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WO2003062391A2 - Proteines structurelle associees au cytosquelette - Google Patents

Proteines structurelle associees au cytosquelette Download PDF

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Publication number
WO2003062391A2
WO2003062391A2 PCT/US2003/001772 US0301772W WO03062391A2 WO 2003062391 A2 WO2003062391 A2 WO 2003062391A2 US 0301772 W US0301772 W US 0301772W WO 03062391 A2 WO03062391 A2 WO 03062391A2
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polynucleotide
seq
polypeptide
amino acid
sequence
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PCT/US2003/001772
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WO2003062391A3 (fr
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Henry Yue
Jennifer A. Griffin
Thomas W. Richardson
Y. Tom Tang
Kavitha Thangavelu
Ian J. Forsythe
Shanya D. Becha
Narinder K. Chawla
April J.A. Hafalia
Anita Swarnakar
Joseph P. Marquis
Ann E. Gorvad
Mariah R. Baughn
Dyung Aina M. Lu
Chandra S. Arvizu
Amy E. Kable
Soo Yeun Lee
Jayalaxmi Ramkumar
Xin Jiang
Alan A. Jackson
Reena Khare
Vicki S. Elliott
Sean A. Bulloch
Yuming Xu
Sally Lee
Patricia M. Lehr-Mason
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Incyte Corporation
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Priority to AU2003207628A priority Critical patent/AU2003207628A1/en
Publication of WO2003062391A2 publication Critical patent/WO2003062391A2/fr
Publication of WO2003062391A3 publication Critical patent/WO2003062391A3/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to novel nucleic acids, structural and cytoskeleton-associated proteins encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of cell proliferative disorders, viral infections, and neurological disorders.
  • the invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and structural and cytoskeleton-associated proteins.
  • the cytoskeleton is a cytoplasmic network of protein fibers that mediate cell shape, structure, and movement.
  • the cytoskeleton supports the cell membrane and forms tracks along which organelles and other elements move in the cytosol.
  • the cytoskeleton is a dynamic structure that allows cells to adopt various shapes and to carry out directed movements.
  • Major cytoskeletal fibers include the microtubules, the microfilaments, and the intermediate filaments.
  • Motor proteins including myosin, dynein, and kinesin, drive movement of or along the fibers.
  • the motor protein dynamin drives the formation of membrane vesicles. Accessory or associated proteins modify the structure or activity of the fibers while cytoskeletal membrane anchors connect the fibers to the cell membrane.
  • Microtubules cytoskeletal fibers with a diameter of about 24 nm, have multiple roles in the cell. Bundles of microtubules form cilia and flagella, which are whip-like extensions of the cell membrane that are necessary for sweeping materials across an epithelium and for swimming of sperm, respectively. Marginal bands of microtubules in red blood cells and platelets are important for these cells' pliability. Organelles, membrane vesicles, and proteins are transported in the cell along tracks of microtubules. For example, microtubules run through nerve cell axons, allowing bidirectional transport of materials and membrane vesicles between the cell body and the nerve terminal. Failure to supply the nerve terminal with these vesicles blocks the transmission of neural signals. Microtubules are also critical to chromosomal movement during cell division. Both stable and short-lived populations of microtubules exist in the cell.
  • Microtubules are polymers of GTP-binding tubulin protein subunits. Each subunit is a heterodimer of ⁇ - and ⁇ - tubulin, multiple isoforms of which exist.
  • the hydrolysis of GTP is linked to the addition of tubulin subunits at the end of a microtubule.
  • the subunits interact head to tail to form protofilaments; the protofilaments interact side to side to form a microtubule.
  • a microtubule is polarized, one end ringed with ⁇ -tubulin and the other with ⁇ -tubulin, and the two ends differ in their rates of assembly.
  • each microtubule is composed of 13 protofilaments although 11 or 15 protofilament-microtubules are sometimes found.
  • Cilia and flagella contain doublet microtubules.
  • Microtubules grow from specialized structures known as centrosomes or microtubule-organizing centers (MTOCs). MTOCs may contain one or two centrioles, which are pinwheel arrays of triplet microtubules.
  • the basal body, the organizing center located at the base of a cilium or flagellum, contains one centriole.
  • Gamma tubulin present in the MTOC is important for nucleating the polymerization of ⁇ - and ⁇ - tubulin heterodimers but does not polymerize into microtubules.
  • the protein pericentrin is found in the MTOC and has a role in microtubule assembly.
  • Microtubule-associated proteins have roles in the assembly and stabilization of microtubules.
  • assembly MAPs can be identified in neurons as well as non-neuronal cells. Assembly MAPs are responsible for cross-linking microtubules in the cytosol. These MAPs are organized into two domains: a basic microtubule-binding domain and an acidic projection domain. The projection domain is the binding site for membranes, intermediate filaments, or other microtubules.
  • assembly MAPs can be further grouped into two types: Type I and Type H Type I MAPs, which include MAP1A and MAPIB, are large, filamentous molecules that co-purify with microtubules and are abundantly expressed in brain and testes.
  • Type I MAPs contain several repeats of a positively-charged amino acid sequence motif that binds and neutralizes negatively charged tubulin, leading to stabilization of microtubules.
  • MAPI A and MAPIB are each derived from a single precursor polypeptide that is subsequently proteolytically processed to generate one heavy chain and one light chain.
  • LC3 Another light chain, is a 16.4 kDa molecule that binds MAP1A, MAPIB, and microtubules. It is suggested that LC3 is synthesized from a source other than the MAPIA or MAPIB transcripts, and that the expression of LC3 may be important in regulating the microtubule binding activity of MAPIA and MAPIB during cell proliferation (Mann, S.S. et al. (1994) J. Biol. Chem. 269:11492-11497).
  • Type II MAPs which include MAP2a, MAP2b, MAP2c, MAP4, and Tau, are characterized by three to four copies of an 18-residue sequence in the microtubule-binding domain.
  • MAP2a, MAP2b, and MAP2c are found only in dendrites
  • MAP4 is found in non-neuronal cells
  • Tau is found in axons and dendrites of nerve cells.
  • Alternative splicing of the Tau mRNA leads to the existence of multiple forms of Tau protein.
  • Tau phosphorylation is altered in neurodegenerative disorders such as Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia and Parkinsonism linked to chromosome 17.
  • the altered Tau phosphorylation leads to a collapse of the microtubule network and the formation of intraneuronal Tau aggregates (Spillantini, M.G. and M. Goedert (1998) Trends Neurosci. 21:428- 433).
  • STOP stable tubule only polypeptide
  • STOP stable tubule only polypeptide
  • a calmodulin-regulated protein that regulates stability
  • STOP proteins function to stabilize the microtubular network. STOP proteins are associated with axonal microtubules, and are also abundant in neurons (Guillaud, L. et al. (1998) J. Cell Biol. 142: 167-179).
  • STOP proteins are necessary for normal neurite formation, and have been observed to stabilize microtubules, in vitro, against cold-, calcium-, or drug-induced dissassembly (Margolis, R.L. et al. (1990) EMBO 9:4095-502).
  • Microfilaments and Associated Proteins Actins are necessary for normal neurite formation, and have been observed to stabilize microtubules, in vitro, against cold-, calcium-, or drug-induced dissassembly (Margolis, R.L. et al. (1990) EMBO 9:4095-502).
  • Microfilaments are vital to cell locomotion, cell shape, cell adhesion, cell division, and muscle contraction. Assembly and disassembly of the microfilaments allow cells to change their morphology. Microfilaments are the polymerized form of actin, the most abundant intracellular protein in the eukaryotic cell. Human cells contain six isoforms of actin. The three ⁇ -actins are found in different kinds of muscle, nonmuscle ⁇ - actin and nonmuscle ⁇ -actin are found in nonmuscle cells, and another ⁇ -actin is found in intestinal smooth muscle cells.
  • G-actin the monomeric form of actin, polymerizes into polarized, helical F- actin filaments, accompanied by the hydrolysis of ATP to ADP.
  • Actin filaments associate to form bundles and networks, providing a framework to support the plasma membrane and determine cell shape. These bundles and networks are connected to the cell membrane.
  • thin filaments containing actin slide past thick filaments containing the motor protein myosin during contraction.
  • a family of actin-related proteins exist that are not part of the actin cytoskeleton, but rather associate with microtubules and dynein. Actin-Associated Proteins
  • Actin-associated proteins have roles in cross-linking, severing, and stabilization of actin filaments and in sequestering actin monomers.
  • actin-associated proteins have multiple functions. Bundles and networks of actin filaments are held together by actin cross-linking proteins. These proteins have two actin-binding sites, one for each filament. Short cross-linking proteins promote bundle formation while longer, more flexible cross-linking proteins promote network formation.
  • Actin-interacting proteins (AJPs) participate in the regulation of actin filament organization.
  • Other actin-associated proteins such as TARA, a novel F-actin binding protein, function in a similar capacity by regulating actin cytoskeletal organization.
  • Calmodulin-like calcium- binding domains in actin cross-linking proteins allow calcium regulation of cross-linking.
  • Group I cross-linking proteins have unique actin-binding domains and include the 30 kD protein, EF-la, fascin, and scruin.
  • Group ⁇ cross-linking proteins have a 7,000-MW actin-binding domain and include villin and dematin.
  • Group HI cross-linking proteins have pairs of a 26,000-MW actin-binding domain and include fimbrin, spectrin, dystrophin, ABP 120, and filamin.
  • Severing proteins regulate the length of actin filaments by breaking them into short pieces or by blocking their ends.
  • Severing proteins include gCAP39, severin (fragmin), gelsolin, and villin.
  • Capping proteins can cap the ends of actin filaments, but cannot break filaments.
  • Capping proteins include CapZ and tropomodulin.
  • the proteins thymosin and profilin sequester actin monomers in the cytosol, allowing a pool of unpolymerized actin to exist.
  • Microtubule and actin filament networks cooperate in processes such as vesicle and organelle transport, cleavage furrow placement, directed cell migration, spindle rotation, and nuclear migration.
  • Microtubules and actin may coordinate to transport vesicles, organelles, and cell fate determinants, or transport may involve targeting and capture of microtubule ends at cortical actin sites.
  • These cytoskeletal systems may be bridged by myosin-kinesin complexes, myosin-CLIP170 complexes, formin-homology (FH) proteins, dynein, the dynactin complex, Kar9p, coronin, ERM proteins, and kelch repeat-containing proteins (for a review, see Goode, B.L.
  • the kelch repeat is a motif originally observed in the kelch protein, which is involved in formation of cytoplasmic bridges called ring canals. A variety of mammalian and other kelch family proteins have been identified. The kelch repeat domain is believed to mediate interaction with actin (Robinson, D.N. and L. Cooley (1997) J. Cell Biol. 138:799-810).
  • ADF/cofilins are a family of conserved 15-18 kDa actin-binding proteins that play a role in cytokinesis, endocytosis, and in development of embryonic tissues, as well as in tissue regeneration and in pathologies such as ischemia, oxidative or osmotic stress.
  • LIM kinase 1 downregulates ADF (Carlier, M.F. et al. (1999) J. Biol. Chem. 274:33827-33830).
  • LIM is an acronym of three transcription factors, Lin-11, Isl-1, and Mec-3, in which the motif was first identified.
  • the LIM domain is a double zinc-finger motif that mediates the protein-protein interactions of transcription factors, signaling, and cytoskeleton-associated proteins (Roof, D . J. et al. (1997) J. Cell Biol. 138:575-588). These proteins are distributed in the nucleus, cytoplasm, or both (Brown, S. et al. (1999) J. Biol. Chem. 274:27083-27091). Recently, ALP (actinin-associated LEVI protein) has been shown to bind alpha-actinin-2 (Bouju, S. et al. (1999) Neuromuscul. Disord. 9:3- 10).
  • Frabin protein is another example of an actin-filament binding protein (Obaishi, H. et al. (1998) J. Biol. Chem. 273: 18697-18700).
  • Frabin FGDl-related F-actin-binding protein
  • FAB actin-filan ent binding
  • DH Dbl homology
  • PH pleckstrin homology
  • Fablp, YOTB, Naclp, and EEA1 early endosomal antigen 1
  • Frabin has shown GDP/GTP exchange activity for Cdc42 small G protein (Cdc42), and indirectly induces activation of Rac small G protein (Rac) in intact cells. Through the activation of Cdc42 and Rac, Frabin is able to induce formation of both filopodia- and lamellipodia- like processes (Ono, Y. et al. (2000) Oncogene 19:3050-3058).
  • Rho family of small GTP-binding proteins are important regulators of actin-dependent cell functions including cell shape change, adhesion, and motility.
  • the Rho family consists of three major subfamilies: Cdc42, Rac, and Rho.
  • Rho family members cycle between GDP-bound inactive and GTP-bound active forms by means of a GDP/GTP exchange factor (GEF) (Umikawa, M. et al. (1999) J. Biol. Chem. 274:25197-25200).
  • GEF GDP/GTP exchange factor
  • the Rho GEF family is crucial for microfilament organization. ⁇ ebulin-related Proteins
  • ⁇ ebulin is a large sarcomeric protein that interacts with actin filaments in skeletal muscle (Wang, K. et al. (1996) J. Biol. Chem. 271:4304-4314).
  • ⁇ ebulin contains 185 or more copies of a 35- residue module that has a consensus sequence and a predicted cc-helical structure.
  • the 35-residue module comprises an actin-binding domain.
  • the 35-residue modules exhibit a seven module super-repeat pattern. This super-repeat pattern is not present in the C-terminal 100 kDa region of nebulin.
  • nebulin contains 8 linker modules and an 8 kDa acidic domain.
  • the C-terminal region is distinct and contains an SH3 domain.
  • nebulin is oriented with its C-terminus located at the Z-line, and its ⁇ -terminus at the pointed slow- growing end of thin filaments in the acto-myosin overlap region.
  • ⁇ ebulin exists as different isoforms which range in size from 600-900 kDa (Kruger, M. et al. (1991) J. Cell. Biol. 115:97-107).
  • the size of nebulin is tissue- and species-specific and is developmentally regulated. Based on the observation that isoform size correlates with the length of thin filaments in skeletal muscle, nebulin is proposed to play a role as a molecular ruler that regulates the length of thin filaments.
  • Each nebulin 35-residue module may associate with one actin monomer; thus, isoforms with different numbers of modules could determine the length of thin filaments.
  • tropomodulin caps actin at the pointed end of thin filaments and maintains filament length by preventing actin monomer dissociation or addition.
  • ⁇ ebulin is absent from cardiac muscle, but related proteins with nebulin-like modules may provide similar functions.
  • ⁇ ebulette for example, is specifically expressed in heart and has a C- terminal region containing twenty-three 35-residue nebulin-like modules (Moncman, CL. and K. Wang (2000) J. Muscle Res. CeU Motil. 21:153-169; Millevoi, S. et al. (1998) J. Mol. Biol. 282:111- 123).
  • the domain structure of nebulette is similar to nebulin, though it is a smaller protein of only 107 kDa.
  • nebulette has an acidic N-terminal domain, a repeat domain containing nebulin-like modules, a linker domain, and an SH3 domain.
  • the repeat domain of nebulette is about one-tenth the size of that of nebulin.
  • the 35-residue modules of nebulette have a consensus motif, and a subfamily of modules 15-22 share a conserved motif. Unlike nebulin, nebulette modules do not display a super-repeat pattern. Nebulette binds to actin as well as other sarcomeric proteins including myosin, calmodulin, tropomyosin, troponin, and -actinin (Moncman, CL. and K.
  • N-RAP Nebulin-related anchoring protein
  • cardiac and skeletal muscle (Luo, G. et al. (1997) Cell Motil. Cytoskeleton 38:75-90). It is a 133 kDa protein found at the ends of myofibrils at muscle myotendon junctions and intercalated disks.
  • the C-terminal region of N-RAP has 27 copies ofthe 35-residue nebulin-like modules. Seventeen ofthe modules are organized in a super-repeat pattern.
  • the N-terminal region contains a cysteine-rich LIM domain. LIM domains bind two zinc ions in two adjacent zinc finger-like structures and are known to mediate protein- protein interactions.
  • N-RAP may mediate interactions between actin filaments of myofibrils and other sarcomeric proteins.
  • N-RAP binds to actin, talin, and vinculin (Luo, G. et al. (1999) Biochemistry 38:6135-6143). It interacts with actin and vinculin through its super-repeat region and with talin through its LIM domain.
  • Talin and vinculin are also located at myotendon junctions and together with N-RAP may provide a link between actin filaments of the myofibril and the sarcolemma and transmit tension from the myofibril to the extracellular matrix.
  • Intermediate filaments are cytoskeletal fibers with a diameter of about 10 nm, intermediate between that of microfilaments and microtubules. IFs serve structural roles in the cell, reinforcing cells and organizing cells into tissues. IFs are particularly abundant in epidermal cells and in neurons. IFs are extremely stable, and, in contrast to microfilaments and microtubules, do not function in cell motility. Five types of IF proteins are known in mammals. Type I and Type II proteins are the acidic and basic keratins, respectively. Heterodimers of the acidic and basic keratins are the building blocks of keratin IFs.
  • Keratins are abundant in soft epithelia such as skin and cornea, hard epithelia such as nails and hair, and in epithelia that line internal body cavities. Mutations in keratin genes lead to epithelial diseases including epidermolysis bullosa simplex, bullous congenital ichthyosiform erythroderma (epidermolytic hyperkeratosis), non-epidermolytic and epidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, pachyonychia congenita, and white sponge nevus. Some of these diseases result in severe skin blistering. (See, e.g., Wawersik, M. et al. (1997) J.
  • Type HI IF proteins include desrnin, glial fibrillary acidic protein, vimentin, and peripherin.
  • Desrnin filaments in muscle cells link myofibrils into bundles and stabilize sarcomeres in contracting muscle.
  • Glial fibrillary acidic protein filaments are found in the glial cells that surround neurons and astrocytes.
  • Nimentin filaments are found in blood vessel endothelial cells, some epithelial cells, and mesenchymal cells such as fibroblasts, and are commonly associated with microtubules. Nimentin filaments may have roles in keeping the nucleus and other organelles in place in the cell.
  • Type IN IFs include the neurofilaments and nestin.
  • ⁇ eurofilaments composed of three polypeptides, ⁇ F-L, ⁇ F- M, and ⁇ F-H, are frequently associated with microtubules in axons. ⁇ eurofilaments are responsible for the radial growth and diameter of an axon, and ultimately for the speed of nerve impulse transmission. Changes in phosphorylation and metabolism of neurofilaments are observed in neurodegenerative diseases including amyotrophic lateral sclerosis, Parkinson's disease, and
  • Type V IFs the larnins, are found in the nucleus where they support the nuclear membrane.
  • IFs have a central ⁇ -helical rod region interrupted by short nonhelical linker segments.
  • the rod region is bracketed, in most cases, by non-helical head and tail domains.
  • the rod regions of intermediate filament proteins associate to form a coiled-coil dimer.
  • a highly ordered assembly process leads from the dimers to the IFs. Neither ATP nor GTP is needed for IF assembly, unlike that of microfilaments and microtubules.
  • IF-associated proteins mediate the interactions of IFs with one another and with other cell structures.
  • IFAPs cross-link IFs into a bundle, into a network, or to the plasma membrane, and may cross-link IFs to the microfilament and microtubule cytoskeleton. Microtubules and IFs are particularly closely associated.
  • IFAPs include BPAG1, plakoglobin, desmoplakin I, desmoplakin EC, plectin, ankyrin, filaggrin, and lamin B receptor. Cytoskeletal-Membrane Anchors
  • Cytoskeletal fibers are attached to the plasma membrane by specific proteins. These attachments are important for maintaining cell shape and for muscle contraction.
  • the spectrin-actin cytoskeleton is attached to the cell membrane by three proteins, band 4.1, ankyrin, and adducin. Defects in this attachment result in abnormally shaped cells which are more rapidly degraded by the spleen, leading to anemia.
  • the spectrin-actin cytoskeleton is also linked to the membrane by ankyrin; a second actin network is anchored to the membrane by filamin.
  • the protein dystrophin links actin filaments to the plasma membrane; mutations in the dystrophin gene lead to Duchenne muscular dystrophy.
  • Focal adhesions are specialized structures in the plasma membrane involved in the adhesion of a cell to a substrate, such as the extracellular matrix (ECM). Focal adhesions form the connection between an extracellular substrate and the cytoskeleton, and affect such functions as cell shape, cell motility and cell proliferation.
  • Transmembrane integrin molecules form the basis of focal adhesions. Upon ligand binding, integrins cluster in the plane of the plasma membrane. Cytoskeletal linker proteins such as the actin binding proteins -actinin, talin, tensin, vinculin, paxillin, and filamin are recruited to the clustering site.
  • integrins mediate aggregation of protein complexes on both the cytosolic and extracellular faces of the plasma membrane, leading to the assembly of the focal adhesion.
  • Many signal transduction responses are mediated via various adhesion complex proteins, including Src, FAK, paxillin, and tensin.
  • IFs are also attached to membranes by cytoskeletal-membrane anchors.
  • the nuclear lamina is attached to the inner surface of the nuclear membrane by the lamin B receptor.
  • Nimentin IFs are attached to the plasma membrane by ankyrin and plectin.
  • Desmosome and hemidesmosome membrane junctions hold together epithelial cells of organs and skin. These membrane junctions allow shear forces to be distributed across the entire epithelial cell layer, thus providing strength and rigidity to the epithelium.
  • IFs in epithelial cells are attached to the desmosome by plakoglobin and desmoplakins. The proteins that link IFs to hemidesmosomes are not known.
  • Desrnin IFs surround the sarcomere in muscle and are linked to the plasma membrane by paranemin, synemin, and ankyrin. Motor Proteins
  • Myosins are actin-activated ATPases, found in eukaryotic cells, that couple hydrolysis of ATP with motion. Myosin provides the motor function for muscle contraction and intracellular movements such as phagocytosis and rearrangement of cell contents during mitotic cell division (cytokinesis).
  • the contractile unit of skeletal muscle termed the sarcomere, consists of highly ordered arrays of thin actin-containing filaments and thick myosin-containing filaments. Crossbridges form between the thick and thin filaments, and the ATP-dependent movement of myosin heads within the thick filaments pulls the thin filaments, shortening the sarcomere and thus the muscle fiber.
  • Myosins are composed of one or two heavy chains and associated light chains.
  • Myosin heavy chains contain an amino-terminal motor or head domain, a neck that is the site of light-chain binding, and a carboxy-terminal tail domain.
  • the tail domains may associate to form an ⁇ -helical coiled coil.
  • Conventional myosins such as those found in muscle tissue, are composed of two myosin heavy- chain subunits, each associated with two light-chain subunits that bind at the neck region and play a regulatory role.
  • Unconventional myosins believed to function in intracellular motion, may contain either one or two heavy chains and associated light chains. There is evidence for about 25 myosin heavy chain genes in vertebrates, more than half of them unconventional. Dynein-related Motor Proteins
  • Dyneins are (-) end-directed motor proteins which act on microtubules. Two classes of dyneins, cytosolic and axonemal, have been identified. Cytosolic dyneins are responsible for translocation of materials along cytoplasmic microtubules, for example, transport from the nerve terminal to the cell body and transport of endocytic vesicles to lysosomes. As well, viruses often take advantage of cytoplasmic dyneins to be transported to the nucleus and establish a successful infection (Sodeik, B. et al. (1997) J. Cell Biol. 136:1007-1021).
  • Nirion proteins of herpes simplex virus 1 interact with the cytoplasmic dynein intermediate chain (Ye, GJ. et al. (2000) J. Virol. 74: 1355-1363). Cytoplasmic dyneins are also reported to play a role in mitosis. Axonemal dyneins are responsible for the beating of flagella and cilia. Dynein on one microtubule doublet walks along the adjacent microtubule doublet. This sliding force produces bending that causes the flagellum or cilium to beat. Dyneins have a native mass between 1000 and 2000 kDa and contain either two or three force-producing heads driven by the hydrolysis of ATP. The heads are linked via stalks to a basal domain which is composed of a highly variable number of accessory intermediate and light chains. Cytoplasmic dynein is the largest and most complex of the motor proteins. Kinesin-related Motor Proteins
  • Kinesins are (+) end-directed motor proteins which act on microtubules.
  • the prototypical kinesin molecule is involved in the transport of membrane-bound vesicles and organelles. This function is particularly important for axonal transport in neurons.
  • Kinesin is also important in all cell types for the transport of vesicles from the Golgi complex to the endoplasmic reticulum. This role is critical for maintaining the identity and functionality of these secretory organelles.
  • Kinesins define a ubiquitous, conserved family of over 50 proteins that can be classified into at least 8 subfamilies based on primary amino acid sequence, domain structure, velocity of movement, and cellular function. (Reviewed in Moore, J.D. and S.A. Endow (1996) Bioessays 18:207-219; and Hoyt, A.M. (1994) Curr. Opin. Cell Biol. 6:63-68.)
  • the prototypical kinesin molecule is a heterotetramer comprised of two heavy polypeptide chains (KHCs) and two light polypeptide chains (KLCs).
  • KHC subunits are typically referred to as "kinesin.” KHC is about 1000 amino acids in length, and KLC is about 550 amino acids in length.
  • Two KHCs dimerize to form a rod-shaped ⁇ molecule with three distinct regions of secondary structure.
  • a globular motor domain that functions in ATP hydrolysis and microtubule binding.
  • Kinesin motor domains are highly conserved and share over 70% identity.
  • an ⁇ -helical coiled-coil region which mediates dimerization.
  • a fan-shaped tail that associates with molecular cargo. The tail is formed by the interaction of the KHC C-termini with the two KLCs.
  • KRPs kinesin-related proteins
  • Some KRPs are required for assembly of the mitotic spindle.
  • Phosphorylation of KRP is required for this activity.
  • Failure to assemble the mitotic spindle results in abortive mitosis and chromosomal aneuploidy, the latter condition being characteristic of cancer cells.
  • centromere protein E localizes to the kinetochore of human mitotic chromosomes and may play a role in their segregation to opposite spindle poles.
  • Dynamin is a large GTPase motor protein that functions as a "molecular pinchase,” generating a mechanochemical force used to sever membranes. This activity is important in forming clathrin-coated vesicles from coated pits in endocytosis and in the biogenesis of synaptic vesicles in neurons. Binding of dynamin to a membrane leads to dynamin' s self-assembly into spirals that may act to constrict a flat membrane surface into a tubule. GTP hydrolysis induces a change in conformation of the dynamin polymer that pinches the membrane tubule, leading to severing of the membrane tubule and formation of a membrane vesicle.
  • dynamin disassembly. Following disassembly the dynamin may either dissociate from the membrane or remain associated to the vesicle and be transported to another region of the cell.
  • Three homologous dynamin genes have been discovered, in addition to several dynamin-related proteins. conserveed dynamin regions are the N-terminal GTP-binding domain, a central pleckstrin homology domain that binds membranes, a central coiled-coil region that may activate dynamin' s GTPase activity, and a C-terminal proline-rich domain that contains several motifs that bind SH3 domains on other proteins.
  • Some dynamin-related proteins do not contain the pleckstrin homology domain or the proline-rich domain. (See McNiven, M.A. (1998) Cell 94: 151-154; Scaife, R.M. and R.L. Margolis (1997) Cell. Signal. 9:395-401.)
  • Microarrays are analytical tools used in bioanalysis.
  • a microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support.
  • Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
  • gene sequencing such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
  • One area in particular in which microarrays find use is in gene expression analysis.
  • 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 specifically related to a particular genetic predisposition, condition, disease, or disorder.
  • Steroid Hormones and Liver Metabolism Steroid Hormones and Liver Metabolism
  • the potential application of gene expression profiling is particularly relevant to measuring the toxic response to potential therapeutic compounds and of the metabolic response to therapeutic agents.
  • Diseases treated with steroids and disorders caused by the metabolic response to treatment with steroids include adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease.
  • Response may be measured by comparing both the levels and sequences expressed in tissues from subjects exposed to or treated with steroid compounds such as mifepristone, progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, betamethasone, or danazol with the levels and sequences expressed in normal untreated tissue.
  • steroid compounds such as mifepristone, progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, betamethasone, or danazol
  • Steroids are a class of lipid-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.
  • Cholesterol for example, is a component of cell membranes that controls membrane fluidity. It is also a precursor for bile acids which solubilize lipids and facilitate absorption in the small intestine during digestion. Vitamin D regulates the absorption of calcium in the small intestine and controls the concentration of calcium in plasma.
  • Steroid hormones produced by the adrenal cortex, ovaries, and testes, include glucocorticoids, mineralocorticoids, androgens, and estrogens.
  • Glucocorticoids for example, increase blood glucose concentrations by regulation of gluconeogenesis in the liver, increase blood concentrations of fatty acids by promoting lipolysis in adipose tissues, modulate sensitivity to catcholamines in the central nervous system, and reduce inflammation.
  • the principal mineralocorticoid, aldosterone, is produced by the adrenal cortex and acts on cells ofthe distal tubules of the kidney to enhance sodium ion reabsorption.
  • Androgens produced by the interstitial cells of Leydig in the testis, include the male sex hormone testosterone, which triggers changes at puberty, the production of sperm and maintenance of secondary sexual characteristics.
  • Female sex hormones, estrogen and progesterone are produced by the ovaries and also by the placenta and adrenal cortex ofthe fetus during pregnancy.
  • Estrogen regulates female reproductive processes and secondary sexual characteristics.
  • Progesterone regulates changes in the endometrium during the menstrual cycle and pregnancy.
  • Progesterone a naturally occurring progestin, is primarily used to treat amenorrhea, abnormal uterine bleeding, or as a contraceptive. Endogenous progesterone is responsible for inducing secretory activity in the endometrium of the estrogen-primed uterus in preparation for the implantation of a fertilized egg and for the maintenance of pregnancy. It is secreted from the corpus luteum in response to luteinizing hormone (LH). The primary contraceptive effect of exogenous progestins involves the suppression of the midcycle surge of LH.
  • LH luteinizing hormone
  • progestins diffuse freely into target cells and bind to the progesterone receptor.
  • Target cells include the female reproductive tract, the mammary gland, the hypothalamus, and the pituitary. Once bound to the receptor, progestins slow the frequency of release of gonadotropin releasing hormone from the hypothalamus and blunt the pre-ovulatory LH surge, thereby preventing follicular maturation and ovulation.
  • Progesterone has minimal estrogenic and androgenic activity. Progesterone is metabolized hepatically to pregnanediol and conjugated with glucuronic acid.
  • MAH Medroxyprogesterone
  • 6 ⁇ -methyl-17-hydroxyprogesterone is a synthetic progestin with a pharmacological activity about 15 times greater than progesterone.
  • MAH is used for the treatment of renal and endometrial carcinomas, amenorrhea, abnormal uterine bleeding, and endometriosis associated with hormonal imbalance.
  • MAH has a stimulatory effect on respiratory centers and has been used in cases of low blood oxygenation caused by sleep apnea, chronic obstructive pulmonary disease, or hypercapnia.
  • Mifepristone also known as RU-486, is an antiprogesterone drug that blocks receptors of progesterone. It counteracts the effects of progesterone, which is needed to sustain pregnancy. Mifepristone induces spontaneous abortion when administered in early pregnancy followed by treatment with the prostaglandin, misoprostol. Further, studies show that mifepristone at a substantially lower dose can be highly effective as a postcoital contraceptive when administered within five days after unprotected intercourse, thus providing women with a "morning-after pill" in case of contraceptive failure or sexual assault. Mifepristone also has potential uses in the treatment of breast and ovarian cancers in cases in which tumors are progesterone-dependent.
  • Mifepristone binds to glucocorticoid receptors and interferes with cortisol binding. Mifepristone also may act as an anti-glucocorticoid and be effective for treating conditions where cortisol levels are elevated such as AIDS, anorexia nervosa, ulcers, diabetes, Parkinson's disease, multiple sclerosis, and Alzheimer's disease.
  • Danazol is a synthetic steroid derived from ethinyl testosterone. Danazol indirectly reduces estrogen production by lowering pituitary synthesis of follicle-stimulating hormone and LH. Danazol also binds to sex hormone receptors in target tissues, thereby exhibiting anabolic, antiestrognic, and weakly androgenic activity. Danazol does not possess any progestogenic activity, and does not suppress normal pituitary release of corticotropin or release of cortisol by the adrenal glands. Danazol is used in the treatment of endometriosis to relieve pain and inhibit endometrial cell growth. It is also used to treat fibrocystic breast disease and hereditary angioedema.
  • Corticosteroids are used to relieve inflammation and to suppress the immune response. They inhibit eosinophil, basophil, and airway epithelial cell function by regulation of cytokines that mediate the inflammatory response. They inhibit leukocyte infiltration at the site of inflammation, interfere in the function of mediators of the inflammatory response, and suppress the humoral immune response. Corticosteroids are used to treat allergies, asthma, arthritis, and skin conditions. Beclomethasone is a synthetic glucocorticoid that is used to treat steroid-dependent asthma, to relieve symptoms associated with allergic or nonallergic (vasomotor) rhinitis, or to prevent recurrent nasal polyps following surgical removal.
  • intranasal beclomethasone is 5000 times greater than those produced by hydrocortisone.
  • Budesonide is a corticosteroid used to control symptoms associated with allergic rhinitis or asthma. Budesonide has high topical anti-inflammatory activity but low systemic activity.
  • Dexamethasone is a synthetic glucocorticoid used in anti-inflammatory or immunosuppressive compositions. It is also used in inhalants to prevent symptoms of asthma. Due to its greater ability to reach the central nervous system, dexamethasone is usually the treatment of choice to control cerebral edema.
  • Dexamethasone is approximately 20-30 times more potent than hydrocortisone and 5-7 times more potent than prednisone.
  • Prednisone is metabolized in the liver to its active form, prednisolone, a glucocorticoid with anti-inflammatory properties.
  • Prednisone is approximately 4 times more potent than hydrocortisone and the duration of action of prednisone is intermediate between hydrocortisone and dexamethasone.
  • Prednisone is used to treat allograft rejection, asthma, systemic lupus erythematosus, arthritis, ulcerative colitis, and other inflammatory conditions.
  • 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.
  • lipocortins phospholipase A 2 inhibitory proteins
  • Lipocortins control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic acid.
  • Proposed mechanisms of action include decreased IgE synthesis, increased number of ⁇ -adrenergic receptors on leukocytes, and decreased arachidonic acid metabolism.
  • allergens bridge the IgE antibodies on the surface of mast cells, which triggers these cells to release chemotactic substances.
  • Mast cell influx and activation therefore, is partially responsible for the inflammation and hyperirritability of the oral mucosa in asthmatic patients. This inflammation can be retarded by administration of corticosteroids.
  • the human C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male with liver tumor), which was selected for strong contact inhibition of growth.
  • HepG2/C3 hepatoma cell line, isolated from a 15-year-old male with liver tumor.
  • the use of a clonal population enhances the reproducibility of the cells.
  • C3A cells have many characteristics of primary human hepatocytes in culture: i) expression of insulin receptor and insulin-like growth factor Et receptor; ii) secretion of a high ratio of serum albumin compared with ⁇ -fetoprotein iii) conversion of ammonia to urea and glutamine; iv) metabolize aromatic amino acids; and v) proliferate in glucose-free and insulin-free medium.
  • the C3A cell line is now well established as an in vitro model of the mature human liver (Mickelson et al. (1995) Hepatology 22:866-875; Nagendra et al. (1997) Am J Physiol 272:G408- G416). Inflammation and Auto-immune Disorders
  • the inflammatory response is a complex vascular reaction mediated by numerous cytokines, chemokines, growth factors, and other signaling molecules expressed by activated endothelial cells (ECs) , vascular smooth muscle cells (VSMCs), and leukocytes. Inflammation provides protection during trauma and infection, but can also lead to pathological conditions such as atherosclerosis.
  • cytokines interleukin (IL)-l and tumor necrosis factor-alpha (TNF ⁇ )
  • IL interleukin
  • TNF ⁇ tumor necrosis factor-alpha
  • leukocytes particularly neutrophils and monocytes/macrophages, accumulate in the extravascular space, where they remove injurious agents by phagocytosis and oxidative killing, a process accompanied by release of toxic factors, such as proteases and reactive oxygen species.
  • Atherosclerosis and the associated coronary artery disease and cerebral stroke represent the most common causes of death in industrialized nations.
  • Atherosclerosis is a pathological condition characterized by a chronic local inflammatory response within the vessel wall of major arteries. Disease progression results in the formation of atherosclerotic lesions, unstable plaques which occasionally rupture, precipitating a catastrophic thrombotic occlusion of the vessel lumen. Although certain key risk factors have been identified, a full molecular characterization that elucidates the causes and identifies all 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, stability, dissolution, rupture and induction of occlusive vessel thrombi.
  • Blood vessel walls are composed of two tissue layers: an endothelial cell (EC) layer which comprises the lumenal surface of the vessel, and an underlying vascular smooth muscle cell (VSMC) layer.
  • EC endothelial cell
  • VSMC vascular smooth muscle cell
  • 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. Inflammation protects the organism during trauma and infection, but can also lead to pathological conditions such as atherosclerosis.
  • the pro-inflammatory cytokines, interleukin (IL)-l and tumor necrosis factor (TNF) are secreted by a small number of activated macrophages or other cells and can set off a cascade of vascular changes, largely through their ability to alter gene expression patterns in ECs and VSMCs.
  • vascular changes include vasodilation and increased permeability of microvasculature, edema, and leukocyte extravasation and transmigration across the vessel wall.
  • leukocytes particularly neutrophils and monocytes/macrophages, accumulate in the extravascular space, where they remove injurious agents by phagocytosis and oxidative killing, 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 NFfcB as well as AP-1, ERF-l, and NF-GMa, leading to alterations in gene expression.
  • Genes known to be differentially regulated in EC by IL-1 and TNF include E selectin, VCAM-1, ICAM-1, PAF, RB ⁇ , IAP-1, MCP-1, eotaxin, ENA-78, G-CSF, A20, ICE, and complement C3 component.
  • BL-3, chemokine Gro ⁇ , and metalloproteinase matrix metallo-elastase were expressed in both RA and IBD.
  • Haley et al. 2000; Circulation 102:2185-2189
  • the overexpression of eotaxin and its receptor CCR3 in atherosclerotic lesions was confirmed by northern analysis.
  • Thiazolidinediones act as agonists for the peroxisome-proliferator-activated receptor gamma (PPAR ⁇ ), a member of the nuclear hormone receptor superfamily. TZDs reduce hyperglycemia, hyperinsulinemia, and hypertension, in part by promoting glucose metabolism and inhibiting gluconeogenesis. Roles for PPAR ⁇ and its agonists have been demonstrated in a wide range of pathological conditions including diabetes, obesity, hypertension, atherosclerosis, polycystic ovarian syndrome, and cancers such as breast, prostate, liposarcoma, and colon cancer.
  • PPAR ⁇ peroxisome-proliferator-activated receptor gamma
  • PPAR ⁇ peroxisome-proliferator-activated receptor gamma
  • Roles for PPAR ⁇ and its agonists have been demonstrated in a wide range of pathological conditions including diabetes, obesity, hypertension, atherosclerosis, polycystic ovarian syndrome, and cancers such as breast, prostate, lip
  • TZDs and other PPAR ⁇ agonists enhance insulin sensitivity are not fully understood, but may involve the ability of PPAR ⁇ to promote adipogenesis.
  • PPAR ⁇ When ectopically expressed in cultured preadipocytes, PPAR ⁇ is a potent inducer of adipocyte differentiation.
  • the relative potency of different TZDs in promoting adipogenesis in vitro is proportional to both their insulin sensitizing effects in vivo, and their ability to bind and activate PPAR ⁇ in vitro.
  • adipocytes derived from omental adipose depots are refractory to the effects of TZDs. It has therefore been suggested that the insulin sensitizing effects of TZDs may result from their ability to promote adipogenesis in subcutaneous adipose depots (Adams et al., supra). Further, dominant negative mutations in the PPAR ⁇ gene have been identified in two non-obese subjects with severe insulin resistance, hypertension, and overt non-insulin dependent diabetes mellitus (NTDDM) (Barroso et al. (1998) Nature 402:880-883). Diabetes
  • NIDDM is the most common form of diabetes mellitus, a chronic metabolic disease that affects 143 million people worldwide. NIDDM is characterized by abnormal glucose and lipid metabolism that result from a combination of peripheral insulin resistance and defective insulin secretion. NIDDM has a complex, progressive etiology and a high degree of heritability. Numerous complications of diabetes including heart disease, stroke, renal failure, retinopathy, and peripheral neuropathy contribute to the high rate of morbidity and mortality.
  • PPAR ⁇ functions as a ligand activated transcription factor.
  • RXR retinoid X receptor
  • PPRE ⁇ response element PPRE
  • the prostaglandin derivative 15-dPGJ2 is a potent endogenous ligand for PPAR ⁇ .
  • PPAR ⁇ is very high in adipose but barely detectable in skeletal muscle, the primary site for insulin stimulated glucose disposal in the body. PPAR ⁇ is also moderately expressed in large intestine, kidney, liver, vascular smooth muscle, hematopoietic cells, and macrophages. The high expression of PPAR ⁇ in adipose suggests that the insulin sensitizing effects of TZDs may result from alterations in the expression of one or more PPAR ⁇ regulated genes in adipose tissue. Identification of PPAR ⁇ target genes will contribute to better drug design and the development of novel therapeutic strategies for diabetes, obesity, and other conditions.
  • adipose tissue is the most important function of adipose tissue.
  • White adipose tissue is the major energy reserve in periods of excess energy use. Its primary purpose is mobilization during energy deprivation. Understanding how various molecules regulate adiposity and energy balance in physiological and pathophysiological situations may lead to the development of novel therapeutics for human obesity.
  • Adipose tissue is also one of the important target tissues for insulin. Adipogenesis and insulin resistance in type II diabetes are linked and present interesting relations. Most patients with type II diabetes are obese and obesity in turn causes insulin resistance.
  • gene expression profiling is also relevant to improving diagnosis, prognosis, and treatment of leukemic disorders. Alzheimer's disease
  • Alzheimer's disease is a progressive neurodegenerative disorder that is characterized by the formation of senile plaques and neurofibrillary tangles containing amyloid beta peptide. These plaques are found in limbic and association cortices ofthe brain, including hippocampus, temporal cortices, cingulate cortex, amygdala, nucleus basalis and locus caeruleus. Early in Alzheimer's pathology, physiological changes are visible in the cingulate cortex (Minoshima, S. et al. (1997) Annals of Neurology 42:85-94). In subjects with advanced Alzheimer's disease, accumulating plaques damage the neuronal architecture in limbic areas and eventually cripple the memory process.
  • Alzheimer's disease Approximately twenty million people worldwide suffer with dementia that results from Alzheimer's disease. The disease can be early onset affecting individuals as young as 30 years of age, or it can be familial or sporadic. Familial Alzheimer's disease was once thought to be inherited strictly as an autosomal dominant trait; however, this view is changing as more genetic determinants are isolated. For example, some normal allelic variants of apolipoprotein E (ApoE), which is found in senile plaques, can either protect against or increase the risk of developing the disease (Strittmatter et al. (1993) Proc Natl Acad Sci 90:1977-1981). Mutations in four genes are known to predispose an individual to Alzheimer's disease:
  • ApoE amyloid precursor protein
  • presenilin-1 presenilin-2
  • presenilin-2 presenilin-2
  • the e4 allele of the ApoE gene confers increased risk for late onset Alzheimer's disease
  • ⁇ -amyloid protein (A ⁇ ) is the major component of senile plaques, and it is normally formed when ⁇ - and ⁇ - secretases cleave APP.
  • a ⁇ ⁇ -amyloid protein
  • Presenilin can be coimmunoprecipitated with APP, and mutations in the presenilin genes increase production of the 42-amino acid peptide form of A ⁇ . These missense point mutations result in a particularly aggressive, early onset form of the disease (Haas and DeStrooper (1999) Science 286:916-919).
  • BACE1 and BACE2 ⁇ -site APP cleaving enzymes 1 and 2 which appear to be ⁇ -secretases, are potential therapeutic targets because of their ability to cleave APP.
  • Vasser et ah (1999; Science 286:735-741) have found that BACE1 is an aspartyl protease with ⁇ -secretase activity which cleaves APP to produce A ⁇ peptide in vitro. It is expressed at moderate levels across all brain regions and is concentrated in neurons but not in glia.
  • BACE2 which has 52% amino acid identity with BACE1, has been described by Saunders et al. (1999; Science 286:1255a).
  • BACE1 maps to the long arm of chromosome 11
  • BACE2 maps to the Down syndrome region of chromosome 21 (Acquati et al. (2000) 468: 59-64; Saunders et al. supra). This location is significant because middle-aged Down syndrome patients have enhanced ⁇ -amyloid deposits.
  • Other members of the BACE family may also participate in this APP cleavage: the amino terminals of A ⁇ peptides appear to be cleaved heterogeneously indicating that there may be several ⁇ -secretases involved in APP processing (Vasser (1999) Science 286:735-741).
  • Fetal Alzheimer antigen FALZ
  • synuclein ⁇ SNCA
  • FALZ Fetal Alzheimer antigen
  • SNCA synuclein ⁇
  • Inheritance of some gene polymorphisms is also linked to increased risk of developing the disease.
  • a polymorphism in the gene encoding ⁇ 2-macroglobulin, a protein that can act as a protease inhibitor is associated with increased risk for developing a late-onset form of Alzheimer's disease.
  • Steroid Hormones The potential application of gene expression profiling is particularly relevant to measuring both the metabolic response and any toxic effects of potential therapeutic compounds. Many diseases can be treated with steroids, but the metabolic response to treatment with steroids can also cause disorders.
  • adenomatosis cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease.
  • Response may be measured by comparing both the levels and sequences expressed in tissues from subjects exposed to or treated with steroid compounds such as mifepristone, progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, or betamethasone, with the levels and sequences expressed in normal untreated tissue.
  • Steroids are a class of lipid-soluble molecules that include 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.
  • Cholesterol for example, is a component of cell membranes that controls membrane fluidity. It is also a precursor for bile acids which solubilize lipids and facilitate absorption in the small intestine during digestion. Vitamin D regulates the absorption of calcium in the small intestine and controls the concentration of calcium in plasma.
  • Steroid hormones produced by the adrenal cortex, ovaries, and testes, include glucocorticoids, mineralocorticoids, androgens, and estrogens.
  • Glucocorticoids for example, increase blood glucose concentrations by regulation of gluconeogenesis in the liver, increase blood concentrations of fatty acids by promoting lipolysis in adipose tissues, modulate sensitivity to catecholamines in the central nervous system, and reduce inflammation.
  • the principal mineralocorticoid, aldosterone, is produced by the adrenal cortex and acts on cells of the distal tubules ofthe kidney to enhance sodium ion reabsorption.
  • Androgens produced by the interstitial cells of Leydig in the testis, include the male sex hormone testosterone, which triggers changes at puberty, the production of sperm, and maintenance of secondary sexual characteristics.
  • Female sex hormones estrogen and progesterone are produced by the ovaries and also by the placenta and adrenal cortex of the fetus during pregnancy.
  • Estrogen regulates female reproductive processes and secondary sexual characteristics.
  • Progesterone regulates changes in the endometrium during the menstrual cycle and pregnancy.
  • 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 naturally occurring progestin, is primarily used to treat amenorrhea, abnormal uterine bleeding, or as a contraceptive.
  • Endogenous progesterone is responsible for inducing secretory activity in the endometrium of the estrogen-primed uterus in preparation for the implantation of a fertilized egg and for the maintenance of pregnancy. It is secreted from the corpus luteum in response to luteinizing hormone (LH).
  • LH luteinizing hormone
  • the primary contraceptive effect of exogenous progestins involves the suppression of the midcycle surge of LH. At the cellular level, progestins diffuse freely into target cells and bind to the progesterone receptor.
  • Target tissues include the female reproductive tract, the mammary gland, the hypothalamus, and the pituitary. Once bound to the receptor, progestins slow the frequency of release of gonadotropin releasing hormone from the hypothalamus and blunt the pre-ovulatory LH surge, thus preventing follicular maturation and ovulation.
  • Progesterone has minimal estrogenic and androgenic activity. Progesterone is metabolized hepatically to pregnanediol and conjugated with glucuronic acid.
  • MAH Medroxyprogesterone
  • 6 -methyl-17-hydroxyprogesterone is a synthetic progestin with a pharmacological activity about 15 times greater than progesterone.
  • MAH is used for the treatment of renal and endometrial carcinomas, amenorrhea, abnormal uterine bleeding, and endometriosis associated with hormonal imbalance.
  • MAH has a stimulatory effect on respiratory centers and has been used in cases of low blood oxygenation caused by sleep apnea, chronic obstructive pulmonary disease, or hypercapnia.
  • Mifepristone also known as RU-486, is an antiprogesterone drug that blocks receptors of progesterone.
  • Mifepristone induces spontaneous abortion when administered in early pregnancy followed by treatment with the prostaglandin, misoprostol. Further, studies show that mifepristone at a substantially lower dose can be highly effective as a postcoital contraceptive when administered within five days after unprotected intercourse. This is the "morning-after pill" used in case of contraceptive failure or sexual assault. Mifepristone also has potential uses in the treatment of breast and ovarian cancers in cases in which tumors are progesterone-dependent.
  • Mifepristone binds to glucocorticoid receptors and interferes with cortisol binding. Mifepristone also may act as an anti-glucocorticoid and be effective for treating conditions where cortisol levels are elevated such as AIDS, anorexia nervosa, ulcers, diabetes, Parkinson's disease, multiple sclerosis, and Alzheimer's disease.
  • Corticosteroids are used to relieve inflammation and to suppress the immune response. They inhibit eosinophil, basophil, and airway epithelial cell function by regulation of cytokines that mediate the inflammatory response. They inhibit leukocyte infiltration at the site of inflammation, interfere in the function of mediators of the inflammatory response, and suppress the humoral immune response. Corticosteroids are used to treat allergies, asthma, arthritis, and skin conditions. Beclomethasone is a synthetic glucocorticoid that is used to treat steroid-dependent asthma, to relieve symptoms associated with allergic or non-allergic (vasomotor) rhinitis, or to prevent recurrent nasal polyps following surgical removal.
  • intranasal beclomethasone is 5000 times greater than those produced by hydrocortisone.
  • Budesonide is a corticosteroid used to control symptoms associated with allergic rhinitis or asthma. Budesonide has high topical anti-inflammatory activity but low systemic activity.
  • Dexamethasone is a synthetic glucocorticoid used in anti-inflammatory or immunosuppressive compositions. It is also used in inhalants to prevent symptoms of asthma. Due to its greater ability to reach the central nervous system, dexamethasone is usually the treatment of choice to control cerebral edema.
  • Dexamethasone is approximately 20-30 times more potent than hydrocortisone and 5-7 times more potent than prednisone.
  • Prednisone is metabolized in the liver to its active form, prednisolone, a glucocorticoid with anti-inflammatory properties.
  • Prednisone is approximately 4 times more potent than hydrocortisone and the duration of action of prednisone is intermediate between hydrocortisone and dexamethasone.
  • Prednisone is used to treat allograft rejection, asthma, systemic lupus erythematosus, arthritis, ulcerative colitis, and other inflammatory conditions.
  • Betamethasone is a synthetic glucocorticoid with anti-inflammatory and immunosuppressive activity and is used to treat psoriasis and fungal infections such as athlete's foot and ringworm.
  • lipocortins phospholipase A 2 inhibitory proteins
  • Lipocortins control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic acid.
  • Proposed mechanisms of action include decreased IgE synthesis, increased number of ⁇ -adrenergic receptors on leukocytes, and decreased arachidonic acid metabolism.
  • allergens bridge the IgE antibodies on the surface of mast cells and trigger these cells to release chemotactic substances.
  • Mast cell influx and activation is partially responsible for the inflammation and hyperirritability of the oral mucosa in asthmatic patients. This inflammation can be retarded by administration of corticosteroids.
  • the human C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male with liver tumor), which was selected for strong contact inhibition of growth. The use of a clonal population enhances the reproducibility of the cells.
  • C3A cells have many characteristics of primary human hepatocytes in culture including i) expression of insulin receptor and insulin-like growth factor ⁇ receptor; ii) secretion of a high ratio of serum albumin compared with ⁇ -fetoprotein iii) conversion of ammonia to urea and glutamine; iv) metabolism of aromatic amino acids; and v) proliferation in glucose-free and insulin-free medium.
  • the C3A cell line is well established as an in vitro model of the mature human liver (Mickelson, J.K. et al. (1995) Hepatology 22:866-875; Nagendra, A.R. et al. (1997) Am. J. Physiol. 272:G408-G416).
  • Gene expression profiling also has potential as an application for improving diagnosis, prognosis, and treatment of disease. For example, both the levels and sequences expressed in tissues from subjects with Alzheimer's disease may be compared with the levels and sequences expressed in normal brain tissue.
  • Alzheimer's disease is a progressive neurodegenerative disorder that is characterized by the formation of senile plaques and neurofibrillary tangles containing amyloid beta peptide. These plaques are found in limbic and association cortices of the brain.
  • the hippocampus is part of the limbic system and plays an important role in learning and memory. In subjects with Alzheimer's disease, accumulating plaques damage the neuronal architecture in limbic areas and eventually cripple the memory process.
  • Alzheimer's disease Approximately twenty million people worldwide suffer with dementia that results from Alzheimer's disease. The disease can be early onset affecting individuals as young as 30 years of age, or it can be familial or sporadic. Familial Alzheimer's disease was once thought to be inherited strictly as an autosomal dominant trait; however, this view is changing as more genetic determinants are isolated. For example, some normal allelic variants of apolipoprotein E (ApoE), which is found in senile plaques, can either protect against or increase the risk of developing the disease (Strittmatter, WJ. et al. (1993) Proc. Natl. Acad. Sci. USA 90:1977-1981). Mutations in four genes are known to predispose an individual to Alzheimer's disease:
  • ApoE amyloid precursor protein
  • presenilin-1 presenilin-2
  • presenilin-2 presenilin-2
  • the e4 allele ofthe ApoE gene confers increased risk for late onset Alzheimer's disease
  • ⁇ -amyloid protein (A ⁇ ) is the major component of senile plaques, and it is normally formed when ⁇ - and ⁇ - secretases cleave APP.
  • a ⁇ ⁇ -amyloid protein
  • Presenilin can be co-immunoprecipitated with APP, and mutations in the presenilin genes increase production of the 42-amino acid peptide form of A ⁇ . These missense point mutations result in a particularly aggressive, early onset form of the disease (Haas, C. and B. DeStrooper (1999) Science 286:916-919).
  • BACE1 and BACE2 ⁇ -site APP cleaving enzymes 1 and 2
  • BACE1 and BACE2 ⁇ -site APP cleaving enzymes 1 and 2
  • Vasser et al. (1999, Science 286:735-741) have found that BACE1 is an aspartyl protease with ⁇ -secretase activity which cleaves APP to produce A ⁇ peptide in vitro. It is expressed at moderate levels across all brain regions and is concentrated in neurons but not in glia.
  • BACE2 which has 52% amino acid identity with BACE1, has been described by Saunders et al. (1999, Science 286: 1255a).
  • BACE1 maps to the long arm of chromosome 11
  • BACE2 maps to the Down syndrome region of chromosome 21 (Acquati, F. et al. (2000) FEBS Letts. 468:59-64; Saunders et al, supra). This location is significant because middle-aged Down syndrome patients have enhanced ⁇ -amyloid deposits.
  • Other members ofthe BACE family may also participate in this APP cleavage: the amino terminals of A ⁇ peptides appear to be cleaved heterogeneously indicating that there may be several ⁇ -secretases involved in APP processing (Vasser et al., supra).
  • Fetal Alzheimer antigen FALZ
  • synuclein- ⁇ SNCA
  • FALZ Fetal Alzheimer antigen
  • SNCA synuclein- ⁇
  • Inheritance of some gene polymorphisms is also linked to increased risk of developing the disease.
  • a polymorphism in the gene encoding ⁇ 2-macroglobulin, a protein that can act as a protease inhibitor is associated with increased risk for developing a late-onset form of Alzheimer's disease.
  • Array technology can also contribute to the understanding of other human disease processes, such as obesity, which is becoming an epidemic in the United States and other developed countries.
  • Obesity represents a significant risk factor for the development of type II diabetes mellitus, cardiovascular disease, high blood pressure, and even some types of cancers. It has become clear in recent years that fat metabolism and insulin signaling are inherently linked, although the molecular processes involved are still being flushed out.
  • Adipose tissue is an important target tissue for insulin. Most patients with type II diabetes are obese and obesity in turn causes insulin resistance. The most important function of adipose tissue is its ability to store and release fat during periods of feeding and fasting.
  • White adipose tissue is the major energy reserve for periods of excess energy use, and its stored energy is mobilized under situations of energy deprivation. Understanding how various molecules regulate adiposity and energy balance in physiological and pathophysiological situations may lead to the development of novel therapeutics for human obesity.
  • gene expression profiling can be applied to the diagnosis, prognosis, and treatment of human cancers, a number of which constitute a significant cause of morbidity and mortality in the United States and in other countries.
  • Monitoring changes in gene expression in patients with cancers will not only contribute to understanding of the mechanisms underlying the disease, but also facilitate analysis of the effects of various therapeutic agents on disease progression.
  • BRCA1 and BRCA2 are known to greatly predispose a woman to breast cancer and may be 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 epithelial cells.
  • EGF epidermal growth factor
  • EGFR epidermal growth factor
  • Overexpression of EGFR, particularly coupled with down-regulation of the estrogen receptor, is a marker of poor prognosis in breast cancer patients.
  • EGFR expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has effects on cell functions related to metastatic potential, such as cell motility, chemotaxis, secretion and differentiation.
  • a human secreted frizzled protein mRNA that is downregulated in breast tumors includes a human secreted frizzled protein mRNA that is downregulated in breast tumors; the matrix Gla protein which is overexpressed is human breast carcinoma cells; Drgl or RTP, a gene whose expression is diminished in colon, breast, and prostate tumors; maspin, a tumor suppressor gene downregulated in invasive breast carcinomas; and CaN19, a member of the S100 protein family, all of which are down regulated in mammary carcinoma cells relative to normal mammary epithelial cells (Zhou, Z. et al. (1998) Int. J. Cancer 78:95-99; Chen, L. et al. (1990) Oncogene 5:1391-1395; Ulrix, W. et al.
  • Cell lines derived from human mammary epithelial cells at various stages of breast cancer provide a useful model to study the process of malignant transformation and tumor progression as it has been shown that these cell lines retain many of the properties of their parental tumors for lengthy culture periods (Wistuba, LL et al. (1998) Clin. Cancer Res. 4:2931-2938). Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epithelial cells at various stages of malignant transformation.
  • Colon cancer evolves through a multi-step process whereby pre-malignant colonocytes undergo a relatively defined sequence of events leading to tumor formation.
  • factors participate in the process of tumor progression and malignant transformation including genetic factors, mutations, and selection.
  • Initiation of colon cancer may result from a primary insult, such as mutation of the APC (adenomatous polyposis coli) gene in stem cells of the colonic crypts, which results in development of a polyp.
  • APC adenomatous polyposis coli
  • Familial adenomatous polyposis is caused by mutations in the APC gene, resulting in truncated or inactive forms ofthe protein.
  • This tumor suppressor gene has been mapped to chromosome 5q.
  • Hereditary nonpolyposis colorectal cancer is caused by mutations in mis-match repair genes.
  • somatic mutations in APC occur in at least 80% of sporadic colon tumors.
  • APC mutations are thought to be the initiating event in the disease.
  • Approximately 50% of colorectal cancers contain activating mutations in ras, while 85% contain inactivating mutations in p53. Changes in all of these genes lead to gene expression changes in colon cancer. Lung cancer
  • Lung cancer is the leading cause of cancer death for men and the second leading cause of cancer death for women in the U.S. Lung cancers are divided into four histopathologically distinct groups. Three groups (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) are classified as non-small cell lung cancers (NSCLCs). The fourth group of cancers is referred to as small cell lung cancer (SCLC). Deletions on chromosome 3 are common in this disease and are thought to indicate the presence of a tumor suppressor gene in this region. Activating mutations in K- ras are commonly found in lung cancer and are the basis of one of the mouse models for the disease. Osteosarcoma
  • Osteosarcoma is the most common malignant 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, followed 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 will 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.
  • Ovarian cancer is the leading cause of death from a gynecologic cancer.
  • the majority of ovarian cancers are derived from epithelial cells, and 70% of patients with epithelial ovarian cancers present with late-stage disease. As a result, the long-term survival rates 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 microsatellite instability. Gene expression patterns likely vary when normal ovary is compared to ovarian tumors. Prostate cancer
  • Prostate cancer is a common malignancy 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 cells 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 cell and transitional cell 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 hyperproliferation of normal luminal and/or basal epithelial cells. Androgen responsive cells become hyperplastic and evolve into early-stage tumors. Although early-stage tumors are often androgen sensitive and respond to androgen ablation, a population of androgen independent cells evolve from the hyperplastic population. These cells represent a more advanced form of prostate tumor that may become invasive and potentially become metastatic to the bone, brain, or lung.
  • a variety of genes may be differentially expressed during tumor progression. For example, loss of heterozygosity (LOH) is frequently observed on chromosome 8p in prostate cancer.
  • LHO loss of heterozygosity
  • Fluorescence in situ hybridization revealed a deletion for at least 1 locus on 8p in 29 (69%) tumors, with a significantly higher frequency of the deletion on 8p21.2-p21.1 in advanced prostate cancer than in localized prostate cancer, implying that deletions on 8p22-p21.3 play an important role in tumor differentiation, while 8p21.2-p21.1 deletion plays a role in progression of prostate cancer (Oba, K. et al. (2001) Cancer Genet. Cytogenet. 124: 20-26).
  • PSA prostate specific antigen
  • PSA is a tissue-specific serine protease almost exclusively produced by prostatic epithelial cells.
  • the quantity of PSA correlates with the number and volume of the prostatic epithelial cells, and consequently, the levels of PSA are an excellent indicator of abnormal prostate growth.
  • Men with prostate cancer exhibit an early linear increase in PSA levels followed 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 well as potential therapeutic targets for prostate cancer.
  • Several growth factors have been shown to play a critical role in tumor development, growth, and progression.
  • the growth factors Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), and Tumor Growth Factor-alpha (TGF- ⁇ ) are important in the growth of normal as well as hyperproliferative prostate epithelial cells, particularly at early stages of tumor development and progression, and affect signaling pathways in these cells in various ways (Lin, J. et al. (1999) Cancer Res. 59:2891-2897; Putz, T. et al. (1999) Cancer Res. 59:227-233).
  • the TGF- ⁇ family of growth factors are generally expressed at increased levels in human cancers and the high expression levels in many cases correlates with advanced stages of malignancy and poor survival (Gold, L.I. (1999) Crit. Rev. Oncology 10:303-360).
  • senescent cells accumulate with age in vivo, contributing to the aging of an organism.
  • senescence suppresses tumorigenesis; and many genes necessary for senescence also function as tumor suppressor genes, such as p53 and the retinoblastoma susceptibility gene.
  • Tumor cells must overcome this checkpoint for proliferation.
  • Most tumors contain cells that have surpassed their replicative limit, i.e. they are immortalized.
  • telomere shortening is correlated with the progressive shortening of telomeres that occurs with each cell division.
  • Expression of the catalytic component of telomerase in cells prevents telomere shortening and immortalizes cells such as fibroblasts and epithelial cells, but not other types of cells, such as CD8+ T cells (Migliaccio, M. et al.(2000) J. Immunol. 165:4978-4984).
  • telomere shortening is controlled by telomere shortening as well as other mechanisms, depending on the type of cell.
  • a number of genes that are differentially expressed between senescent and presenescent cells have been identified as part of ongoing studies to understand the role of senescence in aging and tumorigenesis. Most senescent cells are growth arrested in the GI stage of the cell cycle. While expression of many cell cycle genes is similar in senescent and presenescent cells (Cristofalo, V. J. et al. (1992) Ann. NY Acad. Sci. 663:187-194), expression of others genes such as cyclin-dependent kinases p21 and pl6, which inhibit proliferation, and cyclins Dl and E is elevated in senescent cells.
  • genes that are not directly involved in the cell cycle are also upregulated such as the extracellular matrix proteins fibronectin, procollagen, and osteonectin, and proteases such as collagenase, stromelysin, and cathepsin B (Chen, Q.M. (2000) Ann. NY Acad. Sci. 908:111-125).
  • Genes underexpressed in senescent cells include those that encode heat shock proteins, c-fos, and cdc- 2 (Chen, supra). The further identification of genes controlling the lifespan of cells will be facilitated by array technology.
  • compositions including nucleic acids and proteins, for the diagnosis, prevention, and treatment of cell proliferative disorders, viral infections, and neurological disorders.
  • Various embodiments of the invention provide purified polypeptides, structural and cytoskeleton-associated proteins, referred to collectively as 'SCAP' and individually as 'SCAP-1,' 'SCAP-2,' 'SCAP-3,' 'SCAP-4,' 'SCAP-5,' 'SCAP-6,' 'SCAP-7,' 'SCAP-8,' 'SCAP-9,' 'SCAP-10,' 'SCAP-11,' 'SCAP-12,' 'SCAP-13,' 'SCAP-14,' 'SCAP-15,' 'SCAP-16,' 'SCAP-17,' 'SCAP-18,' 'SCAP-19,' 'SCAP-20,' 'SCAP-21,' 'SCAP-22,' 'SCAP-23,' 'SCAP-24,' 'SCAP-25,' 'SCAP-26,' 'SCAP-27,' 'SCAP-28,' 'SCAP-29,
  • Embodiments also provide methods for utilizing the purified structural and cytoskeleton-associated proteins and/or their encoding polynucleotides for facilitating the drug discovery process, including dete ⁇ nination of efficacy, dosage, toxicity, and pharmacology.
  • Related embodiments provide methods for utilizing the purified structural and cytoskeleton-associated proteins 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: 1- 31, b) a polypeptide comprising a naturally 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 ED NO: 1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31.
  • Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ED NO:l-31.
  • Still 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 ED NO: 1-31, b) a polypeptide comprising a naturally 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 ED NO: 1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31.
  • polynucleotide encodes a polypeptide selected from the group consisting of SEQ ED NO: 1-31. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO-.32-62.
  • Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked 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: 1-31, b) a polypeptide comprising a naturally 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 ED NO: 1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:l-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31.
  • Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. 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 ED NO: 1-31, b) a polypeptide comprising a naturally 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: 1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31.
  • the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • Yet another embodiment provides an isolated antibody which specifically 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: 1-31 , b) a polypeptide comprising a naturally 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 ED NO: 1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31.
  • Still 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 ED NO: 32-62, b) a polynucleotide comprising a naturally 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:32-62, 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 ED NO:32-62, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO: 32-62, 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 polynucle
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide 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.
  • Still 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 ED NO:32-62, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO:32-62, 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 polynu
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof.
  • the method can include detecting the amount of the amplified 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 ED NO: 1-31, b) a polypeptide comprising a naturally 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 ED NO:l-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ DD NO: 1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31, and a pharmaceutically acceptable excipient.
  • the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-31.
  • Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional SCAP, 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: 1-31, b) a polypeptide comprising a naturally 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 ED NO: 1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31.
  • 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 pharmaceutically acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional SCAP, comprising administering to a patient in need of such treatment the composition.
  • Still 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 ED NO:l-31, b) a polypeptide comprising a naturally 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 ED NO: 1-31 , c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31.
  • 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 pharmaceutically acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional SCAP, comprising administering to a patient in need of such treatment the composition.
  • Another embodiment provides a method of screening for a compound that specifically 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 ED NO: 1-31, b) a polypeptide comprising a naturally 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: 1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:l-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31.
  • 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 specifically 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 ED NO: 1-31, b) a polypeptide comprising a naturally 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: 1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-31.
  • 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.
  • Still 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 ED NO: 32-62, 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 ED NO:32-62, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO: 32-62, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent
  • 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 ED NO:32-62, ii) a polynucleotide comprising a naturally 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:32-62, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), 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 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 lists 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 libraries for polynucleotide embodiments.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.
  • Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with allele frequencies in different human populations.
  • SCAP refers to the amino acid sequences of substantially purified SCAP obtained from any species, particularly a mammalian 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 SCAP.
  • Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of SCAP either by directly interacting with SCAP or by acting on components of the biological pathway in which SCAP participates.
  • allelic variant is an alternative form ofthe gene encoding SCAP. Allelic 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 allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally 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 SCAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as SCAP or a polypeptide with at least one functional characteristic of SCAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding SCAP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding SCAP.
  • 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 functionally equivalent SCAP.
  • Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of SCAP is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
  • Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence can refer to an oligopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies well known in the art.
  • PCR polymerase chain reaction
  • antagonists refers to a molecule which inhibits or attenuates the biological activity of SCAP. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of SCAP either by directly interacting with SCAP or by acting on components of the biological pathway in which SCAP participates.
  • antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind SCAP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide 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
  • RNA e.g., a mouse, a rat, or a rabbit
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oligonucleotide 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 libraries.
  • Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
  • RNA aptamer 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 naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a polynucleotide having a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'- deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell 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.
  • the term “biologically active” refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic SCAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells 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,
  • 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 SCAP or fragments of SCAP may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • a stabilizing agent such as a carbohydrate.
  • the probe may be 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
  • 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
  • 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 especially 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.
  • Trp Phe, Tyr Tyr His, Phe, Trp Vji] lie, Leu, Thr
  • Conservative amino acid substitutions generally maintain (a) the structure ofthe polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the 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 chemically 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 allowing acceleration ofthe evolution of new protein functions.
  • a "fragment” is a unique portion of SCAP or a polynucleotide encoding SCAP 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. For example, 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 may be 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 may be preferentially 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 ID NO: 32-62 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ ED NO:32-62, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ED NO:32-62 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ED NO:32-62 from related polynucleotides.
  • the precise length of a fragment of SEQ ED NO:32-62 and the region of SEQ ED NO:32-62 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ED NO: 1-31 is encoded by a fragment of SEQ ED NO:32-62.
  • a fragment of SEQ ED NO: 1-31 can comprise a region of unique amino acid sequence that specifically identifies SEQ ED NO:l-31.
  • a fragment of SEQ ED NO:l-31 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ED NO: 1-31.
  • the precise length of a fragment of SEQ ED NO: 1-31 and the region of SEQ ED NO: 1-31 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 “full length” polynucleotide is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
  • a “full length” polynucleotide sequence encodes a "full length” polypeptide sequence.
  • Homology refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of identical nucleotide matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to align 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 may be, for example:
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ED number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences 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 all encode substantially the same protein.
  • percent identity and % identity refer to the percentage of identical residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
  • Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • percent similarity and % similarity refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aligned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.
  • 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:
  • Gap x drop-off 50
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ED number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length 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.
  • Human artificial chromosomes are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, 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 still retains its original binding ability.
  • 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 allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill 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.
  • stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T ra ) 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
  • RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill 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 Rgt analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • a solid support e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed.
  • insertion and “addition” refer to changes in an amino 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 cellular and systemic defense systems.
  • factors e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • an “immunogenic fragment” is a polypeptide or oligopeptide fragment of SCAP which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of SCAP which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a plurality 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 SCAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of SCAP.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be 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 linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
  • Post-translational modification of an SCAP may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of SCAP.
  • Probe refers to nucleic acids encoding SCAP, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acids.
  • Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
  • Primmers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence, hi 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 ofthe 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).
  • Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
  • the Primer3 primer selection program (available to the public from the Whitehead Institute/MLT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.)
  • the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
  • this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
  • the oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and Russell (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 linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
  • 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 usually 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 radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • 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 all 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 SCAP, nucleic acids encoding SCAP, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small 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 will reduce the amount of labeled A that binds to the antibody.
  • substantially 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 naturally 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, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well 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 cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells 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 cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the 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).
  • a recombinant viral vector such as a lentiviral vector
  • 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.
  • 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 "allelic” (as defined above), "splice,” “species,” or “polymorphic” variant.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing 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 will generally 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. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • SNPs single nucleotide polymorphisms
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity or sequence similarity 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 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity or sequence similarity over a certain defined length of one of the polypeptides.
  • Various embodiments of the invention include new human structural and cytoskeleton-associated proteins (SCAP), the polynucleotides encoding SCAP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative disorders, viral infections, and neurological disorders.
  • SCAP structural and cytoskeleton-associated proteins
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ED). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ED) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ED NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ED) as shown.
  • Column 6 shows the Incyte ED numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention. The full 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 polypeptide embodiments 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 ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ED) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank ED NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ED NO:) of the nearest PROTEOME database homologs.
  • Column 4 shows the probability 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 applicable, all of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention.
  • Columns 1 and 2 show the polypeptide sequence identification number (SEQ ED NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ED) for each polypeptide of the invention.
  • Column 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 MOTIFS program of the GCG sequence analysis software package (Accelrys, Burlington MA).
  • 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 applied.
  • SEQ ID NO:7 is 74% identical, from residue R21 to residue M266, to human skeletal muscle tropomyosin (GenBank ED g339956) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
  • the BLAST probability score is 4.1e-89, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ED NO:7 also has homology to proteins that are localized to the cytoskeleton, are found associated with stress fibers in non- muscle cells and with the contractile apparatus (thin filaments) in muscle cells, are members of an actin-binding and troponin-binding protein family, and are tropomyosin-related proteins, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ED NO:7 also contains a tropomyosin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ED NO:7 is a tropomyosin- related protein.
  • SEQ ED NO: 13 is 100% identical, from residue Ml to residue T72, and from residue G71 to residue A374, to human beta-tubulin (GenBank ED g496887) as determined by the Basic Local Alignment Search Tool (BLAST).
  • BLAST probability score is 1.8e-201, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ED NO: 13 also has homology to proteins that are beta-tubulins and are localized to the cytoskeleton, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ED NO: 13 also contains a tubulin/FtsZ domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and additional BLAST analyses against the PRODOM and DOMO databases provide further corroborative evidence that SEQ ED NO: 13 is a beta-tubulin.
  • HMM hidden Markov model
  • SEQ ID NO: 16 is 86% identical, from residue Ml to residue E651, to human alpha-adducin (GenBank ED g559044) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.8e-296, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ED NO: 16 also has homology to proteins that are localized to the spectrin-actin network on the cytoplasmic surface of the plasma membrane, may promote assembly of spectrin-actin complexes and regulate ion transport, and are alpha-adducin subunits, as determined by BLAST analysis using the PROTEOME database.
  • BLAST Basic Local Alignment Search Tool
  • SEQ ED NO: 16 also contains a class U aldolase and adducin N-terminal domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ED NO: 18 is 100% identical, from residue Ml to residue E190, to moesin B (GenBank ED gl88626) as determined by the Basic Local Alignment Search Tool (BLAST).
  • the BLAST probability score is l.le-99, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ED NO: 18 also has homology to proteins that are members of the protein 4.1-talin-ezrin family of proteins, which are postulated to act as receptors that links the cytoskeleton to the plasma membrane.
  • SEQ ED NO: 18 is homologous to proteins that play roles in assembly of microvilli and cell morphogenesis, are potential auto-antigens in rheumatoid arthritis, and play a role in impaired brain function in Down syndrome, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ED NO:l also contains a FERM domain/Band 4.1 domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM and SMART database of conserved protein families/domains.
  • HMM hidden Markov model
  • SEQ ED NO: 18 is a protein involved in regulating cytoskeletal structure and cell morphology.
  • SEQ ED NO:22 is 57% identical, from residue R16 to residue Q486, to Gallus gallus kinesin light chain (GenBank ED gl208772) as determined by the Basic Local Alignment Search Tool (BLAST).
  • the BLAST probability score is 1.4E-131, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ED NO:22 also has homology to protein a kinesin light chain which is a member of a family of microtubule-associated motor proteins that function in intracellular transport and mitosis; it has very strong similarity to murine Kiel, which is abundantly expressed in the axons of sciatic nerve, and is kinesin light chain, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ED NO:22 also contains a TPR domain and a tetratricopeptide repeats domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM/SMART database of conserved protein families/domains.
  • HMM hidden Markov model
  • SEQ ED NO:27 is 57% identical, from residue P44 to residue P465, to human actin-binding double-zinc-finger protein (GenBank ED g2337952) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 3.4e-158, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ED NO: 27 also has homology to proteins that are localized to the cytoskeleton, have actin-binding activity, and are actin-binding LEVll, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ED NO:27 also contains a LIM domain and a villin headpiece domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)- based PFAM and SMART databases of conserved protein families/domains.
  • HMM hidden Markov model
  • SEQ ED NO:30 is a splice variant of ankyrin G (GenBank ED g608025) as determined by the Basic Local Alignment Search Tool (BLAST).
  • SEQ ED NO:30 also has homology to proteins that are localized to the cytoskeleton, are anchor proteins and play a role in attaching the cytoskeleton to the plasma membrane, as determined by BLAST analysis using the PROTEOME database. SEQ ED NO: 30 also contains ankyrin repeat domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM and SMART databases of conserved protein families/domains.
  • HMM hidden Markov model
  • SEQ ED NO:30 is an ankyrin.
  • SEQ ID NO:l-6, SEQ ED NO:8-12, SEQ ED NO:14-15, SEQ ED NO:17, SEQ ED NO:19- 21, SEQ ED NO:23-26, SEQ ED NO:28-29, and SEQ ED NO:31 were analyzed and annotated in a similar manner.
  • the algorithms and parameters for the analysis of SEQ ID NO: 1-31 are described in Table 7.
  • 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 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ED) for each polynucleotide ofthe invention, and the length of each polynucleotide sequence in basepairs.
  • Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the full length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ED NO:32-62 or that distinguish between SEQ ED NO:32-62 and related polynucleotides.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full 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 may be 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., those sequences including the designation "NP”).
  • the 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 FL_XXXXX_N 1 _N 2 _YYYY_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 applied, and YYYYY is the number of the prediction generated by the algorithm, and N 12 ⁇ ., if present, represent specific exons that may have been manually edited during analysis (See Example V).
  • the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
  • a polynucleotide sequence identified as LXXXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAA ⁇ A being the GenBank identification number ofthe human genomic sequence to which the "exon-stretching" algorithm was applied, 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).
  • a RefSeq identifier (denoted by "NM,” “NP,” or “NT”) may be used in place of the GenBank identifier (i.e., gBBBBB).
  • GenBank identifier i.e., 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 following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table
  • Table 5 shows the representative cDNA libraries for those full length polynucleotides which were assembled using Incyte cDNA sequences.
  • the representative cDNA library is the icyte cDNA library 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 libraries 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 allele frequencies in different human populations.
  • Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ED NO:) and the corresponding Incyte project identification number (PED) for polynucleotides of the invention.
  • Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST ED), and column 4 shows the identification number for the SNP (SNP ED).
  • 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 full- length polynucleotide sequence (CB1 SNP).
  • SCAP variants can have at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity to the SCAP amino acid sequence, and can contain at least one functional or structural characteristic of SCAP.
  • polynucleotides which encode SCAP also encompass polynucleotides which encode SCAP.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO:32-62, which encodes SCAP.
  • the polynucleotide sequences of SEQ ED NO:32-62 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 SCAP.
  • such a variant polynucleotide will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding SCAP.
  • a particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ED NO:32-62 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 ED NO:32-62. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of SCAP.
  • a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding SCAP.
  • a splice variant may have portions which have significant sequence identity to a polynucleotide encoding SCAP, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing during mRNA processing.
  • a splice 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 SCAP over its entire length; however, portions of the splice variant will 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 SCAP.
  • a polynucleotide comprising a sequence of SEQ ED NO:38 and a polynucleotide comprising a sequence of SEQ ED NO:52 are splice variants of each other;
  • a polynucleotide comprising a sequence of SEQ ED NO:47 and a polynucleotide comprising a sequence of SEQ ED NO:48 are splice variants of each other;
  • a polynucleotide comprising a sequence of SEQ ED NO:54 and a polynucleotide comprising a sequence of SEQ ED NO:57 are splice variants of each other.
  • Any one of the splice variants described above can encode a polypeptide which contains at least one functional or structural characteristic of SCAP.
  • polynucleotides which encode SCAP and its variants are generally capable of hybridizing to polynucleotides encoding naturally occurring SCAP under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding SCAP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be 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 utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of polynucleotides which encode SCAP and SCAP derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic polynucleotide may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art.
  • synthetic chemistry may be used to introduce mutations into a polynucleotide encoding SCAP 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 ID NO: 32-62 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 well 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 (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad CA).
  • sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied 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 well known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853).
  • the nucleic acids encoding SCAP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • various 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 amplify unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods Applic. 2:318-322).
  • Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (Triglia, T. et al.
  • a third method involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119).
  • multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before perforrning 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 may be designed using commercially 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
  • OLIGO 4.06 primer analysis software National Biosciences, Plymouth MN
  • anneal to the template at temperatures of about 68°C to 72°C
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary 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/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
  • appropriate software e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems
  • polynucleotides or fragments thereof which encode SCAP may be cloned in recombinant DNA molecules that direct expression of SCAP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionally equivalent polypeptides may be produced and used to express SCAP.
  • the polynucleotides of the invention can be engineered using methods generally known in the art in order to alter SCAP-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 oligonucleotides may be used to engineer the nucleotide sequences.
  • oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDLNG (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, F.C 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 SCAP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
  • MOLECULARBREEDLNG 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, F.C et al
  • DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
  • polynucleotides encoding SCAP may be synthesized, in whole or in part, using one or more chemical methods well 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).
  • SCAP 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 solid-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 may be achieved using the ABI 431 A peptide synthesizer (Applied Biosystems).
  • amino acid sequence of SCAP may be 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 naturally occurring polypeptide.
  • the peptide may be substantially purified by preparative high performance liquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392421).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
  • the polynucleotides encoding SCAP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • 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 SCAP.
  • Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding SCAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems (Sambrook and Russell, 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 cell systems infected with viral expression vectors (e.g., baculovirus)
  • plant cell systems transformed with viral expression vectors e.g
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of polynucleotides to the targeted organ, tissue, or cell 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, EM. and N. Somia (1997) Nature 389:239- 242).
  • the invention is not limited by the host cell employed.
  • cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding SCAP.
  • routine cloning, subcloning, and propagation of polynucleotides encoding SCAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Invitrogen).
  • PBLUESCRIPT Stratagene, La Jolla CA
  • PSPORT1 plasmid Invitrogen.
  • these vectors may be 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 SCAP may be used.
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of SCAP.
  • 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 intracellular 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 SCAP. Transcription of polynucleotides encoding SCAP 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. 3:17-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105).
  • 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. 3:181)
  • plant promoters
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196).
  • mammalian cells a number of viral-based expression systems may be utilized.
  • polynucleotides encoding SCAP may be ligated 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 SCAP in host cells (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer
  • RSV Rous sarcoma virus
  • SV40 or EBV-based vectors may also be used for high-level protein expression.
  • Human artificial chromosomes may also be employed to deliver 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 delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, JJ. et al. (1997) Nat. Genet. 15:345-355). For long term production of recombinant proteins in mammalian systems, stable expression of
  • SCAP in cell lines is preferred.
  • polynucleotides encoding SCAP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed 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 allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
  • selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in ik " and apf cells, respectively (Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also, antimetabolite, 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 chlorsulfuron and phosphinotricin acetyltransferase, respectively (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14).
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ - glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be 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 SCAP is inserted within a marker gene sequence
  • transformed cells containing polynucleotides encoding SCAP can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding SCAP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain the polynucleotide encoding SCAP and that express SCAP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, 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 SCAP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding SCAP include oligolabelmg, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • polynucleotides encoding SCAP, or any fragments thereof may be 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 radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with polynucleotides encoding SCAP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode SCAP may be designed to contain signal sequences which direct secretion of SCAP through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
  • Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational 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 cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • natural, modified, or recombinant polynucleotides encoding SCAP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric SCAP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of SCAP activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (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 immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the SCAP encoding sequence and the heterologous protein sequence, so that SCAP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
  • synthesis of radiolabeled SCAP may be 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.
  • test compounds may be used to screen for compounds that specifically bind to SCAP.
  • One or more test compounds may be screened for specific binding to SCAP.
  • 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to SCAP.
  • Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.
  • variants of SCAP can be used to screen for binding of test compounds, such as antibodies, to SCAP, a variant of SCAP, or a combination of SCAP and/or one or more variants SCAP.
  • a variant of SCAP can be used to screen for compounds that bind to a variant of SCAP, but not to SCAP having the exact sequence of a sequence of SEQ ED NO:l-31.
  • SCAP variants used to perform such screening can have a range of about 50% to about 99% sequence identity to SCAP, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
  • a compound identified in a screen for specific binding to SCAP can be closely related to the natural ligand of SCAP, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Coligan, J.E. et al. (1991) Current Protocols in Immunology l(2):Chapter 5).
  • the compound thus identified can be a natural ligand of a receptor SCAP (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 SCAP can be closely related to the natural receptor to which SCAP binds, at least a fragment of the receptor, or a fragment ofthe receptor including all or a portion of the ligand binding site or binding pocket.
  • the compound may be a receptor for SCAP which is capable of propagating a signal, or a decoy receptor for SCAP which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336).
  • the compound can be rationally designed using known techniques.
  • Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked 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 SCAP, fragments of SCAP, or variants of SCAP.
  • the binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of SCAP.
  • an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of SCAP.
  • an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of SCAP.
  • anticalins can be screened for specific binding to SCAP, fragments of SCAP, or variants of SCAP.
  • Anticalins are ligand-binding proteins that have been constructed based on a lipocalin 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 lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end.
  • loops form the natural ligand-binding site of the lipocalins, 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 specifically bind to, stimulate, or inhibit SCAP involves producing appropriate cells which express SCAP, either as a secreted protein or on the cell membrane.
  • Preferred cells can include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing SCAP or cell membrane fractions which contain SCAP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either SCAP 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 SCAP, either in solution or affixed to a solid support, and detecting the binding of SCAP to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
  • the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support.
  • An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors.
  • examples of such 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 amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands (Matthews, D J. and J.A. Wells. (1994) Chem. Biol. 1:25-30).
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (Cunningham, B.C. and J.A. Wells (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 ligand
  • SCAP, fragments of SCAP, or variants of SCAP may be used to screen for compounds that modulate the activity of SCAP.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for SCAP activity, wherein SCAP is combined with at least one test compound, and the activity of SCAP in the presence of a test compound is compared with the activity of SCAP in the absence of the test compound. A change in the activity of SCAP in the presence of the test compound is indicative of a compound that modulates the activity of SCAP.
  • a test compound is combined with an in vitro or cell-free system comprising SCAP under conditions suitable for SCAP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of SCAP may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds may be screened.
  • polynucleotides encoding SCAP or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells.
  • ES embryonic stem
  • Such techniques are well known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337).
  • mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture.
  • the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244: 1288-1292).
  • 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. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
  • Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
  • Polynucleotides encoding SCAP may also be manipulated in vitro in ES cells derived from human blastocysts.
  • Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding SCAP can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
  • knockin technology a region of a polynucleotide encoding SCAP is injected into animal ES cells, and the injected sequence integrates into the animal cell genome.
  • Transformed cells are injected into blastulae, and the blastulae are implanted as described above.
  • Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress SCAP e.g., by secreting SCAP 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
  • SCAP appears to play a role in cell proliferative disorders, viral infections, and neurological disorders.
  • disorders associated with increased SCAP expression or activity it is desirable to decrease the expression or activity of SCAP.
  • disorders associated with decreased SCAP expression or activity it is desirable to increase the expression or activity of SCAP.
  • SCAP 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 SCAP.
  • disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and a cancer including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver
  • a vector capable of expressing SCAP 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 SCAP including, but not limited to, those described above.
  • a composition comprising a substantially purified SCAP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SCAP including, but not limited to, those provided above.
  • an agonist which modulates the activity of SCAP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SCAP including, but not limited to, those listed above.
  • an antagonist of SCAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of SCAP.
  • disorders include, but are not limited to, those cell proliferative disorders, viral infections, and neurological disorders described above.
  • an antibody which specifically binds SCAP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express SCAP.
  • a vector expressing the complement of the polynucleotide encoding SCAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of SCAP including, but not limited to, those described above.
  • any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be 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 synergistically 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.
  • An antagonist of SCAP may be produced using methods which are generally known in the art.
  • purified SCAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind SCAP.
  • Antibodies to SCAP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library.
  • neutralizing antibodies i.e., those which inhibit dimer formation
  • Single chain antibodies may be potent enzyme inhibitors and may have application 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, llamas, humans, and others may be immunized by injection with SCAP or with any fragment or oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially preferable.
  • the oligopeptides, peptides, or fragments used to induce antibodies to SCAP have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are substantially identical to a portion of the amino acid sequence of the natural protein. Short stretches of SCAP 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 SCAP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell 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, RJ. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120).
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries (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 libraries or panels of highly specific binding reagents as disclosed in the literature (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 SCAP 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 libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al. (1989) Science 246:1275-1281).
  • immunoassays may be 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 well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between SCAP and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering SCAP epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
  • K a is defined as the molar concentration of SCAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
  • K a association constant
  • the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular SCAP 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 SCAP-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 SCAP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach, ERL Press, Washington DC; Liddell, 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 quality and suitability of such preparations for certain downstream applications.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generally employed in procedures requiring precipitation of SCAP-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available (Catty, supra; Coligan et al., supra).
  • polynucleotides encoding SCAP 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 oligonucleotides) to the coding or regulatory regions ofthe gene encoding SCAP.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oligonucleotides
  • antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding SCAP (Agrawal, S., ed. (1996) Antisense Therapeutics. Humana Press, Totawa NJ).
  • Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein (Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102:469-475; Scanlon, KJ. et al. (1995) 9: 1288-1296).
  • Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A.D. (1990) Blood 76:271; Ausubel et al., supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63:323-347).
  • viral vectors such as retrovirus and adeno-associated virus vectors
  • Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, JJ. (1995) Br. Med; Bull. 51:217-225; Boado, RJ. et al. (1998) J. Pharm. Sci. 87:1308- 1315; Morris, M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736).
  • polynucleotides encoding SCAP may be used for somatic or germline gene therapy.
  • Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCED)-Xl disease characterized by X- linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C et al.
  • SCED severe combined immunodeficiency
  • ADA adenosine deaminase
  • diseases or disorders caused by deficiencies in SCAP are treated by constructing mammalian expression vectors encoding SCAP and introducing these vectors by mechanical means into SCAP-deficient cells.
  • Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) Cell 91:501-510; Boulay, J.-L. and H. Recipon (1998) Curr.
  • Expression vectors that may be effective for the expression of SCAP include, but are not limited to, the PCDNA 3.1, EPLTAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCPJPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
  • SCAP may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSN), SN40 virus, thy idine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. ⁇ atl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.N. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PNGRXR and PI ⁇ D;
  • the FK506/rapamycin inducible promoter or the RU486/mifepristone inducible promoter (Rossi, F.M.N. and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding SCAP from a normal individual.
  • liposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen
  • PERFECT LIPID TRANSFECTION KIT available from Invitrogen
  • transformation is performed using the calcium phosphate method (Graham, F.L. and AJ. Eb (1973) Virology 52:456467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845).
  • the introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
  • diseases or disorders caused by genetic defects with respect to SCAP expression are treated by constructing a retrovirus vector consisting of (i)the polynucleotide encoding SCAP under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus czs-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commercially 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 cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J.
  • VPCL vector producing cell line
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4 + T- cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020- 7029; Bauer, G. et al.
  • an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding SCAP to cells which have one or more genetic abnormalities with respect to the expression of SCAP.
  • the construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference.
  • a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding SCAP to target cells which have one or more genetic abnormalities with respect to the expression of SCAP.
  • the use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing SCAP to cells of the central nervous system, for which HSV has a tropism.
  • the construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art.
  • a replication-competent herpes simplex virus (HSV) type 1 -based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395).
  • 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"), wliich 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 cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
  • HSV vectors see also Goins, W.F. et al. (1999; J. Virol.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding SCAP to target cells.
  • SFV Semliki Forest Virus
  • the specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
  • the methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art. Oligonucleotides derived from the transcription initiation site, e.g., between about positions
  • 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, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding SCAP.
  • RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA molecules encoding SCAP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues. RNA molecules may be modified to increase intracellular stability and half-life.
  • flanking sequences at the 5' and/or 3' ends of the molecule 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 ofthe molecule.
  • This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • RNAi RNA interference
  • PTGS post-transcriptional gene silencing
  • RNAi is a post-transcriptional mode of gene silencing in which double-stranded RNA (dsRNA) introduced into a targeted cell specifically suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantially reduces the expression of the targeted gene.
  • dsRNA double-stranded RNA
  • PTGS can also be accomplished by use of DNA or DNA fragments as well. RNAi methods are described by Fire, A. et al.
  • PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene delivery and/or viral vector delivery methods described herein or known in the art.
  • RNAi can be induced in mammalian cells by the use of small interfering RNA also known as siRNA.
  • siRNA small interfering RNA also known as siRNA.
  • SiRNA are shorter segments of dsRNA (typically about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease.
  • SiRNA appear to be the mediators of the RNAi effect in mammals.
  • the most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3' overhangs.
  • the use of siRNA for inducing RNAi in mammalian cells is described by Elbashir, S.M. et al. (2001; Nature 411:494-498).
  • SiRNA can either be generated indirectly by introduction of dsRNA into the targeted cell, or directly by mammalian transfection methods and agents described herein or known in the art (such as liposome-mediated transfection, viral vector methods, or other polynucleotide delivery/introductory methods).
  • Suitable SiRNAs can be selected by examining a transcript of the target polynucleotide (e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occurrence of each nucleotide and the 3' adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being preferred.
  • mRNA target polynucleotide
  • Regions to be avoided for target siRNA sites include the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex.
  • the selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be eliminated from consideration.
  • the selected SiRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commercially available methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin TX).
  • long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA.
  • This can be accomplished using expression vectors that are engineered to express hairpin RNAs (shRNAs) using methods known in the art (see, e.g., Brummelkamp, T.R. et al. (2002) Science 296:550-553; and Paddison, PJ. et al. (2002) Genes Dev. 16:948-958).
  • shRNAs can be delivered to target cells using expression vectors known in the art.
  • An example of a suitable expression vector for delivery of siRNA is the PSECENCER1.0-U6 (circular) plasmid (Ambion).
  • the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis.
  • Expression levels of the mRNA of a targeted gene can be determined by northern analysis methods using, for example, the NORTHERNMAX-GLY kit (Ambion); by microarray methods; by PCR methods; by real time PCR methods; and by other RNA/polynucleotide assays known in the art or described herein.
  • Expression levels of the protein encoded by the targeted gene can be determined by Western analysis using standard techniques known in the art.
  • An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding SCAP.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, 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 specifically inhibits expression of the polynucleotide encoding SCAP may be therapeutically useful, and in the treatment of disorders associated with decreased SCAP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding SCAP may be therapeutically useful.
  • one or more test compounds may be 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, commercially-available or proprietary library of naturally-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 library of chemical compounds created combinatorially or randomly.
  • a sample comprising a polynucleotide encoding SCAP is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding SCAP are assayed by any method commonly known in the art.
  • 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 SCAP.
  • 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. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide.
  • 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 cell line such as HeLa cell (Clarke, ML. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13).
  • 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 cell line such as HeLa cell (Clarke, ML. et al. (2000) Biochem. Biophys
  • a particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) 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 cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo.
  • oligonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462- 466).
  • any of the therapeutic methods described above may be applied 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 generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
  • Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
  • Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
  • Such compositions may consist of SCAP, antibodies to SCAP, and mimetics, agonists, antagonists, or inhibitors of SCAP.
  • compositions described herein may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • Compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast- acting formulations is well-known in the art. In the case of macromolecules (e.g.
  • 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 well within the capability of those skilled in the art.
  • Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising SCAP or fragments thereof.
  • liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule.
  • SCAP or a fragment thereof may be joined to a short cationic N- terminal portion from the HJV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • An animal 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 therapeutically effective dose refers to that amount of active ingredient, for example SCAP or fragments thereof, antibodies of SCAP, and agonists, antagonists or inhibitors of SCAP, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (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 cell 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 little 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 will be determined by the practitioner, in light 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 the half-life 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 delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specifically bind SCAP may be used for the diagnosis of disorders characterized by expression of SCAP, or in assays to monitor patients being treated with SCAP or agonists, antagonists, or inhibitors of SCAP.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for SCAP include methods which utilize the antibody and a label to detect SCAP in human body fluids or in extracts of cells 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.
  • SCAP SCAP-specific kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinas, and fragments thereof.
  • the polynucleotides which may be used include oligonucleotides, 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 SCAP may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of SCAP, and to monitor regulation of SCAP levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding SCAP or closely related molecules may be used to identify nucleic acid sequences which encode SCAP.
  • 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 amplification will determine whether the probe identifies only naturally occurring sequences encoding SCAP, allelic 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 SCAP encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ED NO:32-62 or from genomic sequences including promoters, enhancers, and introns of the SCAP gene.
  • Means for producing specific hybridization probes for polynucleotides encoding SCAP include the cloning of polynucleotides encoding SCAP or SCAP derivatives into vectors for the production of mRNA probes.
  • vectors are known in the art, are commercially 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 radionuclides 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 like.
  • Polynucleotides encoding SCAP may be used for the diagnosis of disorders associated with expression of SCAP.
  • disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and a cancer including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancrea
  • Polynucleotides encoding SCAP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered SCAP expression. Such qualitative or quantitative methods are well known in the art.
  • polynucleotides encoding SCAP may be used in assays that detect the presence of associated disorders, particularly those mentioned above.
  • Polynucleotides complementary to sequences encoding SCAP 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. After a suitable incubation period, 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 SCAP in the sample indicates the presence of the associated disorder.
  • Such 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 established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding SCAP, under conditions suitable for hybridization or amplification. 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 substantially 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 establish 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 ofthe 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 treatment earlier, thereby preventing the development or further progression of the cancer.
  • oligonucleotides designed from the sequences encoding SCAP may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding SCAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding SCAP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oligonucleotide primers derived from polynucleotides encoding SCAP may be used to detect single nucleotide polymorphisms (SNPs).
  • SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
  • Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • oligonucleotide primers derived from polynucleotides encoding SCAP are used to amplify DNA using the polymerase chain reaction (PCR).
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
  • SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
  • sequence database analysis methods termed in silico SNP (isSNP) are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle cell 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 life-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-lipoxygenase pathway.
  • Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well 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).
  • Methods which may also be used to quantify the expression of SCAP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves (Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236).
  • the speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oligonucleotides or longer fragments derived from any ofthe 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 may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor 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. In particular, 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. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • SCAP, fragments of SCAP, or antibodies specific for SCAP 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 cell type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484; hereby expressly incorporated by reference herein).
  • a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type.
  • the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, cell lines, 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 cell 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 well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog.
  • test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
  • These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity (see, for example, Press
  • 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 transcript 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 cell 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 cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell 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 visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
  • the optical density of each protein spot is generally proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment.
  • the proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry.
  • the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data may be obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for SCAP to quantify the levels of SCAP expression. Ln one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem.
  • Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
  • Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J.
  • proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
  • 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 application WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150- 2155; Heller, MJ. et al. (1997) U.S. Patent No. 5,605,662).
  • Various types of microarrays are well known and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A Practical Approach, Oxford University Press, London).
  • nucleic acid sequences encoding SCAP may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be 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 potentially cause undesired cross hybridization during chromosomal mapping.
  • sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries (Harrington, JJ. 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 libraries
  • 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 may be 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 Mendelian Inheritance in Man (OMEM) World Wide Web site. Correlation between the location of the gene encoding SCAP 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.
  • FISH Fluorescent in situ hybridization
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 lq22-23, 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.
  • SCAP catalytic or immunogenic fragments, or oligopeptides thereof
  • SCAP can be used for screening libraries 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 solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between SCAP 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 application WO84/03564).
  • This method large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with SCAP, or fragments thereof, and washed. Bound SCAP is then detected by methods well known in the art. Purified SCAP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support. Ln another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding SCAP specifically compete with a test compound for binding SCAP.
  • nucleotide sequences which encode SCAP 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 limited to, such properties as the triplet genetic code and specific base pair interactions.
  • poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • RNA was provided with RNA and constructed the corresponding cDNA libraries.
  • cDNA was synthesized and cDNA libraries 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 oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • 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 ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen, Carlsbad CA), PCDNA2.1 plasmid (Invitrogen), PBK-CMV plasmid (Stratagene), PCR2- TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof.
  • Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XL1- BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Invitrogen.
  • Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: 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. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C
  • plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
  • PICOGREEN dye Molecular Probes, Eugene OR
  • FLUOROSKAN II fluorescence scanner Labsystems Oy, Helsinki, Finland.
  • Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
  • Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (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 validated 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.
  • Incyte cDNA sequences or translations thereof were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae,
  • HMM hidden Markov model
  • PFAM PFAM
  • INCY PFAM
  • TIGRFAM TIGRFAM
  • 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 families; 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 full length.
  • Full 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.
  • Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
  • Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable 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, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
  • Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
  • 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.
  • the encoded polypeptides were analyzed by querying against PFAM models for structural and cytoskeleton-associated proteins. Potential structural and cytoskeleton-associated proteins were also identified by homology to Incyte cDNA sequences that had been annotated as structural and cytoskeleton-associated proteins. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases.
  • 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 public cDNA coverage ofthe Genscan-predicted sequences, thus providing evidence for transcription.
  • Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence.
  • Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example in. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
  • a chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
  • GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial D ⁇ A 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.
  • sequences which were used to assemble SEQ ED ⁇ O:32-62 were compared with sequences from the Incyte LDFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ED NO:32-62 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 public 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 all sequences of that cluster, including its particular SEQ ED NO:, to that map location.
  • SHGC Stanford Human Genome Center
  • WIGR Whitehead Institute for Genome Research
  • 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.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (Sambrook and Russell, 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 normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quality in a BLAST alignment.
  • 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 SCAP are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example E_). Each cDNA sequence is derived from a cDNA library constructed from a human tissue.
  • Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified mixed; or urinary tract.
  • the number of libraries in each category is counted and divided by the total number of libraries across all categories.
  • each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease- specific expression of cDNA encoding SCAP.
  • cDNA sequences and cDNA library/tissue information are found in the LEFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII. Extension of SCAP Encoding Polynucleotides
  • Full length polynucleotides are produced by extension of an appropriate fragment of the full length molecule using oligonucleotide 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 hai ⁇ in structures and primer-primer dimerizations was avoided.
  • Selected human cDNA libraries 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 well 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 well of an opaque fluorimeter plate (Coming Costar, Acton MA), allowing the DNA to bind to the reagent.
  • the plate was scanned in a Fluoroskan EC (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Biosciences).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • sonicated or sheared prior to religation into pUC 18 vector
  • the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
  • Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-well plates in ,
  • the cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the following parameters: 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 reamplified using the same conditions as described above.
  • SNPs Single nucleotide polymorphisms
  • LIFESEQ database Incyte Genomics
  • Sequences from the same gene were clustered together and assembled as described in Example III, allowing the identification of all sequence variants in the gene.
  • An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecall errors by requiring a minimum Phred quality score of 15, and removed sequence alignment errors and errors resulting from improper ttimrning of vector sequences, chimeras, and splice variants.
  • Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele 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), all African Americans.
  • the Hispanic population comprised 324 individuals (162 male, 162 female), all 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. Allele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no allelic variance in this population were not further tested in the other three populations.
  • Hybridization probes derived from SEQ ID NO:32-62 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pahs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of
  • [ ⁇ - 32 P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences). An aliquot 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 following endonucleases: Ase I, Bgl U, Eco RI, Pst I, Xba I, or Pvu E (DuPont NEN).
  • the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, 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 visualized using autoradiography or an alternative imaging means and compared. XI. Microarrays
  • the linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, 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 solid with a non-porous surface (Schena, M., ed. (1999) DNA Microarrays: A Practical Approach. Oxford University Press, London). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon 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 well known to those of ordinary skill 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; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).
  • Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well 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 may be used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed.
  • microarray preparation and usage is described in detail below.
  • Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oligo-(dT) cellulose method.
  • Each poly (A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oligo-(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. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ⁇ l 5X SSC/0.2% SDS.
  • SpeedVAC SpeedVAC
  • Sequences ofthe present invention are used to generate array elements.
  • Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
  • PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g.
  • Amplified array elements are then purified using SEPHACRYL-400 (Amersham Biosciences). Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Coming) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
  • Array elements are applied 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 capillary printing element by a high-speed robotic apparatus.
  • the apparatus then deposits about 5 nl of array element sample per slide.
  • Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
  • Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C followed by washes in 0.2% SDS and distilled water as before.
  • 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 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm 2 coverslip.
  • the arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide.
  • the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5X SSC in a comer 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% SDS), three times for 10 minutes each at 45°C in a second wash buffer (0.1X SSC), and dried.
  • Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an
  • Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY).
  • the slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective.
  • the 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
  • a mixed gas multiline laser excites the two fluorophores sequentially. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals.
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
  • the sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.
  • the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (AID) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore' s emission spectrum.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte Genomics). Array elements that exhibit at least about a two-fold change in expression, a signal-to- background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentially expressed.
  • SEQ ED NO:49, SEQ ED NO:50, and SEQ ED NO:52 showed differential expression in endothelial cells treated with TNF ⁇ alone or TNF ⁇ + IL-l ⁇ , as determined by microarray expression analysis.
  • the human endothelial cell lines HUVEC (human umbilical cord vascular endothelial cells), HMVECdNeo (human aortic endothelial cells from microvasculature of the skin), and ECV304 (an immortalized human endothelial cell line) are commonly used as model systems to study the activation of vascular endothelium by inflammatory factors, which plays a role in a number of human diseases such as atherosclerosis.
  • SEQ ED NO:49 showed an increased expression in HUVEC and HMVECdNeo cell lines of at least two-fold, peaking after 8 hours of TNF ⁇ treatment, when compared to untreated cells.
  • Treatment of HUVEC or ECV304 cells with TNF ⁇ + EL-l ⁇ resulted in a similar two-fold increase in expression of SEQ ED NO:49, and also in expression of SEQ D NO:52.
  • HUVEC cells treated with TNF ⁇ + IL-l ⁇ demonstrated a two-fold decrease in expression of SEQ ED NO:51.
  • SEQ ID NO:49, SEQ ED NO:51, and SEQ ED NO:52 can each be used for one or more of the following: i) monitoring treatment of atherosclerosis and other inflammatory conditions, ii) diagnostic assays for atherosclerosis and other inflammatory conditions, and iii) developing therapeutics and/or other treatments for atherosclerosis and other inflammatory conditions.
  • SEQ ED NO:49-52 showed differential expression in a number of human breast cancer cell lines and donor samples, as determined by microarray expression analysis.
  • a number of cell lines were examined, representing various stages of tumor progression.
  • HMEC human mammary epithelial cells
  • MCF-10A cell line from a donor with fibrocystic breast disease
  • the tumor cell lines examined have been generally grouped into three classes. Class I cell lines include the MCF7 adenocarcinoma cell line and the Hs578T ductal carcinoma cell line, both of which are non-metastatic tumor cell lines.
  • Class JL includes the MDA-mb-435S cell line, established from a metastatic ductal carcinoma, and the T-47D cell line, derived from an infiltrating ductal carcinoma, which are both non-tumorigenic in mice.
  • the BT-474 and BT-483 invasive ductal carcinoma lines both form tumors in mice, as do the cell lines derived from metastatic adenocarcinomas MDA-mb-468, Sk-BR-3, BT-20, and MDA-mb-231, with BT-20 cell line generating stage EC adenocarcinomas, and MDA-mb-231 generating stage DL adenocarcinomas in mice.
  • These lines represent Class IJL cell lines.
  • SEQ ED NO:49 expression in the Class I cell line MCF7 was decreased 3.5-fold compared to starved HMEC cells, 22-fold compared to untreated HMEC cells, and between 5- and 9-fold compared to the MCF-10A cell line.
  • SEQ ID NO:49 expression was decreased 10-fold compared to HMEC cells, and at least 6-fold compared to MCF-10A cells.
  • SEQ ID NO:49 expression was decreased by between 2- and 18-fold in comparison to HMEC cells, and between 2- and 6-fold compared to MCF-IOA cells.
  • SEQ ED NO:49 expression was reduced at least 2.5-fold in tumor tissue isolated from a lobular breast carcinoma, when compared to matched normal breast tissue from the same donor.
  • SEQ ED NO:50 expression in several cell lines was compared to the gene expression levels in HMEC cells.
  • SEQ ID NO:50 expression was reduced at least 2-fold in the Class I cell line MCF7.
  • the Class II cell line T-47D expression of SEQ ED NO:50 was decreased at least 6-fold.
  • SEQ ED NO:50 expression was reduced between 2- and 3-fold.
  • SEQ ED NO:51 expression in the Class I cell lines MCF7 and Hs578T was reduced between 3- and 7-fold when compared to HMEC cell gene expression levels, and at least 2-fold in comparison to expression levels in MCF-IOA cells.
  • Ln Class II cell lines (T-47D and MDA-mb-435S), SEQ ED NO: 51 gene expression was decreased at least 5-fold, and between 3- and 6-fold, in comparison to the expression levels detected in HMEC and MCF-IOA cells, respectively.
  • the fibrocystic cell line MCF-IOA showed a 2-fold reduction in SEQ ID NO:52 expression, and several Class HI cell lines (specifically BT-20, BT-474, and BT- 483) had a 2- to 3-fold reduction in SEQ ID NO:52 gene expression as well.
  • SEQ ID NO:49 and SEQ ID NO:51 expression levels were shown to change in response to the addition of growth factors and inhibitors of signaling proteins by using microarray expression analysis.
  • Various growth factors and downstream signal transduction proteins play important roles in the progression of breast and other types of cancer.
  • Abnormal cell proliferation rates in breast tumors have been associated with increased expression of epidermal growth factor receptor and Her2/ErbB2 receptor tyrosine kinases (RTKs). These RTKs stimulate mitogenesis in part by activation of the ras ⁇ Raf ⁇ MEK ⁇ MAPK cascade.
  • Expression of SEQ HD NO:49 was examined in MCF7 cells treated with 50 ng/ml EGF at various time points.
  • SEQ ID NO:49 expression levels reached their peak decrease of 2.5-fold, in comparison to untreated MCF7 cells.
  • expression levels of SEQ ED NO:51 in MCF-IOA cells treated with 50 ng/ml EGF decreased 2.5-fold after 12 hours when compared to untreated MCF- 10A cells.
  • treatment of these cells with an EGFR inhibitor AG1478 (tyrphostin) at 250 nM resulted in a 2.5-fold increase in SEQ ED NO:51 expression after 48 hours.
  • SEQ ED NO:51 expression levels increased at least 2-fold in a dose- and time-dependent manner in MCF-IOA cells treated with the MEK inhibitor PD98059.
  • SEQ ED NO:51 showed differential expression in cells cultured with Matrigel, a reconstituted basement membrane matrix isolated from a mouse sarcoma rich in extracellular matrix proteins, as determined by microarray expression analysis. Examination of the morphogenesis of cells in the presence of Matrigel can facilitate understanding ofthe contribution that the microenvhonment makes to development and metastasis of cancer, and how manipulation of the interaction between cancer cells and the microenvironment may lead to novel therapeutic targets.
  • BT-20 cells one of the Class HI cell lines described above that leads to formation of stage H adenocarcinomas in mice, were grown in Matrigel matrix for a period of 1.7 weeks, and then colonies analyzed for expression levels of SEQ ID NO:51. In comparison to BT-20 cells not exposed to Matrigel matrix, SEQ HD NO:51 expression levels decreased at least 2-fold upon growth in the extracellular matrix-rich environment.
  • SEQ ED NO:50 and SEQ ED NO: 51 demonstrated differential expression in breast cancer model cell lines upon treatment with UV radiation (UVR), as determined by microarray expression analysis.
  • UVR UV radiation
  • DNA lesions can accumulate and lead to genomic instability and development of cancer.
  • Deficient DNA repair capacity appears to be a predisposing factor in familial breast cancer and in some sporadic breast cancers.
  • DNA-damaging agents are often used as anti-tumor therapies, targeting the faster-growing cancer cells for destruction by inducing lethal DNA lesions. Thus, it is important to understand the effects of DNA damaging agents, such as UVR, on the activation of DNA repair systems in cells.
  • MDA-mb-231 cells a Class HL cell line described above that leads to formation of stage EQ adenocarcinomas in mice, were treated with various doses of UVR, and the expression levels of SEQ ED NO:50 were examined at various time points thereafter.
  • SEQ ED NO:50 showed a 6-fold decrease in expression 24 hours after UVR (5 mJ/cm 2 ), when compared to un- radiated MDA-mb-231 cells.
  • SEQ ID NO:50 also showed at least a 2-fold decrease in expression 8 hours after UVR (1-5 mJ/cm 2 ) in the Class EQ cell lines BT-20, described above, when compared to untreated BT-20 cells.
  • SEQ ED NO:51 expression showed a dose-dependent increase 30 minutes after UVR (range between 0.5 and 5.0 mJ/cm 2 ) of between 2- and 3-fold in MCF-IOA fibrocystic cells, compared to untreated cells.
  • SEQ ED NO:34 showed differential expression associated with breast cancer as determined by microarray analysis.
  • the gene expression profile of a nonmalignant mammary epithelial cell line was compared to the gene expression profiles of breast carcinoma cell lines representing different stages of tumor progression.
  • BT-20 a breast carcinoma cell line derived in vitro from the cells emigrating out of thin slices of tumor mass isolated from a 74-year-old female
  • BT-474 a breast ductal carcinoma cell line that was isolated from a solid, invasive ductal carcinoma of the breast obtained from a 60-year-old woman
  • BT-483 a breast ductal carcinoma cell line that was isolated from a papillary invasive ductal tumor obtained from a 23-year-old normal, menstruating, parous female with a family history of breast cancer
  • Hs 578T a breast ductal carcinoma cell line isolated from a 74-year-old female with breast carcinoma
  • MCF7 a nonmalignant breast adenocarcinoma cell line isolated from the pleural effusion of a 69- year-old female
  • MCF-IOA a breast mammary gland (luminal ductal characteristics) cell
  • SEQ ED NO:37 showed at least a two-fold increase in expression in MDA-mb-231 breast carcinoma cells treated with the combination of the protein kinase C activator, phorbol myristate acetate (PMA), and the calcium ionophore, ionomycin, compared to untreated cells as determined by microarray analysis.
  • PMA is a broad activator of the protein kinase C-dependent pathways. Ionomycin permits the entry of calcium into cells and increases the cytosolic calcium concentration. PMA and ionomycin activate major signaling pathways in mammalian cells.
  • MDA- mb-231 is a breast tumor cell line isolated from the pleural effusion of a 51-year old female.
  • SEQ ID NO:37 increased at least two-fold in the MDA-mb-231 breast carcinoma cell line compared to the HMEC breast epithelial cell line.
  • the expression of SEQ ED NO: 37 also increased at least two-fold in the MDA-mb-435S breast carcinoma cell line compared to the nonmalignant MCF-IOA cell line.
  • MDA-mb435S is a spindle-shaped strain that evolved from the parent line (435) isolated by R. Cailleau from pleural effusion of a 31-year-old female with metastatic, ductal adenocarcinoma of the breast.
  • SEQ ED NO:37 showed at least a twofold decrease in expression in MCF-IOA cells treated with TNF- ⁇ at concentrations of 1-10 ng/ml for 48 hours compared with untreated cells. Therefore, in various embodiments, SEQ ID NO:34, SEQ ID NO:37, and SEQ ID NO:49-52 can each be used for one or more of the following: i) monitoring treatment of breast cancer, ii) diagnostic assays for breast cancer, and iii) developing therapeutics and/or other treatments for breast cancer. Prostate cancer
  • SEQ ID NO:49-51 demonstrated differential expression in a number of prostate cancer model cell lines, as determined by microarray analysis.
  • PrEC prostate epithelial cells
  • PZ-HPV-7 cells peripheral zone epithelial cells of the prostate transformed with HPV18
  • LNCaP cells isolated from a lymph node metastasis of a prostate carcinoma
  • PC3 cells isolated from a metastatic site in the bone of a donor with grade EV prostate adenocarcinoma
  • DU 145 cells derived from a metastasis to the brain from a donor with prostate carcinoma
  • MDAPCa2b cells isolated from a bone metastasis from a donor with adenocarcinoma of the prostate.
  • Expression of SEQ ED NO:49 was decreased at least 3-fold in LNCaP cells, when compared to expression levels in PrEC and PZ-HPV-7 cells, and decreased over 10-fold in MDAPCa2b cells in comparison to PrEC expression levels.
  • SEQ ED NO:50 expression levels were reduced at least 2-fold in LNCaP, PC3, and DU 145 cells when compared to PrEC gene expression levels, and at least 2-fold in LNCaP cells as compared to PZ-HPV-7 cells.
  • SEQ ED NO:51 gene expression levels when compared to PrEC levels, were decreased between 3- and 7-fold in DU 145 cells, between 10- and 13-fold in LNCaP cells, between 6- and 11-fold in PC3 cells, and 5-fold in MDAPCa2b cells, depending on the media conditions used to culture the cells.
  • SEQ ED NO:51 expression levels were also decreased, by at least 8-fold in DU 145 cells, 14-fold in LNCaP cells, and 6-fold in PC3 cells.
  • Senescence is a normal mechanism of tumor suppression, which limits cell proliferation and provides protection against cancer.
  • the proliferative lifespan of most cells is limited by "replicative senescence", a type of cell cycle arrest induced after a preset number of cell divisions.
  • the accumulation of senescent cells as humans age may actually synergize with accumulated genetic mutations, and increase the risk of developing some cancers.
  • PrEC cells can be used as a model for progressively senescent cells, and the changes in gene expression can be analyzed by microarray analysis. PrEC cells at passage 3 represent early progenitor cells, while passages 4 through 7 represent progressively more senescent cells, and PrEC cells at passage 8 are fully senescent.
  • SEQ ED NO:50 was decreased at least 2-fold in passage 8 PrEC cells when compared to early progenitor cells at passage 3.
  • SEQ ED NO:51 gene expression levels showed a progressive decrease as cells become more senescent, with a 2-fold decrease at passage 6, a 3-fold decrease at passage 7, and a 3.4-fold decrease at passage 8, when compared to passage 3 PrEC cells. Therefore, in various embodiments, SEQ ED NO:49-51 can each be used for one or more of
  • SEQ ED NO: 37 showed at least a two-fold decrease in expression in colon tissue from patients with colon cancer and colon polyps compared to matched microscopically normal tissue from the same donors as determined by microarray analysis.
  • Normal colon tissue from an 83 year-old donor (gender unknown) was compared to colon tumor tissue from the same donor (Huntsman Cancer Institute, Salt Lake City, UT).
  • Normal colon tissue from a 59 year-old male was compared to colon adenoma hyperplastic polyp tissue from the same donor Huntsman Cancer Institute).
  • SEQ ED NO:37 can each be used for one or more of the following: i) monitoring treatment of colon cancer, ii) diagnostic assays for colon cancer, and iii) developing therapeutics and/or other treatments for colon cancer.
  • Other cancers i) monitoring treatment of colon cancer, ii) diagnostic assays for colon cancer, and iii) developing therapeutics and/or other treatments for colon cancer.
  • SEQ ED NO:49-51 also showed differential expression in matched normal and tumor tissue samples from various donors with colon, lung, and ovarian cancers, as determined by microarray expression analysis.
  • SEQ ED NO:49 and SEQ ED NO:51 demonstrated at least a 2-fold decrease in expression in tissue from a colon polyp when compared to normal tissue from the same donor colon.
  • SEQ ED NO:51 had at least a 2-fold decrease in expression in several colon carcinoma tumor samples, when compared to matched normal tissues from the same donors.
  • SEQ ED NO:49 showed a 2.5-fold decrease in expression, in comparison to normal lung tissue from the same donor.
  • SEQ ID NO:51 was increased between 2.5- and 7-fold in squamous cell carcinoma tissue from several donor lungs, when compared to matched normal lung tissue from the same donors.
  • SEQ ID NO:49 and SEQ ED NO:50 both demonstrated at least a 2-fold decrease in expression in adenocarcinoma tissue from a donor with ovarian cancer, as compared to normal ovarian tissue from the same donor.
  • SEQ ED NO:32 showed differential expression associated with lung cancer, as determined by microarray analysis.
  • SEQ ED NO:32 showed at least a two-fold decrease in expression in lung tissue from patients with lung squamous cell carcinoma compared to matched microscopically normal tissue from the same donors as determined by microarray analysis.
  • SEQ ED NO:32 can be used for one or more of the following: i) monitoring treatment of lung cancer, ii) diagnostic assays for lung cancer, and iii) developing therapeutics and/or other treatments for lung cancer.
  • SEQ ED NO:49-51 can be used for one or more of the following: i) monitoring treatment of colon, lung, and ovarian cancers, ii) diagnostic assays for colon, lung, and ovarian cancers, and iii) developing therapeutics and/or other treatments for colon, lung, and ovarian cancers.
  • SEQ ED NO:52 showed differential expression in a number of osteosarcoma tumor samples, as measured by microarray expression analysis. Tumor samples of various types were taken from the tibias, femurs, or lung metastases of donors diagnosed with osteosarcoma and matched with normal bone or lung tissue from the same donors.
  • SEQ ED NO:52 showed a 2.5-fold decrease in expression in "soft" tumor tissue from donor with telangiectatic osteosarcoma when compared to normal femur tissue.
  • SEQ ED NO:52 showed between a 2- and 6- fold decrease in expression in "bony" tumor tissue, tumor tissue, and cultured tumor cells isolated from a donor with chondroblastic osteosarcoma, when compared with expression levels in normal femur tissue from the same donor. Additionally, “bony” and “soft” tumor tissue, tumor tissue, and cultured tumor cells isolated from the tibia of a donor with spindle cell osteosarcoma demonstrated a 3- to 9-fold decrease in expression of SEQ ED NO:52 when contrasted with normal tibia cells.
  • Tumor tissue from a patient with fibroblastic osteosarcoma when compared with normal femur tissue from this same patient, had a 3.5-fold decrease in SEQ ED NO:52 gene expression.
  • Another donor with chondroblastic osteosarcoma was examined, and found to have a 14-fold decrease in SEQ ED NO:52 expression in the tumor tissue when compared to normal femur tissue.
  • SEQ ED NO:52 expression was decreased 9.5-fold in tumor tissue, and nearly 3-fold in tumor cells, isolated from a donor with chondro-/osteoblastic osteosarcoma, when compared to normal femur cells from the same donor.
  • SEQ ED NO:52 can be used for one or more of the following: i) monitoring treatment of osteosarcomas, ii) diagnostic assays for osteosarcomas, and iii) developing therapeutics and/or other treatments for osteosarcomas.
  • the expression of SEQ ED NO:40 was decreased by at least two-fold in lung squamous cell carcinoma tissue as compared to normal lung tissue from a 73-year-old male donor.
  • the expression of SEQ ED NO:42 was increased by at least two-fold in lung squamous cell carcinoma tissue as compared to normal tissue from a 75-year-old female donor.
  • the expression of SEQ ED NO:40 was decreased by at least two-fold in lung squamous cell carcinoma tissue as compared to normal lung tissue from a 73-year-old male donor.
  • the expression of SEQ ED NO:42 was increased by at least two-fold in lung squamous cell carcinoma tissue as compared to normal tissue from a 75-year-old female donor.
  • NO:44 was decreased by at least two-fold in colon tumor tissue as compared to normal tissue from an 83-year-old donor.
  • Gene expression profiles of nonmalignant mammary epithelial cell lines were compared to gene expression profiles of a carcinoma cell line.
  • MDA-mb-231 a breast tumor cell line isolated from the pleural effusion of a 51-year-old female was compared to two non-malignant cell lines: MCF-IOA, a breast mammary gland (luminal ductal characteristics) cell line isolated from a 36- year-old woman with fibrocystic breast disease, and HMEC, a primary breast epithelial cell line isolated from a normal donor.
  • SEQ ED NO:44 was increased by at least two-fold in the carcinoma cell line as compared to the non-malignant cell lines. Therefore, in various embodiments, SEQ ED NO:40, SEQ ED NO:42 and SEQ ED NO:44 can each be used for one or more of the following: i) monitoring treatment of lung, breast, and colon cancer, ii) diagnostic assays for lung, breast, and colon cancer, and iii) developing therapeutics and/or other treatments for lung, breast, and colon cancer. Obesity In another example, SEQ ED NO:49 gene expression levels were altered in differentiating adipocytes isolated from various donors, as determined by microarray expression analysis.
  • the decrease in SEQ ID NO:49 expression was sustained at a level of at least 2-fold for up to 2.1 weeks, when contrasted with levels of expression in untreated adipocytes. Similar results were obtained when adipocytes from the obese donor were examined, with at least a 3-fold decrease in SEQ ID NO:49 gene expression levels after 48 hours, and a sustained 2-fold decrease in expression for up to 2.1 weeks, when comparing cells cultured in differentiation medium with untreated cells.
  • the expression of SEQ ID NO:37 was decreased at least two-fold in human adipocytes from either normal or obese donors that were treated with PPAR- ⁇ agonist and human insulin compared to non-treated adipocytes from the same donors.
  • Human primary subcutaneous preadipocytes were isolated from adipose tissue of a normal 28-year-old healthy female with a body mass index (BMI) of 23.59 and from an obese 40-year-old healthy female with a body mass index (BMI) of 32.47.
  • the preadipocytes were cultured and induced to differentiate into adipocytes by culturing them in a differentiation medium containing PPAR- ⁇ agonist and human insulin.
  • Human preadipocytes were treated with human insulin and PPAR- ⁇ agonist for three days and subsequently were switched to medium containing insulin alone for a total duration of 24 hours, 48 hours, four days, 8 days or 15 days before the cells were collected for analysis. Differentiated adipocytes were compared to untreated preadipocytes maintained in culture in the absence of inducing agents. Between 80% and 90% of the preadipocytes finally differentiated to adipocytes as observed under phase contrast microscope. In another example, SEQ ED NO:44 showed differential expression associated with obesity, as determined by microarray analysis. Human primary subcutaneous pre-adipocytes were isolated from adipose tissue of a 28-year-old healthy female with body mass index (BMI) of 23.59.
  • BMI body mass index
  • SEQ ID NO:37, SEQ ED NO:44 and SEQ ED NO:49 can each be used for one or more of the following: i) monitoring treatment of obesity and related disorders, ii) diagnostic assays for obesity and related disorders, and iii) developing therapeutics and/or other treatments for obesity and related disorders.
  • SEQ ID NO:49 and SEQ ID NO:50 showed differential expression in various regions of the brain isolated from a donor with sever Alzheimer's disease (AD), as determined by microarray analysis.
  • AD is a progressive dementia characterized neuropathologically by the presence of amyloid ⁇ -peptide-containing plaques and neurofibrillary tangles in specific brain regions.
  • neurons and synapses are lost and inflammatory responses are activated in microglia and astrocytes.
  • Gene expression profiling of mild, moderate, and severe AD cases will aid in defining the molecular mechanisms responsible for functional loss. Specific dissected brain regions from a donor with severe AD were compared with similar dissected regions from the brains of normal donors.
  • SEQ ID NO:49 gene expression was found to be upregulated at least 2-fold in severe AD-affected brain regions when compared to normal brain tissue from matched regions.
  • SEQ ED NO: 50 expression levels demonstrated at least a 2-fold decrease in brain regions from the severe AD patient; these regions included the anterior hippocampus and archeocortex thereof, and the globus pallidus striatum.
  • SEQ ED NO:50 expression was decreased in a sample from a patient with mild AD, in the dentate nucleus of the vermis of the cerebellum, compared to levels in this region from a normal donor.
  • SEQ ID NO:46 showed differential expression associated with Alzheimer's disease.
  • the expression of SEQ ID NO:46 was decreased at least two-fold in brain amygdala and hippocampus tissue from patients with Alzheimer's disease compared to normal brain tissue dissected from these regions as determined by microarray analysis.
  • SEQ ID NO:46 did not show differential expression in brain striatum, globus pallidus and cerebellum, dentate nucleus, vermis regions in tissue comparisons.
  • Brain tissue from a normal 61-year-old female was compared to brain tissue from a 68-year-old female with mild Alzheimer's disease and to brain tissue from a 79-year-old female with severe Alzheimer's disease.
  • SEQ ID NO:46 and SEQ ED NO:49 can each be used for one or more of the following: i) monitoring treatment of AD, ii) diagnostic assays for AD, and iii) developing therapeutics and/or other treatments for AD.
  • Steroid hormones i) monitoring treatment of AD, ii) diagnostic assays for AD, and iii) developing therapeutics and/or other treatments for AD.
  • SEQ ED NO:50 showed differential expression in hepatoma cells in response to a variety of steroid hormone treatments, as determined by microarray analysis. Analysis of the effects of such treatments on gene expression levels allow characterization of the anti- inflammatory responses, as well as the adverse effects, related to steroid use in treatment of various diseases.
  • SEQ ID NO:50 can be used for one or more of the following: i) monitoring of steroid treatment., ii) diagnostic assays for the effects of steroid treatment, and iii) developing therapeutics and/or other treatments associated with steroids and liver functioning. Inflamation
  • SEQ ID NO: 34 showed at least a 2-fold decrease in expression in vascular endothelial tissue treated with TNF- ⁇ compared with untreated vascular endothelial tissue, as determined by microarray analysis.
  • HUVEC cells were maintained in culture at 37° C in IMDM media with 10% FCS, and 5% CO 2 . Cultures were treated with recombinant human TNF- ⁇ (R&D Systems, Minneapolis MN) at 10 ng/ml for 1, 4, or 24 hours.
  • SEQ ED NO:34 can be used for one or more of the following: i) monitoring treatment of inflammatory disorders, cardiovascular disorders such as atherosclerosis, hypertension, and complications of thrombolysis and balloon angioplasty, and endothelial cell function, ii) diagnostic assays for inflammatory disorders, cardiovascular disorders such as atherosclerosis, hypertension, and complications of thrombolysis and balloon angioplasty, and endothelial cell function, and iii) developing therapeutics and/or other treatments for inflammatory disorders, cardiovascular disorders such as atherosclerosis, hypertension, and complications of thrombolysis and balloon angioplasty, and endothelial cell function.
  • SEQ ID NO: 34 showed differential expression in human C3A liver cell cultures treated with steroids compared to untreated cells as determined by microarray analysis.
  • Early confluent human liver C3A cells were treated with mifepristone, progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, betamethasone, or danazol at concentrations of 1 ⁇ M, 10 ⁇ M, and 100 ⁇ M for 1, 3, and 6 hours.
  • SEQ ED NO:34 showed at least a 2-fold increase in expression in C3A cells treated with progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, or betamethasone.
  • SEQ ED NO:34 can be used for one or more of the following: i) monitoring treatment of liver disorders associated with steroid therapy as well as liver, endocrine, reproductive, and inflammatory diseases, ii) diagnostic assays for liver disorders associated with steroid therapy as well as liver, endocrine, reproductive, and inflammatory diseases, and iii) developing therapeutics and/or other treatments for liver disorders associated with steroid therapy as well as liver, endocrine, reproductive, and inflammatory diseases.
  • Immune disorders i) monitoring treatment of liver disorders associated with steroid therapy as well as liver, endocrine, reproductive, and inflammatory diseases.
  • SEQ ED NO:42 and SEQ ED NO:44 showed differential expression associated with activation and differentiation of immune cells, as determined by microarray analysis. Comparisons of treated and untreated cell lines derived from patients with either acute monocytic (THP-1) or chronic myelogenic (K562) leukemia were made. The cell lines were activated by treatment with PMA or PMA and ionomycin, respectively. In PMA and ionomycin-treated K562 cells, expression of SEQ ED NO:44 was increased by at least two-fold as compared to untreated K562 cells. The expression of SEQ ED NO:42 was decreased by at least 6.5-fold in PMA-treated THP-1 cells as compared to untreated THP-1 cells.
  • SEQ ED NO:42 and SEQ ED NO:44 can each be used for one or more of the following: i) monitoring treatment of leukemic disorders, ii) diagnostic assays for leukemic disorders, and iii) developing therapeutics and/or other treatments for leukemic disorders.
  • the expression of SEQ ED NO:46 showed at least a two-fold increase in human peripheral blood mononuclear cells (PBMCs) treated with staphylococcal exotoxin, rapamycin, or interleukin-4 compared to untreated cells.
  • PBMCs contain the major cellular components of the immune system, including about 52% lymphocytes, 20% NK cells, 25% monocytes, and 3% various cells such as dendritic cells and progenitor cells.
  • Staphylococcal exotoxins specifically activate human T cells expressing an appropriate TCR-N ⁇ chain. Rapamycin is a macrolide antibiotic that prevents allograft rejection and is a potent inhibitor of cell proliferation.
  • Rapamycin suppresses T-cell activation by impairing the response to lymphokines such as interleukin-2 and interleukin-4.
  • Interleukin-4 is a pleiotropic cytokine produced by activated T cells, mast cells, and basophils, and acts as a B cell differentiation and stimulatory factor.
  • PBMCs from the blood of healthy donors were stimulated with 1 ng/ml Staphylococcal exotoxins in the presence or absence of 1 ⁇ M rapamycin or 10 ng/ml interleukin-4 for 2, 4, 24, and 72 hours. PBMCs were also treated with rapamycin alone.
  • SEQ ED NO:46 can be used for one or more of the following: i) monitoring treatment of immune disorders, ii) diagnostic assays for immune disorders, and iii) developing therapeutics and/or other treatments for immune disorders.
  • SEQ ID NO:59 showed tissue-specific expression as determined by microarray analysis.
  • RNA samples isolated from a variety of normal human tissues were compared to a common reference sample. Tissues contributing to the reference sample were selected for their ability to provide a complete distribution of RNA in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), small intestine (9%), spleen (3%), stomach (6%), testis (9%), and uterus (5%).
  • the normal tissues assayed were obtained from at least three different donors. RNA from each donor was separately isolated and individually hybridized to the microarray. Since these hybridization experiments were conducted using a common reference sample, differential expression values are directly comparable from one tissue to another.
  • the expression of SEQ ED NO:59 was increased by at least two-fold in skeletal muscle as compared to the reference sample. Therefore, SEQ ED NO:59 can be used as a tissue marker for skeletal muscle.
  • oligonucleotide Sequences complementary to the SCAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring SCAP. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of SCAP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the SCAP-encoding transcript.
  • SCAP Stac-lac
  • 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 SCAP upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
  • SCAP Stenorized calif ornica nuclear polyhedrosis vims
  • AcMNPN Autographica calif ornica nuclear polyhedrosis vims
  • the nonessential polyhedrin gene of baculovims is replaced with cD ⁇ A encoding SCAP 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 cD ⁇ A transcription.
  • baculoviras Recombinant baculoviras is used to infect Spodopterafrugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculoviras (Engelhard, E.K. et al. (1994) Proc. ⁇ atl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937- 1945).
  • SCAP is synthesized as a fusion protein with, e.g., glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crade cell lysates.
  • GST a 26- kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences). Following purification, the GST moiety can be proteolytically cleaved from SCAP at specifically engineered sites.
  • FLAG an 8-amino acid peptide
  • 6- His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins
  • SCAP function is assessed by expressing the sequences encoding SCAP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegaloviras promoter.
  • 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • an automated, laser optics-based technique is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death.
  • the influence of SCAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding SCAP and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding SCAP and other genes of interest can be analyzed by northern analysis or microarray techniques. XV. Production of SCAP Specific Antibodies
  • SCAP substantially purified using polyacrylamide gel electrophoresis PAGE; see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols.
  • the SCAP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art (Ausubel et al., supra, ch. 11).
  • oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma-
  • Naturally occurring or recombinant SCAP is substantially purified by immunoaffinity chromatography using antibodies specific for SCAP.
  • An immunoaffinity column is constmcted by covalently coupling anti-SCAP antibody to an activated chromatographic resin, such as
  • Media containing SCAP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of SCAP (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/SCAP 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 SCAP is collected.
  • SCAP SCAP, or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539).
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled SCAP, washed, and any wells with labeled SCAP complex are assayed. Data obtained using different concentrations of SCAP are used to calculate values for the number, affinity, and association of SCAP with the candidate molecules.
  • molecules interacting with SCAP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • SCAP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • a microtubule motility assay for SCAP measures motor protein activity. Ln this assay, recombinant SCAP is immobilized onto a glass slide or similar substrate. Taxol-stabilized bovine brain microtubules (commercially available) in a solution containing ATP and cytosolic extract are perfused onto the slide. Movement of microtubules as driven by SCAP motor activity can be visualized and quantified using video-enhanced light microscopy and image analysis techniques. SCAP activity is directly proportional to the frequency and velocity of microtubule movement.
  • an assay for SCAP measures the formation of protein filaments in vitro.
  • a solution of SCAP at a concentration greater than the "critical concentration" for polymer assembly is applied to carbon-coated grids. Appropriate nucleation sites may be supplied in the solution.
  • the grids are negative stained with 0.7% (w/v) aqueous uranyl acetate and examined by electron microscopy. The appearance of filaments of approximately 25 nm (microtubules), 8 nm (actin), or 10 nm (intermediate filaments) is a demonstration of SCAP activity.
  • SCAP activity is measured by the binding of SCAP to protein filaments.
  • 35 S-Met labeled SCAP sample is incubated with the appropriate filament protein (actin, tubulin, or intermediate filament protein) and complexed protein is collected by immunoprecipitation using an antibody against the filament protein. The irnmunoprecipitate is then ran out on SDS-PAGE and the amount of SCAP bound is measured by autoradiography.

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Abstract

La présente invention concerne, sous divers aspects, des protéines structurelles humaines liées au cytosquelette et des polynucléotides identifiant et codant pour des protéines structurelles humaines liées au cytosquelette. Des modes de réalisation de l'invention proposent également des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes, et des antagonistes. D'autres modes de réalisation de l'invention proposent des procédés pour le diagnostic, le traitement, ou la prévention de troubles liés à l'expression anormale des protéines structurelles humaines liées au cytosquelette.
PCT/US2003/001772 2002-01-18 2003-01-16 Proteines structurelle associees au cytosquelette WO2003062391A2 (fr)

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US20110086042A1 (en) * 2005-09-30 2011-04-14 Qingshen Gao Centrosomal Proteins, Nucleic Acids and Method of Use Thereof
US20110300141A1 (en) * 2005-06-17 2011-12-08 Balu Chakravarthy Novel Alphabeta-Binding protein and its peptide derivatives and uses thereof
US8871737B2 (en) 2010-09-22 2014-10-28 Alios Biopharma, Inc. Substituted nucleotide analogs
US8916538B2 (en) 2012-03-21 2014-12-23 Vertex Pharmaceuticals Incorporated Solid forms of a thiophosphoramidate nucleotide prodrug
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EP3988563A3 (fr) * 2016-08-26 2022-07-27 Immatics Biotechnologies GmbH Nouveaux peptides et échafaudages destinés à être utilisés en immunothérapie contre le carcinome à cellules squameuses de la tête et du cou et d'autres cancers
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US20220396841A1 (en) * 2019-12-02 2022-12-15 Viome Life Sciences, Inc. Detection and elimination of aberrant cells

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