ch. 1, 3, and 15). A variety of expression vector host systems may be utilized to contain and express polynucleotides encoding TRICH.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceH systems transformed with viral expression vectors (e.g., cauHflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems (Sambrook, supra; Ausubel et al., supra; Van Heeke, G. and S.M. Schuster (1989) J, Biol. Chem.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceH systems transformed with viral expression vectors
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids maybe used for deHvery of polynucleotides to the targeted organ, tissue, or ceU population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; BuUer, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31:219-226; Verma, I.M. and N. Somia (1997) Nature 389:239-242).
  • the invention is not Hmited by the host ceH employed.
  • a number of cloning and expression vectors maybe selected depending upon the use intended for polynucleotides encoding TRICH.
  • routine cloning, subcloning, and propagation of polynucleotides encoding TRICH can be achieved using a multifunctional E. coli vector such as PBLUESCRTPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Invitrogen).
  • PBLUESCRTPT Stratagene, La JoUa CA
  • PSPORT1 plasmid Invitrogen.
  • vectors maybe useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509).
  • vectors which direct high level expression of TRICH may be used.
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter maybe used.
  • Yeast expression systems may be used for production of TRICH.
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intraceUular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, CA. et al. (1994) Bio/Technology 12:181-184).
  • Plant systems may also be used for expression of TRICH. Transcription of polynucleotides encoding TRICH may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:1631). Alternatively, plant promoters such as the smaU subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; BrogHe, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. CeU Differ. 17:85-105). These constructs can be introduced into plant ceUs by direct DNA transformation or pathogen-mediated transfection (The McGraw HiU Yearbook of Science and Technology (1992) McGraw HiU, New York NY, pp. 191-196).
  • a number of viral-based expression systems may be utilized.
  • polynucleotides encoding TRICH may be Hgated • into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses TRICH in host ceUs (Logan, J. and T. Shehk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammaHan host ceUs.
  • SV40 or EBV-based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs may also be employed to deHver larger fragments of DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and deHvered via conventional deHvery methods (Hposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355).
  • TRICH in ceU Hues For long term production of recombinant proteins in mammaHan systems, stable expression of TRICH in ceU Hues is preferred.
  • polynucleotides encoding TRICH can be transformed into ceH lines using expression vectors which may contain viral origins of repHcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. FoUowing the introduction of the vector, ceUs maybe aUowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence aUows growth and recovery of ceUs which successfully express the introduced sequences.
  • Resistant clones of stably transformed ceHs may be propagated using tissue culture techniques appropriate to the ceU type.
  • any number of selection systems may be used to recover transformed ceU lines. These include, but are not Hmited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltiansferase genes, for use in tk and apr ceUs, respectively (Wigler, M. et al. (1977) CeH 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823). Also, antimetaboHte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and pat confer resistance to chlorsuHuron and phosphinotricin acetyltransferase, respectively
  • trpB and hisD which alter ceUular requirements for metaboHtes
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ - glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin maybe used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131).
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding TRICH is inserted within a marker gene sequence
  • transformed ceHs containing polynucleotides encoding TRICH can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding TRICH under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaHy indicates expression of the tandem gene as weU.
  • host ceUs that contain the polynucleotide encoding TRICH and that express TRICH may be identified by a variety of procedures known to those of skiU in the art. These procedures include, but are not Hmited to, DNA-DNA or DNA-RNA hybridizations, PCR ampHfication, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of TRICH using either specific polyclonal or monoclonal antibodies are known in the art.
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated ceU sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding TRICH include oHgolabeling, nick translation, end-labeling, or PCR ampHfication using a labeled nucleotide.
  • polynucleotides encoding TRICH, or any fragments thereof maybe cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 RNA polymerase
  • reporter molecules or labels which may be used for ease of detection include radionucHdes, enzymes, fluorescent, chemilummescent, or chromogenic agents, as weH as substrates, cofactors, inhibitors, magnetic particles, and the Hke.
  • Host ceUs transformed with polynucleotides encoding TRICH may be cultured under conditions suitable for the expression and recovery of the protein from ceU culture.
  • the protein produced by a transformed ceU may be secreted or retained intraceUularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode TRICH may be designed to contain signal sequences which direct secretion of TRICH through a prokaryotic or eukaryotic ceU membrane.
  • a host ceH strain maybe chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not Hmited to, acetylation, carboxylation, glycosylation, phosphorylation, Hpidation, and acylation.
  • Post-tianslational processing which cleaves a "prepro” or “pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host ceUs which have specific ceUular machinery and characteristic mechanisms for post-tianslational activities (e.g., CHO, HeLa, MDCK, HEK293, and WD 8) are available from the American Type Culture CoUection (ATCC, Manassas VA) and maybe chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture CoUection
  • Manassas VA American Type Culture CoUection
  • natural, modified, or recombinant polynucleotides encoding TRICH may be Hgated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric TRICH protein containing a heterologous moiety that can be recognized by a commerciaUy available antibody may facilitate the screening of peptide Hbraries for inhibitors of TRICH activity.
  • Heterologous protein and peptide • moieties may also facilitate purification of fusion proteins using commerciaUy available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-tiansferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobiHzed glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commerciaUy available monoclonal and polyclonal antibodies that specificaUy recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the TRICH encoding sequence and the heterologous protein sequence, so that TRICH may be cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). A variety of commerciaUy available kits may also be used to faciHtate expression and purification of fusion proteins.
  • synthesis of radiolabeled TRICH maybe achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
  • TRICH fragments of TRICH, or variants of TRICH may be used to screen for compounds that specificaUy bind to TRICH.
  • One or more test compounds maybe screened for specific binding to TRICH.
  • 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to TRICH.
  • Examples of test compounds can include antibodies, anticalins, oHgonucleotides, proteins (e.g., Hgands or receptors), or smaU molecules.
  • variants of TRICH can be used to screen for binding of test compounds, such as antibodies, to TRICH, a variant of TRICH, or a combination of TRICH and/or one or more variants TRICH.
  • a variant of TRICH can be used to screen for compounds that bind to a variant of TRICH, but not to TRICH having the exact sequence of a sequence of SEQ DD NO:l-26.
  • TRICH variants used to perform such screening can have a range of about 50% to about 99% sequence identity to TRICH, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
  • a compound identified in a screen for specific binding to TRICH can be closely related to the natural Hgand of TRICH, e.g., a Hgand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (CoHgan, J.E. et al. (1991) Current Protocols in Immunology l(2):Chapter 5).
  • the compound thus identified can be a natural Hgand of a receptor TRICH (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132- 140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
  • a compound identified in a screen for specific binding to TRICH can be closely related to the natural receptor to which TRICH binds, at least a fragment of the receptor, or a fragment of the receptor including aU or a portion of the Hgand binding site or binding pocket.
  • the compound may be a receptor for TRICH which is capable of propagating a signal, or a decoy receptor for TRICH which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Curr. Opin. CeU Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328- 336).
  • the compound can be rationaUy designed using known techniques.
  • Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer Hnked to the Fc portion of human IgG j (Taylor, P.C. et al. (2001) Curr. Opin. Immunol. 13:611-616).
  • TNF tumor necrosis factor
  • two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to TRICH, fragments of TRICH, or variants of TRICH.
  • the binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of TRICH.
  • an antibody can be selected such that its binding specificity aUows for preferential identification of specific fragments or variants of TRICH.
  • an antibody can be selected such that its binding specificity aUows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of TRICH.
  • anticalins can be screened for specific binding to TRICH, fragments of TRICH, or variants of TRICH.
  • Anticalins are Hgand-binding proteins that have been constructed based on a Hpocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177-R184; Skerra, A. (2001) J. Biotechnol. 74:257-275).
  • the protein architecture of Hpocalins can include a beta-barrel having eight antiparaUel beta-strands, which supports four loops at its open end.
  • These loops form the natural Hgand-binding site of the Hpocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities.
  • the amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
  • screening for compounds which specificaUy bind to, stimulate, or inhibit TRICH involves producing appropriate ceUs which express TRICH, either as a secreted protein or on the ceH membrane.
  • Preferred ceHs include ceUs from mammals, yeast, Drosophila, or E. coli.
  • CeUs expressing TRICH or ceU membrane fractions which contain TRICH are then contacted with a test compound and binding, stimulation, or inhibition of activity of either TRICH or the compound is analyzed.
  • An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
  • the assay may comprise the steps of combining at least one test compound with TRICH, either in solution or affixed to a soHd support, and detecting the binding of TRICH to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor. AdditionaUy, the assay maybe carried out using ceU-free preparations, chemical Hbraries, or natural product mixtures, and the test compound(s) maybe free in solution or affixed to a soHd support.
  • An assay can be used to assess the abiHty of a compound to bind to its natural Hgand and/or to inhibit the binding of its natural Hgand to its natural receptors.
  • assays include radio- labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No. 6,372,724.
  • one or more arnino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its abiHty to bind to its natural Hgands (Matthews, D.J. and J.A. WeHs. (1994) Chem. Biol. 1:25-30).
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a Hgand) to improve or alter its abiHty to bind to its natural receptors (Cunningham, B.C. and J.A. WeUs (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-10988).
  • a polypeptide compound such as a Hgand
  • TRICH, fragments of TRICH, or variants of TRICH may be used to screen for compounds that modulate the activity of TRICH.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for TRICH activity, wherein TRICH is combined with at least one test compound, and the activity of TRICH in the presence of a test compound is compared with the activity of TRICH in the absence of the test compound. A change in the activity of TRICH in the presence of the test compound is indicative of a compound that modulates the activity of TRICH.
  • a test compound is combined with an in vitro or ceU-free system comprising TRICH under conditions suitable for TRICH activity, and the assay is performed.
  • a test compound which modulates the activity of TRICH may do so indirectly and need not come in direct contact with the test compound. At least one and up to a pluraHty of test compounds maybe screened.
  • polynucleotides encoding TRICH or their mammaHan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceHs.
  • ES embryonic stem
  • mouse ES ceUs such as the mouse 129/SvJ ceU line
  • the ES ceUs are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • the vector integrates into the corresponding region of the host genome by homologous recombination.
  • 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.
  • Transformed ES ceUs are identified and microinjected into mouse ceU blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgicaUy 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.
  • Polynucleotides encoding TRICH may also be manipulated in vitro in ES ceUs derived from human blastocysts.
  • Human ES ceHs have the potential to differentiate into at least eight separate ceU lineages including endoderm, mesoderm, and ectodermal ceU types. These ceU Hneages differentiate into, for example, neural ceUs, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding TRICH can also be used to create "knockin" humanized animals (pigs) or tiansgenic animals (mice or rats) to model human disease.
  • knockin technology a region of a polynucleotide encoding TRICH is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome.
  • Transformed ceUs are injected into blastulae, and the blasrulae 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.
  • a mammal inbred to overexpress TRICH e.g., by secreting TRICH in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74). THERAPEUTICS
  • TRICH appears to play a role in transport, neurological, muscle, immunological and ceU proHferative disorders.
  • TRICH or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of TRICH.
  • disorders include, but are not Hmited to, a transport disorder such as akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia, cystic fibrosis, Becker's muscular dystrophy, BeU's palsy, Charcot-Marie Tooth disease, diabetes mellitus, diabetes insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic paralysis, normokalemic periodic paralysis, Parkinson's disease, maHgnant 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, bradyarrytbmi
  • a transport disorder
  • a vector capable of expressing TRICH or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of TRICH including, but not Hmited to, those described above.
  • composition comprising a substantiaUy purified TRICH in conjunction with a suitable pharmaceutical carrier maybe administered to a subject to treat or prevent a disorder associated with decreased expression or activity of TRICH including, but not Hmited to, those provided above.
  • an agonist which modulates the activity of TRICH may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of TRICH including, but not Hmited to, those Hsted above.
  • an antagonist of TRICH may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of TRICH.
  • disorders include, but are not Hmited to, those transport, neurological, muscle, immunological and ceU proHferative disorders described above.
  • an antibody which specificaUy binds TRICH may be used directly as an antagonist or indirectly as a targeting or deHvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express TRICH.
  • a vector expressing the complement of the polynucleotide encoding TRICH may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of TRICH including, but not Hmited to, those described above.
  • any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments maybe administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergisticaUy to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • TRICH An antagonist of TRICH may be produced using methods which are generaUy known in the art.
  • purified TRICH maybe used to produce antibodies or to screen Hbraries of pharmaceutical agents to identify those which specificaUy bind TRICH.
  • Antibodies to TRICH may also be generated using methods that are weU known in the art. Such antibodies may include, but are not Hmited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Hbrary.
  • NeutraHzing antibodies i.e., those which inhibit dimer formation
  • Single chain antibodies e.g., from camels or Hamas
  • Single chain antibodies maybe potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).
  • various hosts including goats, rabbits, rats, mice, camels, dromedaries, Uamas, humans, and others maybe immunized by injection with TRICH or with any fragment or oHgopeptide thereof which has immunogenic properties.
  • various adjuvants maybe used to increase immunological response.
  • Such adjuvants include, but are not Hmited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dmitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especiaUy preferable.
  • the oHgopeptides, peptides, or fragments used to induce antibodies to TRICH have an amino acid sequence consistmg of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these oHgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of TRICH amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to TRICH maybe prepared using any technique which provides for the production of antibody molecules by continuous ceU lines in culture. These include, but are not Hmited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole, S.P. et al. (1984) Mol. CeU Biol. 62:109-120).
  • chimeric antibodies such as the spHcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).
  • techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce TRICH-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition maybe generated by chain shuffling from random combinatorial immunoglobulin Hbraries (Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137).
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin Hbraries or panels of highly specific binding reagents as disclosed in the Hterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for TRICH may also be generated.
  • fragments include, but are not limited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab ⁇ )2 fragments.
  • Fab expression Hbraries maybe constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al. (1989) Science 246:1275-1281).
  • immunoassays maybe used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are weU known in the art.
  • Such immunoassays typicaUy involve the measurement of complex formation between TRICH and its specific antibody.
  • a two-site, monoclonal-based immunoassay utiHzing monoclonal antibodies reactive to two non-interfering TRICH epitopes is generaUy used, but a competitive binding assay may also be employed (Pound, supra).
  • K a is defined as the molar concentration of TRICH-antibody complex divided by the molar concentrations of free antigen and free antibody under equiHbrium conditions.
  • K a association constant
  • the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular TRICH epitope, represents a true measure of affinity.
  • High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the TRICH- antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of TRICH, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, E L Press, Washington DC; LiddeU, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quaHty and suitabiHty of such preparations for certain downstream appHcations.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generaUy employed in procedures requiring precipitation of TRICH-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quaHty and usage in various appHcations, are generaUy available (Catty, supra; CoHgan et al., supra).
  • polynucleotides encoding TRICH may be used for therapeutic purposes.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oHgonucleotides) to the coding or regulatory regions of the gene encoding TRICH.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oHgonucleotides
  • antisense oHgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding TRICH (Agrawal, S., ed. (1996) Antisense Therapeutics. Humana Press, Totawa NJ).
  • Antisense sequences can be deHvered intraceHularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the ceUular sequence encoding the target protein (Slater, J.E. et al. (1998) J. AUergy CHn. Immunol. 102:469-475; Scanlon, K.J. et al. (1995) 9:1288-1296).
  • Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (MiUer, A.D.
  • polynucleotides encoding TRICH maybe 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 (SCDD)-Xl disease characterized by X- Hnked 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.
  • SCDD severe combined immunodeficiency
  • ADA adenosine deaminase
  • TRICH hepatitis B or C virus
  • fungal parasites such as Candida albicans and Paracoccidioides bras ⁇ liensis
  • protozoan parasites such as Plasmodiumfalciparum and Trypanosoma cruzi.
  • diseases or disorders caused by deficiencies in TRICH are treated by constructing mammaHan expression vectors encoding TRICH and introducing these vectors by mechanical means into TRICH-deficient ceUs.
  • Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual ceHs, (ii) ballistic gold particle deHvery, (ui) Hposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) CeU 91:501-510; Boulay, J.-L and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).
  • PCDNA 3.1 EPITAG
  • PRCCMV2, PREP PREP
  • PVAX PCR2-TOPOTA vectors
  • PCMV-SCRIPT PCMV-TAG
  • PEGSH/PERV Stratagene, La JoUa CA
  • PTET-OEF PTET-ON
  • PTRE2 PTRE2-LUC PTK-HYG Clontech, Palo Alto CA.
  • TRICH maybe expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (n) an inducible promoter (e.g., the tetracycHne-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
  • TRANSFECTION KIT available from Invitiogen
  • aUow one with ordinary skiU in the art to deHver polynucleotides to target ceUs in culture and require minimal effort to optimize experimental parameters.
  • transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845).
  • the introduction of DNA to primary ceUs requires modification of these standardized mammaHan transfection protocols.
  • diseases or disorders caused by genetic defects with respect to TRICH expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding TRICH under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (u) appropriate RNA packaging signals, and (in) a Rev-responsive element (RRE) along with additional retiovirus cts-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commerciaUy available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
  • the vector is propagated in an appropriate vector producing ceU line (VPCL) that expresses an envelope gene with a tropism for receptors on the target ceHs 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 A.D. MiUer (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al.
  • VSVg vector producing ceU line
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging ceU lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of ceUs (e.g., CD4 + T-ceUs), and the return of transduced ceUs to a patient are procedures weU known to persons skiUed in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al.
  • an adenovirus-based gene therapy deHvery system is used to deHver polynucleotides encoding TRICH to ceUs which have one or more genetic abnormaHties with respect to the expression of TRICH.
  • the construction and packaging of adenovirus-based vectors are weH known to those with ordinary skiH in the art.
  • RepHcation 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).
  • PotentiaUy useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference.
  • Adenovirus vectors for gene therapy For adenoviral vectors, see also Antinozzi, P.A. et al. (1999; Annu. Rev. Nutr. 19:511-544) and Verma, I.M. and N. Somia (1997; Nature 18:389:239-242).
  • a herpes-based, gene therapy deHvery system is used to deHver polynucleotides encoding TRICH to target ceUs which have one or more genetic abnormaHties with respect to the expression of TRICH.
  • the use of herpes simplex virus (HSV)-based vectors may be especiaUy valuable for introducing TRICH to ceUs of the central nervous system, for which HSV has a tropism.
  • the construction and packaging of herpes-based vectors are weU known to those with ordinary skiU in the art.
  • a repHcation-competent herpes simplex virus (HSV) type 1-based vector has been used to deHver a reporter gene to the eyes of primates (Liu, X.
  • HSV-1 virus vector has also been disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
  • U.S. Patent No. 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 ceU 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.
  • 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).
  • the manipulation of cloned herpesvirus sequences, the generation of recombinant virus foUowing the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of ceUs with herpesvirus are techniques weU known to those of ordinary skiU in the art.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deHver polynucleotides encoding TRICH to target ceUs.
  • SFV Semliki Forest Virus
  • SFV Semliki Forest Virus
  • SFV Semliki Forest Virus
  • alphavirus RNA repHcation a subgenomic RNA is generated that normaUy encodes the viral capsid proteins.
  • inserting the coding sequence for TRICH into the alphavirus genome in place of the capsid-coding region results in the production of a large number of TRICH-coding RNAs and the synthesis of high levels of TRICH in vector transduced ceUs.
  • alphavirus infection is typicaUy associated with ceU lysis within a few days
  • the abiHty to estabHsh a persistent infection in hamster normal kidney ceHs (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic repHcation of alphaviruses can be altered to suit the needs of the gene therapy appHcation (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the wide host range of alphaviruses wiU aU ow the introduction of TRICH into a variety of ceU types.
  • the specific transduction of a subset of ceUs in a population may require the sorting of ceUs prior to transduction.
  • the methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA tiansfections, and performing alphavirus infections, are weU known to those with ordinary skiU in the art.
  • OHgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
  • inhibition can be achieved using triple heHx base-pairing methodology.
  • Triple heHx pairing is useful because it causes inhibition of the abiHty of the double heHx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • Recent therapeutic advances using triplex DNA have been described in the Hterature (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura PubHshing, Mt. Kisco NY, pp. 163-177).
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules
  • Ribozymes may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding TRICH.
  • RNA sequences of between 15 and 20 ribonucleotides may be evaluated for secondary structural features which may render the oHgonucleotide inoperable.
  • the suitabiHty of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oHgonucleotides using ribonuclease protection assays.
  • RNA molecules may be generated by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemicaUy synthesizing oHgonucleotides such as soHd phase phosphoramidite chemical synthesis.
  • RNA molecules maybe generated by in vitro and in vivo transcription of DNA molecules encoding TRICH. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
  • these cDNA constructs that synthesize complementary RNA, constitutively or induc ⁇ bly, can be introduced into ceU Hues, ceUs, or tissues.
  • RNA molecules may be modified to increase intraceUular stability and half-Hfe. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3 'ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding TRICH.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oHgonucleotides, antisense oHgonucleotides, triple heHx-forming oHgonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
  • a compound which specificaUy inhibits expression of the polynucleotide encoding TRICH may be therapeuticaUy useful, and in the treatment of disorders associated with decreased TRICH expression or activity, a compound which specificaUy promotes expression of the polynucleotide encoding TRICH may be therapeuticaUy useful.
  • At least one, and up to a pluraHty, of test compounds maybe screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commerciaUy-available or proprietary Hbrary of naturaUy-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a Hbrary of chemical compounds created combinatoriaUy or randomly.
  • a sample comprising a polynucleotide encoding TRICH is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabiHzed ceU, or an in vitro ceU-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding TRICH are assayed by any method commonly known in the art.
  • TypicaUy the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding TRICH.
  • the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human ceU line such as HeLa ceU (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
  • a particular embodiment of the present invention involves screening a combinatorial Hbrary of oHgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oHgonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691). Many methods for introducing vectors into ceUs or tissues are available and equaHy suitable for use in vivo, in vitro, and ex vivo.
  • oHgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oHgonucleotides
  • vectors For ex vivo therapy, vectors maybe introduced into stem ceHs taken from the patient and clonaHy propagated for autologous transplant back into that same patient. DeHvery by transfection, by Hposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462- 466).
  • any of the therapeutic methods described above maybe appHed to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
  • An additional embodiment of the invention relates to the administration of a composition which generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
  • Excipients may include, for example, sugars, starches, ceHuloses, gums, and proteins.
  • Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack PubHshing, Easton PA).
  • Such compositions may consist of TRICH, antibodies to TRICH, and mimetics, agonists, antagonists, or inhibitors of TRICH.
  • compositions utiHzed in this invention may be administered by any number of routes including, but not Hmited to, oral, intravenous, intramuscular, intra-arterial, intiameduHary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • compositions for pulmonary administration may be prepared in Hquid or dry powder form. These compositions are generaUy aerosoHzed immediately prior to inhalation by the patient.
  • smaU molecules e.g. traditional low molecular weight organic drugs
  • aerosol deHvery of fast- acting formulations is weU-known in the art.
  • macromolecules e.g. larger peptides and proteins
  • recent developments in the field of pulmonary deHvery via the alveolar region of the lung have enabled the practical deHvery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848).
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is weU within the capability of those skiUed in the art.
  • SpeciaHzed forms of compositions maybe prepared for direct intraceUular deHvery of macromolecules comprising TRICH or fragments thereof.
  • Hposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular deHvery of the macromolecule.
  • TRICH or a fragment thereof may be joined to a short cationic N- terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the ceUs of aU tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeuticaUy effective dose can be estimated initiaUy either in ceU culture assays, e.g., of neoplastic ceUs, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • An ar ⁇ mal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeuticaUy effective dose refers to that amount of active ingredient, for example
  • Therapeutic efficacy and toxicity maybe deteimined by standard pharmaceutical procedures in ceU cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD S0 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 /ED 50 ratio.
  • Compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from ceU culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with Httle or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • the exact dosage wiU be determined by the practitioner, in Hght of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on thehalf-Hfe and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of deHvery is provided in the Hterature and generaUy available to practitioners in the art. Those skiUed in the art wiU employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, deHvery of polynucleotides or polypeptides wiH be specific to particular ceUs, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specificaUy bind TRICH may be used for the diagnosis of disorders characterized by expression of TRICH, or in assays to monitor patients being treated with TRICH or agonists, antagonists, or inhibitors of TRICH.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for TRICH include methods which utiHze the antibody and a label to detect TRICH inhuman body fluids or in extracts of ceUs or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • TRICH TRICH
  • ELISAs RIAs
  • FACS FACS-activated cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic mas, and others.
  • the amount of standard complex formation may be quantitated by various methods, such as photometric means.
  • Quantities of TRICH expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • polynucleotides encoding TRICH may be used for diagnostic purposes.
  • the polynucleotides which maybe used include oHgonucleotides, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of TRICH may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of TRICH, and to monitor regulation of TRICH levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding TRICH or closely related molecules may be used to identify nucleic acid sequences which encode TRICH.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or ampHfication wiU determine whether the probe identifies only naturaUy occurring sequences encoding TRICH, aUeHc variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the TRICH encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ DD NO:27-52 or from genomic sequences including promoters, enhancers, and rntions of the TRICH gene.
  • Means for producing specific hybridization probes for polynucleotides encoding TRICH include the cloning of polynucleotides encoding TRICH or TRICH derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commerciaUy available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionucHdes such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the Hke.
  • Polynucleotides encoding TRICH may be used for the diagnosis of disorders associated with expression of TRICH.
  • disorders include, but are not Hmited to, a transport disorder such as akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia, cystic fibrosis, Becker's muscular dystrophy, BeU's palsy, Charcot-Marie Tooth disease, diabetes meUitus, diabetes insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic paralysis, normokalemic periodic paralysis, Parkinson's disease, maHgnant 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,
  • Polynucleotides encoding TRICH maybe used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-Hke assays; and in microarrays utiHzing fluids or tissues from patients to detect altered TRICH expression. Such quaHtative or quantitative methods are weU known in the art.
  • polynucleotides encoding TRICH may be used in assays that detect the presence of associated disorders, particularly those mentioned above.
  • Polynucleotides complementary to sequences encoding TRICH may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes.
  • the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding TRICH in the sample indicates the presence of the associated disorder.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is estabHshed. This maybe accompHshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding TRICH, under conditions suitable for hybridization or ampHfication.
  • Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantiaUy purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabHsh the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive tieatment earHer, thereby preventing the development or further progression of the cancer.
  • oHgonucleotides designed from the sequences encoding TRICH may involve the use of PCR. These oHgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro. OHgomers wiU preferably contain a fragment of a polynucleotide encoding TRICH, or a fragment of a polynucleotide complementary to the polynucleotide encoding TRICH, and wiU be employed under optimized conditions for identification of a specific gene or condition. OHgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oHgonucleotide primers derived from polynucleotides encoding TRICH maybe 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.
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • oHgonucleotide primers derived from polynucleotides encoding TRICH are used to ampHfy DNA using the polymerase chain reaction (PCR).
  • the DNA maybe derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the Hke.
  • 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.
  • the oHgonucleotide primers are fluorescently labeled, which aUows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
  • AdditionaUy sequence database analysis methods, termed in siHco SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs maybe detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • SNPs maybe used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes meUitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceU anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as Hfe-threatening toxicity.
  • N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-Hpoxygenase pathway.
  • Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weH as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. and Z. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin. Neurobiol. 11:637-641).
  • TRICH TRICH-Relabeling or biotinylating nucleotides
  • coampHfication of a control nucleic acid and interpolating results from standard curves
  • the speed of quantitation of multiple samples maybe accelerated by rurrning the assay in a high-throughput format where the oHgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oHgonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray.
  • the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information ma be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to momtor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
  • this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient.
  • therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • TRICH fragments of TRICH, or antibodies specific for TRICH may be used as elements on a microarray.
  • the microarray may be used to monitor or measure protein- protein interactions, drug-target interactions, and gene expression profiles, as described above.
  • a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or ceU type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or ceU 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 (SeiHiamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484; hereby expressly incorporated by reference herein).
  • a transcript image maybe generated by hybridizing the polynucleotides of the present invention or their complements to the totaHty of transcripts or reverse transcripts of a particular tissue or ceU type.
  • the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a pluraHty of elements on a microarray.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, ceU Hues, biopsies, or other biological samples.
  • the transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a ceH line.
  • Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as weH as toxicological testing of industrial and naturaUy-occurring environmental compounds.
  • AU compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechamsms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Sterner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471).
  • f a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
  • the toxicity of a test compound can be assessed by treating a biological sample 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 tianscript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
  • proteome refers to the global pattern of protein expression in a particular tissue or ceU type.
  • 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 ceU's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU type.
  • 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 visuaHzed in the gel as discrete and uniquely positioned spots, typicaUy by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
  • the optical density of each protein spot is generaUy proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment.
  • the proteins in the spots are partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage foHowed by mass spectrometry.
  • the identity of the protein in a spot maybe determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data maybe obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for TRICH to quantify the levels of TRICH expression.
  • 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. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection maybe 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 be analyzed in paraUel 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 J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reHable and informative in such cases.
  • 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 polypeptides of the present invention.
  • 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 polypeptides 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.
  • Microarrays may be prepared, used, and analyzed using methods known in the art (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 appHcation WO95/251116; Shalon, D. et al. (1995) PCT appHcation WO95/35505; HeUer, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; HeUer, M.J. et al. (1997) U.S. Patent No. 5,605,662).
  • Various types of microarrays are weU known and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A Practical Approach, Oxford University Press, London).
  • nucleic acid sequences encoding TRICH may be used to generate hybridization probes useful in mapping the naturaUy occurring genomic sequence.
  • Either coding or noncoding sequences maybe used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentiaUy cause undesired cross hybridization during chromosomal mapping.
  • the sequences maybe mapped to a particular chromosome, to a specific region of a chromosome, or to artificial cliromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA Hbraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, BJ. (1991) Trends Genet. 7:149-154).
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA Hbraries
  • nucleic acid sequences may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357).
  • RFLP restriction fragment length polymorphism
  • Fluorescent in situ hybridization maybe correlated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online MendeHan Inheritance in Man (OMLM) World Wide Web site. Correlation between the location of the gene encoding TRICH on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
  • OMLM Online MendeHan Inheritance in Man
  • In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using estabHshed chromosomal markers, maybe used for extending genetic maps.
  • physical mapping techniques such as linkage analysis using estabHshed chromosomal markers
  • linkage analysis using estabHshed chromosomal markers maybe used for extending genetic maps.
  • the placement of a gene on the chromosome of another mammaHan species, such as mouse may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation (Gatti, R.A. et al. (1988) Nature 336:577-580).
  • the nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • TRICH its catalytic or irnmunogenic fragments, or oHgopeptides thereof can be used for screening Hbraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a soHd support, borne on a ceH surface, or located intiaceUularly. The formation of binding complexes between TRICH and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al. (1984) PCT appHcation WO84/03564).
  • large numbers of different smaU test compounds are synthesized on a soHd substrate.
  • the test compounds are reacted with TRICH, or fragments thereof, and washed.
  • Bound TRICH is then detected by methods weU known in the art.
  • Purified TRICH can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobiHze it on a soHd support.
  • nucleotide sequences which encode TRICH may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not Hmited to, such properties as the triplet genetic code and specific base pair interactions.
  • Incyte cDNAs were derived from cDNA Hbraries described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • TRIZOL Invitrogen
  • poly(A)+ RNA was isolated using oHgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • Stratagene was provided with RNA and constructed the corresponding cDNA Hbraries. Otherwise, cDNA was synthesized and cDNA Hbraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al, supra, ch. 5). Reverse transcription was initiated using oHgo d(T) or random primers. Synthetic oHgonucleotide adapters were Hgated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • the cDNA was size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
  • cDNAs were Hgated into compatible restriction enzyme sites of the polyHnker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK- CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or plNCY (Incyte Genomics), or derivatives thereof.
  • a suitable plasmid e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK- CMV plasmid (Stratagene),
  • coli ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Invitrogen.
  • Plasmids were purified using at least one of the foUowing: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E. A.L. PREP 96 plasmid purification kit from QIAGEN. FoUowing precipitation, plasmids were resuspended in 0.1 ml of distiUed water and stored, with or without lyophiHzation, at 4°C
  • plasmid DNA was ampHfied from host ceU lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-weU plates, and the concentration of ampHfied plasmid DNA was quantified fluorometricaUy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN D fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
  • Incyte cDNA recovered in plasmids as described in Example D were sequenced as foUows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (AppHed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the
  • cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or suppHed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppHed Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (AppHed 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 (Ausubel et al, supra, ch. 7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VDI.
  • the polynucleotide sequences derived from Incyte cDNAs were vaHdated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof were then queried against a selection of pubHc databases such as the GenBank primate, rodent, mammaHan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, LNCY, and TIGRFAM (Haft, D.H.
  • pubHc databases such as the GenBank primate, rodent, mammaHan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM
  • HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244).
  • HMM is a probabilistic approach which analyzes consensus primary structures of gene famiHes; see, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.
  • the queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
  • the Incyte cDNA sequences were assembled to produce full length polynucleotide sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to fuU length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
  • the fuU length polynucleotide sequences were tianslated to derive the corresponding fuU length polypeptide sequences.
  • a polypeptide may begin at any of the methionine residues of the fuU length translated polypeptide.
  • FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM, LNCY, and TIGRFAM; and HMM-based protein domain databases such as SMART.
  • GenBank protein databases Genpept
  • PROTEOME databases
  • BLOCKS BLOCKS
  • PRINTS DOMO
  • PRODOM hidden Markov model
  • Prosite Prosite
  • HMM-based protein family databases such as PFAM, LNCY, and TIGRFAM
  • HMM-based protein domain databases such as SMART.
  • FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda CA)
  • Polynucleotide and polypeptide sequence aHgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence aHgnment program (DNASTAR), which also calculates the percent identity between aHgned sequences.
  • Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of
  • Incyte cDNA and full length sequences and provides appHcable descriptions, references, and threshold parameters.
  • the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, aU of which are incorporated by reference herein in their entirety, and the fourth column presents, where appHcable, the scores, probabiHty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabiHty value, the greater the identity between two sequences).
  • Genscan is a FASTA database of polynucleotide and polypeptide sequences.
  • the maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode transporters and ion channels, the encoded polypeptides were analyzed by querying against PFAM models for transporters and ion channels.
  • Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubHc databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or pubHc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.
  • FuU length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubHc cDNA sequences using the assembly process described in Example DI. Alternatively, fuH length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
  • Partial DNA sequences were extended to fuU length with an algorithm based on BLAST analysis.
  • First, partial cDNAs assembled as described in Example ID were queried against pubHc databases such as the GenBank primate, rodent, mammaHan, vertebrate, and eukaryote databases using the BLAST program.
  • the nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV.
  • a chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the tianslated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
  • HSPs high-scoring segment pairs
  • GenBank protein homolog the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubHc human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene. VI. Chromosomal Mapping of TRICH Encoding Polynucleotides
  • sequences which were used to assemble SEQ DD NO:27-52 were compared with sequences from the Incyte LIFESEQ database and pubHc domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ DD NO:27-52 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from pubHc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of aH sequences of that cluster, including its particular SEQ DD NO:, to that map location.
  • pubHc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped
  • Map locations are represented by 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.
  • centiMorgan cM
  • centiMorgan 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 Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the pubHc, such as the NCBI "GeneMap'99" World Wide Web site
  • 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 ceU type or tissue have been bound (Sambrook, supra, ch. 7; Ausubel et al, supra, ch. 4).
  • 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 normaHzed value between 0 and 100, and is calculated as foUows: the BLAST score is multipHed 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 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 quaHty in a BLAST aHgnment. 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.
  • polynucleotides encoding TRICH are analyzed with respect to the tissue sources from which they were derived. For example, some fuU length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example ID). Each cDNA sequence is derived from a cDNA Hbrary constructed from a human tissue.
  • Each human tissue is classified into one of the foUowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitaHa, female; genitaHa, male; germ ceUs; hemic and immune system; Hver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of Hbraries in each category is counted and divided by the total number of Hbraries across aU categories.
  • each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceUHne, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of Hbraries in each category is counted and divided by the total number of Hbraries across aU categories.
  • the resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding TRICH.
  • cDNA sequences and cDNA Hbrary/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
  • FuU length polynucleotides are produced by extension of an appropriate fragment of the fuU length molecule using oHgonucleotide primers designed from this fragment.
  • One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3 ' extension of the known fragment.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate 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 was avoided.
  • Selected human cDNA Hbraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
  • the concentration of DNA in each weU was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l of undiluted PCR product into each weU of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing the DNA to bind to the reagent.
  • the plate was scanned in a Fluoroskan D (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • Step 1 94 °C, 3 min
  • Step 2 94°C, 15 sec
  • Step 3 60°C, 1 min
  • Step 4 72°C, 2 min
  • Step 5 steps 2, 3, and 4 repeated 29 times
  • Step 6 72°C, 5 min
  • Step 7 storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reampHfied using the same conditions as described above.
  • SNPs single nucleotide polymorphisms
  • Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze aUele frequencies at the SNP sites in four different human populations.
  • the Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three deciualan, and two Amish individuals.
  • the African population comprised 194 individuals (97 male, 97 female), aU African Americans.
  • the Hispanic population comprised 324 individuals (162 male, 162 female), aH Mexican Hispanic.
  • the Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
  • AHele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no aHeHc variance in this population were not further tested in the other three populations.
  • Hybridization probes derived from SEQ DD NO:27-52 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oHgonucleotides, consisting of about 20 base pairs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments.
  • OHgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oHgomer, 250 Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oHgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextoanbead column (Amersham Biosciences).
  • An aHquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the foUowing endonucleases: Ase I, Bgl D, Eco RI, Pst I, Xba L or Pvu D (DuPont NEN).
  • the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & SchueU, Durham NH). Hybridization is carried out for 16 hours at 40 °C To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuaHzed using autoradiography or an alternative imaging means and compared.
  • the linkage or synthesis of array elements upon a microarray can be achieved utiHzing photoHthography, piezoelectric printing (ink-jet printing; see, e.g., Baldeschweiler et al, supra), mechanical microspotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and soHd with a non-porous surface (Schena, M., ed. (1999) DNA Microarrays: A Practical Approach, Oxford University Press, London). Suggested substiates include siHcon, siHca, glass sHdes, glass chips, and siHcon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using available methods and machines weH known to those of ordinary skiU in the art and may contain any appropriate number of elements (Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; MarshaU, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).
  • FuH length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oHgomers thereof may comprise the elements of the microarray. Fragments or oHgomers suitable for hybridization can be selected using software weU known in the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass spectrometry maybe used for detection of hybridization.
  • RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oHgo-(dT) ceHulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-tianscriptase, 0.05 pg/ ⁇ l oHgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBRIGHT kits (Incyte Genomics).
  • Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. 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. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech, Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol.
  • Microarray Preparation Sequences of the present invention are used to generate array elements. Each array element is ampHfied from bacterial ceHs containing vectors with cloned cDNA inserts. PCR ampHfication uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are ampHfied in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g.
  • AmpHfied array elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
  • Purified array elements are immobilized on polymer-coated glass sHdes.
  • Glass microscope sHdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distiUed water washes between and after treatments.
  • Glass sHdes are etched in 4% hydrofluoric acid (VWR
  • Array elements are appHed to the coated glass substrate using a procedure described in U.S. Patent No. 5,807,522, incorporated herein by reference.
  • 1 ⁇ l of the array element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capiUary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per sHde.
  • Microarrays are UV-crosslinked using a STRATALINKER UV-crossHnker (Stratagene).
  • Microarrays are washed at room temperature once in 0.2% SDS and three times in distiUed 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 foUowed by washes in 0.2%
  • PBS phosphate buffered saline
  • Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
  • the sample mixture is heated to 65° C for 5 rninutes and is aHquoted onto the microarray surface and covered with an 1.8 cm 2 coversHp.
  • the arrays are transferred to a waterproof chamber having a cavity just sHghtly larger than a microscope sHde.
  • the chamber is kept at 100% humidity internaUy by the addition of 140 ⁇ l 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 (IX SSC, 0.1 %
  • 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 Hght is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY).
  • the sHde containing the array is placed on a computer-controUed 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.
  • a mixed gas multiline laser excites the two fluorophores sequentiaUy. Emitted Hght is spHt, based on wavelength, into two photomultipHer tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultipHer 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 typicaUy 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 typicaUy caHbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the array contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.
  • the caHbration is done by labeling samples of the caHbrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultipHer tube is digitized using a 12-bit R ⁇ -835H analog-to-digital (AID) conversion board (Analog Devices, Inc., Norwood MA) instaUed 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 Genomics). Array elements that exhibited at least about a two-fold change in expression, a signal-to-background ratio of at least 2.5, and an element spot size of at least 40% were identified as differentiaUy expressed.
  • SEQ DD NO:30 showed differential expression in bone osteosarcoma tissues versus normal osteocytes, as determined by microarray analysis.
  • Messenger RNA from normal human osteoblasts was compared with mRNA from biopsy specimens, osteosarcoma tissues, primary cultures, or metastasized tissues.
  • a normal osteoblast primary culture, NHOst 5488, was chosen as the reference in the initial experiments.
  • One basic set of experiments is defined as the comparison of mRNA from biopsy specimen with that of definitive surgical specimen from the same patient.
  • Extended study of this basic set includes mRNA from primary ceU cultures of the definitive surgical specimen, muscle, or cartilage tissue from the same patient. Biopsy specimens, definitive surgical specimens, or lung metastatic tissues from different individuals were also included to reveal individual variability.
  • the expression of SEQ DD NO:30 was increased by at least two-fold in bone osteosarcoma tissues relative to normal osteocytes. Therefore, in various embodiments, SEQ DD NO:30 can be used for one or more of the foUowing: i) monitoring tieatment of bone cancer, H) diagnostic assays for bone cancer, and Hi) developing therapeutics and/or other treatments for bone cancer.
  • SEQ DD NO:33 showed differential expression in lung squamous carcinoma tissues versus normal lung tissues as determined by microarray analysis. In matched tissue experiments, the expression of SEQ DD NO:33 was decreased by at least two-fold in lung squamous carcinoma tissues relative to grossly uninvolved normal lung tissues from the same donors. Therefore, in various embodiments, SEQ DD NO:33 can be used for one or more of the foUowing: i) monitoring treatment of lung cancer, H) diagnostic assays for lung cancer, and Hi) developing therapeutics and/or other treatments for lung cancer.
  • SEQ DD NO:33 also showed differential expression in ovarian adenocarcinoma tissues versus normal ovarian tissues as determined by microarray analysis.
  • the expression of SEQ DD NO:33 was decreased by at least two-fold in ovarian adenocarcinoma tissues relative to grossly uninvolved normal ovarian tissues from the same donor. Therefore, in various embodiments, SEQ DD NO:33 can be used for one or more of the foUowing: i) monitoring treatment of ovarian cancer, H) diagnostic assays for ovarian cancer, and Hi) developing therapeutics and/or other treatments for ovarian cancer.
  • SEQ DD NO:33 and SEQ DD NO:40 showed differential expression in association with immune and inflarnmatory responses as determined by microarray analysis.
  • the expression of SEQ DD NO:33 was increased by at least two-fold in human umbiHcal vein ceUs treated with PMA and ionomycin relative to untreated human umbiHcal vein ceHs.
  • Human umbiHcal vein ceUs are derived from the endotheHum of the human umbiHcal vein, and have been used as an experimental model for investigating the functional biology of human endotheHal ceUs in vitro.
  • PMA is a broad activator of protein kinase C-dependent pathways and ionomycin is a calcium ionophore that permits the entry of calcium in the ceU, hence increasing the cytosoHc calcium concentration.
  • SEQ DD NO:40 was increased by at least 2.5-fold in vascular endotheHal tissue treated with TNF ⁇ and IL-l ⁇ compared with untreated vascular endotheHal tissue, as determined by microarray analysis.
  • Human coronary artery endotheHal ceHs and human coronary artery smooth muscle ceUs obtained from the same donor were cultured in tissue culture flasks in EndotheHum Growth Medium or Smooth Muscle Growth Medium, respectively (BioWhittaker). Cultures at 85% confluency were either treated with recombinant human TNF ⁇ and IL-l ⁇ (R&D Systems, MinneapoHs MN) at 10 ng/ml each for 24 hours at 37° C or were left untreated.
  • SEQ DD NO:33 and SEQ DD NO:40 can each be used for one or more of the foUowing: i) monitoring treatment of immune/ flammatory responses, H) diagnostic assays for immune/inflammatory responses, and in) developing therapeutics and/or other treatments for immune/inflammatory responses.
  • SEQ DD NO:38 showed differential expression in breast carcinoma ceU lines versus primary mammary epitheHal ceUs as determined by microarray analysis.
  • the breast carcinoma ceU lines include MCF7, a breast adenocarcinoma ceU Hne derived from the pleural effusion of a 69-year-old female; T-47D, a breast carcinoma ceU Hne derived from a pleural effusion from a 54-year-old female with an infiltrating ductal carcinoma of the breast; Sk-BR-3, a breast adenocarcinoma ceU line isolated from a maHgnant pleural effusion of a 43 -year-old female; MDA- mb-231, a metastatic breast tumor ceU Hne derived from the pleural effusion of a 51-year-old female with metastatic breast carcinoma; and MDA-mb-435S, a spindle shaped strain that evolved from a ceU Hne isolated from the
  • the primary mammary epitheHal ceU Hne HMEC was derived from normal human mammary tissue (Clonetics, San Diego, CA). AU ceU cultures were propagated in a chemicaUy- defined medium, according to the suppHer's recommendations and grown to 70-80% confluence prior to RNA isolation.
  • the microarray experiments showed that the expression of SEQ DD NO:38 was decreased by at least two-fold in aU five breast carcinoma lines (MCF7, T-47D, Sk-BR-3, MDA-mb- 231, and MDA-mb-435S) relative to primary mammary epitheHal ceUs. Therefore, in various embodiments, SEQ DD NO:38 can be used for one or more of the foUowing: i) monitoring tieatment of breast cancer, H) diagnostic assays for breast cancer, and Hi) developing therapeutics and/or other treatments for breast cancer.
  • SEQ DD NO:38 also showed differential expression in certain prostate carcinoma ceU Hues versus normal prostate epitheHal ceUs as determined by microarray analysis.
  • the prostate carcinoma ceU Hues include CA-HPV-10, DU 145, LNCaP, PC-3, and MDAPCa2b.
  • CA-HPV-7 was derived from ceUs from a 63 year old male with prostate adenocarcinoma and was transformed by transfection with HPV18 DNA.
  • DU 145 was isolated from a metastatic site in the brain of a 69 year old male with widespread metastatic prostate carcinoma.
  • LNCaP prostate carcinoma ceU Hne isolated from a lymph node biopsy of a 50 year old male with metastatic prostate carcinoma. LNCaP expresses PSA, produces prostate acid phosphatase, and expresses androgen receptors.
  • PC-3 a prostate adenocarcinoma ceU Hne, was isolated from a metastatic site in the bone of a 62 year old male with grade JN prostate adenocarcinoma.
  • MDAPCa2b is a prostate adenocarcinoma ceU Hne isolated from a metastatic site in the bone of a 63 year old male.
  • the MDAPCa2b ceU Hne expresses PSA and androgen receptor and is androgen sensitive.
  • the normal epitheHal ceU Hne, PrEC is a primary prostate epitheHal ceU Hne isolated from a normal donor.
  • the expression of SEQ DD ⁇ O:38 was decreased by at least two-fold in three out of five prostate carcinoma lines (DU 145, LNCaP, and PC-3) relative to ceUs from the normal prostate epitheHal ceU Hne, PrEC.
  • SEQ DD NO:38 can be used for one or more of the foUowing: i) monitoring treatment of prostate cancer, H) diagnostic assays for prostate cancer, and Hi) developing therapeutics and/or other treatments for prostate cancer.
  • SEQ DD NO:38 and SEQ DD NO:43 showed differential expression in toxicology studies as determined by microarray analysis. The expression of SEQ DD NO:43 was increased by at least two-fold in C3 A hepatoblastoma ceUs treated with 1-100 ⁇ M beclomethazone as compared with untreated C3 A hepatoblastoma ceUs.
  • the human C3 A ceU Hne is a clonal derivative of HepG2/C3 (hepatoma ceU Hne, isolated from a 15-year-old male with Hver tumor), which was selected for strong contact inhibition of growth.
  • HepG2/C3 hepatoma ceU Hne, isolated from a 15-year-old male with Hver tumor.
  • the use of a clonal population enhances the reproducibiHty of the ceHs.
  • C3A ceHs have many characteristics of primary human hepatocytes in culture: i) expression of insulin receptor and insuHn-Hke growth factor D receptor; u) secretion of a high ratio of serum albumin compared with ⁇ -fetoprotein Hi) conversion of ammonia to urea and glutamine; iv) metaboHsm of aromatic --mino acids; and v) proHferation in glucose-free and insulin-free medium.
  • the C3A ceUHne is now weH estabHshed as an in vitro model of the mature human Hver (Mickelson et al. (1995) Hepatology 22:866-875; Nagendra et al (1997) Am J Physiol 272:G408-G416).
  • C3A ceUs were treated with 1-100 ⁇ M beclomethazone for lhr, 3hr, 6hr and compared with untreated ceHs.
  • the expression of SEQ DD NO:34 was increased by at least two-fold in early confluent C3A ceUs treated with progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, or betamethasone, for 1, 3, or 6 hours, as compared to untreated C3A ceUs.
  • SEQ DD NO:38 was decreased by at least two-fold in early confluent C3A ceUs treated with progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, or betamethasone, for 1, 3, or 6 hours, as compared to untreated C3A ceUs.
  • the effects of steroids on Hver metaboHsm are important to the understanding of the pharmacodynamics of drugs.
  • SEQ DD NO:34, SEQ DD NO:38 and SEQ DD NO:43 can each be used for one or more of the foUowing: i) monitoring treatment of Hver toxicity, diseases and disorders, H) diagnostic assays for Hver toxicity, diseases and disorders, and Hi) developing therapeutics and/or other treatments for Hver toxicity, diseases and disorders.
  • the expression of SEQ DD NO:48 was differentiaUy expressed in a specific region of human brain tissue as compared to pooled brain tissue control. Characterization of region-specific gene expression in the human brain provides a context and background for molecular neurobiology research in general. This knowledge may provide insight into the genetic basis of brain structure and function.
  • the expression of SEQ DD NO:48 was decreased by at least two-fold in normal human amygdala, entorhinal cortex, brain tissue as compared to the normal human pooled brain tissue used as a control.
  • SEQ DD NO:48 can be used for one or more of the foUowing: i) monitoring treatment of neurological disorders, u) diagnostic assays for neurological disorders, and Hi) developing therapeutics and/or other treatments for neurological disorders.
  • XII Complementary Polynucleotides Sequences complementary to the TRICH-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring TRICH. Although use of oHgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with smaUer or with larger sequence fragments.
  • oHgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of TRICH.
  • a complementary oHgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary oHgonucleotide is designed to prevent ribosomal binding to the TRICH-encoding transcript.
  • TRICH Transcription factor
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not Hmited to, the tip-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 TRICH upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
  • TRICH in eukaryotic ceUs is achieved by infecting msect or mammaHan ceU Hues with recombinant Autographica califomica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographica califomica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding TRICH 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.
  • baculovirus Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus (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).
  • TRICH is synthesized as a fusion protein with, e.g., glutathione S-tiansferase (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 ceU lysates.
  • GST glutathione S-tiansferase
  • FLAG or 6-His a peptide epitope tag
  • GST a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobiHzed glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences).
  • the GST moiety can be proteolyticaUy cleaved from TRICH at specificaUy engineered sites.
  • FLAG an 8-amino acid peptide
  • 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 et al. (supra, ch. 10 and 16). Purified TRICH obtained by these methods can be used directly in the assays shown in Examples XVD, XVDI, and XLX, where appHcable. XIV. Functional Assays
  • TRICH function is assessed by expressing the sequences encoding TRICH at physiologicaHy elevated levels in mammaHan ceU culture systems.
  • cDNA is subcloned into a mammaHan expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT plasmid (Invitiogen, Carlsbad CA) and PCR3.1 plasmid (Invitiogen), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU line, for example, an endotheHal or hematopoietic ceU Hne, using either Hposome formulations or electroporation.
  • a marker protein provides a means to distinguish transfected ceHs from nontransfected ceUs and is a reHable 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.
  • FCM Flow cytometty
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward Hght scatter and 90 degree side Hght scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceH surface and intraceUular 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 ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY).
  • TRICH The influence of TRICH on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encoding TRICH and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected ceUs are efficiently separated from nontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the ceUs using methods weU known by those of skiU in the art. Expression of mRNA encoding TRICH and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • XV Production of TRICH Specific Antibodies TRICH substantiaUy purified using polyacrylamide gel electrophoresis (PAGE; see, e.g.,
  • oHgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (AppHed Biosystems) using EMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleinL dobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al, supi' ⁇ ). Rabbits are immunized with the oHgopeptide-KLH complex in complete Freund's adjuvant.
  • ABI 431 A peptide synthesizer AppHed Biosystems
  • KLH Sigma- Aldrich, St. Louis MO
  • MBS N-maleinL dobenzoyl-N-hydroxysuccinimide ester
  • Resulting antisera are tested for antipeptide and anti-TRICH activity by, for example, binding the peptide or TRICH to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • TRICH Media containing TRICH are passed over the immunoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of TRICH (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/TRICH 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 TRICH is coUected.
  • Candidate molecules previously arrayed in the weUs of a multi-weU plate are incubated with the labeled TRICH, washed, and any weUs with labeled TRICH complex are assayed. Data obtained using different concentrations of TRICH are used to calculate values for the number, affinity, and association of TRICH with the candidate molecules.
  • molecules interacting with TRICH are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commerciaUy available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • TRICH 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 aU interactions between the proteins encoded by two large Hbraries of genes (Nandabalan, K. et al (2000) U.S. Patent No. 6,057,101).
  • Molecules which interact with TRICH may include transporter substrates, agonists or antagonists, modulatory proteins such as G ⁇ proteins (Reimann, supra) or proteins involved in TRICH locaHzation or clustering such as MAGUKs (Craven, supra).
  • TRICH, or biologicaUy active fragments thereof are labeled with 125 I 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 weUs of a multi-weU plate are incubated with the labeled TRICH, washed, and any weUs with labeled TRICH complex are assayed. Data obtained using different concentrations of TRICH are used to calculate values for the number, affinity, and association of TRICH with the candidate molecules.
  • proteins that interact with TRICH are isolated using the yeast 2-hybrid system
  • TRICH or fragments thereof, are expressed as fusion proteins with the DNA binding domain of Gal4 or lexA, and potential interacting proteins are expressed as fusion proteins with an activation domain. Interactions between the TRICH fusion protein and the TRICH interacting proteins (fusion proteins with an activation domain) reconstitute a tiansactivation function that is observed by expression of a reporter gene.
  • Yeast 2-hybrid systems are commerciaUy available, and methods for use of the yeast 2-hybrid system with ion channel proteins are discussed in Niethammer, M. and M. Sheng (1998, Meth. Enzymol. 293:104-122).
  • TRICH 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 aU interactions between the proteins encoded by two large Hbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • TRICH agonists or antagonists may be tested for activation or inhibition of TRICH ion channel activity using the assays described in section XVHL
  • TRICH can be expressed by transforming a mammaHan ceH Hne such as COS7, HeLa or CHO with a eukaryotic expression vector encoding TRICH. Eukaryotic expression vectors are commerciaUy available, and the techniques to introduce them into ceUs are weU known to those skilled in the art.
  • a second plasmid which expresses any one of a number of marker genes, such as ⁇ - galactosidase, is co-transformed into the ceUs to aUow rapid identification of those ceUs which have taken up and expressed the foreign DNA. The ceUs are incubated for 48-72 hours after tiansformation under conditions appropriate for the ceU line to aUow expression and accumulation of TRICH and ⁇ -galactosidase.
  • Transformed ceUs expressing ⁇ -galactosidase are stained blue when a suitable colorimetric substrate is added to the culture media under conditions that are weU known in the art. Stained ceUs are tested for differences in membrane conductance by electrophysiological techniques that are weU known in the art. Untransformed ceUs, and/or ceUs transformed with either vector sequences alone or ⁇ -galactosidase sequences alone, are used as controls and tested in paraUel CeUs expressing TRICH wiU have higher anion or cation conductance relative to control ceUs. The contribution of TRICH to conductance can be confirmed by incubating the ceUs using antibodies specific for TRICH. The antibodies wiU bind to the extiaceUular side of TRICH, thereby blocking the pore in the ion channel, and the associated conductance.
  • TRICH ion channel activity of TRICH is measured as current flow across a TRICH- containing Xenopus laevis oocyte membrane using the two-electrode voltage-clamp technique (Ishi et al, supra; Jegla, T. and L. Salkoff (1997) J. Neurosci. 17:32-44).
  • TRICH is subcloned into an appropriate Xenopus oocyte expression vector, such as pBF, and 0.5-5 ng of mRNA is injected into mature stage IN oocytes. Injected oocytes are incubated at 18 °C for 1-5 days.
  • IntraceUular solution containing 116 mM K-gluconate, 4 mM KCl, and 10 mM Hepes (pH 7.2).
  • the intraceUular solution is supplemented with varying concentrations of the TRICH mediator, such as cAMP, cGMP, or Ca +2 (in the form of CaCl 2 ), where appropriate.
  • Electrode resistance is set at 2-5 M ⁇ and electrodes are fiHed with the intraceUular solution lacking mediator. Experiments are performed at room temperature from a holding potential of 0 mV. Voltage ramps (2.5 s) from -100 to 100 mV are acquired at a sampling frequency of 500 Hz. Current measured is proportional to the activity of TRICH in the assay.
  • Transport activity of TRICH is assayed by measuring uptake of labeled substrates into Xenopus laevis oocytes.
  • Oocytes at stages V and VI are injected with TRICH mR ⁇ A (10 ng per oocyte) and incubated for 3 days at 18°C in OR2 medium (82.5mM ⁇ aCl, 2.5 mM KCl, ImM CaCl 2 , lmM MgCl 2 , ImM ⁇ - ⁇ HPO ⁇ 5 mM Hepes, 3.8 mM ⁇ aOH , 50 ⁇ g/ml gentamycin, pH 7.8) to aUow expression of TRICH.
  • Oocytes are then transferred to standard uptake medium (lOOmM ⁇ aCl, 2 mM KCl, ImM CaCl 2 , ImM MgCl 2 , 10 mM Hepes/Tris pH 7.5).
  • Uptake of various substrates e.g., amino acids, sugars, drugs, ions, and neurotransmitters
  • labeled substrate e.g. radiolabeled with 3 H, fluorescently labeled with rhodamine, etc.
  • uptake is terminated by washing the oocytes three times in Na + -free medium, measuring the incorporated label, and comparing with controls.
  • TRICH activity is proportional to the level of internaHzed labeled substrate.
  • ATPase activity associated with TRICH can be measured by hydrolysis of radiolabeled ATP- [ ⁇ - 32 P], separation of the hydrolysis products by chromatographic methods, and quantitation of the recovered 32 P using a scintiUation counter.
  • the reaction mixture contains ATP-[ ⁇ - 32 P] and varying amounts of TRICH in a suitable buffer incubated at 37 °C for a suitable period of time.
  • the reaction is terminated by acid precipitation with trichloroacetic acid and then neutraHzed with base, and an aHquot of the reaction mixture is subjected to membrane or filter paper-based chromatography to separate the reaction products.
  • the amount of 32 P Hberated is counted in a scintiUation counter.
  • the amount of radioactivity recovered is proportional to the ATPase activity of TRICH in the assay.
  • Lipocalin activity of TRICH is measured by Hgand fluorescence enhancement spectrofluorometry (Lin et al. (1997) Molecular Vision 3:17).
  • Hgands include retinol (Sigma, St. Louis MO) and 16-anthryloxy-palmitic acid (16-AP) (Molecular Probes Inc., Eugene OR).
  • Ligand is dissolved in 100% ethanol and its concentration is estimated using known extinction coefficents (retinol: 46,000 A/M cm at 325 nm; 16-AP: 8,200 A/M/cm at 361 nm).
  • TRICH is expressed in a eukaryotic ceH Hne such as CHO (Chinese Hamster Ovary) or HEK
  • Ion channel activity of the transformed ceUs is measured in the presence and absence of candidate agonists or antagonists. Ion channel activity is assayed using patch clamp methods weU known in the art or as described in Example XVTD. Alternatively, ion channel activity is assayed using fluorescent techniques that measure ion flux across the ceU membrane (VeHcelebi, G. et al. (1999) Meth. Enzymol. 294:20-47; West, M.R. and CR. MoUoy
  • Candidate agonists or antagonists may be selected from known ion channel agonists or antagonists, peptide Hbraries, or combinatorial chemical Hbraries.

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Abstract

Divers modes de réalisation de cette invention concernent des transporteurs humains et des canaux ioniques (TRICH), ainsi que des polynucléotides qui identifient et codent ces transporteurs et canaux ioniques (TRICH). Certains modes de réalisation de ladite invention ont trait à des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes, et des antagonistes. D'autres modes de réalisation ont pour objet des méthodes de diagnostic, de traitement ou de prévention de troubles liés à l'expression aberrante des transporteurs et des canaux ioniques (TRICH).
PCT/US2002/026323 2001-08-17 2002-08-16 Transporteurs et canaux ioniques WO2003016493A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1428877A1 (fr) * 2001-09-17 2004-06-16 Takeda Chemical Industries, Ltd. Nouvelle proteine et adn associe
WO2003074656A3 (fr) * 2002-02-28 2005-06-16 Immunex Corp Polypeptides antiports sodium/proton induits par rank ligand
WO2005097822A1 (fr) * 2004-04-09 2005-10-20 University Of Manitoba Identification de la sequence precise d'acides amines de l'epitope identifie par l'anticorps monoclonal igg1b12 puissant de neutralisation anti-vih 1 humain
WO2007084715A2 (fr) * 2006-01-19 2007-07-26 Caprion Pharmaceuticals, Inc. Tat-031 et procédés de confirmation et de traitement du cancer
US10393757B2 (en) 2010-12-28 2019-08-27 Dainippon Sumitomo Pharma Co., Ltd. Diagnostic drug and diagnostic method for Alzheimer's disease

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WO2019046233A1 (fr) * 2017-08-30 2019-03-07 Bristol-Myers Squibb Company Procédé de surveillance de réponses pharmacodynamiques médiées par l'administration in vivo de glucocorticoïdes

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WO2001034804A1 (fr) * 1999-11-12 2001-05-17 Lexicon Genetics Incorporated Proteines membranaires humaines et polynucleotides codant pour elles

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1428877A1 (fr) * 2001-09-17 2004-06-16 Takeda Chemical Industries, Ltd. Nouvelle proteine et adn associe
EP1428877A4 (fr) * 2001-09-17 2004-11-17 Takeda Chemical Industries Ltd Nouvelle proteine et adn associe
WO2003074656A3 (fr) * 2002-02-28 2005-06-16 Immunex Corp Polypeptides antiports sodium/proton induits par rank ligand
WO2005097822A1 (fr) * 2004-04-09 2005-10-20 University Of Manitoba Identification de la sequence precise d'acides amines de l'epitope identifie par l'anticorps monoclonal igg1b12 puissant de neutralisation anti-vih 1 humain
WO2007084715A2 (fr) * 2006-01-19 2007-07-26 Caprion Pharmaceuticals, Inc. Tat-031 et procédés de confirmation et de traitement du cancer
WO2007084715A3 (fr) * 2006-01-19 2008-04-10 Caprion Pharmaceuticals Inc Tat-031 et procédés de confirmation et de traitement du cancer
US10393757B2 (en) 2010-12-28 2019-08-27 Dainippon Sumitomo Pharma Co., Ltd. Diagnostic drug and diagnostic method for Alzheimer's disease

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