CA2435260A1 - Receptors and membrane-associated proteins - Google Patents

Abstract

The invention provides human receptors and membrane-associated proteins (REMAP) and polynucleotides which identify and encode REMAP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, o r preventing disorders associated with aberrant expression of REMAP.

Description

RECEPTORS AND MEMBRANE-ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of receptors and membrane-s associated proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, autoimmune/inflammatory, neurological, metabolic, developmental, and endocrine disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of receptors and membrane-associated proteins.
BACKGROUND OF THE INVENTION
Signal transduction is the general process by which cells respond to extracellular signals.
Signal transduction across the plasma membrane begins with the binding of a signal molecule, e.g., a hormone, neurotransmitter, or growth factor, to a cell membrane receptor. The receptor, thus activated, triggers an intracellular biochemical cascade that ends with the activation of an intracellular target molecule, such as a transcription factor. This process of signal transduction regulates all types of cell functions including cell proliferation, differentiation, and gene transcription.
Biological membranes surround organelles, vesicles, and the cell itself.
Membranes are highly selective permeability barriers made up of lipid bilayer sheets composed of phosphoglycerides, fatty acids, cholesterol, phospholipids, glycolipids, proteoglycans, and proteins. Membranes contain ion pumps, ion channels, and specific receptors for external stimuli which transmit biochemical signals across the membranes. These membranes also contain second messenger proteins which interact with these pumps, channels, and receptors to amplify and regulate transmission of these signals.
Plasma Membrane Proteins Plasma membrane proteins (MPs) are divided into two groups based upon methods of protein extraction from the membrane. Extrinsic or peripheral membrane proteins can be released using extremes of ionic strength or pH, urea, or other disruptors of protein interactions. Intrinsic or integral membrane proteins are released only when the lipid bilayer of the membrane is dissolved by detergent.
The majority of known integral membrane proteins are transmembrane proteins (TM) which are characterized by an extracellular, a transmembrane, and an intracellular domain. TM domains are typically comprised of 15 to 25 hydrophobic amino acids which are predicted to adopt an a-helical conformation. TM proteins are classified as bitopic (Types I and II) and polytopic (Types III and IV) (Singer, S.J. (1990) Annu. Rev. Cell Biol. 6:247-96). Bitopic proteins span the membrane once while polytopic proteins contain multiple membrane-spanning segments. TM proteins carry out a variety of important cellular functions, including acting as cell-surface receptor proteins involved in signal transduction. These functions are represented by growth and differentiation factor receptors, and receptor-interacting proteins such as Drosophila pecanex and frizzled proteins, LIV-1 protein, NF2 protein, and GNS 1/SUR4 eukaryotic integral membrane proteins. TM proteins also act as transporters of ions or metabolites, such as gap junction channels (connexins), and ion channels, and as cell anchoring proteins, such as lectins, integrins, and fibronectins. TM
proteins are found in vesicle organelle-forming molecules, such as caveolins; or cell recognition molecules, such as cluster of differentiation (CD) antigens, glycoproteins, and mucins.
Many MPs contain amino acid sequence motifs that serve to localize proteins to specific subcellular sites. Examples of these motifs include PDZ domains, KDEL, RGD, NGR, and GSL
sequence motifs, von Willebrand factor A (vWFA) domains, and EGF-like domains.
RGD, NGR, and GSL motif containing peptides have been used as drug delivery agents in targeted cancer treatment of tumor vasculature (Arap, W. et al. (1998) Science, 279:377-380).
Furthermore, MPs may also contain amino acid sequence motifs that serve to interact with extracellular or intracellular molecules, such as carbohydrate recognition domains (CRD).
Chemical modification of amino acid residue side chains alters the manner in which MPs interact with other molecules, for example, phospholipid membranes. Examples of such chemical modifications to amino acid residue side chains are covalent bond formation with glycosaminoglycans, oligosaccharides, phospholipids, acetyl and palmitoyl moieties, ADP-ribose, phosphate, and sulphate groups.
RNA encoding membrane proteins may have alternative splice sites which give rise to proteins encoded by the same gene but with different messenger RNA and amino acid sequences.
Splice variant membrane proteins may interact with other ligand and protein isoforms.
Receptors The term receptor describes proteins that specifically recognize other molecules. The category is broad and includes proteins with a variety of functions. The bulk of receptors are cell surface proteins which bind extracellular ligands and produce cellular responses in the areas of growth, differentiation, endocytosis, and immune response. Other receptors facilitate the selective transport of proteins out of the endoplasmic reticulum and localize enzymes to particular locations in the cell. The team may also be applied to proteins which act as receptors for ligands with known or unknown chemical composition and which interact with other cellular components. For example, the steroid hormone receptors bind to and regulate transcription of DNA.
Cell surface receptors are typically integral plasma membrane proteins. These receptors recognize hormones such as catecholamines; peptide hormones; growth and differentiation factors;
small peptide factors such as thyrotropin-releasing hormone; galanin, somatostatin, and tachykinins;

and circulatory system-borne signaling molecules. Cell surface receptors on immune system cells recognize antigens, antibodies, and major histocompatibility complex (MHC)-bound peptides. Other cell surface receptors bind ligands to be internalized by the cell. This receptor-mediated endocytosis functions in the uptake of low density lipoproteins (LDL), transferrin, glucose- or mannose-terminal glycoproteins, galactose-terminal glycoproteins, immunoglobulins, phosphovitellogenins, fibrin, proteinase-inhibitor complexes, plasminogen activators, and thrombospondin (Lodish, H. et al. (1995) Molecular Cell Biolo~y, Scientific American Books, New York NY, p. 723;
Mikhailenko, I. et al.
(1997) J. Biol. Chem. 272:6784-6791).
Receptor Protein I~inases Many growth factor receptors, including receptors for epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, as well as the growth modulator a-thrombin, contain intrinsic protein kinase activities. When growth factor binds to the receptor, it triggers the autophosphorylation of a serine, threonine, or tyrosine residue on the receptor. These phosphorylated sites are recognition sites for the binding of other cytoplasmic signaling proteins. These proteins participate in signaling pathways that eventually link the initial receptor activation at the cell surface to the activation of a specific intracellular target molecule. In the case of tyrosine residue autophosphorylation, these signaling proteins contain a common domain referred to as a Src homology (SH) domain. SH2 domains and SH3 domains are found in phospholipase C-y, PI-3-I~ p85 regulatory subunit, Ras-GTPase activating protein, and pp60G5'°
(Lowenstein, E.J. et al. (1992) Cell 70:431-442). The cytokine family of receptors share a different common binding domain and include transmembrane receptors for growth hormone (GH), interleukins, erythropoietin, and prolactin.
Other receptors and second messenger-binding proteins have intrinsic serine/threonine protein kinase activity. These include activin/TGF-~3/BMP-superfamily receptors, calcium- and diacylglycerol-activated/phospholipid-dependant protein kinase (PK-C), and RNA-dependant protein kinase (PK-R). In addition, other serine/threonine protein kinases, including nematode Twitchin, have fibronectin-like, immunoglobulin C2-like domains.
G-protein coupled receptors The G-protein coupled receptors (GPCRs), encoded by one of the largest families of genes yet identified, play a central role in the transduction of extracellular signals across the plasma membrane. GPCRs have a proven history of being successful therapeutic targets.
GPCRs are integral membrane proteins characterized by the presence of seven hydrophobic transmembrane domains which together form a bundle of antipaxallel alpha (a) helices. GPCRs range in size from under 400 to over 1000 amino acids (Strosberg, A.D. (1991) Eur.
J. Biochem. 196:1-10;
Coughlin, S.R. (1994) Curr. Opin. Cell Biol. 6:191-197). The amino-terminus of a GPCR is extracellular, is of variable length, and is often glycosylated. The carboxy-terminus is cytoplasmic and generally phosphorylated. Extracellular loops alternate with intracellular loops and link the transmembrane domains. Cysteine disulfide bridges linking the second and third extracellular loops may interact with agonists and antagonists. The most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops. The transmembrane domains account, in part, for structural and functional features of the receptor. In most cases, the bundle of a helices forms a ligand-binding pocket. The extracellular N-terminal segment, or one or more of the three extracellular loops, may also participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor. In turn, the large, third intracellular loop of the activated receptor interacts with a heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, including the activation of second messengers such as cyclic AMP (cAMP), phospholipase C, and inositol triphosphate, and the interaction of the activated GPCR with ion channel proteins. (See, e.g., Watson, S. and S.
Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego CA, pp. 2-6; Bolander, F.F. (1994) Molecular Endocrinolo~y, Academic Press, San Diego CA, pp. 1G2-176;
Baldwin, J.M. (1994) Curr. Opin. Cell Biol. 6:180-190.) GPCRs include receptors for sensory signal mediators (e.g., light and olfactory stimulatory molecules); adenosine, y-aminobutyric acid (GABA), hepatocyte growth factor, melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine and norepinephrine, histamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin); chemokines; lipid mediators of inflammation (e.g., prostaglandins and prostanoids, platelet activating factor, and leukotrienes); and peptide hormones (e.g., bombesin, bradykinin, .calcitonin, C5a anaphylatoxin, endothelin, follicle-stimulating hormone (FSH), gonadotropic-releasing hormone (GnRI~, neurokinin, and thyrotropin-releasing hormone (TRH), and oxytocin). GPCRs which act as receptors for stimuli that have yet to be identified are known as orphan receptors.
The largest family of GPCRs consists of the rhodopsin-like GPCRs, wluch transmit diverse extracellular signals including hormones, neurotransmitters, and light.
Rhodopsin is a photosensitive GPCR found in animal retinas. In vertebrates, rhodopsin molecules axe embedded in membranous stacks found in photoreceptor (rod) Bells. Each rhodopsin molecule responds to a photon of light by triggering a decrease in cGMP levels which leads to the closure of plasma membrane sodium channels. In this manner, a visual signal is converted to a neural impulse.
Other rhodopsin-like GPCRs axe directly involved in responding to neurotransmitters. These GPCRs include the receptors for adrenaline (adrenergic receptors), acetylcholine (muscarinic receptors), adenosine, galanin, and glutamate (N-methyl-D-aspartate/NMDA receptors). (Reviewed in Watson, S. and S. Arkinstall (1994) The G-Protein Linked Receptor Facts Book, Academic Press, San Diego CA, pp. 7-9, 19-22, 32-35, 130-131, 214-216, 221-222; Habert-Ortoli, E. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:9780-9783.) The largest subfamily of GPCRs, the olfactory receptors, are also members of the rhodopsin-like GPCR family. These receptors function by transducing odorant signals.
Numerous distinct olfactory receptors are required to distinguish different odors. Each olfactory sensory neuron expresses only one type of olfactory receptor, and distinct spatial zones of neurons expressing distinct receptors are found in nasal passages. For example, the RAlc receptor which was isolated from a rat brain library, has been shown to be limited in expression to very distinct regions of the brain and a defined zone of the olfactory epithelium (Raining, K. et al. (1998) Receptors Channels 6:141-151).
The olfactory mucosa also appears to possess an additional group of odorant-binding proteins which recognize and bind separate classes of odorants. For example, cDNA
clones from rat have been isolated which correspond to mRNAs highly expressed in olfactory mucosa but not detected in other tissues. The proteins encoded by these clones are homologous to proteins that bind lipopolysaccharides or polychlorinated biphenyls, and the different proteins appear to be expressed in specific areas of the mucosal tissue. These proteins are believed to interact with odorants before or after specific recognition by odorant receptors, perhaps acting as selective signal filters (Deax, T.N. et al. (1991) EMBO J. 10:2813-2819; Vogt, R.G. et al. (1991) J. Neurobiol. 22:74-84).
Members of the secretin-like GPCR subfamily have as their ligands peptide hormones such as secretin, calcitonin, glucagon, growth hormone-releasing hormone, parathyroid hormone, and vasoactive intestinal peptide. For example, the secretin receptor responds to secretin, a peptide hormone that stimulates the secretion of enzymes and ions in the pancreas and small intestine (Watson, supra, pp. 278-283). Secretin receptors are about 450 amino acids in length and are found in the plasma membrane of gastrointestinal cells. Binding of secretin to its receptor stimulates the production of cAMP.
Examples of secretin-like GPCRs implicated in inflammation and the immune response include the EGF module-containing, mucin-like hormone receptor (Emr1) and CD97 receptor proteins. CD97 is predominantly expressed in leukocytes and is markedly upregulated on activated B
and T cells (McKnight, A.J. and S. Gordon (1998) J. Leukoc. Biol. 63:271-280).
These GPCRs are members of the recently characterized EGF-TM7 receptors subfamily. These seven transmembrane hormone receptors exist as heterodimers in vivo and contain between three and seven potential calcium-binding EGF-like motifs. The EGF motif is about forty amino acid residues in length and includes six conserved cysteine residues, and a calcium-binding site near the N-terminus of the signature sequence. Post-translational hydroxylation of aspartic acid or asparagine residues has been associated with EGF-like domains in several proteins (Prosite PDOC00010 Aspartic acid and asparagine hydroxylation site).

A number of proteins that contain calcium-binding EGF-like domain signature sequences are involved in growth and differentiation. Examples include bone morphogenic protein 1, which induces the formation of cartilage and bone; crumbs, which is a Drosonhila epithelial development protein; Notch and a number of its homologs, which are involved in neural growth and differentiation, and transforming growth factor beta-1 binding protein (Expasy PROSITE document PDOC00913;
Soler, C. and Carpenter, G., in Nicola, N.A. (1994) The Cytokine Facts Book, Oxford University Press, Oxford, UK, pp 193-197). EGF-like domains mediate protein-protein interactions for a variety of proteins. For example, EGF-like domains in the ECM glycoprotein fibulin-1 have been shown to mediate both self association and binding to fibronectin (Tram H. et al.
(1997) J. Biol. Chem.
272:22600-22606). Point mutations in the EGF-like domains of ECM proteins have been identified as the cause of human disorders such as Marfan syndrome and pseudochondroplasia (Maurer, P. et al.
(1996) Curr. Opin. Cell Biol. 8:609-617).
GPCR mutations, which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra,). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene. Furthermore, somatic activating mutations in the thyrotropin receptor have been reported to cause hyperfunctioning thyroid adenomas, suggesting that certain GPCRs susceptible to constitutive activation may behave as protooncogenes (Parma, J. et al.
(1993) Nature 365:649-651). GPCR receptors for the following ligands also contain mutations associated with human disease: luteinizing hormone (precocious puberty);
vasopressin VZ (X-linked nephrogenic diabetes); glucagon (diabetes and hypertension); calcium (hyperparathyroidism, hypocalcuria, hypercalcemia); parathyroid hormone (short limbed dwarfism); ~i3-adrenoceptor (obesity, non-insulin-dependent diabetes mellitus); growth hormone releasing hormone (dwarfism);
and adrenocorticotropin (glucocorticoid deficiency) (Wilson, S. et al. (1998) Br. J. Pharmocol.
125:1387-1392; Stadel, J.M. et al. (1997) Trends Phaxmacol. Sci. 18:430-437).
GPCRs are also involved in depression, schizophrenia, sleeplessness, hypertension, anxiety, stress, renal failure, and several cardiovascular disorders (Horn, F. and G. Vriend (1998) J. Mol. Med.
76:464-468).
In addition, within the past 20 years several hundred new drugs have been recognized that are directed towards activating or inhibiting GPCRs. The therapeutic targets of these drugs span a wide range of diseases and disorders, including cardiovascular, gastrointestinal, and central nervous system disorders as well as cancer, osteoporosis and endometriosis (Wilson, supra;
Stadel, supra). For example, the dopamine agonist L-dopa is used to treat Parkinson's disease, while a dopamine antagonist is used to treat schizophrenia and the early stages of Huntington's disease. Agonists and antagonists of adrenoceptors have been used for the treatment of asthma, high blood pressure, other cardiovascular disorders, and anxiety; muscarinic agonists are used in the treatment of glaucoma and tachycardia; serotonin 5HT1D antagonists are used against migraine; and histamine Hl antagonists are used against allergic and anaphylactic reactions, hay fever, itching, and motion sickness (Horn, supra).
Nuclear Hormone Receptors The nuclear hormone receptors, also known as the nuclear receptors or the intracellular receptors, constitute a protein superfamily whose members are both receptors and transcriptional regulators. Nuclear hormone receptors rely on both their receptor function and their transcriptional regulatory function to affect a broad array of biological processes, including development, homeostasis, cell proliferation, and cell differentiation. (Reviewed in Mangelsdorf, D.J. et al. (1995) Cell 83:835-840; Wen, D.X. and D.P. McDonnell (1995) Curr. Opin. Biotechnol.
6:582-589;
Perlmann, T. and R.M. Evans (1997) Cell 90:391-397; Tenbaum, S. and A.
Baniahmad (1997) Int. J.
Biochem. Cell Biol. 29:1325-1341; Moras, D. and H. Gronemeyer (1998) Curr.
Opin. Cell Biol.
10:384-391; Willy, P.J. and D.J. Mangelsdorf (1998) in: Hormones and Signaling (ed: B.W.
O'Malley) vol. 1, Academic Press, San Diego CA, pp. 307-358; Weatherman, R.V.
et al. (1999) Annu. Rev. Biochem. 68:559-581.) Nuclear hormone receptors as receptors Generally, the term receptor describes a protein that specifically recognizes other molecules.
As receptors, nuclear hormone receptors specifically recognize and bind to their cognate ligands.
Although nuclear hormone receptors are located intracellularly, many receptors are extracellular cell surface proteins which bind extracellular ligands. Such extracellular receptors produce cellular responses affecting growth, differentiation, endocytosis, and the immune response. Other receptors facilitate the selective transport of proteins out of the endoplasmic reticulum and localize enzymes to particular regions of the cell. Transcriptional regulation by nuclear hormone receptors, propagation of cellular signals by extracellular receptors, and transport and localization of proteins by other receptors, all rely upon specific interactions between the receptors and a variety of cellular components. In many cases, the identity of the cognate ligand to which a receptor binds is unknown.
Such receptors are termed orphan receptors. This term also applies to those nuclear hormone receptors which carry out their transcriptional regulatory functions without binding any ligands.
Nuclear hormone receptors as transcriptional re u1 Multicellular organisms are comprised of diverse cell types that differ dramatically both in structure and function. The identity of a cell is determined by its characteristic pattern of gene expression, and different cell types express overlapping but distinctive sets of genes throughout development. Spatial and temporal regulation of gene expression is critical for the control of cell proliferation, cell differentiation, apoptosis, and other processes that contribute to organismal development. As transcriptional regulators, nuclear hormone receptors play key roles in controlling these fundamental biological processes. Other transcriptional regulators affect gene expression in response to extracellular signals that mediate cell-cell communication and that coordinate the activities of different cell types.
In general, transcriptional regulators such as nuclear hormone receptors initiate, activate, repress, or terminate gene transcription by binding to the promoter, enhancer, and upstream regulatory regions of a gene in a sequence-specific manner. However, some transcriptional regulators bind regulatory elements within or downstream of a gene's coding region. Transcriptional regulatory proteins may bind to a specific region of DNA singly, or in a complex with other accessory factors. (Reviewed in Lewin, B. (1990) in: Genes IV, Oxford University Press, New York NY, and Cell Press, Cambridge MA, pp. 554-570.) Mechanism of nuclear hormone receptor function In the unliganded state, a nuclear hormone receptor exists in association with a multiprotein complex of chaperones, including heat shock proteins such as hsp90 and immunophilins such as hsp56. These chaperones maintain the ligand-free receptor in an inactive state which is amenable to binding of free ligand, and prevent the ligand-free receptor from translocating to the nucleus. Upon activation by its cognate ligand, the receptor may form a homodimer or heterodimer which translocates to the nucleus, binds to specific DNA sequences, and exerts its transcriptional regulatory function. In order to effectively carry out its regulatory roles, an activated nuclear hormone receptor dissociates from a histone deacetylase-containing corepressor complex and associates with a histone acetyltransferase-containing coactivator complex (Xu, L. et al. (1999) Curr.
Opin. Genet. Dev. 9:140-147). The association of the activated receptor with coactivator proteins results in remodeling of chromatin so that it adopts an open transcriptionally active state, providing access to the transcriptional regulatory elements of the activated nuclear receptor (Lemon, B.D. and L.P. Freedman (1999) Curr. Opin. Genet. Dev. 9:499-504).
Structure of nuclear hormone receptors Nuclear hormone receptors function as signal transducers by converting hormonal signals into transcriptional responses. In general, nuclear hormone receptors consist of a variable amino-terminal domain, a highly conserved DNA-binding domain, and a conserved C-terminal ligand-binding domain. In the steroid-binding nuclear hormone receptors, the amino-terminal domain harbors a trans-activation element termed AF-1. Some nuclear hormone receptors also contain a trans-activation element in the ligand-binding domain termed AF-2. The DNA-binding and ligand-binding domains of nuclear hormone receptors may contain dimerization elements, and the DNA-binding domain may contain a nuclear localization signal (Weatherman, R.V. et al. (1999) Annu. Rev.
Biochem. 68:559-581).
The DNA-binding domain of nuclear hormone receptors is composed of two zinc finger motifs which mediate recognition of specific DNA sequences. A zinc finger motif contains periodically spaced cysteine and histidine residues which coordinate Zn+2.
Examples of this sequence pattern include the C2H2-type, C4-type, and C3HC4-type ("RING" finger) zinc fingers, and the PHD
domain (Lewin, supra; Aasland, R. et al. (1995) Trends Biochem. Sci. 20:56-59). A zinc finger motif contains an a helix and an antiparallel !3 sheet whose proximity and conformation are maintained by the zinc ion. Contact with DNA is made by the arginine preceding the oc helix and by the second, third, and sixth residues of the a helix. Zinc finger motifs may be repeated in a tandem array within a protein such that the a helix of each zinc finger in the protein makes contact with the major groove of the DNA double helix. This repeated contact between the protein and the DNA
produces a strong and specific DNA-protein interaction. The strength and specificity of the interaction can be regulated by the number of zinc forger motifs within the protein. Although zinc fingers were originally identified in DNA-binding proteins as regions that interact directly with DNA, they have since been found in proteins that do not bind to DNA. (See, e.g., Lodish, H. et al. (1995) Molecular Cell Biolo~y, Scientific American Books, New York NY, pp. 447-451.) The ligand-binding domain of nuclear hormone receptors is responsible for binding to ligands, coactivator proteins, and coreprescor proteins. This domain is composed of three layers of a helices, with the central layer consisting of two helices containing many hydrophobic side chains (Moms, D. and H. Gronemeyer (1998) Curr. Opin. Cell Biol. 10:384-391). These two central a helices thus create a hydrophobic pocket which is the site of ligand binding.
A ligand bound in this hydrophobic ligand-binding site is completely buried inside the receptor protein and is not exposed to solvent. This suggests that large conformational changes in the ligand-binding domain would accompany binding of a ligand. One of the a helices of the ligand-binding domain provides many of the inter-subunit contacts in dimers of nuclear receptors. This a helix contacts the ligand when it is bound in the ligand-binding pocket, suggesting that ligand binding can affect formation of receptor dimers (Weatherman, R.V. et al. (1999) Annu. Rev. Biochem. 68:559-581).
Classes of nuclear hormone receptors Nuclear hormone receptors can be grouped into three broad classes: the steroid receptors, the RXR-heterodimeric receptors, and the orphan nuclear hormone receptors. The steroid receptors bind to steroid hormones, and this class includes the androgen receptor, mineralocorticoid receptor, estrogen receptor, glucocorticoid receptor, and progesterone receptor. The RXR-heterodimeric receptors bind to nonsteroid ligands, and this class includes the thyroid hormone receptor, retinoic acid receptor, vitamin D receptor, ecdysone receptor, and peroxisome proliferator activated receptor.
The orphan nuclear hormone receptors include steroidogenic factor 1, nerve growth factor-induced receptor, and X-linked orphan receptor DAX-1.
The steroid hormone receptors are activated upon binding to specific steroid hormones. The conformational change induced by ligand binding leads to dissociation of the receptor from heat shock proteins and formation of receptor homodimers which recognize specific palindromic DNA
sequences called hormone response elements (HREs). Upon binding to an HRE, a steroid hormone receptor homodimer can regulate the transcription of target genes.
For example, the progesterone receptor (PR) is a steroid hormone receptor which is activated by progesterone, a 4-pregnene-3,20-dione derived from cholesterol which is a critical oscillating component of the female reproductive cycle. These oscillations correlate with anatomical and morphological changes including menstruation and pregnancy. The activities of progesterone are mediated through PR. In the cytoplasm, PR associates with several other proteins and factors known as the PR heterocomplex. This heterocomplex includes heat shock proteins and immunophilins such as hsp70, hsp90, hsp27, p59 (hsp56), p48, and p23 (Johnson, J.L. et al. (1994) Mol. Cell. Biol.
14:1956-1963). Upon binding progesterone, activated PR translocates to the nucleus, binds to canonical DNA transcriptional elements, and regulates progesterone-regulated genes implicated in differentiation and the cell cycle (Moutsatsou, P and C.E. Sekeris (1997) Ann.
N.Y. Acad. Sci.
816:99-115). The PR antagonist RU 486, which can be used to terminate a pregnancy, is an example of a commercial therapeutic targeted toward a steroid hormone receptor.
The RXR-heterodimeric nuclear receptors are distinguished from the steroid hormone receptors in that members of the former group bind to their target DNA
sequences upon formation of heterodimers with retinoid X receptors (RXRs) (Mangelsdorf, D.J. and R.M.
Evans (1995) Cell 83:841-850). Three different isoforms of RXR have been identified (Minucci, S.
and I~. Ozato (1996) Curr. Opin. Genet. Dev. 6:567-574). The retinoic acid receptors (RARs) are examples of RXR-heterodimeric nuclear receptors. Retinoic acid (RA) is a biologically active metabolite of vitamin A (retinol), a fat-soluble vitamin found mainly in fish liver oils, liver, egg yolk, butter, and cream. While 9-cis-RA binds to RARs and RXRs, all-trans-RA binds only to RXRs.
RAR/RXR
heterodimers bind with high affinity to specific DNA sequences known as retinoic acid response elements (RAREs), thus acting as regulators of RA-dependent transcription.
Peroxisome proliferator activated receptors (PPARs) are therapeutically important RXR-heterodimeric nuclear receptors which are induced by fatty acids and eicosanoid~. There are three known isotypes of PPAR, each with specific expression patterns, and these PPARs are involved in the regulation of genes involved in systemic homeostatic of glucose and lipids (I~liewer, S.A. and T.M.
Willson (1998) Curr. Opin. Genet. Dev. 8:576-581; Michalik, L. and W. Wahli (1999) Curr. Opin.
Biotechnol. 10:564-570). As such, PPARs are therapeutic targets for disorders such as diabetes, dyslipidemia, and obesity (Smith, S.A. (1996) Pharmacol. Rev. Commun. 8:57-64;
Willson, T.M. and W. Wahli (1997) Curr. Opin. Chem. Biol. 1:235-241; Barroso, I. et al. (1999) Nature 402:880-883).
The orphan nuclear receptors either have no known activating ligand, or can exert their transcriptional regulatory activities without benefit of ligand binding. For example, in Caenorhabditis ele~ans, the X-chromosome encoded nuclear hormone receptor homologue SEX-1 regulates transcription of the sex determination gene xol-1 (Carmi, I, et al. (1998) Nature 396:168-173). Rather than relying on ligand binding, SEX-1 acts as a transcriptional regulator in a dose-dependent manner, in effect controlling sexual differentiation through an X-chromosome-counting mechanism.
Some nuclear hormone receptors lack the conventional DNA-binding domain typically associated with the nuclear hormone receptor family. DAX-1 is one such nuclear hormone receptor lacking the conventional DNA-binding domain, and mutations in DAX-1 have been shown to cause X-linked adrenal hypoplasia congenita (Zanaria, E.F. et al. (1994) Nature 372:635-641). DAX-1 is an orphan nuclear receptor which interacts directly with steroidogenic factor 1 (SF-1) (Ito, M. et al.
(1997) Mol. Cell. Biol. 17:1476-1483), and DAX-1 is capable of modulating the action of SF-1 in sex-specific gene expression (Nachtigal, M.W. et al. (1998) Cell 445-454). SF-1 is an orphan nuclear receptor which acts as a transcription factor for several steroidogenic enzyme genes in the adrenal gland and gonads (Lala, D.S. et al. (1992) Mol. Endocrinol. 6:1249-1258;
Lynch, J.P. et al. (1993) Mol. Endocrinol. 7:776-786; Clemens, J.W. et al. (1994) Endocrinology 134:1499-1508), and can also regulate several genes expressed in pituitary gonadotrope cells (Barnhart, K.M. and P.L. Mellon (1994) Mol. Endocrinol. 8:878-885; Ingraham, H.A. et al. (1994) Genes Dev.
8:2302-2312;
Halvorson, L.M. et al. (1996) J. Biol. Chem. 271:6645-6650; Keri, R.A. and J.H. Nilson (1996) J.
Biol. Chem. 271:10782-10785).
SF-1 also acts as a potent transactivator of small heterodimer partner (SHP;
short heterodimer partner) (Lee, Y.K. et al. (1999) J. Biol. Chem. 274:20869-20873). SHP is another example of a nuclear hormone receptor lacking the conventional DNA-binding domain (Seol, W.
et al. (1996) Science 272:1336-1339; Lee, H.-K. et al. (1998) J. Biol. Chem. 273:14398-14402). SHP interacts with many members of the nuclear hormone receptor family, including retimoid receptors, estrogen receptor, thyroid hormone receptor, and the orphan receptor CAR. SHP acts as an inhibitor of estrogen receptor-mediated transcriptional activation by competing with coactivators for binding to estrogen receptor (Johansson, L. et al. (1999) J. Biol. Chem. 274:345-353).
SHP also inhibits transactivation by the orphan receptor hepatocyte nuclear factor 4, and by retinoid X receptor (Lee, Y.K. et al. (2000) Mol. Cell. Biol. 20:187-195).
Consequences of defective transcription regulation Many neoplastic disorders in humans can be attributed to inappropriate gene expression.
Malignant cell growth may result from either excessive expression of tumor promoting genes or insufficient expression of tumor suppxessor genes (Cleary, M.L. (1992) Cancer Surv. 15:89-104).
Chromosomal translocations may also produce chimeric loci which fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. Such an arrangement likely results in inappropriate gene transcription, potentially contributing to malignancy.

In addition, the immune system responds to infection or trauma by activating a cascade of events that coordinate the progressive selection, amplification, and mobilization of cellular defense mechanisms. A complex and balanced program of gene activation and repression is involved in this process. However, hyperactivity of the immune system as a result of improper or insufficient regulation of gene expression may result in considerable tissue or organ damage. This damage is well documented in immunological responses associated with arthritis, allergens, heart attack, stroke, and infections. (See, e.g., Isselbacher et al. (1996) Harnson's Principles of Internal Medicine, 13/e, McGraw Hill, Inc. and Teton Data Systems Software.) Furthermore, the growth of multicellular organisms is based upon the induction and coordination of cell differentiation at the appropriate stages of development.
Central to this process is differential gene expression, which confers the distinct identities of cells and tissues throughout the body. Failure to regulate gene expression during development could result in developmental disorders.
Ligand-Gated Receptor Ion Channels Ligand-gated receptor ion channels fall into two categories. The first category, extracellular ligand-gated receptor ion channels (ELGs), rapidly transduce neurotransmitter-binding events into electrical signals, such as fast synaptic neurotransmission. ELG function is regulated by post-translational modification. The second category, intracellular ligand-gated receptor ion channels (II,Gs), are activated by.many intracellular second messengers and do not require post-translational modifications) to effect a channel-opening response.
ELGs depolarize excitable cells to the threshold of action potential generation.. In non-excitable cells, ELGs permit a limited calcium ion-influx during the presence of agonist. ELGs include channels directly gated by neurotransmitters such as acetylcholine, L-glutamate, glycine, ATP, serotonin, GABA, and histamine. ELG genes encode proteins having strong structural and functional similarities. lLGs are encoded by distinct and unrelated gene families and include receptors for cAMP, cGMP, calcium ions, ATP, and metabolites of arachidonic acid.
Macrophage Scavenger Receptors Macrophage scavenger receptors with broad ligand specificity may participate in the binding of low density lipoproteins (LDL) and foreign antigens. Scavenger receptors types I and II are trimeric membrane proteins with each subunit containing a small N-terminal intracellular domain, a transmembrane domain, a large extracellular domain, and a C-terminal cysteine-rich domain. The extracellular domain contains a short spacer domain, an a-helical coiled-coil domain, and a triple helical collagenous domain. These receptors have been shown to bind a spectrum of ligands, including chemically modified lipoproteins and albumin, polyribonucleotides, polysaccharides, phospholipids, and asbestos (Matsumoto, A. et al. (1990) Proc. Natl. Acad.
Sci. USA 87:9133-9137;

Elomaa, O. et al. (1995) Cell 80:603-609). The scavenger receptors are thought to play a key role in atherogenesis by mediating uptake of modified LDL in arterial walls, and in host defense by binding bacterial endotoxins, bacteria, and protozoa.
T-Cell Receptors T cells play a dual role in the immune system as effectors and regulators, coupling antigen recognition with the transmission of signals that induce cell death in infected cells and stimulate proliferation of other immune cells. Although a population of T cells can recognize a wide range of different antigens, an individual T cell can only recognize a single antigen and only when it is presented to the T cell receptor (TCR) as a peptide complexed with a major histocompatibility molecule (MHC) on the surface of an antigen presenting cell. The TCR on most T
cells consists of immunoglobulin-like integral membrane glycoproteins containing two polypeptide subunits, a and (3, of similar molecular weight. Both TCR subunits have an extracellular domain containing both variable and constant regions, a transmembrane domain that traverses the membrane once, and a short intracellular domain (Saito, H. et al. (1984) Nature 309:757-762). The genes for the TCR subunits are constructed through somatic rearrangement of different gene segments.
Interaction of antigen in the proper MHC context with the TCR initiates signaling cascades that induce the proliferation, maturation, and function of cellular components of the immune system (Weiss, A. (1991) Annu. Rev.
Genet. 25: 487-510). Rearrangements in TCR genes and alterations in TCR
expression have been noted in lymphomas, leukemias, autoimmune disorders, and immunodeficiency disorders (Aisenberg, A.C. et al. (1985) N. Engl. J. Med. 313:529-533; Weiss, supra).
Selectins Selectins, or LEC-CAMS, comprise a specialized lectin subfamily involved primarily in inflammation and leukocyte adhesion (reviewed in Lasky, L.A. (1991) J. Cell.
Biochem. 45:139-146).
Selectins mediate the recruitment of leukocytes from the circulation to sites of acute inflammation and are expressed on the surface of vascular endothelial cells in response to cytokine signaling.
Selectins bind to specific ligands on the leukocyte cell membrane and enable the leukocyte to adhere to and migrate along the endothelial surface. Binding of selectin to its ligand leads to polarized rearrangement of the actin cytoskeleton and stimulates signal transduction within the leukocyte .
(Brenner, B. et al. (1997) Biochem. Biophys. Res. Commun. 231:802-807; Hidari, K.I. et al. (1997) J.
Biol. Chem. 272:28750-28756). Members of the selectin family possess three characteristic motifs: a lectin or carbohydrate recognition domain; an epidermal growth factor-like domain; and a variable number of short consensus repeats (scr or "sushi" repeats). Sushi domains, also known as complement control protein (CCP) modules, or short consensus repeats (SCR), occur in a wide variety of complement and adhesion proteins (Norman, D.G. et al. (1991) J.
Mol. Biol. 219:717-725).
Netrin Receptors The netrins are a family of molecules that function as diffusible attractants and repellants to guide migrating cells and axons to their targets within the developing nervous system. The netrin receptors include the C. elegans protein UNC-5, as well as homologues recently identified in vertebrates (Leonardo, E.D. et al. (1997) Nature 386:833-838). These receptors are members of the immunoglobulin superfamily, and also contain a characteristic domain called the ZU5 domain.
Mutations in the mouse member of the netrin receptor family, Rcm (rostral cerebellar malformation) result in cerebellar and midbrain defects as an apparent result of abnormal neuronal migration (Ackerman, S.L. et al. (1997) Nature 386:838-842).
VPS 10 Domain Containing Receptors The members of the VPS 10 domain containing receptor family all contain a domain with homology to the yeast vacuolar sorting protein 10 (VPS10) receptor. This family includes the mosaic receptor SorLA, the neurotensin receptor sortilin, and SorCS, which is expressed during mouse embryonal and early postnatal nervous system development (Hermey, G. et al.
(1999) Biochem.
Biophys. Res. Commun. 266:347-351; Hermey, G. et al. (2001) Neuroreport 12:29-32). A recently identified member of this family, SorCS2, is highly expressed in the developing and mature mouse central nervous system. Its main site of expression is the floor plate, and high levels are also detected transiently in brain regions including the dopaminergic brain nuclei and the dorsal thalamus (Rezgaoui, M. (2001) Mech. Dev. 100:335-338).
Membrane-Associated Proteins Tetraspan Family Proteins The transmembrane 4 superfamily (TM4SF) or tetraspan family is a multigene family encoding type III integral membrane proteins (Wright, M.D. and Tomlinson, M.G.
(1994) Immunol.
Today 15:588). The TM4SF is comprised of membrane proteins which traver$e the cell membrane four times. Members of the TM4SF include platelet and endothelial cell membrane proteins, melanoma-associated antigens, leukocyte surface glycoproteins, colonal carcinoma antigens, tumor-associated antigens, and surface proteins of the schistosome parasites (Jankowski, S.A. (1994) Oncogene 9:1205-1211). Members of the TM4SF share about 25-30% amino acid sequence identity with one another. A number of TM4SF members have been implicated in signal transduction, control of cell adhesion, regulation of cell growth and proliferation, including development and oncogenesis, and cell motility, including tumor cell metastasis. Expression of TM4SF
proteins is associated with a variety of tumors and the level of expression may be altered when cells are growing or activated.
Tumor Antigens Tumor antigens are surface molecules that are differentially expressed in tumor cells relative to normal cells. Tumor antigens distinguish tumor cells immunologically from normal cells and provide diagnostic and therapeutic targets for human cancers (Takagi, S. et al. (1995) Int. J. Cancer 61:706-715; Liu, E. et al. (1992) Oncogene 7:1027-1032).
Ion Channels Ion channels are found in the plasma membranes of virtually every cell in the body. For example, chloride channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ions across epithelial membranes.
When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, chloride channels also regulate organelle pH. (See, e.g., Greger, R. (1988) Annu. Rev. Physiol.
50:111-122.) Electrophysiological and pharmacological properties of chloride channels, including ion conductance, current-voltage relationships, and sensitivity to modulators, suggest that different chloride channels exist in muscles, neurons, fibroblasts, epithelial cells, and lymphocytes.
Many channels have sites for phosphorylation by one or more protein kinases including protein kinase A, protein kinase C, tyrosine kinase, and casein kinase II, all of which regulate ion channel activity in cells. Inappropriate phosphorylation of proteins in cells has been linked to changes in cell cycle progression and cell differentiation. Changes in the cell cycle have been linked to induction of apoptosis or cancer.
Changes in cell differentiation have been linked to diseases and disorders of the reproductive system, immune system, and skeletal muscle.
Cerebellar granule neurons possess a non-inactivating potassium current which modulates firing frequency upon receptor 'stimulation by neurotransmitters and controls the resting membrane potential. Potassium channels that exhibit non-inactivating currents include the ether a go-go (EAG) channel. A membrane protein designated KCR1 specifically binds to rat EAG by means of its C-terminal region and regulates the cerebellar non-inactivating potassium current. KCRl is predicted to contain 12 transmembrane domains, with intracellular amino and carboxyl termini. Structural characteristics of these transmembrane regions appear to be similar to those of the transporter superfamily, but no homology between KCR1 and known transporters was found, suggesting that KCR1 belongs to a novel class of transporters. KCR1 appears to be the regulatory component of non-inactivating potassium channels (Hoshi, N. et al. (1998) J. Biol. Chem.
273:23080-23085).
ABC Transporters ATP-binding cassette (ABC) transporters, also called the "traffic ATPases", are a superfamily of membrane proteins that mediate transport and channel functions in prokaryotes and eukaryotes (Higgins, C.F. (1992) Annu~ Rev. Cell Biol. 8:67-113). ABC proteins share a similar overall structure and significant sequence homology. All ABC proteins contain a conserved domain of approximately two hundred amino acid residues which includes one or more nucleotide binding domains. Mutations in ABC transporter genes are associated with various disorders, such as hyperbilirubinemia II/Dubin-Johnson syndrome, recessive Stargardt's disease, X-linked adrenoleukodystrophy, multidrug resistance, celiac disease, and cystic fibrosis.

Semaphorins and Neuropilins Semaphorins are a large group of axonal guidance molecules consisting of at least 30 different members and are found in vertebrates, invertebrates, and even certain viruses. All semaphorins contain the sema domain which is approximately 500 amino acids in length. Neuropilin, a semaphorin receptor, has been shown to promote neurite outgrowth in vitro.
The extracellular region of neuropilins consists of three different domains: CUB, discoidin, and MAM domains. The CUB and the MAM motifs of neuropilin have been suggested to have roles in protein-protein interactions and are thought to be involved in the binding of semaphorins through the sema and the C-terminal domains (reviewed in Raper, J.A. (2000) Curr. Opin. Neurobiol.
10:88-94).
Membrane Proteins Associated with Intercellular Communication Intercellular communication is essential for the development and survival of multicellular organisms. Cells communicate with one another through the secretion and uptake of protein signaling molecules. The uptake of proteins into the cell is achieved by endocytosis, in which the interaction of signaling molecules with the plasma membrane surface, often via binding to specific receptors, results in the formation of plasma membrane-derived vesicles that enclose and transport the molecules into the cytosol. The secretion of proteins from the cell is achieved by exocytosis, in which molecules inside of the cell are packaged into membrane-bound transport vesicles derived from the trar2s Golgi network. These vesicles fuse with the plasma membrane and release their contents into the surrounding extracellular space. Endocytosis and exocytosis result in the removal and addition of plasma membrane components, and the recycling of these components is essential to maintain the integrity, identity, and functionality of both the plasma membrane and internal membrane-bound compartments.
Nogo has been identified as a component of the central nervous system myelin that prevents axonal regeneration in adult vertebrates. Cleavage of the Nogo-66 receptor and other glycophosphatidylinositol-linked proteins from axonal surfaces renders neurons insensitive to Nogo-66, facilitating potential recovery from CNS damage (Fournier, A.E. et al.
(2001) Nature 409:341-346).
The slit proteins are extracellulax matrix proteins expressed by cells at the ventral midline of the nervous system. Slit proteins are ligands for the repulsive guidance receptor Roundabout (Robo) and thus play a role in repulsive axon guidance (Brose, K. et al. (1999) Cell 96:795-806).
Lysosomes are the site of degradation of intracellular material during autophagy and of extracellular molecules following endocytosis. Lysosomal enzymes are packaged into vesicles which bud from the traps-Golgi network. These vesicles fuse with endosomes to form the mature lysosome in which hydrolytic digestion of endocytosed material occurs. Lysosomes can fuse with autophagosomes to form a unique compartment in which the degradation of organelles and other intracellular components occurs.
Protein sorting by transport vesicles, such as the endosome, has important consequences for a variety of physiological processes including cell surface growth, the biogenesis of distinct intracellular organelles, endocytosis, and the controlled secretion of hormones and neurotransmitters (Rothman, J.E. and Wieland, F.T. (1996) Science 272:227-234). In particular, neurodegenerative disorders and other neuronal pathologies are associated with biochemical flaws during endosomal protein sorting or endosomal biogenesis (Mayer R.J. et al. (1996) Adv. Exp.
Med. Biol. 389:261-269).
Peroxisomes are organelles independent from the secretory pathway. They are the site of many peroxide-generating. oxidative reactions in the cell. Peroxisomes are unique among eukaryotic organelles in that their size, number, and enzyme content vary depending upon organism, cell type, and metabolic needs (Waterham, H.R. and Cregg, J.M. (1997) BioEssays 19:57-66). Genetic defects in peroxisome proteins which result in peroxisomal deficiencies have been linked to a number of human pathologies, including Zellweger syndrome, rhizomelic chonrodysplasia punctata, X-linked adrenoleukodystrophy, aryl-CoA oxidase deficiency, bifunctional enzyme deficiency, classical Refsum's disease, DHAP alkyl transferase deficiency, and acatalasemia (Moser, H.W. and Moser, A.B. (1996) Ann. NY Acad. Sci. 804:427-441). In addition, Gartner, J. et al.
(1991; Pediatr. Res.
29:141-146) found a 22 kDa integral membrane protein associated with lower density peroxisome-like subcellular fractions in patients with Zellweger syndrome.
Normal embryonic development and control of germ cell maturation is modulated by a number of secretory proteins which interact with their respective membrane-bound receptors. Cell fate during embryonic development is determined by members of the activin/TGF-(3 superfamily, cadherins, IGF-2, and other morphogens. In addition, proliferation, maturation, and redifferentiation of germ cell and reproductive tissues are regulated, for example, by IGF-2, inhibins, activins, and follistatins (Petraglia, F. (1997) Placenta 18:3-8; Mather, J.P. et al. (1997) Proc. Soc. Exp. Biol. Med.
215:209-222). Transforming growth factor beta (TGFbeta) signal transduction is mediated by two receptor Ser/Thr kinases acting in series, type II TGFbeta receptor and (TbetaR-II) phosphorylating type I TGFbeta receptor (TbetaR-I). TbetaR-I-associated protein-1 (TRECAP-1), which distinguishes between quiescent and activated forms of the type I transforming growth factor beta receptor, has been associated with TGFbeta signaling (Charng, M.J et al. (1998) J. Biol.
Chem. 273:9365-9368).
Retinoic acid receptor alpha (RAR alpha) mediates retinoic-acid induced maturation and~has been implicated in myeloid development. Genes induced by retinoic acid during granulocytic differentiation include E3, a hematopoietic-specific gene that is an immediate target for the activated RAR alpha during myelopoiesis (Scott, L.M. et al. (1996) Blood 88:2517-2530).
The ~.-opioid receptor (MOR) mediates the actions of analgesic agents including morphine, codeine, methadone, and fentanyl as well as heroin. MOR is functionally coupled to a G-protein-activated potassium channel (Mestek A. et al. (1995) J. Neurosci. 15:2396-2406). A variety of MOR
subtypes exist. Alternative splicing has been observed with MOR-1 as with a number of G
protein-coupled receptors including somatostatin 2, dopamine D2, prostaglandin EP3, and serotonin receptor subtypes 5-hydroxytryptamine4 and 5-hydroxytryptamine7 (Pan, Y.X. et al. ( 1999) Mol.
Pharm. 56:396-403).
Peripheral and Anchored Membrane Proteins Some membrane proteins are not membrane-spanning but are attached to the plasma membrane via membrane anchors or interactions with integral membrane proteins.
Membrane anchors are covalently joined to a protein post-translationally and include such moieties as prenyl, myristyl, and glycosylphosphatidyl inositol groups. Membrane localization of peripheral and anchored proteins is important for their function in processes such as receptor-mediated signal transduction. For example, prenylation of Ras is required for its localization to the plasma membrane and for its normal and oncogenic functions in signal transduction.
Extracellular Messengers Intercellular communication is essential for the growth and survival of multicellular organisms, and in particular, for the function of the endocrine, nervous, and immune systems. In addition, intercellular communication is critical for developmental processes such as tissue construction and organogenesis, in which cell proliferation, cell differentiation, and morphogenesis must be spatially and temporally regulated in a precise and coordinated manner. Cells communicate with one another through the secretion and uptake of diverse types of signaling molecules such as hormones, growth factors, neuropeptides, and cytokines.
Hormones Hormones are signaling molecules that coordinately regulate basic physiological processes from embryogenesis throughout adulthood. These processes include metabolism, respiration, reproduction, excretion, fetal tissue differentiation and organogenesis, growth and development, homeostasis, and the stress response. Hormonal secretions and the nervous system are tightly integrated and interdependent. Hormones are secreted by endocrine glands, primarily the hypothalamus and pituitary, the thyroid and parathyroid, the pancreas, the adrenal glands, and the ovaries and testes.
The secretion of hormones into the circulation is tightly controlled. Hormones are often secreted in diurnal, pulsatile, and cyclic patterns. Hormone secretion is regulated by perturbations in blood biochemistry, by other upstream-acting hormones, by neural impulses, and by negative feedback loops. Blood hormone concentrations are constantly monitored and adjusted to maintain optimal, steady-state levels. Once secreted, hormones act only on those target cells that express specific receptors.
Most disorders of the endocrine system are caused by either hyposecretion or hypersecretion of hormones. Hyposecretion often occurs when a hormone's gland of origin is damaged or otherwise impaired. Hypersecretion often results from the proliferation of tumors derived from hoimone-secreting cells. Inappropriate hormone levels may also be caused by defects in regulatory feedback loops or in the processing of hormone precursors. Endocrine malfunction may also occur when the target cell fails to respond to the hormone.
Hormones can be classified biochemically as polypeptides, steroids, eicosanoids, or amines.
Polypeptide hormones, which include diverse hormones such as insulin and growth hormone, vary in size and function and are often synthesized as inactive precursors that are processed intracellularly into mature, active forms. Amine hormones, which include epinephrine and dopamine, are amino acid derivatives that function in neuroendocrine signaling. Steroid hormones, which include the cholesterol-derived hormones estrogen and testosterone, function in sexual development and reproduction. Eicosanoid hormones, which include prostaglandins and prostacyclins, are fatty acid derivatives that function in a variety of processes. Most polypeptide hormones and some amine hormones are soluble in the circulation where they are highly susceptible to proteolytic degradation within seconds after their secretion. Steroid hormones and eicosanoid hormones are insoluble and must be transported in the circulation by carrier proteins. The following discussion will focus primarily on polypeptide hormones.
Hormones secreted by the hypothalamus and pituitary gland play a critical role in endocrine function by coordinately regulating hormonal secretions from other endocrine glands in response to neural signals. Hypothalamic hormones include thyrotropin-releasing hormone, gonadotropin-releasing hormone, somatostatin, growth-hormone releasing factor, corticotropin-releasing hormone, substance P, dopamine, and prolactin-releasing hormone. These hormones directly regulate the secretion of hormones from the anterior lobe of the pituitary. Hormones secreted by the anterior pituitary include adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone, somatotropic hormones such as growth hormone and prolactin, glycoprotein hormones such as thyroid-stimulating hormone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), (3-lipotropin, and (3-endorphins. These hornones regulate hormonal secretions from the thyroid, pancreas, and adrenal glands, and act directly on the reproductive organs to stimulate ovulation and spermatogenesis. The posterior pituitary synthesizes and secretes antidiuretic hormone (ADH, vasopressin) and oxytocin.
Disorders of the hypothalamus and pituitary often result from lesions such as primary brain tumors, adenomas, infarction associated with pregnancy, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immunological disorders, and complications due to head trauma. Such disorders have profound effects on the function of other endocrine glands, Disorders associated with hypopituitarism include hypogonadism, Sheehan syndrome, diabetes insipidus, I~allman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism. Disorders associated with hyperpituitaxism include acromegaly, giantism, and syndrome of inappropriate ADH secretion (SIADH), often caused by benign adenomas.
Hormones secreted by the thyroid and parathyroid primarily control metabolic rates and the regulation of serum calcium levels, respectively. Thyroid hormones include calcitonin, somatostatin, and thyroid hormone. The parathyroid secretes parathyroid hormone. Disorders associated with hypothyroidism include goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism. Disorders associated with hyperthyroidism include thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease. Disorders associated with hyperparathyroidism include Conn disease (chronic hypercalemia) leading to bone resorption and parathyroid hyperplasia.
Hormones secreted by the pancreas regulate blood glucose levels by modulating the rates of carbohydrate, fat, and protein metabolism. Pancreatic hormones include insulin, glucagon, amylin, y-aminobutyric acid, gastrin, somatostatin, and pancreatic polypeptide. The principal disorder associated with pancreatic dysfunction is diabetes mellitus caused by insufficient insulin activity.
Diabetes mellitus is generally classified as either Type I (insulin-dependent, juvenile diabetes) or Type II (non-insulin-dependent, adult diabetes). The treatment of both forms by insulin replacement therapy is well known. Diabetes mellitus often leads to acute complications such as hypoglycemia (insulin shock), coma, diabetic ketoacidosis, lactic acidosis, and chronic complications leading to disorders of the eye, kidney, skin, bone, joint, cardiovascular system, nervous system, and to decreased resistance to infection.
The anatomy, physiology, and diseases related to hormonal function are reviewed in McCance, I~.L. and Huether, S.E. (1994) Pathophysiology: The Biological Basis for Disease in Adults and Children, Mosby-Year Book, Inc., St. Louis, MO; Greenspan, F.S. and Baxter, J.D.
(1994) Basic and Clinical Endocrinolo~y, Appleton and Lange, East Norwalk, CT.
Growth Factors Growth factors are secreted proteins that mediate intercellular communication.
Unlike hormones, which travel great distances via the circulatory system, most growth factors are primarily local mediators that act on neighboring cells. Most growth factors contain a hydrophobic N-terminal signal peptide sequence which directs the growth factor into the secretory pathway. Most growth factors also undergo post-translational modifications within the secretory pathway. These modifications can include proteolysis, glycosylation, phosphorylation, and intramolecular disulfide bond formation. Once secreted, growth factors bind to specific receptors on the surfaces of neighboring target cells, and the bound receptors trigger intracellular signal transduction pathways.
These signal transduction pathways elicit specific cellular responses in the target cells. These responses can include the modulation of gene expression and the stimulation or inhibition of cell division, cell differentiation, and cell motility.
Growth factors fall into at least two broad and overlapping classes. The broadest class includes the large polypeptide growth factors, which are wide-ranging in their effects. These factors include epidermal growth factor~(EGF), fibroblast growth factor (FGF), transforming growth factor- (3 (TGF-(3), insulin-like growth factor (IGF), nerve growth factor (NGF), and platelet-derived growth factor (PDGF), each defining a family of numerous related factors. ~ The large polypeptide growth factors, with the exception of NGF, act as mitogens on diverse cell types to stimulate wound healing, bone synthesis and remodeling, extracellular matrix synthesis, and proliferation of epithelial, epidermal, and connective tissues. Members of the TGF-(3, EGF, and FGF
families also function as inductive signals in the differentiation of embryonic tissue. NGF functions specifically as a neurotrophic factor, promoting neuronal growth and differentiation.
Another class of growth factors includes the hematopoietic growth factors, which are narrow in their target specificity. These factors stimulate the proliferation and differentiation of blood cells such as B-lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils, basophils, neutrophils, macrophages, and their stem cell precursors. These factors include the colony-stimulating factors (G-CSF, M-CSF, GM-CSF, and CSF1-3), erythropoietin, and the cytokines. The cytokines are specialized hematopoietic factors secreted by cells of the immune system and are discussed in detail below.
Growth factors play critical roles in neoplastic transformation of cells in vitro and in tumor progression in vivo. Overexpression of the large polypeptide growth factors promotes the proliferation and transformation of cells in culture. Inappropriate expression of these growth factors by tumor cells in vivo may contribute to tumor vascularization and metastasis.
Inappropriate activity of hematopoietic growth factors can result in anemias, leukemias, and lymphomas. Moreover, growth factors are both structurally and functionally related to oncoproteins, the potentially cancer-causing products of proto-oneogenes. Certain FGF and PDGF family members are themselves homologous to oncoproteins, whereas receptors for some members of the EGF, NGF, and FGF
families are encoded by proto-oncogenes. Growth factors also affect the transcriptional regulation of both proto-oncogenes and oncosuppressor genes. (Pimentel, E. (1994) Handbook of Growth Factors, CRC Press, Ann Arbor, MI; McKay, I. and Leigh, L, eds. (1993) Growth Factors:
A Practical Approach, Oxford University Press, New York, NY; Habenicht, A., ed. (1990) Growth Factors, Differentiation Factors, and Cytokines, Springer-Verlag, New York, NY.) In addition, some of the large polypeptide growth factors play crucial roles in the induction of the primordial germ layers in the developing embryo. This induction ultimately results in the formation of the embryonic mesoderm, ectoderm, and endoderm which in turn provide the framework for the entire adult body plan. Disruption of this inductive process would be catastrophic to embryonic development.
Small Peptide Factors - Neuropeptides and Vasomediators Neuropeptides and vasomediators (NP/VM) comprise a family of small peptide factors, typically of 20 amino acids or less. These factors generally function in neuronal excitation and inhibition of vasoconstriction/vasodilation, muscle contraction, and hormonal secretions from the brain and other endocrine tissues. Included in this family are neuropeptides and neuropeptide hormones such as bombesin, neuropeptide Y, neurotensin, neuromedin N, melanocortins, opioids, galanin, somatostatin, tachykvlins, urotensin II and related peptides involved in smooth muscle stimulation, vasopressin, vasoactive intestinal peptide, and circulatory system-borne signaling molecules such as angiotensin, complement, calcitonin, endothelins, formyl-methionyl peptides, glucagon, cholecystokinin, gastrin, and many of the peptide hormones discussed above. NP/VMs can transduce signals directly, modulate the activity or release of other neurotransmitters and hormones, and act as catalytic enzymes in signaling cascades. The effects of NP/VMs range from extremely brief to long-lasting. (Reviewed in Martin, C.R. et al. (1985) Endocrine Ph, si~y, Oxford University Press, New York NY, pp. 57-62.) Cytokines Cytokines comprise a family of signaling molecules that modulate the immune system and the inflammatory response. Cytokines are usually secreted by leukocytes, or white blood cells, in response to injury or infection. Cytokines function as growth and differentiation factors that act primarily on cells of the immune system such as B- and T-lymphocytes, monocytes, macrophages, and granulocytes. Like other signaling molecules, cytokines bind to specific plasma membrane receptors and trigger intracellular signal transduction pathways which alter gene expression patterns.
There is considerable potential for the use of cytokines in the treatment of inflammation and immune system disorders.
Cytokine structure and function have been extensively characterized in vitro.
Most cytokines are small polypeptides of about 30 kilodaltons or less. Over 50 cytokines have been identified from human and rodent sources. Examples of cytokine subfamilies include the interferons (IFN- a, -(3, and -y), the interleukins (1L1-IL13), the tumor necrosis factors (TNF-a and -(3), and the chemokines.
Many cytokines have been produced using recombinant DNA techniques, and the activities of individual cytokines have been determined in vitro. These activities include regulation of leukocyte proliferation, differentiation, and motility. ' The activity of an individual cytokine in vitro may not reflect the full scope of that cytokine's activity in vivo. Cytokines are not expressed individually in vivo but are instead expressed in combination with a multitude of other cytokines when the organism is challenged with a stimulus.
Together, these cytokines collectively modulate the immune response in a manner appropriate for that particular stimulus. Therefore, the physiological activity of a cytokine is determined by the stimulus itself and by complex interactive networks among co-expressed cytokines which may demonstrate both synergistic and antagonistic relationships.
Chemokines comprise a cytokine subfamily with over 30 members. (Reviewed in Wells, T.N.C. and Peitsch, M.C. (1997) J. Leukoc. Biol. 61:545-550.) Chemokines were initially identified as chemotactic proteins that recruit monocytes and macrophages to sites of inflammation. Recent evidence indicates that chemokines may also play key roles in hematopoiesis and HIV-1 infection.
Chemokines axe small proteins which range from about 6-15 kilodaltons in molecular weight.
Chemokines are further classified as C, CC, CXC, or CX3C based on the number and position of critical cysteine residues. The CC chemokines, for example, each contain a conserved motif consisting of two consecutive cysteines followed by two additional cysteines which occur downstream at 24- and 16-residue intervals, respectively (ExPASy PROSITE
database, documents PS00472 and PDOC00434). The presence and spacing of these four cysteine residues are highly conserved, whereas the intervening residues diverge significantly. However, a conserved tyrosine located about 15 residues downstream of the cysteine doublet seems to be important for chemotactic activity. Most of the human genes encoding CC chemokines are clustered on chromosome 17, although there are a few examples of CC chemokine genes that map elsewhere.
Other chemokines include lymphotactin (C chemokine); macrophage chemotactic and activating factor (MCAF/MCP-1;
CC chemokine); platelet factor 4 and IL-8 (CXC chemokines); and fractalkine and neurotractin (CX3C chemokines). (Reviewed in Luster, A.D. (1998) N. Engl. J. Med. 338:436-445.) Chromogranins and secretogranins are acidic proteins present in the secretory granules of endocrine and neuro-endocrine cells (Huttner, W.B. et al. (1991) Trends Biochem. Sci. 16:27-30) (Simon, J.-P. et al. (1989) Biochem. J. 262:1-13). Granins may be precursors of biologically-active peptides, or they may be helper proteins in the packaging of peptide hormones and neuropeptides -their precise role is unclear.
The discovery of new receptors and membrane-associated proteins, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative, autoimmune/inflammatory, neurological, metabolic, developmental, and endocrine disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of receptors and membrane-associated proteins.

SUMMARY OF THE INVENTION
The invention features purified polypeptides, receptors and membrane-associated proteins, referred to collectively as "REMAP" and individually as "REMAP-1," "REMAP-2,"
"REMAP-3,"
"REMAP-4," "REMAP-5," "REMAP-6," "REMAP-7," "REMAP-8," "REMAP-9," "REMAP-10,"
"REMAP-11," "REMAP-12," "REMAP-13," "REMAP-14," and "REMAP-15." In one aspect, the invention 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
N0:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90%
identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
ID NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID N0:1-15.
The invention further 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 m N0:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ
ID N0:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-15, and d) an irninunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-15.
In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-15. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID N0:16-30.
Additionally, the invention 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 ~ N0:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-15. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D NO: l-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ >D N0:1-15. 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.
Additionally, the invention 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 m NO:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ )D NO: l-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-15.
The invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ m N0:16-30, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ m N0:16-30, 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).
In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ )D N0:16-30, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90°Io identical to a polynucleotide sequence selected from the group consisting of SEQ >D N0:16-30, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe 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, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ m N0:16-30, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ m N0:16-30, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
The invention further provides a composition 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 m NO:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-15, c) a biologically active fragment of a polypeptide having an anuno acid sequence selected from the group consisting of SEQ m NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-15, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ m NO:1-15. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional REMAP, comprising administering to a patient in need of such treatment the composition.
The invention also 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 m NO:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-15. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional REMAP, comprising administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-15. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional REMAP, comprising administering to a patient in need of such treatment the composition.
The invention further 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 ID NO:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15. 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.
The invention further 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 m NO:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D NO:
l-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO: l-15. 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.
The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID N0:16-30, 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.
The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:16-30, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:16-30, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID
N0:16-30, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:16-30, 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).
Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount 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.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.

Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides 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 sequences of the invention, 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 sequences of the invention, along with selected fragments of the polynucleotide sequences.
Table 5 shows the representative cDNA library for polynucleotides of the invention.
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 the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

DEFINITIONS
"REMAP" refers to the amino acid sequences of substantially purified REMAP
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, marine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of REMAP. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of REMAP either by directly interacting with REMAP or by acting on components of the biological pathway in which REMAP
participates.
An "allelic variant" is an alternative form of the gene encoding REMAP.
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 REMAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as REMAP or a polypeptide with at least one functional characteristic of REMAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding REMAP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding REMAP. 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 REMAP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of REMAP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and 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.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or 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 sequence.
Amplification is generally carried out using polymerise chain reaction (PCR) technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of REMAP. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of REMAP either by directly interacting with REMAP or by acting on components of the biological pathway in which REMAP participates.
The term "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 REMAP 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) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KI,H). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "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 rnay 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'-NHZ), 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. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.) The term "intramer" refers to an aptamer which is expressed in vivo. For example, 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).
The term "spiegelmer" refers to an aptamer wluch 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.
The term "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of 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. Likewise, "immunologically active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic REMAP, 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, 3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotide sequences encoding REMAP or fragments of REMAP 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. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

"Consensus sequence" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELV1EW fragment assembly system (GCG, Madison Wn or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
"Conservative 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.
Original Residue Conseryative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the 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.
The term "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 of the evolution of new protein functions.
A "fragment" is a unique portion of REMAP or the polynucleotide encoding REMAP
which is identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 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.
For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ ID N0:16-30 comprises a region of unique polynucleotide sequence that specifically identifies SEQ m N0:16-30, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:16-30 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ m N0:16-30 from related polynucleotide sequences. The precise length of a fragment of SEQ
ID N0:16-30 and the region of SEQ ID N0:16-30 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 >D NO:1-15 is encoded by a fragment of SEQ >D N0:16-30. A
fragment of SEQ ID NO:1-15 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-15. For example, a fragment of SEQ ID NO: l-15 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-15.
The precise length of a fragment of SEQ ID NO: l-15 and the region of SEQ ID NO:1-15 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a translation initiation colon (e.g., methionine) followed by an open reading frame and a translation termination colon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLASTI. 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. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off.' SO
Expect: 10 Word Size: 1l Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over whichpercentage 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.
The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions.
Such conservative substitutions, explained in more detail above, generally preserve the charge and-hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Operz Gap: 1l and Extension Gap: 1 penalties Gap x drop-off.' S0 Expect: 10 Word Size: 3 Filter: on Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length 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" (HACs) 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.
The term "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 steps) 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.
Generally, 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 (Tin) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al.
(1989) Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 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%.
Typically, 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, may also be used under particular circumstances, such as for 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.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence 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).
The words "insertion" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"hnmune 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, wluch may affect cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of REMAP
which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "imrnunogenic fragment" also includes any polypeptide or oligopeptide fragment of REMAP which is useful in any of the antibody production methods disclosed herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of REMAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of REMAP.
The phrases "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 wluch 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.
"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. For instance, 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.
"Peptide 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 REMAP 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 REMAP.
"Probe" refers to nucleic acid sequences encoding REMAP, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerise chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MTT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.

This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
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 (LTTRs). 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.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of containing REMAP, nucleic acids encoding REMAP, 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.
The terms "specific 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.
The term "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 60°Io free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
A "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. Txansformation 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. The term "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 oxganism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (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 of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides 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.
A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 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 of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human receptors and membrane-associated proteins (REMAP), the polynucleotides encoding REMAP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, autoimmune/inflammatory, neurological, metabolic, developmental, and endocrine disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Tncyte project identification number (Incyte Project m). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ m NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide m) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ >D NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide m) as shown.

Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBanle protein (genpept) database. Columns 1 and 2 show the polypeptide sequence identification~number (Polypeptide SEQ D7 NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide 1D) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog.
Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s).
Column 5 shows the annotation of the GenBank 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 >D NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) 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 (Genetics Computer Group, Madison WI). Column 6 shows amino acid residues comprising signature sequences, domains, and motifs. Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are receptors and membrane-associated proteins.
For example, SEQ >D NO:1 is 97% identical to rat retinoic acid receptor alpha 2 isoform (GenBank m g3213188) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.) The BLAST probability score is 8.5e-245, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ >D N0:1 also contains a nuclear hormone receptor lignad-binding domain and a C4 type zinc finger 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 PROFILESCAN
analyses provide further corroborative evidence that SEQ )D N0:1 is a retinoic acid receptor. In an alternative example, SEQ ID N0:2 is 31 % identical, from residue A3 to residue H575, to human multiple membrane spanning receptor TRC8 (GenBank m g3395787) as determined by BLAST
with a probability score of 9.7e-61. (See Table 2.) Data from additional BLAST
analyses provide further corroborative evidence that SEQ ID N0:2 is a multiple membrane spanning receptor. In an alternative example, SEQ )D N0:5 is 7G% identical, from residue V4 to residue A479, to rat potential ligand-binding protein (GenBank ID g57734) as determined by BLAST with a probability score of 6.3e-187. (See Table 2.) Data from additional BLAST analyses provide further corroborative evidence that SEQ >D N0:5 is an olfactory ligand binding protein. In an alternative example, SEQ >D

NO:11 is 77% identical, from residue M1 to residue F310, to Canis familiaris olfactory receptor CfOLF2 (GenBank ID g1314663) as determined by BLAST with a probability score of 9.7e-129.
(See Table 2.) SEQ ID N0:11 also contains a 7-transmembrane receptor (rhodopsin family) active site domain as determined by searching for statistically significant matches in the HMM-based PFAM
database. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ll~ NO:11 is a G-protein coupled receptor. In an alternative example, SEQ ID N0:15 is 99% identical, from residue M5 to residue M328, and is 89% identical, from residue V313 to residue E410, to a human protein which is an ortholog of the potential ligand-binding protein RYA3 (GenBank ID g11877275) as determined by BLAST with a probability score of 3.3e-207. (See Table 2.) Data from BLIMPS and additional BLAST analyses provide further corroborative evidence that SEQ ID N0:15 is a ligand-binding protein. SEQ ID
NO:3-4, SEQ ID
N0:6-10, and SEQ ID N0:12-14 were analyzed and annotated in a similar manner.
The algorithms and parameters for the analysis of SEQ ll~ NO:1-15 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present invention 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 ID) for each polynucleotide of the 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 sequences of the invention, and of fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ID
N0:16-30 or that distinguish between SEQ ID N0:16-30 and related polynucleotide sequences.
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. Alternatively, the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotide sequences. In addition, 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"). Alternatively, 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"). Alternatively, 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. For example, a polynucleotide sequence identified as FL XXXXXX_NI IVz_YYYYY_N3 N4 represents a "stitched" sequence in which XXXXXX
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 NI,z,3...~ if present, represent specific exons that may have been manually edited during analysis (See Example V).
Alternatively, the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm. For example, a polynucleotide sequence identified as FLXXXXXX_gAAAAA_gBBBBB_1 N is a "stretched" sequence, with XXXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the 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). In instances where a RefSeq sequence was used as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier (denoted by "NM," "NP," or "NT") may be used in place of the GenBank identifier (i.e., gBBBBB).
Alternatively, 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).
Prefix Type of analysis andJor examples of programs GNN, GFG,Exon prediction from genomic sequences using, for example, ENST GENSCAN (Stanford University, CA, USA) or FGENES

(Computer Genomics Group, The Sanger Centre, Cambridge, UI~).

GBI Hand-edited analysis of genomic sequences.

FL Stitched or stretched genomic sequences (see Example V).

INCY Full length transcript and exon prediction from mapping of EST

sequences to the genome. Genomic location and EST composition data are combined to predict the exons and resulting transcript.

In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA
identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.

The invention also encompasses REMAP variants. A preferred REMAP variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the REMAP amino acid sequence, and which contains at least one functional or structural characteristic of REMAP.
The invention also encompasses polynucleotides which encode REMAP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:16-30, which encodes REMAP. The polynucleotide sequences of SEQ )D N0:16-30, 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 a variant of a polynucleotide sequence encoding REMAP.
In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding REMAP. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID
N0:16-30 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 ID N0:16-30. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of REMAP.
In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide sequence encoding REMAP. A splice variant may have portions which have significant sequence identity to the polynucleotide sequence encoding REMAP, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons 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 the polynucleotide sequence encoding REMAP
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 sequence encoding REMAP. For example, a polynucleotide comprising a sequence of SEQ ll~ N0:30 is a splice variant of a polynucleotide comprising a sequence of SEQ ID N0:20. Any one of the splice variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of REMAP.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding REMAP, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurnng gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standaxd~triplet genetic code as applied to the polynucleotide sequence of naturally occurring REMAP, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode REMAP and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring REMAP
under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding REMAP 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. Other reasons for substantially altering the nucleotide sequence encoding REMAP and its derivatives without altering the encoded amino acid sequences include the production of 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 DNA sequences which encode REMAP
and REMAP derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding REMAP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID
N0:16-30 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol.
152:507-511.) Hybridization conditions, 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 Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably, 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 (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biolog~and Biotechnoloey, Wiley VCH, New York NY, pp.
856-853.) The nucleic acid sequences encoding REMAP 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. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., 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. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, 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.
When screening for full length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. 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. In particular, 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. Outputllight 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.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode REMAP may be cloned in recombinant DNA molecules that direct expression of REMAP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express REMAP.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter REMAP-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. For example, 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 MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, 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 REMAP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding REMAP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.

7:225-232.) Alternatively, REMAP itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Pro erties, WH Freeman, New York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the amino acid sequence of REMAP, or any part thereof, may be altered during direct synthesis andlor 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. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.) In order to express a biologically active REMAP, the nucleotide sequences encoding REMAP
or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements fox 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 polynucleotide sequences encoding REMAP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding REMAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the I~ozak sequence. In cases where sequences encoding REMAP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding REMAP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9, 13, and 16.) A variety of expression vectorlhost systems may be utilized to contain and express sequences encoding REMAP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See, e.g., Sambrook, supra;
Ausubel, supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc.
Natl. Acad. Sci. USA
81:3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
(See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M.
et al. (1993) Proc.
Natl. Acad. Sci. USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al. (1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding REMAP. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding REMAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding REMAP into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, 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. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of REMAP are needed, e.g. for the production of antibodies, vectors which direct high level expression of REMAP may be used.
For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of REMAP. 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 astoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel, 1995, s_ upra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et al. (1994) Bio/Technology 12:181-184.) Plant systems may also be used for expression of REMAP. Transcription of sequences encoding REMAP 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.
6:307-31I). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp. 191-196.) .
In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding REMAP
may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses REMAP in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors rnay also be used for high-level protein expression.
Human artificial chromosomes (HACs) 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. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.) For long term production of recombinant proteins in mammalian systems, stable expression of REMAP in cell lines is preferred. For example, sequences encoding REMAP 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.
Any number of 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 tk- and apr' cells, respectively.
(See, e.g., 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. For example, dhfr confers resistance to methotrexate; faeo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., 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.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech),13 glucuronidase and its substrate 13-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.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding REMAP is inserted within a marker gene sequence, transformed cells containing sequences encoding REMAP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding REMAP 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.
In general, host cells that contain the nucleic acid sequence encoding REMAP
and that express REMAP 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 REMAP
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). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on REMAP is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.) A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding REMAP
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, the sequences encoding REMAP, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable 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 nucleotide sequences encoding REMAP 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. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode REMAP may be designed to contain signal sequences which direct secretion of REMAP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences 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.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding REMAP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric REMAP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of REMAP
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-r~2yc, 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 REMAP encoding sequence and the heterologous protein sequence, so that REMAP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled REMAP 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, 35S-methionine.
REMAP of the present invention.or fragments thereof may be used to screen for compounds that specifically bind to REMAP. At least one and up to a plurality of test compounds may be screened for specific binding to REMAP. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the natural ligand of REMAP, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which REMAP
binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express REMAP, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosonhila, or E. coli. Cells expressing REMAP or cell membrane fractions which contain REMAP
are then contacted with a test compound and binding, stimulation, or inhibition of activity of either REMAP
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. For example, the assay may comprise the steps of combining at least one test compound with REMAP, either in solution or affixed to a solid support, and detecting the binding of REMAP to the compound.

Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compounds) may be free in solution or affixed to a solid support.
REMAP of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of REMAP. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for REMAP activity, wherein REMAP is combined with at least one test compound, and the activity of REMAP in the presence of a test compound is compared with the activity of REMAP in the absence of the test compound. A change in the activity of REMAP. in the presence of the test compound is indicative of a compound that modulates the activity of REMAP. Alternatively, a test compound is combined with an in vitro or cell-free system comprising REMAP under conditions suitable for REMAP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of REMAP 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.
In another embodiment, polynucleotides encoding REMAP or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337.) For example, mouse ES cells, such as the mouse 1291SvJ 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). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding REMAP 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 REMAP can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding REMAP is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells are injected into blastulae, and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
Alternatively, a mammal inbred to overexpress REMAP, e.g., by secreting REMAP
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
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of REMAP and receptors and membxane-associated pxoteins. In addition, examples of tissues expressing REMAP can be found in Table 6. Therefore, REMAP appears to play a role in cell proliferative, autoimmune/inflammatory, neurological, metabolic, developmental, and endocrine disorders. In the treatment of disorders associated with increased REMAP
expression or activity, it is desirable to decrease the expression or activity of REMAP. In the treatment of disorders associated with decreased REMAP expression or activity, it is desirable to increase the expression or activity of REMAP.
Therefore, in one embodiment, REMAP 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 REMAP. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a metabolic disorder such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, fatty hepatocirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal storage diseases, mannosidosis, neuraminidase deficiency, obesity, osteoporosis, phenylketonuria, pseudovitamin D-deficiency rickets, disorders of carbohydrate metabolism such as congenital type II
dyserythropoietic anemia, diabetes, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, galactose epimerase deficiency, glycogen storage diseases, lysosomal storage diseases, fructosuria, pentosuria, and inherited abnormalities of pyruvate metabolism, disorders of lipid metabolism such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GMZ gangliosidosis, and cexoid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin:cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease, SandhofF's disease, hyperlipidemia, hyperlipemia, and lipid myopathies, and disorders of copper metabolism such as Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type IX
diabetes; a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, a seizure disorder such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; and an endocrine disorder such as a disorder of the hypothalamus and/or pituitary resulting from lesions such as a primary brain tumor, adenoma, infarction associated with pregnancy, hypophysectomy, aneurysm, vascular malformation, thrombosis, infection, immunological disorder, and complication due to head trauma, a disorder associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterex-Siwe disease, sarcoidosis, empty sella syndxome, and dwarfism, a disorder associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma, a disorder associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism, a disorder associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease, a disorder associated with hyperparathyroidism including Conn disease (chronic hypercalemia), a pancreatic disorder such as Type I or Type II diabetes mellitus and associated complications, a disorder associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease, a disorder associated with gonadal steroid hormones such as: in women, abnormal prolactin production, infertility, endometriosis, perturbation of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteoporosis, and, in men, Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, a hypergonadal disorder associated with Leydig cell tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 a-reductase, and gynecomastia.
In another embodiment, a vector capable of expressing REMAP 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 REMAP including; but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified REMAP 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 REMAP
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of REMAP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of REMAP including, but not limited to, those listed above.
In a further embodiment, an antagonist of REMAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of REMAP. Examples of such disorders include, but are not limited to, those cell proliferative, autoimmune/inflammatory, neurological, metabolic, developmental, and endocrine disorders described above. In one aspect, an antibody which specifically binds REMAP 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 REMAP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding REMAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of REMAP including, but not limited to, those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention 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 REMAP may be produced using methods which are generally known in the art. In particular, purified REMAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind REMAP.
Antibodies to REMAP 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) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with REMAP or with any fragment or oligopeptide thereof which has irmnunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, arid surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to REMAP 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 identical to a portion of the amino acid sequence of the natural protein. Short stretches of REMAP 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 REMAP 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. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
hnmunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.) In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (I985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce REMAP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., 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. (See, e.g., 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 REMAP may also be generated.
For example, such 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. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.) Various 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 REMAP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering REMAP epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for REMAP.
Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of REMAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple REMAP epitopes, represents the average affinity, or avidity, of the antibodies for REMAP. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular REMAP epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 10'2 L/mole axe preferred for use in immunoassays in which the REMAP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of REMAP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL, Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For ' 35 example, 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 REMAP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.) In another embodiment of the invention, the polynucleotides encoding REMAP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, 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 of the gene encoding REMAP. Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding REMAP. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.) In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g., Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon, I~.J. et al. (1995) 9(13):1288-1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C, et al. (1997) Nucleic Acids Res.
25(14):2730-2736.) In another embodiment of the invention, polynucleotides encoding REMAP 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 (SCE)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inhexited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falci ap rum and Trypanosoma cruzi). In the case where a genetic deficiency in REMAP expression or regulation causes disease, the expression of REMAP from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in REMAP are treated by constructing mammalian expression vectors encoding REMAP
and introducing these vectors by mechanical means into REMAP-deficient cells.
Mechanical transfer technologies fox 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; Ivics, Z. (1997) Cell 91:501-510;
Boulay, J-L. and H.
Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of REMAP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
REMAP
may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769;
Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and P1ND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding REMAP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require nninimal effort to optimize experimental parameters. In the alternative, transfoi-~nation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-4.67), or by electroporation (Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to REMAP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding REMAP under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on~published data (Riviere, I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A, et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1.998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4+ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G, et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding REMAP to cells which have one or more genetic abnormalities with respect to the expression of REMAP. 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. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding REMAP to target cells which have one or more genetic abnormalities with respect to the expression of REMAP. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing REMAP to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well lmown to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy.
Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well lrnown to those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding REMAP to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for REMAP into the alphavirus genome in place of the capsid-coding region results in the production of a large number of REMAP-coding RNAs and the synthesis of high levels of REMAP in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of REMAP into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.

Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and hnmunolo~ic Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, 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.
For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding REMAP.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short 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.
Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA
sequences encoding REMAP. 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. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. 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.
An additional embodiment of the invention encompasses a method fox screening for a compound which is effective in altering expression of a polynucleotide encoding REMAP.
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. Thus, in the treatment of disorders associated with increased REMAP expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding REMAP may be therapeutically useful, and in the treatment of disorders associated with decreased REMAP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding REMAP may be therapeutically useful.
At least one, and up to a plurality, of 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 REMAP 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 REMAP are assayed by any method commonly known in the art. Typically, 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 REMAP. 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 Schizosaccharom ces 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, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A
particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified o~igonucleotides) 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. For ex vivo therapy, 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. (See, e.g., Goldman, C.K. et al. (1997) Nat.
Biotechno1.15:462-466.) 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, fox example, sugars, starches, celluloses, gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the latest edition of Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of REMAP, antibodies to REMAP, and mimetics, agonists, antagonists, or inhibitors of REMAP.
The compositions utilized in tlus invention 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.
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. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
Compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended puzpose. 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 REMAP or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, REMAP or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwaxze, S.R. et al. (1999) Science 285:1569-1572).
For any compound, 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 REMAP or fragments thereof, antibodies of REMAP, and agonists, antagonists or inhibitors of REMAP, 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 EDSO (the dose therapeutically effective in 50% of the population) or LDSO (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 LDSO/EDSO 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 EDso 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 O. l ,ug to 100,000 ,ug, 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
In another embodiment, antibodies which specifically bind REMAP may be used for the diagnosis of disorders characterized by expression of REMAP, or in assays to monitor patients being treated with REMAP or agonists, antagonists, or inhibitors of REMAP.
Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics.
Diagnostic assays for REMAP include methods which utilize the antibody and a label to detect REMAP 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.
A variety of protocols for measuring REMAP, including ELISAs, RIAs, and FAGS, are known in the art and provide a basis for diagnosing altered or abnormal levels of REMAP expression.
Normal or standard values for REMAP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to REMAP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means.
Quantities of REMAP
expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding REMAP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, 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 REMAP
may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of REMAP, and to monitor regulation of REMAP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding REMAP or closely related molecules may be used to identify nucleic acid sequences which encode REMAP. 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 REMAP, allelic variants, or related sequences.
35- Probes may also be used for the detection of related sequences, and may have at least 50%

sequence identity to any of the REMAP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:16-30 or from genomic sequences including promoters, enhancers, and introns of the REMAP
gene.
Means for producing specific hybridization probes for DNAs encoding REMAP
include the cloning of polynucleotide sequences encoding REMAP or REMAP derivatives into vectors for the production of mRNA probes. Such 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
polymerises and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 355, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding REMAP may be used for the diagnosis of disorders associated with expression of REMAP. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; an autoimmunelinflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a metabolic disorder such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, fatty hepatocirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal storage diseases, mannosidosis, neuraminidase deficiency, obesity, osteoporosis, phenylketonuria, pseudovitamin D-deficiency rickets, disorders of carbohydrate metabolism such as congenital type II
dyserythropoietic anemia, diabetes, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, galactose epimerase deficiency, glycogen storage diseases, lysosomal storage diseases, fructosuria, pentosuria, and inherited abnormalities of pyruvate metabolism, disorders of lipid metabolism such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GMz gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin:cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, and lipid myopathies, and disorders of copper metabolism such as Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type IX
diabetes; a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wihns' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, a seizure disorder such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; and an endocrine disorder such as a disorder of the hypothalamus and/or pituitary resulting from lesions such as a primary brain tumor, adenoma, infarction associated with pregnancy, hypophysectomy, aneurysm, vascular malformation, thrombosis, infection, immunological disorder, and complication due to head trauma a disorder associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism, a disorder associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma, a disorder associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism, a disorder associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease, a disorder associated with hyperparathyroidism including Conn disease (chronic hypercalemia), a pancreatic disorder such as Type I or Type II diabetes mellitus and associated complications, a disorder associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease, a disorder associated with gonadal steroid hormones such as: in women, abnormal prolactin production, infertility, endometriosis, perturbation of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteoporosis, and, in men, Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, a hypergonadal disorder associated with Leydig cell tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 a-reductase, and gynecomastia. The polynucleotide sequences encoding REMAP 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 REMAP
expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding REMAP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding REMAP 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 nucleotide sequences encoding REMAP 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.
In order to provide a basis for the diagnosis of a disorder associated with expression of REMAP, 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 REMAP, 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 sarilples from patients who are symptomatic for a disorder.
Deviation from standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, 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.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding REMAP 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 REMAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding REMAP, and will be employed undex 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.
In a particular aspect, oligonucleotide primers derived from the polynucleotide sequences encoding REMAP may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans. Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from the polynucleotide sequences encoding REMAP are used to amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. Additionally, sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
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.
For example, a variation in 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 ALOXS 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;
I~wok, 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 REMAP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Tmmunol. 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 quantitatidn.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences 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.
In another embodiment, REMAP, fragments of REMAP, or antibodies specific for REMAP
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. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity.

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. 24:153-159; Steiner, S.
and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families.
Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expxession of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity. (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, su~a). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for REMAP
to quantify the levels of REMAP expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). 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. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the 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. (See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acid. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acid. Sci. USA 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types of nnicroarrays are well known and thoroughly described in DNA Microarrays: A Practical Approach, M. Schena, ed.
(1999) Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding REMAP
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
Either coding ox 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. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134;
and Trask, B.J.
(1991) Trends Genet. 7:149-154.) Once mapped, the nucleic acid sequences of the invention 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). (See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl.
Acid. Sci. USA 83:7353-7357.) Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e.g.; 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 (OMIM) World Wide Web site. Correlation between the location of the gene encoding REMAP 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.
In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, 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 IO 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 l 1q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide 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.
In another embodiment of the invention, REMAP, its catalytic or immunogenic fragments, or oligopeptides thereof 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 REMAP 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. (See, e.g., Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with REMAP, or fragments thereof, and washed. Bound REMAP is then detected by methods well known in the art.
Purified REMAP
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.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding REMAP specifically compete with a test compound for binding REMAP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with REMAP.
In additional embodiments, the nucleotide sequences which encode REMAP 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.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above and below, including U.S. Ser. No. 60/262,838, U.S. Ser. No. 60/265,927, U.S. Ser. No.
60/271,196, U.S. Ser.
No. 60/274,549, and U.S. Ser. No. 60/334,179, are expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, 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). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE
(Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XLl-BIueMRF, or SOLR from Stratagene or DHSa, DHlOB, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones 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 Iysis.
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 Plasnnid 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.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis 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 rnicrodispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI
protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VllI.

The polynucleotide sequences derived from Incyte eDNAs 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. The 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 norve~icus, Mus musculus, Caenorhabditis ele~ans, Saccharomyces cerevisiae, Schizosaccharom cues pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); and hidden Markov model (HMM)-based protein family databases such as PFAM. (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 HM1VIER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV
and V) were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences. Alternatively, a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide. 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, and hidden Markov model (HMM)-based protein family databases such as PFAM. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL
algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
Table 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).
The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ
m N0:16-30. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative receptors and membrane-associated proteins were initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA
sequences from a variety of organisms (See Burge, C. and S. Karlin (1997) J.
Mol. Biol. 268:78-94, and 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 colon.
The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode receptors and membrane-associated proteins, the encoded polypeptides were analyzed by querying against PFAM models for receptors and membrane-associated proteins. Potential receptors and membrane-associated proteins were also identified by homology to Incyte cDNA sequences that had been annotated as receptors and membrane-associated proteins. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA
or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When 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 III. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity. For example, if an~interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals thus identified were then "stitched" together by the stitching algorithm in the order that they appear along.their parent sequences to generate the longest possible sequence, as well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.
"Stretched" Sequences Partial DNA sequences were extended to full length with an algorithm based on BLAST
analysis. First, partial cDNAs assembled as described in Example III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. The 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 DNA sequences were therefore "stretched" or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.
VI. Chromosomal Mapping of REMAP Encoding Polynucleotides The sequences which were used to assemble SEQ ID N0:16-30 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID N0:16-30 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 ID NO:, to that map location.
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. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI "GeneMap'99" World Wide Web site (http://www.ncbi.nlin.nih.gov/genemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity 5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding REMAP 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 III). 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. Similarly, each human tissue i's 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 REMAP. cDNA sequences and cDNA
library/tissue information are found in the LIF'ESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of REMAP Encoding Polynucleotides Full length polynucleotide sequences were also 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 hairpin 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.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH4)ZS04, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerise (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step l: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68 ° C, 5 min; Step 7: storage at 4 ° C.
The concentration of DNA in each well was determined by dispensing 100 ~.1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 ~.1 of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 ,u1 to 10 ~1 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 Pharmacia Biotech). For shotgun sequencing, 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 Pharmacia Biotech), treated with Pfu DNA polymerise (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 LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise (Amersham Pharmacia Biotech) and Pfu DNA polymerise (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.
Samples were diluted with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the above procedure or are used to obtain 5' regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in REMAP Encoding Polynucleotides Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ )I? N0:16-30 using the 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 trimming of vector sequences, chimeras, and splice variants. An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP. Clone error filters used statistically generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering error filters used statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed duplicates and SNPs found in immunoglobulins or T-cell receptors.
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 Venezualan, 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.
X. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:16-30 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, 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 ~sCi of [y 3zP] adenosine triphosphate (Amersham Pharmacia Biotech), 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 Pharmacia Biotech).
An aliquot containing 10' 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 II, Eco RI, Pst I, Xba I, or Pvu II (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, 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 (1999), supra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers.
Alternatively, 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, W, chemical, or mechanical bonding procedures. A typical array may be produced using available methods and machines well known to those of ordinaxy skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalom 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.
After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element.
Alternatively, 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. In one embodiment, nucroarray preparation and usage is described in detail below.
Tissue or Cell Sample Preparation 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/~.1 oligo-(dT) primer (2lmer), 1X
first strand buffer, 0.03 units/,ul RNase inhibitor, 500 ~M dATP, 500 ,uM
dGTP, 500 ,uM dTTP, 40 ~,M dCTP, 40 ~,M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte). 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
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 mI of glycogen (1 mg/ml), 60 m1 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.
Microarra~paration Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ~,g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C oven.
Array.elements are applied to the coated glass substrate using a procedure described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~,l of the array element DNA, at an average concentration of 100 ngl~,l, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are ITV-crosslinked using a STRA.TALINI~ER W-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2% SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 ~1 of sample mixture consisting of 0.2 ,ug 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 cmz 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 I40 ~.I of 5X SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45 ° C in a first wash buffer (IX SSC, O.I% SDS), three times for IO 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 CyS. The excitation laser Iight 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 I.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT 81477, 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 CyS. 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. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC
computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Tncyte).
XII. Complementary Polynucleotides Sequences complementary to the REMAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring REMAP.
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 REMAP.
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 REMAP-encoding transcript. .
XIII. Expression of REMAP
Expression and purification of REMAP is achieved using bacterial or virus-based expression systems. For expression of REMAP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the try-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express REMAP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of REMAP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autog-raphica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding REMAP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.

Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.) In most expression systems, REMAP is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from REMAP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified REMAP obtained by these methods can be used directly in the assays 1S shown in Examples XVII, XVIII, and XIX, where applicable.
XIV. Functional Assays REMAP function is assessed by expxessing the sequences encoding REMAP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ,ug 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 ,ug of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein 2S provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA
with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow CytometrX, Oxford, New York NY.
The influence of REMAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding REMAP and either CDG4 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 REMAP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XV. Production of REMAP Specific Antibodies REMAP 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 rabbits and to produce antibodies using standard protocols.
Alternatively, the REMAP 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. (See, e.g., Ausubel, 1995, supra, ch. 11.) Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to I~LH (Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-REMAP activity by, for example, binding the peptide or REMAP to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XVI. Purification of Naturally Occurring REMAP Using Specific Antibodies Naturally occurring or recombinant REMAP is substantially purified by immunoaffinity chromatography using antibodies specific for REMAP. An immunoaffinity column is constructed by covalently coupling anti-REMAP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing REMAP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of REMAP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/REMAP 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 REMAP is collected.
XVII, Identification of Molecules Which Interact with REMAP
REMAP, or biologically active fragments thereof, are labeled with izsI Bolton-Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a mufti-well plate are incubated with the labeled REMAP, washed, and any wells with labeled REMAP complex are assayed. Data obtained using different concentrations of REMAP are used to calculate values for the number, affinity, and association of REMAP with the candidate molecules.
Alternatively, molecules interacting with REMAP 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).
REMAP 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).
XVIII. Demonstration of REMAP Activity An assay for REMAP activity measures the expression of REMAP on the cell surface. cDNA
encoding REMAP is transfected into an appropriate mammalian cell line. Cell surface proteins are labeled with biotin as described (de la Fuente, M.A. et al. (1997) Blood 90:2398-2405).
Immunoprecipitations are performed using REMAP-specific antibodies, and immunoprecipitated samples are analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of REMAP expressed on the cell surface.
In the alternative, an assay for REMAP activity is based on a prototypical assay for ligand/receptor-mediated modulation of cell proliferation. This assay measures the rate of DNA
synthesis in Swiss mouse 3T3 cells. A plasmid containing polynucleotides encoding REMAP is added to quiescent 3T3 cultured cells using transfection methods well known in the art. The transiently transfected cells axe then incubated in the presence of [3H]thymidine, a radioactive DNA
precursor molecule. Varying amounts of REMAP ligand are then added to the cultured cells.
Incorporation of [3H]thymidine into acid-precipitable DNA is measured over an appropriate time interval using a radioisotope counter, and the amount incorporated is directly proportional to the amount of newly synthesized DNA. A linear dose-response curve over at least a hundred-fold REMAP ligand concentration range is indicative of receptor activity. One unit of activity per milliliter is defined as the concentration of REMAP producing a 50% response level, where 100%
represents maximal incorporation of [3H]thymidine into acid-precipitable DNA
(McKay, I. and T.
Leigh, eds. (1993) Growth Factors: A Practical Approach, Oxford University Press, New York NY, p.
73.) Tn a further alternative, the assay for REMAP activity is based upon the ability of GPCR
family proteins to modulate G protein-activated second messenger signal transduction pathways (e.g., CAMP; Gaudin, P. et al. (1998) J. Biol. Chem. 273:4990-4996). A plasmid encoding full length REMAP is transfected into a mammalian cell line (e.g., Chinese hamster ovary (CHO) or human embryonic kidney (HEK-293) cell lines) using methods well-known in the art.
Transfected cells are grown in 12-well trays in culture medium for 48 hours, then the culture medium is discarded, and the attached cells are gently washed with PBS. The cells are then incubated in culture medium with or without ligand for 30 minutes, then the medium is removed and cells lysed by treatment with 1 M
perchloric acid. The cAMP levels in the lysate are measured by radioimmunoassay using methods °well-known in the art. Changes in the levels of cAMP in the lysate from cells exposed to ligand compared to those without ligand are proportional to the amount of REMAP
present in the transfected cells.
To measure changes in inositol phosphate levels, the cells are grown in 24-well plates containing 1x105 cells/well and incubated with inositol-free media and [3H]myoinositol, 2 ~,Ci/well, for 48 hr. The culture medium is removed, and the cells washed with buffer containing 10 mM LiCI
followed by addition of ligand. The reaction is stopped by addition of perchloric acid. Inositol phosphates are extracted and separated on Dowex AGl-X8 (Bio-Rad) anion exchange resin, and the total labeled inositol phosphates counted by liquid scintillation. Changes in the levels of labeled inositol phosphate from cells exposed to ligand compared to those without ligand are proportional to the amount of REMAP present in the transfected cells.
In a further alternative, the ion conductance capacity of REMAP is demonstrated using an electrophysiological assay. REMAP is expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with a eukaryotic expression vector encoding REMAP.
Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art. A small amount of a second plasmid, which expresses any one of a number of marker genes such as (3-galactosidase, is co-transformed into the cells in order to allow rapid identification of those cells which have taken up and expressed the foreign DNA. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of REMAP and (3-galactosidase. Transformed cells expressing (3-galactosidase are stained blue when a suitable colorimetric substrate is added to the culture media under conditions that are well known in the art. Stained cells are tested for differences in membrane conductance due to various ions by electrophysiological techniques that are well known in the art.
Untransformed cells, and/or cells transformed with either vector sequences alone or (3-galactosidase sequences alone, are used as controls and tested in parallel. The contribution of REMAP to canon or anion conductance cay be shown by incubating the cells using antibodies specific for either REMAP.
The respective antibodies will bind to the extracellular side of REMAP, thereby blocking the pore in the ion channel, and the associated conductance.
In a further alternative, REMAP transport activity is assayed by measuring uptake of labeled substrates into Xenopus laevis oocytes. Oocytes at stages V and VI are injected with REMAP mRNA
(10 ng per oocyte) and incubated for 3 days at 18 °C in OR2 medium (82.5 mM NaCI, 2.5 mM KCI, 1 mM CaClz, 1 mM MgCIz, 1 mM Na.,HP04, 5 mM Hepes, 3.8 mM NaOH , 50 ~,g/ml gentamycin, pH
7.8) to allow expression of REMAP protein. Oocytes are then transferred to standard uptake medium (I00 mM NaCI, 2 mM KCI, I mM CaCl2, I nnM MgCl2, 10 mM HepeslTris pH 7.5).
Uptake of various substrates (e.g., amino acids, sugars, drugs, and neurotransmitters) is initiated by adding a 3H
substrate to the oocytes. After incubating for 30 minutes, uptake is terminated by washing the oocytes three times in Na+-free medium, measuring the incorporated 3H, and comparing with controls. REMAP activity is proportional to the level of internalized 3H
substrate.
In a further alternative, REMAP protein kinase (PK) activity is measured by phosphorylation of a protein substrate using gamma-labeled [32P]-ATP and quantitation of the incorporated radioactivity using a gamma radioisotope counter. REMAP is incubated with the protein substrate, [32P]-ATP, and an appropriate kinase buffer. The 32P incorporated into the product is separated from free [3zP]-ATP by electrophoresis and the incorporated 32P is counted. The amount of 32P recovered is proportional to the PK activity of REMAP in the assay. A determination of the specific amino acid residue phosphorylated is made by phosphoamino acid analysis of the hydrolyzed protein.
XIX. Identification of REMAP Ligands REMAP is expressed in a eukaryotic cell line such as CHO (Chinese Hamster Ovary) or HEK
(Human Embryonic Kidney) 293 which have a good history of GPCR expression and which contain a wide range of G-proteins allowing for functional coupling of the expressed REMAP to downstream effectors. The transformed cells are assayed for activation of the expressed receptors in the presence of candidate ligands. Activity is measured by changes in intracellular second messengers, such as cyclic AMP or Caz~. These may be measured directly using standard methods well known in the art, or by the use of reporter gene assays in which a luminescent protein (e.g.
firefly luciferase or green fluorescent protein) is under the transcriptional control of a promoter responsive to the stimulation of protein kinase C by the activated receptor (Milligan, G. et al. (1996) Trends Pharmacol. Sci. 17:235-237). Assay technologies are available for both of these second messenger systems to allow high throughput readout in mufti-well plate format, such as the adenylyl cyclase activation FlashPlate Assay (NEN Life Sciences Products), or fluorescent Ca2+ indicators such as Fluo-4 AM (Molecular Probes) in combination with the FLIPR fluorimetric plate reading system (Molecular Devices). In cases where the physiologically relevant second messenger pathway is not known, REMAP may be coexpressed with the G-proteins Gaisns which have been demonstrated to couple to a wide range of G-proteins (Offermanns, S. and M.I. Simon (1995) J. Biol. Chem. 270:15175-15180), in order to funnel the signal transduction of the REMAP through a pathway involving phospholipase C and Caz+
mobilization. Alternatively, REMAP may be expressed in engineered yeast systems which lack endogenous GPCRs, thus providing the advantage of a null background for REMAP
activation screening. These yeast systems substitute a human GPCR and Ga protein for the corresponding components of the endogenous yeast pheromone receptor pathway. Downstream signaling pathways are also modified so that the normal yeast response to the signal is converted to positive growth on selective media or to reporter gene expression (Broach, J.R. and J. Thorner (1996) Nature 384 (suppl.):14-16). The receptors are screened against putative ligands including known GPCR ligands and other naturally occurring bioactive molecules. Biological extracts from tissues, biological fluids and cell supernatants are also screened.
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

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cd Pr P; P-i P-i U ~ ~ H ~, <110> INCYTE GENOMICS, TNC.
LEE, Ernestine A.
WALIA, Narinder K.
BAUGHN, Mariah R.
AZIMZAI, Yalda TANG, Y. Tom YUE, Henry THANGAVELU, Kavitha XU, Yuming ARVIZU, Chandra WARREN, Bridget A.
YAO, Monique G.
AU-YOUNG, Janice HAFALIA, April J.A.
ELLIOTT, Vicki S.
KALLICK, Deborah A.
GANDHI, Ameena R.
RICHARDSON, Thomas W.
KHAN, Farrah A.
LU, Yan SWARNAKAR, Anita RAMKUMAR, Jayala~ani NGUYEN, Danniel B.
GRAUL, Richard LU, Dyung Aina M.
<120> RECEPTORS AND MEMBRANE-ASSOCIATED PROTEINS
<130> PI-0346 PCT
<140> To Be Assigned <141> Herewith <150> 60/262,838; 60/265,927; 60/271,196; 60/274,549; 60/334,179 <151> 2001-01-19; 2001-02-02; 2001-02-23; 2001-03-09; 2001-11-28 <160> 30 <170> PERL Program <210> 1 <211> 457 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 71924779CD1 <400> 1 Met Tyr G1u Ser Val Glu Val G1y Gly Pro Thr Pro Asn Pro Phe Leu Val Val Asp Phe Tyr Asn G1n Asn Arg Ala Cys Leu Leu Pro Glu Lys Gly Leu Pro A1a Pro G1y Pro Tyr Ser Thr Pro Leu Arg Thr Pro Leu Trp Asn Gly Ser Asn His Ser Ile Glu Thr Gln Ser Ser Ser Ser Glu Glu Ile Val Pro Ser Pro Pro Ser Pro Pro Pro Leu Pro Arg Ile Tyr Lys Pro Cys Phe Val Cys Gln Asp Lys Ser Ser Gly Tyr His Tyr Gly Val Ser Ala Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Ile Gln Lys Asn Met Val Tyr Thr Cys His Arg Asp Lys Asn Cys Ile Ile Asn Lys Val Thr Arg Asn Arg Cys Gln Tyr Cys Arg Leu Gln Lys Cys Phe Glu Val Gly Met Ser Lys Glu Ser Va1 Arg Asn Asp Arg Asn Lys Lys Lys Lys Glu Val Pro Lys Pro Glu Cys Ser Glu Ser Tyr Thr Leu Thr Pro Glu Val Gly Glu Leu Ile Glu Lys Val Arg Lys Ala His Gln Glu Thr Phe Pro Ala Leu Cys Gln Leu Gly Lys Tyr Thr Thr Asn Asn Ser Ser Glu Gln Arg Val Ser Leu Asp Ile Asp Leu Trp Asp Lys Phe Ser Glu Leu Ser Thr Lys Cys Ile Ile Lys Thr Val Glu Phe Ala Lys Gln Leu Pro Gly Phe Thr Thr Leu Thr Ile Ala Asp Gln Ile Thr Leu Leu Lys Ala Ala Cys Leu Asp Ile Leu Ile Leu Arg Ile Cys Thr Arg Tyr Thr Pro Glu Gln Asp Thr Met Thr Phe Ser Asp Gly Leu Thr Leu Asn Arg Thr Gln Met His Asn Ala Gly Phe Gly Pro Leu Thr Asp Leu Val Phe Ala Phe Ala Asn Gln Leu Leu Pro Leu Glu Met Asp Asp Ala Glu Thr Gly Leu Leu Ser Ala Ile Cys Leu Ile Cys Gly Asp Arg Gln Asp Leu Glu Gln Pro Asp Arg Val Asp Met Leu Gln Glu Pro Leu Leu Glu Ala Leu Lys Val Tyr Val Arg Lys Arg Arg Pro Ser Arg Pro His Met Phe Pro Lys Met Leu Met Lys Ile Thr Asp Leu Arg Ser Ile Ser Ala Lys Gly Ala Glu Arg Va1 Ile Thr Leu Lys Met Glu I1e Pro Gly Ser Met Pro Pro Leu Ile Gln Glu Met Leu Glu Asn Ser Glu Gly Leu Asp Thr Leu Ser Gly Gln Pro Gly Gly Gly Gly Arg Asp Gly Gly Gly Leu Ala Pro Pro Pro Gly Ser Cys Ser Pro Ser Leu Ser Pro Ser Ser Asn Arg Ser Ser Pro Ala Thr His Ser Pro <210> 2 <211> 663 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2319430CD1 <400> 2 Met Ala A1a Lys Glu Lys Leu Glu Ala Val Leu Asn Val Ala Leu Arg Val Pro Ser Ile Met Leu Leu Asp Val Leu Tyr Arg Trp Asp Val Ser Ser Phe Phe Gln Gln Ile Gln Arg Ser Ser Leu Ser Asn Asn Pro Leu Phe Gln Tyr Lys Tyr Leu Ala Leu Asn Met His Tyr 50 ~ 55 60 Val Gly Tyr Ile Leu Ser Val Val Leu Leu Thr Leu Pro Arg Gln His Leu Val Gln Leu Tyr Leu Tyr Phe Leu Thr Ala Leu Leu Leu Tyr Ala Gly His Gln Ile Ser Arg Asp Tyr Val Arg Ser Glu Leu Glu Phe Ala Tyr G1u Gly Pro Met Tyr Leu Glu Pro Leu Ser Met Asn Arg Phe Thr Thr Ala Leu Ile Gly Gln Leu Val Val Cys Thr Leu Cys Ser Cys Val Met Lys Thr Lys Gln Ile Trp Leu Phe Ser Ala His Met Leu Pro Leu Leu Ala Arg Leu Cys Leu Val Pro Leu Glu Thr Ile Val Ile Ile Asn Lys Phe Ala Met Ile Phe Thr Gly Leu Glu Val Leu Tyr Phe Leu Gly Ser Asn Leu Leu Val Pro Tyr Asn Leu Ala Lys Ser Ala Tyr Arg Glu Leu Val Gln Val Val Glu Val Tyr Gly Leu Leu Ala Leu Gly Met Ser Leu Trp Asn Gln Leu Va1 Val Pro Val Leu Phe Met Val Phe Trp Leu Val Leu Phe Ala Leu Gln Ile Tyr Ser Tyr Phe Ser Thr Arg Asp Gln Pro Ala Ser Arg Glu Arg Leu Leu Phe Leu Phe Leu Thr Ser Ile Ala Glu.Cys Cys Ser Thr Pro Tyr Ser Leu Leu Gly Leu Val Phe Thr Val Ser Phe Val A1a Leu G1y Val Leu Thr Leu Cys Lys Phe Tyr Leu Gln Gly Tyr Arg Ala Phe Met Asn Asp Pro Ala Met Asn Arg Gly Met Thr Glu Gly Val Thr Leu Leu Ile Leu Ala Val Gln Thr G1y Leu Ile Glu Leu Gln Val Val His Arg Ala Phe Leu Leu Ser Ile Ile Leu Phe Ile Val Val Ala Ser Ile Leu Gln Ser Met Leu Glu Ile Ala Asp Pro Ile Val Leu Ala Leu Gly Ala Ser Arg Asp Lys Ser Leu Trp Lys His Phe Arg Ala Val Ser Leu Cys Leu Phe Leu Leu Val Phe Pro Ala Tyr Met Ala Tyr Met Ile Cys Gln Phe Phe His Met Asp Phe Trp Leu Leu Ile Ile Ile Ser Ser Ser Ile Leu Thr 410 41.5 420 Ser Leu Gln Val Leu Gly Thr Leu Phe Ile Tyr Val Leu Phe Met Val Glu Glu Phe Arg Lys Glu Pro Val Glu Asn Met Asp Asp Val Ile Tyr Tyr Val Asn Gly Thr Tyr Arg Leu Leu Glu Phe Leu Val Ala Leu Cys Val Val Ala Tyr Gly Val Ser G1u Thr Ile Phe Gly Glu Trp Thr Val Met Gly Ser Met Ile Ile Phe Ile His Ser Tyr Tyr Asn Val Trp Leu Arg Ala Gln Leu Gly Trp Lys Ser Phe Leu Leu Arg Arg Asp Ala Val Asn Lys Ile Lys Ser Leu Pro Ile Ala Thr Lys Glu Gln Leu Glu Lys His Asn Asp I1e Cys Ala Ile Cys Tyr Gln Asp Met Lys Ser Ala Val Ile Thr Pro Cys Ser His Phe Phe His Ala Gly Cys Leu Lys Lys Trp Leu Tyr Val Gln Glu Thr Cys Pro Leu Cys His Cys His Leu Lys Asn Ser Ser Gln Leu Pro Gly Leu Gly Thr Glu Pro Val Leu Gln Pro His Ala Gly Ala Glu 590 595 . 600 Gln Asn Val Met Phe Gln Glu Gly Thr Glu Pro Pro Gly Gln Glu His Thr Pro Gly Thr Arg Ile Gln Glu Gly Ser Arg Asp Asn Asn Glu Tyr Ile Ala Arg Arg Pro Asp Asn Gln G1u Gly Ala Phe Asp Pro Lys Glu Tyr Pro His Ser Ala Lys Asp Glu Ala His Pro Val Glu Ser Ala <210> 3 <211> 504 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7291877CD1 <400> 3 Met Lys Gly Ile Arg Lys Gly Glu Ser Arg Ala Lys Glu Ser Lys Pro Trp Glu Pro Gly Lys Arg Arg Cys Ala Lys Cys Gly Arg Leu Asp Phe Ile Leu Met Lys Lys Met Gly Ile Lys Ser Gly Phe Thr Phe Trp Asn Leu Val Phe Leu Leu Thr Val Ser Cys Val Lys Gly Phe Ile Tyr Thr Cys Gly Gly Thr Leu Lys Gly Leu Asn Gly Thr Ile Glu Ser Pro Gly Phe Pro Tyr Gly Tyr Pro Asn Gly A1a Asn Cys Thr Trp Val Ile Ile Ala Glu Glu Arg Asn Arg Ile Gln Ile Val Phe Gln Ser Phe Ala Leu Glu Glu Glu Tyr Asp Tyr Leu Ser Leu Tyr Asp Gly His Pro His Pro Thr Asn Phe Arg Thr Arg Leu Thr Gly Phe His Leu Pro Pro Pro Val Thr Ser Thr Lys Ser Val Phe Ser Leu Arg Leu Thr Ser Asp Phe Ala Val Ser Ala His Gly Phe Lys Val Tyr Tyr Glu Glu Leu Gln Ser Ser Ser Cys Gl.y Asn Pro Gly Val Pro Pro Lys Gly Val Leu Tyr Gly Thr Arg Phe Asp Val Gly Asp Lys Ile Arg Tyr Ser Cys Val Thr Gly Tyr Ile Leu Asp Gly His Pro Gln Leu Thr Cys Ile Ala Asn Ser Val Asn Thr Ala Ser Trp Asp Phe Pro Val Pro Ile Cys Arg Ala Glu Asp Ala Cys Gly Gly Thr Met Arg Gly Ser Ser G1y Ile Ile Ser Ser Pro Ser Phe Pro Asn Glu Tyr His Asn Asn Ala Asp Cys Thr Trp Thr Ile Val Ala Glu Pro Gly Asp Thr Ile Ser Leu Ile Phe Thr Asp Phe Gln Met G1u Glu Lys Tyr Asp Tyr Leu Glu Ile Glu Gly Ser Glu Pro Pro Thr Ile Trp Leu Ser Gly Met Asn Ile Pro Pro Pro Ile Ile Ser Asn Lys Asn Trp Leu Arg Leu His Phe Val Thr Asp Ser Asn His Arg Tyr Arg Gly Phe Ser Ala Pro Tyr Gln Gly Ser Ser Thr Leu Thr His Thr Thr Ser Thr Gly Glu Leu Glu Glu His Asn Arg Thr Thr Thr Gly Ala Ile Ala Val Ala Ser Thr Pro Ala Asp Va1 Thr Val Ser Ser Val Thr Ala Val Thr Ile His Arg Leu Ser Glu Glu Gln Arg Val Gln Val Thr Ser Leu Arg Asn Ser Gly Leu Asp Pro Asn Thr Ser Lys Asp Gly Leu Ser Pro His Pro Ala Asp Thr Gln Ser Thr Arg Arg Arg Pro Arg His Ala G1u Gln Ile Glu Arg Thr Lys Glu Leu Ala Val Val Thr His Arg Gly His Cys Asn Arg VaI Glu Asp Ile Glu Lys Pro Ile Leu VaI Val Gln Asp Arg Phe Cys Lys Met Asn Ser Asp Gln Ser Thr Lys Glu Val Thr 470 ' 475 480 Val Cys Met Gln Arg Val Ser Leu Leu Ser Tyr Phe Phe Asn Glu Leu Val Asn Asn Arg Lys Pro Ile Ala <210> 4 <211> 1114 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1218126CD1 <400> 4 Met Ala Pro Thr Leu Phe Gln Lys Leu Phe Ser Lys Arg Thr G1y Leu Gly Ala Pro Gly Arg Asp Ala Arg Asp Pro Asp Cys Gly Phe Ser '~rp Pro Leu Pro Glu Phe Asp Pro Ser Gln Ile Arg Leu Ile Val Tyr Gln Asp Cys Glu Arg Arg Gly Arg Asn Val Leu Phe Asp Ser Ser Val Lys Arg Arg Asn Glu Asp Ile Ser Va1 Ser Lys Leu Cys Ser Asp Ala Gln Val Lys Val Phe Gly Lys Cys Cys Gln Leu Lys Pro Gly Gly Asp Ser Ser Ser Ser Leu Asp Ser Ser Val Thr Ser Ser Ser Asp Ile Lys Asp Gln Cys Leu Lys Tyr Gln Gly Ser Arg Cys Ser Ser Asp Ala Asn Met Leu Gly Glu Met Met Phe Gly Ser Val Ala Met Ser Tyr Lys Gly Ser Thr Leu Lys Ile His Gln Ile Arg Ser Pro Pro Gln Leu Met Leu Ser Lys Val Phe Thr Ala Arg Thr Gly Ser Ser Ile Cys Gly Ser Leu Asn Thr Leu Gln Asp Ser Leu Glu Phe Ile Asn Gln Asp Asn Asn Thr Leu Lys A1a Asp Asn Asn Thr Val Tle Asn Gly Leu Leu Gly Asn Ile Ala Ser Leu Ser Ser Leu Leu Tle Thr Pro Phe Pro Ser Pro Asn Ser Ser Leu Thr Arg Ser Cys Ala Ser Ser Tyr Gln Arg Arg Trp Arg Arg Ser Gln Thr Thr Ser Leu Glu Asn Gly Val Phe Pro Arg Trp Ser Ile Glu Glu Ser Phe Asn Leu Ser Asp Glu Ser Cys Gly Pro Asn Pro Gly Ile Val Arg Lys Lys Lys Ile Ala Ile Gly Val Ile Phe Ser Leu Ser Lys Asp Glu Asp Glu Asn Asn Lys Phe Asn Glu Phe Phe Phe Ser His Phe Pro Leu Phe Glu Ser Tyr Met Asn Lys Leu Lys Ser Ala Ile G1u Gln Ala Met Lys Met Ser Arg Arg Ser A1a Asp Ala Ser Gln Arg Ser Leu Ala Tyr Asn Arg Ile Val Asp Ala Leu Asn G1u Phe Arg Thr Thr Ile Cys Asn Leu Tyr Thr Met Pro Arg Ile G1y Glu Pro Val Trp Leu Thr Met Met Ser Gly Thr Pro Glu Lys Asn His Leu Cys Tyr Arg Phe Met Lys Glu Phe Thr Phe Leu Met Glu Asn Ala Ser Lys Asn Gln Phe Leu Pro Ala Leu Ile Thr Ala Val Leu Thr Asn His Leu Ala Trp Val Pro Thr Val Met Pro Asn Gly Gln Pro Pro Ile Lys Ile Phe Leu Glu Lys His Ser Ser G1n Ser Val Asp Met Leu A1a Lys Thr His Pro Tyr Asn Pro Leu Trp A1a Gln Leu Gly Asp Leu Tyr Gly Ala Ile Gly Ser Pro Val Arg Leu Ala Arg Thr Val Val Va1 Gly Lys Arg Gln Asp Met Val Gln Arg Leu Leu Tyr Phe Leu Thr Tyr Phe Ile Arg Cys Ser Glu Leu Gln Glu Thr His Leu Leu Glu Asn Gly Glu Asp Glu Ala Ile Val Met Pro Gly Thr Val Ile Thr Thr Thr Leu Glu Lys Gly Glu Ile GIu Glu Ser Glu Tyr Val Leu Val Thr Met His Arg Asn Lys Ser Ser Leu Leu Phe Lys Glu Ser Glu Glu Ile Arg Thr Pro Asn Cys Asn Cys Lys Tyr Cys Ser His Pro Leu Leu Gly Gln Asn Val Glu Asn Ile Ser Gln Gln Glu Arg Glu Asp Ile Gln Asn Ser Ser Lys Glu Leu Leu Gly Ile Ser Asp Glu Cys Arg Met Ile Ser Pro Ser Asp Cys Gln Glu Glu Asn Ala Val Asp Val Lys Gln Tyr Arg Asp Lys Leu Arg Thr Cys Phe Asp Ala Lys Leu Glu Thr Val Val Cys Thr Gly Ser Val Pro Val Asp Lys Cys Ala Leu Ser Glu Ser Gly Leu Glu Ser Thr Glu Glu Thr Trp Gln Ser Glu Lys Leu Leu Asp Ser Asp Ser His Thr Gly Lys Ala Met Arg Ser Thr Gly Met Val Val Glu Lys Lys Pro Pro Asp Lys Ile Val Pro Ala Ser Phe Ser Cys Glu Ala Ala Gln Thr Lys Val Thr Phe Leu I1e Gly Asp Ser Met Ser Pro Asp Ser Asp Thr Glu Leu Arg Ser Gln Ala Val Val Asp Gln Ile Thr Arg His His Thr Lys Pro Leu Lys Glu Glu Arg Gly Ala Ile Asp Gln His Gln Glu Thr Lys Gln Thr Thr Lys Asp Gln Ser Gly Glu Ser Asp Thr Gln Asn Met Val Ser Glu Glu Pro Cys Glu Leu Pro Cys Trp Asn His Ser Asp Pro Glu Ser Met Ser Leu Phe Asp Glu Tyr Phe Asn Asp Asp Ser Ile Glu Thr Arg Thr I1e Asp Asp Val Pro Phe Lys Thr Ser Thr Asp Ser Lys Asp His Cys Cys Met Leu Glu Phe Ser Lys Ile Leu Cys Thr Lys Asn Asn Lys Gln Asn Asn Glu Phe Cys Lys Cys Ile Glu Thr Val Pro Gln Asp Ser Cys Lys Thr Cys Phe Pro Gln Gln Asp Gln Arg Asp Thr Leu Ser Ile Leu Val Pro His Gly Asp Lys Glu Ser Ser Asp Lys Lys Ile Ala Val Gly Thr Glu Trp Asp Ile Pro Arg Asn Glu Ser Ser Asp Ser Ala Leu Gly Asp Ser Glu Ser Glu Asp Thr G1y His Asp Met Thr Arg Gln Val Ser Ser Tyr Tyr Gly Gly Glu Gln Glu Asp Trp Ala Glu Glu Asp Glu Ile Pro Phe Pro Gly Ser Lys Leu Ile Glu Val Ser Ala Val Gln Pro Asn Ile Ala Asn Phe Gly Arg Ser Leu Leu Gly Gly Tyr Cys Ser Ser Tyr Va1 Pro Asp Phe Val Leu Gln Gly Ile Gly Ser Asp Glu Arg Phe Arg Gln Cys Leu Met Ser Asp Leu Ser His Ala Val Gln His Pro Val Leu Asp Glu Pro Ile Ala Glu Ala Val Cys Ile Ile Ala Asp Met Asp Lys Trp Thr Val Gln Val Ala Ser Ser Gln Arg Arg Val Thr Asp Asn Lys Leu Gly Lys Glu Val Leu Val Ser Ser Leu Val Ser Asn Leu Leu His Ser Thr Leu Gln Leu Tyr Lys His Asn Leu Ser Pro Asn Phe Cys Val Met His Leu Glu Asp Arg Leu Gln G1u Leu Tyr Phe Lys Ser Lys Met Leu Ser Glu Tyr Leu Arg Gly Gln Met Arg Val His Val Lys Glu Leu Gly Val Val Leu Gly Ile Glu Ser Ser Asp Leu Pro Leu Leu Ala Ala Val Ala Ser Thr His Ser Pro Tyr Val Ala 1100 1.105 1110 G1n Ile Leu Leu <210> 5 <221> 479 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7479161CD1 <400> 5 Met Gln Pro Val Met Leu Ala Leu Trp Ser Leu Leu Leu Leu Trp Gly Leu Ala Thr Pro Cys Gln Glu Leu Leu Glu Thr Val Gly Thr Leu Ala Arg Ile Asp Lys Asp Glu Leu Gly Lys Ala Ile G1n Asn Ser Leu Val Gly Glu Pro Ile Leu Gln Asn Val Leu Gly Ser Val Thr AIa Val Asn Arg Gly Leu Leu G1y Ser Gly Gly Leu Leu Gly Gly Gly Gly Leu Leu Gly His Gly Gly Val Phe Gly Val Val Glu Glu Leu Ser Gly Leu Lys Tle Glu Glu Leu Thr Leu Pro Lys Val Leu Leu Lys Leu Leu Pro Gly Phe Gly Val Gln Leu Ser Leu His Thr Lys Val Gly Met His Cys Ser Gly Pro Leu Gly Gly Leu Leu GIn Leu Ala Ala Glu Val Asn Val Thr Ser Arg Val AIa Leu Ala Val Ser Ser Arg Gly Thr Pro Ile Leu Ile Leu Lys Arg Cys Ser Thr Leu Leu Gly His Ile Ser Leu Phe Ser Gly Leu Leu Pro Thr Pro Leu Phe Gly Val Val Glu Gln Met Leu Phe Lys Va1 Leu Pro Gly Leu Leu Cys Pro Val Va1 Asp Ser Val Leu Gly Val Val Asn Glu Leu Leu Gly Ala Val Leu Gly Leu Val Ser Leu Gly Ala Leu Gly Ser Val Glu Phe Ser Leu Ala Thr Leu Pro Leu Ile Ser Asn Gln Tyr Ile Glu Leu Asp Ile Asn Pro Ile Val Lys Ser Val Ala G1y Asp Ile Ile Asp Phe Pro Lys Ser Arg Ala Pro Ala Lys Va1 Pro Pro Lys Lys Asp His Thr Ser Gln Val Met Val Pro Leu Tyr Leu Phe Asn Thr Thr Phe Gly Leu Leu Gln Thr Asn Gly Ala Leu Asp Met Asp Ile Thr Pro Glu Leu Val Pro Ser Asp Val Pro Leu Thr Thr Thr Asp Leu Ala Ala Leu Leu Pro Glu Ala Leu Gly Lys 320 325 330 .
Leu Pro Leu His Gln Gln Leu Leu Leu Phe Leu Arg Val Arg Glu Ala Pro Thr Val Thr Leu His Asn Lys Lys Ala Leu Val Ser Leu Pro Ala Asn Ile His Val Leu Phe Tyr Val Pro Lys Gly Thr Pro Glu Ser Leu Phe Glu Leu Asn Ser Val Met Thr Val Arg Ala Gln Leu Ala Pro Ser Ala Thr Lys Leu His I1e Ser Leu Ser Leu Glu Arg Leu Ser Val Lys Val Ala Ser Ser Phe Thr His Ala Phe Asp Gly Ser Arg Leu Glu Glu Trp Leu Ser His Val Val Gly Ala Val Tyr Ala Pro Lys Leu Asn Val Ala Leu Asp Val Gly Ile Pro Leu Pro Lys Val Leu Asn Ile Asn Phe Ser Asn Ser Val Leu Glu Ile Va1 Glu Asn Ala Val Ala Ala Leu Tyr Val Leu Val Val Ala <210> 6 <211> 1774 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7722591CD1 <400> 6 Met Ala Pro Val Ser Met Leu Ser Pro Ala Ser Ser Leu Ser His Pro Ala Gly Ala Tyr Arg G1y Thr Ser Gln Gly Pro Ser Val Gly Val Thr Ala Pro Cys Gly Val Gly Glu Gly Leu Gly Ala Ser Arg Gly Pro A1a Leu Pro Val Trp Ala Tyr Ala Arg Cys Pro Asp Val Asp Glu Cys Arg Leu Gly Leu Ala Arg Cys His Pro Arg Ala Thr Cys Leu Asn Thr Pro Leu Ser Tyr Glu Cys His Cys Gln Arg Gly Tyr Gln Gly Asp Gly Ile Ser His Cys Asn Arg Thr Cys Leu Glu Asp Cys Gly His Gly Val Cys Ser Gly Pro Pro Asp Phe Thr Cys Val Cys Asp Leu Gly Trp Thr Ser Asp Leu Pro Pro Pro Thr Pro Ala Pro Gly Pro Pro Ala Pro Arg Cys Ser Arg Asp Cys Gly Cys Ser Phe His Ser His Cys Arg Lys Arg Gly Pro Gly Phe Cys Asp Glu Cys Gln Asp Trp Thr Trp Gly Glu His Cys Glu Arg Cys Arg Pro Gly Ser Phe Gly Asn Ala Thr Gly Ser Arg Gly Cys Arg Pro Cys Gln Cys Asn Gly His G1y Asp Pro Arg Arg Gly His Cys Asp Asn Leu Ser Gly Leu Cys Phe Cys G1n Asp His Thr Glu Gly Ala His Cys Gln Leu Cys Ser Pro Gly Tyr Tyr Gly Asp Pro Arg Ala Gly Gly Ser Cys Phe Arg Glu Cys Gly Gly Arg Ala Leu Leu Thr Asn Val Ser Ser Val Ala Leu Gly Ser Arg Arg Val Gly Gly Leu Leu Pro Pro Gly Gly Gly Ala Ala Arg Ala Gly Pro Gly Leu Ser Tyr Cys Val Trp Val Val Ser Ala Thr Glu Glu Leu Gln Pro Cys Ala Pro Gly Thr Leu Cys Pro Pro Leu Thr Leu Thr Phe Ser Pro 305 ~ 310 315 Asp Ser Ser Thr Pro Cys Thr Leu Ser Tyr Val Leu Ala Phe Asp Gly Phe Pro Arg Phe Leu Asp Thr Gly Val Val Gln Ser Asp Arg Ser Leu Ile Ala Ala Phe Cys Gly Gln Arg Arg Asp Arg Pro Leu Thr Va1 Gln Ala Leu Ser Gly Leu Leu Val Leu His Trp Glu Ala Asn Gly Ser Ser Ser Trp G1y Phe Asn Ala Ser Val Gly Ser Ala Arg Cys Gly Ser Gly Gly Pro Gly Ser Cys Pro Val Pro Gln Glu Cys Val Pro Gln Asp Gly Ala A1a Gly A1a Gly Leu Cys Arg Cys Pro G1n G1y Trp Ala Gly Pro His Cys Arg Met Ala Leu Cys Pro Glu Asn Cys Asn Ala His Thr Gly Ala Gly Thr Cys Asn Gln Ser Leu Gly Val Cys Ile Cys Ala Glu Gly Phe Gly Gly Pro Asp Cys Ala Thr Lys Leu Asp Gly Gly Gln Leu Val Trp Glu Thr Leu Met Asp Ser Arg Leu Ser Ala Asp Thr Ala Ser Arg Phe Leu His Arg Leu Gly His Thr Met Val Asp Gly Pro Asp Ala Thr Leu Trp Met Phe Gly G1y Leu G1y Leu Pro Gln Gly Leu Leu Gly Asn Leu Tyr Arg Tyr Ser Val Ser Glu Arg Arg Trp Thr Gln Met Leu Ala Gly Ala Glu Asp Gly Gly Pro Gly Pro Ser Pro Arg Ser Phe His Ala Ala Ala Tyr Val Pro Ala Gly Arg Gly Ala Met Tyr Leu Leu Gly Gly Leu Thr Ala Gly Gly Val Thr Arg Asp Phe Trp Val Leu Asn Leu Thr Thr Leu Gln Trp Arg Gln Glu Lys Ala Pro Gln Thr Val Glu Leu Pro Ala Va1 Ala Gly His Thr Leu Thr Ala Arg Arg Gly Leu Ser Leu Leu Leu Val Gly Gly Tyr Ser Pro Glu Asn Gly Phe Asn Gln Gln Leu Leu Glu Tyr Gln Leu Ala Thr Gly Thr Trp Va1 Ser Gly Ala Gln Ser Gly Thr Pro Pro Thr Gly Leu Tyr Gly His Ser Ala Val Tyr His Glu Ala Thr Asp Ser Leu Tyr Val Phe Gly Gly Phe Arg Phe His Val Glu Leu Ala A1a Pro Ser Pro Glu Leu Tyr Ser Leu His Cys Pro Asp Arg Thr Trp Ser Leu Leu Ala Pro Ser Gln Gly Ala Lys Pro Arg Pro Arg Leu Phe His Ala Ser Ala Leu Leu Gly Asp Thr Met Val Val Leu Gly Gly Arg Ser Asp Pro Asp Glu Phe Ser Ser Asp Val Leu Leu Tyr Gln Val Asn Cys Asn Ala Trp Leu Leu Pro Asp Leu Thr Arg Ser Ala Ser Val Gly Pro Pro Met Glu Glu Ser Val Ala His Ala Val Ala Ala Va1 Gly Ser Arg Leu Tyr Ile Ser Gly Gly Phe Gly Gly Val Ala Leu Gly Arg Leu Leu Ala Leu Thr Leu Pro Pro Asp Pro Cys Arg Leu Leu Ser Ser Pro Glu A1a Cys Asn Gln Ser Gly Ala Cys Thr Trp Cys His Gly Ala Cys Leu Ser Gly Asp Gln Ala His Arg Leu Gly Cys Gly Gly Ser Pro Cys Ser Pro Met Pro Arg Ser Pro Glu Glu Cys Arg Arg Leu Arg Thr Cys Ser Glu Cys Leu Ala Arg His Pro Arg Thr Leu Gln Pro Gly Asp Gly Glu Ala Ser Thr Pro Arg Cys Lys Trp Cys Thr Asn Cys Pro Glu Gly Ala Cys Ile Gly Arg Asn Gly Ser Cys Thr Ser Glu Asn Asp Cys Arg Ile Asn Gln Arg Glu Va1 Phe Trp Ala Gly Asn Cys Ser Glu A1a Ala Cys Gly Ala Ala Asp Cys Glu Gln Cys Thr Arg Glu Gly Lys Cys Met Trp Thr Arg Gln Phe Lys Arg Thr Gly Glu Thr Arg Arg Ile Leu Ser Val Gln Pro Thr Tyr Asp Trp Thr Cys Phe Ser His Ser Leu Leu Asn Val Ser Pro Met Pro Val Glu Ser Ser Pro Pro Leu Pro Cys Pro Thr Pro Cys His Leu Leu Pro Asn Cys Thr Ser Cys Leu Asp Ser Lys Gly Ala Asp Gly Gly Trp Gln His Cys Val Trp Ser Ser Ser Leu Gln Gln Cys Leu Ser Pro Ser Tyr Leu Pro Leu Arg Cys Met Ala Gly Gly Cys Gly Arg Leu Leu Arg Gly Pro Glu Ser Cys Ser Leu Gly Cys Ala Gln Ala Thr Gln Cys Ala Leu Cys Leu Arg Arg Pro His Cys Gly Trp Cys Ala Trp Gly Gly Gln Asp Gly Gly Gly Arg Cys Met Glu G1y Gly Leu Ser Gly Pro Arg Asp Gly Leu Thr Cys Gly Arg Pro Gly Ala Ser Trp Ala Phe Leu Ser Cys Pro Pro Glu Asp Glu Cys Ala Asn Gly His His Asp Cys Asn Glu Thr Gln Asn Cys His Asp Gln Pro His Gly Tyr Glu Cys Ser Cys Lys Thr Gly Tyr Thr Met Asp Asn Met Thr Gly Leu Cys Arg Pro Val Cys Ala Gln Gly Cys Val Asn Gly Ser Cys Val Glu Pro Asp His Cys Arg Cys His Phe Gly Phe Va1 G1y Arg Asn Cys Ser Thr Glu Cys Arg Cys Asn Arg His Ser Glu Cys Ala Gly Val Gly Ala Arg Asp His Cys Leu Leu Cys Arg Asn His Thr Lys G1y Ser His Cys Glu Gln Cys Leu Pro Leu Phe Val Gly Ser Ala Val Gly Gly Gly Thr Cys Arg Pro Cys His Ala Phe Cys Arg Gly Asn Ser His Ile Cys Ile Ser Arg Lys Glu Leu G1n Met Ser Lys Gly Glu Pro Lys Lys Tyr Ser Leu Asp Pro Glu Glu Ile Glu Asn Trp Val Thr Glu Gly Pro Ser Glu Asp Glu Ala Val Cys Val Asn Cys Gln Asn Asn Ser Tyr Gly Glu Lys Cys Glu Ser Cys Leu Gln G1y Tyr Phe Leu Leu Asp Gly Lys Cys Thr Lys Cys Gln Cys Asn G1y His Ala Asp Thr Cys Asn Glu Gln Asp Gly Thr Gly Cys Pro Cys Gln Asn Asn Thr G1u Thr Gly Thr Cys Gln Gly Ser Ser Pro Ser Asp Arg Arg Asp Cys Tyr Lys Tyr Gln Cys Ala Lys Cys Arg G1u Ser Phe His Gly Ser Pro Leu Gly Gly Gln Gln Cys Tyr Arg Leu Ile Ser Val Glu Gln Glu Cys Cys Leu Asp Pro Thr Ser Gln Thr Asn Cys Phe His Glu Pro Lys Arg Arg Ala Leu Gly Pro Gly Arg Thr Val Leu Phe Gly Val G1n 1'400 1405 1410 Pro Lys Phe Thr Asn Val Asp Ile Arg Leu Thr Leu Asp Val Thr Phe Gly Ala Va1 Asp Leu Tyr Val Ser Thr Ser Tyr Asp Thr Phe Val Val Arg Val Ala Pro Asp Thr Gly Val His Thr Val His Ile G1n Pro Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro Ala Asp Gly Gly Pro Arg Gly Ala Gly Asp Pro Gly Gly Ala Gly Ala Ser Ser Gly Pro Gly Ala Pro Ala Glu Pro Arg Val Arg Glu Val Trp Pro Arg Gly Leu Ile Thr Tyr Val Thr Val Thr Glu Pro Ser Ala Val Leu Val Va1 Arg Gly Val Arg Asp Arg Leu Val Ile Thr Tyr Pro His Glu His His Ala Leu Lys Ser Ser Arg Phe Tyr Leu Leu Leu Leu Gly Val Gly Asp Pro Ser G1y Pro Gly Ala Asn Gly Ser Ala Asp Ser Gln Gly Leu Leu Phe Phe Arg Gln Asp Gln Ala His Ile Asp Leu Phe Val Phe Phe Ser Val Phe Phe Ser Cys Phe Phe Leu Phe Leu Ser Leu Cys Val Leu Leu Trp Lys Ala Lys Gln Ala Leu Asp Gln Arg Gln Glu Gln Arg Arg His Leu Gln Glu Met Thr Lys Met Ala Ser Arg Pro Phe Ala Lys Val Thr Val Cys Phe Pro Pro Asp Pro Thr Ala Pro Ala Ser Ala Trp Lys Pro Ala Gly Leu Pro Pro Pro Ala Phe Arg Arg Ser Glu Pro Phe Leu Ala Pro Leu Leu Leu Thr Gly Ala Gly Gly Pro Trp Gly Pro Met Gly Gly Gly Cys Cys Pro Pro Ala Ile Pro Ala Thr Thr Ala Gly Leu Arg Ala Gly Pro Ile Thr Leu Glu Pro Thr Glu Asp Gly Met Ala Gly Val Ala Thr Leu Leu Leu Gln Leu Pro Gly Gly Pro His Ala Pro Asn Gly Ala Cys Leu Gly Ser Ala Leu Val Thr Leu Arg His Arg Leu His Glu Tyr Cys Gly G1y Gly Gly Gly Ala Gly Gly Ser Gly His Gly Thr Gly Ala Gly Arg Lys Gly Leu Leu Ser Gln Asp Asn Leu Thr Ser Met Ser Leu <210> 7 <211> 393 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2173285CD1 <400> 7 Met Phe Ser Val Glu Ser Leu Glu Arg Ala Glu Leu Cys Glu Ser Leu Leu Thr Trp Ile Gln Thr Phe Asn Val Asp Ala Pro Cys Gln Thr Val Glu Asp Leu Thr Asn Gly Val Val Met Ala Gln Val Leu Gln Lys 21e Asp Pro Ala Tyr Phe Asp Glu Asn Trp Leu Asn Arg Ile Lys Thr Glu Val Gly Asp Asn Trp Arg Leu Lys Ile Ser Asn Leu Lys Lys Ile Leu Lys Gly Ile Leu Asp Tyr Asn His Glu Ile Leu Gly Gln Gln Ile Asn Asp Phe Thr Leu Pro Asp Val Asn Leu Ile Gly Glu His Ser Asp Ala Ala Glu Leu Gly Arg Met Leu Gln Leu Ile Leu Gly Cys Ala Val Asn Cys Glu Gln Lys Gln Glu Tyr Ile Gln Ala Ile Met Met Met Glu Glu Ser Val Gln His Val Val Met Thr Ala Ile Gln Glu Leu Met Ser Lys Glu Ser Pro Val Ser Ala Gly Asn Asp Ala Tyr Val Asp Leu Asp Arg Gln Leu Lys Lys Thr Thr Glu Glu Leu Asn Glu Ala Leu Ser Ala Lys Glu Glu T1e Ala Gln Arg Cys His Glu Leu Asp Met Gln Val Ala Ala Leu Gln Glu Glu Lys Ser Ser Leu Leu Ala Glu Asn Gln Val Leu Met Glu Arg Leu Asn Gln Ser Asp Ser Ile Glu Asp Pro Asn Ser Pro Ala Gly Arg Arg His Leu Gln Leu Gln Thr Gln Leu Glu Gln Leu Gln Glu Glu Thr Phe Arg Leu Glu Ala Ala Lys Asp Asp Tyr Arg Ile Arg Cys Glu Glu Leu Glu Lys Glu Ile Ser G1u Leu Arg Gln Gln Asn Asp Glu Leu Thr Thr Leu Ala Asp Glu Ala Gln Ser Leu Lys Asp Glu Ile Asp VaI Leu Arg His Ser Ser Asp Lys VaI Ser Lys Leu Glu Gly Gln Val Glu Ser Tyr Lys Lys Lys Leu Glu Asp Leu Gly Asp Leu Arg Arg Gln Val Lys Leu Leu Glu Glu Lys Asn Thr Met Tyr Met Gln Asn Thr Val Ser Leu Glu Glu Glu Leu Arg Lys Ala Asn Ala Ala Arg Ser Gln Leu Glu Thr Tyr Lys Arg Gln Val Lys Glu Thr Gln His Leu Asp Asp Gly Phe Arg Gln Ala Leu Ser Tyr Asp Met <210> 8 <211> 311 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7487619CD1 <400> 8 Met Ser Asn Ala Ser Leu Val Thr Ala Phe Ile Leu Thr Gly Leu l 5 10 15 Pro His Ala Pro Gly Leu Asp Ala Pro Leu Phe Gly Ile Phe Leu Val Val Tyr Val Leu Thr Val Leu G1y Asn Leu Leu Ile Leu Leu Val Ile Arg Va1 Asp Ser His Leu His Thr Pro Met Tyr Tyr Phe Leu Thr Asn Leu Ser Phe Ile Asp Met Trp Phe Ser Thr Val Thr Val Pro Lys Met Leu Met Thr Leu Val Ser Pro Ser Gly Arg Ala Ile Ser Phe His Ser Cys Val Ala Gln Leu Tyr Phe Phe His Phe Leu Gly Ser Thr Glu Cys Phe Leu Tyr Thr Val Met Ala Tyr Asp Arg Tyr Leu Ala Ile Ser Tyr Pro Leu Arg Tyr Thr Ser Met Met Thr Gly Arg Ser Cys Thr Leu Leu Ala Thr Ser Thr Trp Leu Ser Gly Ser Leu His Ser Ala Val Gln Ala Ile Leu Thr Phe His Leu 155 160 ' 165 Pro Tyr Cys Gly Pro Asn Trp I1e Gln His Tyr Leu Cys Asp Ala 170 175 . 180 Pro Pro Ile Leu Lys Leu Ala Cys Ala Asp Thr Ser Ala Ile Glu Thr Val Ile Phe Val Thr Val Gly Ile Val Ala Ser Gly Cys Phe Val Leu Ile Val Leu Ser Tyr Val Ser Ile Val Cys Ser Ile Leu Arg Ile Arg Thr Ser Glu Gly Lys His Arg Ala Phe Gln Thr Cys Ala Ser His Cys Ile Val Val Leu Cys Phe Phe Gly Pro Gly Leu Phe Ile Tyr Leu Arg Pro Gly Ser Arg Lys Ala Val Asp Gly Val Va1 Ala Va1 Phe Tyr Thr Val Leu Thr Pro Leu Leu Asn Pro Val Val Tyr Thr Leu Arg Asn Lys Glu Val Lys Lys Ala Leu Leu Lys Leu Lys Asp Lys Val Ala His Ser Gln Ser Lys <210> 9 <211> 318 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7487607CD1 <400> 9 Met Glu Trp G1u Asn His Thr Ile Leu Val Glu Phe Phe Leu Lys Gly Leu Ser Gly His Pro Arg Leu Glu Leu Leu Phe Phe Val Leu 21e Phe Ile Met Tyr Val Val Tle Leu Leu G1y Asn Gly Thr Leu Ile Leu I1e Ser Ile Leu Asp Pro His Leu His Thr Pro Met Tyr Phe Phe Leu Gly Asn Leu Ser Phe Leu Asp Ile Cys Tyr Thr Thr Thr Ser Ile Pro Ser Thr Leu Val Ser Phe Leu Ser Glu Arg Lys Thr Ile Ser Leu Ser Gly Cys Ala Va1 Gln Met Phe Leu Gly Leu Ala Met Gly Thr Thr Glu Cys Va1 Leu Leu G1y Met Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys Asn Pro Leu Arg Tyr Pro Ile Ile Met Ser Lys Asp Ala Tyr Val Pro Met Ala Ala Gly Ser Trp Ile 21e Gly Ala Val Asn Ser Ala Val Gln Ser Val Phe Val Val Gln Leu Pro Phe Cys Arg Asn Asn Ile Ile Asn His Phe Thr Cys Glu Ile Leu Ala Val Met Lys Leu Ala Cys Ala Asp Ile Ser Asp Asn Glu Phe Ile Met Leu Val Ala Thr Thr Leu Phe Ile Leu Thr Pro Leu Leu Leu Ile Ile Val Ser Tyr Thr Leu Ile Ile Val Ser Ile Phe Lys Ile Ser Ser Ser Glu Gly Arg Ser Lys Ala Ser Ser Thr Cys Ser Ala His Leu Thr Val Val Ile Ile Phe Tyr Gly Thr Ile Leu Phe Met Tyr Met Lys Pro Lys Ser Lys Glu Thr Leu Asn Ser Asp Asp Leu Asp Ala Thr Asp Lys Tle Ile Ser Met Phe Tyr Gly Val Met Thr Pro Met Met Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Glu Ala Val Lys His Leu Leu Asn Arg Arg Phe Phe Ser Lys <210> 10 <211> 311 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7487616CD1 <400> 10 Met Ser Asn Ala Ser Leu Val Thr Ala Phe Ile Leu Thr Gly Leu Pro His Ala Pro Gly Leu Asp Ala Pro Leu Phe Gly Ile Phe Leu Val Va1 Tyr Val Leu Thr Val Leu Gly Asn Leu Leu Ile Leu Leu Val Ile Arg Val Asp Ser His Leu His Thr Pro Met Tyr Tyr Phe Leu Thr Asn Leu Ser Phe Ile Asp Met Trp Phe Ser Thr Va1 Thr Val Pro Lys Met Leu Met Thr Leu Val Ser Pro Ser Gly Arg Thr Ile Ser Phe His Ser Cys Val Ala Gln Leu Tyr Phe Phe His Phe Leu Gly Ser Thr Glu Cys Phe Leu Tyr Thr Val Met Ser Tyr Asp Arg Tyr Leu Ala Ile Ser Tyr Pro Leu Arg Tyr Thr Asn Met Met Thr Gly Arg Ser Cys Ala Leu Leu Ala Thr Gly Thr Trp Leu Ser Gly Ser Leu His Ser Ala Val Gln Thr Ile Leu Thr Phe His Leu Pro Tyr Cys GIy Pro Asn Gln Ile Gln His Tyr Phe Cys Asp Ala Pro Pro Ile Leu Lys Leu Ala Cys Ala Asp Thr Ser Ala Asn Glu Met Val Ile Phe Val Asn Ile Gly Leu Val Ala Ser Gly Cys Phe Val Leu Ile Val Leu Ser Tyr Val Ser Ile Val Cys Ser Ile Leu Arg Ile Arg Thr Ser Glu Gly Arg His Arg Ala Phe Gln Thr Cys Ala Ser His Cys Ile Val Val Leu Cys Phe Phe Gly Pro Gly Leu Phe Ile Tyr Leu Arg Pro Gly Ser Arg Asp A1a Leu His Gly Val Val Ala Val Phe Tyr Thr Thr Leu Thr Pro Leu Phe Asn Pro Val Val Tyr Thr Leu Arg Asn Lys Glu Val Lys Lys Ala Leu Leu Lys Leu Lys Asn Gly Ser Val Phe Ala Gln Gly Glu <210> 11 <211> 310 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7483204CD1 <400> 11 Met Asp Trp Glu Asn Cys Ser Ser Leu Thr Asp Phe Phe Leu Leu l 5 10 15 Gly Ile Thr Asn Asn Pro Glu Met Lys Val Thr Leu Phe Ala Val Phe Leu Ala Va1 Tyr Ile Ile Asn Phe Ser Ala Asn Leu Gly Met Ile Val~Leu Ile Arg Met Asp Tyr Gln Leu His Thr Pro Met Tyr Phe Phe Leu Ser His Leu Ser Phe Cys Asp Leu Cys Tyr Ser Thr Ala Thr Gly Pro Lys Met Leu Val Asp Leu Leu Ala Lys Asn Lys Ser Ile Pro Phe Tyr Gly Cys Ala Leu Gln Phe Leu Val Phe Cys Ile Phe Ala Asp Ser Glu Cys Leu Leu Leu Ser Val Met Ala Phe Asp Arg Tyr Lys Ala Ile Ile Asn Pro Leu Leu Tyr Thr Val Asn Met Ser Ser Arg Val Cys Tyr Leu Leu Leu Thr Gly Val Tyr Leu Val Gly I1e Ala Asp Ala Leu Ile His Met Thr Leu Ala Phe Arg Leu Cys Phe Cys Gly Ser Asn Glu Ile Asn His Phe Phe Cys Asp 21e Pro Pro Leu Leu Leu Leu Ser Cys Ser Asp Thr Gln Val Asn Glu Leu Val Leu Phe Thr Val Phe Gly Phe Ile Glu Leu Ser Thr Ile Ser Gly Va1 Phe Ile Ser Tyr Cys Tyr Ile Ile Leu Ser Val Leu Glu Ile His Ser Ala Glu Gly Arg Phe Lys Ala Leu Ser Thr Cys Thr Ser His Leu Ser Ala Val Ala Ile Phe G1n Gly Thr Leu Leu Phe Met Tyr Phe Arg Pro Ser Ser Ser Tyr Ser Leu Asp Gln Asp Lys Met Thr Ser Leu Phe Tyr Thr Leu Val Val Pro Met Leu Asn Pro Leu I1e Tyr Ser Leu Arg Asn Lys Asp Val Lys Glu Ala Leu Lys Lys Leu Lys Asn Glu Ile Leu Phe <210> 12 <211> 316 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7472099CD1 <400> 12 Met Ser Ala Ser Ser 21e Thr Ser Thr His Pro Thr Ser Phe Leu Leu Met Gly Ile Pro Gly Leu Glu His Leu His Ile Trp Ile Ser Ile Pro Phe Ser Ala Tyr Thr Leu Ala Leu Leu Gly Asn Cys Thr Leu Leu Leu Ile Ile Gln Ala Asp~Ala Ala Leu His Glu Pro Ile Tyr Leu Phe Leu Ala Met Leu Ala Ala Ile Asp Leu Val Leu Ser Ser Ser Ala Leu Pro Lys Met Leu Ala Ile Phe Trp Phe Arg Asp Arg Glu Ile Asn Phe Phe Ala Cys Leu Val Gln Met Phe Phe Leu His Ser Phe Ser Ile Met Glu Ser Ala Val Leu Leu A1a Met Ala Phe Asp Arg Tyr Val Ala Ile Cys Lys Pro Leu His Tyr Thr Thr Val Leu Thr Gly Ser Leu Ile Thr Lys Ile Gly Met Ala Ala Val Ala Arg Ala Val Thr Leu Met Thr Pro Leu Pro Phe Leu Leu Arg Cys Phe His Tyr Cys Arg Gly Pro Val Ile Ala Arg Cys Tyr Cys Glu His Met Ala Val Val Arg Leu Ala Val Gly Thr Leu Gly Phe Asn Asn Ile Tyr Gly I1e Ala Val Ala Met Phe Ile Gly Val Leu Asp Leu Phe Phe Ile Ile Leu Ser Tyr Ile Phe Ile Leu Gln Ala Val Leu Gln Leu Ser Ser Gln Glu Ala Arg Tyr Lys Ala Phe Gly Thr Cys Val Ser His Ile Gly Ala Ile Leu Ala Phe Tyr Thr Pro Ser Val Ile Ser Ser Val Met His Arg,Val Ala Arg Cys Ala Val Pro His Val His Ile Leu Leu Ala Asn Phe Tyr Leu Leu Phe Pro Pro Met Val Asn Pro Ile Ile Tyr Gly Val Lys Thr Lys Gln Ile Arg Asp Ser Leu Gly Ser Ile Pro Glu Lys Gly Cys Val Asn Arg Glu <210> 13 <211> 318 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7485443CD1 <400> 13 Met Glu Trp Glu Asn His Thr Ile Leu Val Glu Phe Phe Leu Lys Gly Leu Ser Gly His Pro Arg Leu Glu Leu Leu Phe Phe Val Leu I1e Phe I1e Met Tyr Val Val Tle Leu Leu Gly Asn Gly Thr Leu Ile Leu Ile Ser Ile Leu Asp Pro His Leu His Thr Pro Met Tyr Phe Phe Leu Gly Asn Leu Ser Phe Leu Asp Ile Cys Tyr Thr Thr 65~ 70 75 Thr Ser Ile Pro Ser Thr Leu Val Ser Phe Leu Ser Glu Arg Lys Thr Ile Ser Phe Ser Gly Cys Ala Val Gln Met Phe Leu Gly Leu Ala Met Gly Thr Thr Glu Cys Val Leu Leu Gly Met Met Ala Phe Asp Arg Tyr Val Ala Ile Cys Asn Pro Leu Arg Tyr Pro Ile Ile Met Ser Lys Asn Ala Tyr Val Pro Met Ala Val Gly Ser Trp Phe Ala Gly Ile Val Asn Ser Ala Val Gln Thr Thr Phe Val Val Gln Leu Pro Phe Cys Arg Lys Asn Val Ile Asn His Phe Ser Cys Glu Ile Leu Ala Val Met Lys Leu Ala Cys Ala Asp Ile Ser Gly Asn Glu Phe Leu Met Leu Val Ala Thr Ile Leu Phe Thr Leu Met Pro Leu Leu Leu Ile Val Ile Ser Tyr Ser Leu Ile Ile Ser Ser Ile Leu Lys Ile His Ser Ser Glu Gly Arg Ser Lys Ala Phe Ser Thr Cys Ser Ala His Leu Thr Val Val Ile Ile Phe Tyr Gly Thr Ile Leu Phe Met Tyr Met Lys Pro Lys Ser Lys G1u Thr Leu Asn Ser Asp Asp Leu Asp Ala Thr Asp Lys Ile Ile Ser Met Phe Tyr Gly Val Met Thr Pro Met Met Asn Pro Leu I1e Tyr Ser Leu Arg Asn Lys Asp Val Lys Glu Ala Va1 Lys His Leu Pro Asn Arg Arg Phe Phe Ser Lys <210> 14 <211> 321 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3090414CD1 <400> 14 Met Leu Thr Leu Asn Lys Thr Asp Leu I1e Pro Ala Ser Phe I1e Leu Asn Gly Val Pro Gly Leu Glu Asp Thr Gln Leu Trp Ile Ser Phe Pro Phe Cys Ser Met Tyr Val Val Ala Met Val Gly Asn Cys Gly Leu Leu Tyr Leu Ile His Tyr Glu Asp Ala Leu His Lys Pro Met Tyr Tyr Phe Leu Ala Met Leu Ser Phe Thr Asp Leu Val Met Cys Ser Ser Thr Ile Pro Lys Ala Leu Cys Ile Phe Trp Phe His Leu Lys Asp Ile Gly Phe Asp Glu Cys Leu Val Gln Met Phe Phe Ile His Thr Phe Thr Gly Met Glu Ser Gly Val Leu Met Leu Met Ala Leu Asp Arg Tyr Val Ala Ile Cys Tyr Pro Leu Arg Tyr Ser Thr Tle Leu Thr Asn Pro Val Tle Ala Lys Val Gly Thr Ala Thr Phe Leu Arg Gly Val Leu Leu Ile Ile Pro Phe Thr Phe Leu Thr Lys Arg Leu Pro Tyr Cys Arg G1y Asn Ile Leu Pro His Thr Tyr Cys Asp His Met Ser Val Ala Lys Leu Ser Cys Gly Asn Val Lys Val Asn Ala Ile Tyr Gly Leu Met Val Ala Leu Leu Ile Trp Gly Phe Asp Ile Leu Cys Ile Thr Ile Ser Tyr Thr Met Ile Leu Arg Ala Val Val Ser Leu Ser Ser Ala Asp Ala Arg Gln Lys Ala Phe Asn Thr Cys Thr Ala His Ile Cys Ala Ile Val Phe Ser Tyr Thr Pro Ala Phe Phe Ser Phe Phe Ser His Arg Phe Gly Glu His Ile Ile Pro Pro Ser Cys His Ile Ile Val Ala Asn Ile Tyr Leu Leu Leu Pro Pro Thr Met Asn Pro Ile Va1 Tyr Gly Val Lys Thr Lys Gln Ile Arg Asp Cys Val Ile Arg Ile Leu Ser Gly Ser Lys Asp Thr Lys Ser Tyr Ser Met <210> 15 <211> 422 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No; 7503710CD1 <400> 15 Met Gln Pro Val Met Leu Ala Leu Trp Ser Leu Leu Leu Leu Trp Gly Leu Ala Thr Pro Cys Gln Glu Leu Leu Glu Thr Val Gly Thr Leu Ala Arg Ile Asp Lys Asp Glu Leu Gly Lys Ala Ile Gln Asn Ser Leu Val Gly Glu Pro Ile Leu Gln Asn Val Leu Gly Ser Val Thr Ala Val Asn Arg G1y Leu Leu Gly Ser Gly Gly Leu Leu Gly Gly Gly Gly Leu Leu Gly His Gly Gly Val Phe Gly Val Val Glu Glu Leu Ser Gly Leu Lys Ile Glu Glu Leu Thr Leu Pro Lys Val Leu Leu Lys Leu Leu Pro Gly Phe Gly Val Gln Leu Ser Leu His Thr Lys Val Gly Met His Cys Ser G1y Pro Leu Gly Gly Leu Leu Gln Leu Ala Ala Glu Val Asn Val Thr Ser Arg Val Ala Leu Ala Val Ser Ser Arg G1y Thr Pro Ile Leu Tle Leu Lys Arg Cys Ser Thr Leu Leu Gly His Ile Ser Leu Phe Ser Gly Leu Leu Pro Thr Pro Leu Phe Gly Val Val Glu Gln Met Leu Phe Lys Val Leu Pro Gly Leu Leu Cys Pro Val Val Asp Ser Val Leu Gly Val Val Asn Glu Leu Leu Gly Ala Val Leu Gly Leu Val Ser Leu Gly Ala Leu Gly Ser Val Glu Phe Ser Leu Ala Thr Leu Pro Leu Ile Ser Asn Gln Tyr Ile Glu Leu Asp Ile Asn Pro Ile Val Lys Ser Va1 Ala Gly Asp Ile Ile Asp Phe Pro Lys Ser Arg Ala Pro Ala Lys Val Pro Pro Lys Lys Asp His Thr Ser Gln Val Met Val Pro Leu Tyr Leu Phe Asn Thr Thr Phe Gly Leu Leu Gln Thr Asn Gly Ala Leu Asp Met Asp Ile Thr Pro Glu Leu Val Pro Ser Asp VaI Pro Leu Thr Thr Thr Asp Leu Ala Ala Leu Leu Pro Glu Val Met Thr Val Arg Ala Gln Leu Ala Pro Ser Ala Thr Lys Leu His Ile Ser Leu Ser Leu Glu Arg Leu Ser Val Lys Val Ala Ser Ser Phe Thr His A1a Phe Asp Gly Ser Arg Leu Glu Glu Trp Leu Ser His Val Val Gly Ala Val Tyr Ala Pro Lys Leu Asn Val Ala Leu Asp Val Gly Ile Pro Leu Pro Lys Val Leu Asn Ile Asn Phe Ser Asn Ser Val Leu Glu I1e Val Glu Asn Ala Val Ala Ala Leu Tyr Val Leu Val Val Ala <210> 16 <211> 2192 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 71924779CB1 <400> 16 aggtggcgcg taccaccaga gaccgagcga gtcgccagct gcccctggcc tggcgggggc 60 ggaaccgcgc gggatcccca cccccacccg gaatcctcgc cacggagaat ccctggagaa 120 gccccggatc cccggctggg aggaggaagt gctcgttgac ccccagcccc gcgctgatcc 180 cgcccccggc ctgcggactt ggggagccgc tgtactctgc ctcggacgcc acgagactct 240 agacgggagt cccctcgagg tgaagccgct gagttcccgg gccccgccag gcttccctgg 300 gagagccgac ggaccccccc tcccagcaca cacaacttcc ctgcttttca ccgggactgg 360 cggagcggcc ggcggactta gacgcgggga cttcagggca gggggcgccc cctgcccggg 420 tcaccagtcg gggcgagggg acgtctcctc tcccccagct gctctgctcg gatggcgccg 480 ccggctgagt gacgggggcg gcgcgcagga cttcccagct cggacctctt gccttcgagg 540 ggaaagatgt acgagagtgt agaagtgggg ggtcccaccc ctaatccctt cctagtggtg 600 gatttttata accagaaccg ggcctgtttg ctcccagaga aggggctccc cgccccgggt 660 ccgtactcca ccccgctccg gactccgctt tggaatggct caaaccactc cattgagacc 720 cagagcagca gttctgaaga gatagtgccc agccctccct cgccaccccc tctaccccgc 780 atctacaagc cttgctttgt ctgtcaggac aagtcctcag gctaccacta tggggtcagc 840 gcctgtgagg gctgcaaggg cttcttccgc cgcagcatcc agaagaacat ggtgtacacg 900 tgtcaccggg acaagaactg catcatcaac aaggtgaccc ggaaccgctg ccagtactgc 960 cgactgcaga agtgctttga agtgggcatg tccaaggagt ctgtgagaaa cgaccgaaac 1020 aagaagaaga aggaggtgcc caagcccgag tgctctgaga gctacacgct gacgccggag 1080 gtgggggagc tcattgagaa ggtgcgcaaa gcgcaccagg aaaccttccc tgccctctgc 1140 cagctgggca aatacactac gaacaacagc tcagaacaac gtgtctctct ggacattgac 1200 ctctgggaca agttcagtga actctccacc aagtgcatca ttaagactgt ggagttcgcc 1260 aagcagctgc ccggcttcac caccctcacc atcgccgacc agatcaccct cctcaaggct 1320 gcctgcctgg acatcctgat cctgcggatc tgcacgcggt acacgcccga gcaggacacc 1380 atgaccttct cggacgggct gaccctgaac cggacccaga tgcacaacgc tggcttcggc 1440 cCCCtcaccg acctggtctt tgccttcgcc aaccagctgc tgcccctgga gatggatgat 1500 gcggagacgg ggctgctcag cgccatctgc ctcatctgcg gagaccgcca ggacctggag 1560 cagccggacc gggtggacat gctgcaggag ccgctgctgg aggcgctaaa ggtctacgtg 1620 cggaagcgga ggcccagccg cccccacatg ttccccaaga tgctaatgaa gattactgac 1680 ctgcgaagca tcagcgccaa gggggctgag cgggtgatca cgctgaagat ggagatcccg 1740 ggctccatgc cgcctctcat ccaggaaatg ttggagaact cagagggcct ggacactctg 1800 agcggacagc cggggggtgg ggggcgggac gggggtggcc tggccccccc gccaggcagc 1860 tgtagcccca gcctcagccc cagctccaac agaagcagcc cggccaccca ctccccgtga 1920 ccgcccacgc cacatggaca cagccctcgc cctccgcccc ggcttttctc tgctttctac 1980 cagacattgt gaccccgcac cagccctggc cccactgcct ccgggcagta ctggcgactt 2040 ccctggggac ggggagggag gagaagatct tggacagagg ctggcctcag ggatgcctgt 2100 cccagctggt gaataaagcg aggcgaagat gagccggccg gttcaaaggt cgggccgttc 2160 aaacctgccg ccatttaaga aggggcccaa as 2192 <210> 17 <211> 3614 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2319430CB1 <400> 17 cgttgagggc aacccagagg ccatgccccg ctcctcccct ctatcccgct ccggctgtta 60 agggctcggc cccgagcgcc tctgcccgcg gacgatggtg accgtacggg ccgggccact 120 gccgctgcct ccgcctcccc agagagccac atccgaggct cggcgcagaa gagccgccgc 180 tgtgaccgtg ccgtaccggc cccctgcctc cgcccgagga gaacgggagg gcgggcgaga 240 gagccgggga gttgcggagc ccgccccccg gcagcgccgc tcccccggga gggagtcccc 300 cagcctgagg tcttctccca gaaaaaaaaa aagaaaaaaa aaaacaacat ggctgcaaag 360 gagaaactgg aggcagtgtt aaatgtggcc ctgagggtgc caagcatcat gctgttggat 420 gtcctgtaca gatgggatgt cagctccttt ttccagcaga tccaaagaag tagccttagt 480 aataaccctc ttttccagta taagtatttg gctcttaata tgcattatgt aggttatatc 540 ttaagtgtgg tgctgctaac attgcccagg cagcatctgg ttcagcttta tctatatttt 600 ttgactgctc tgctcctcta tgctggacat caaatttcca gggactatgt tcggagtgaa 660 ctggagtttg cctatgaggg accaatgtat ttagaacctc tctctatgaa tcggtttacc 720 acagccttaa taggtcagtt ggtggtgtgt actttatgct cctgtgtcat gaaaacaaag 780 cagatttggc tgttttcagc tcacatgctt cctctgctag cacgactctg ccttgttcct 840 ttggagacaa ttgttatcat caataaattt gctatgattt ttactggatt ggaagttctc 900 tattttcttg ggtctaatct tttggtacct tataaccttg ctaaatctgc atacagagaa 960 ttggttcagg tagtggaggt atatggcctt ctcgccttgg gaatgtccct gtggaatcaa 1020 ctggtagtcc ctgttctttt catggttttc tggctcgtct tatttgctct tcagatttac 1080 tcctatttca gtactcgaga tcagcctgca tcacgtgaga ggcttctttt cctttttctg 1140 acaagtattg cggaatgctg cagcactcct tactctcttt tgggtttggt cttcacggtt 1200 tcttttgttg ccttgggtgt tctcacactc tgcaagtttt acttgcaggg ttatcgagct 1260 ttcatgaatg atcctgccat gaatcggggc atgacagaag gagtaacgct gttaatcctg 1320 gcagtgcaga ctgggctgat agaactgcag gttgttcatc gggcattctt gctcagtatt 1380 atccttttca ttgtcgtagc ttctatccta cagtctatgt tagaaattgc agatcctatt 1440 gttttggcac tgggagcatc tagagacaag agcttgtgga aacacttccg tgctgtaagc 1500 ctttgtttat ttttattggt attccctgct tatatggctt atatgatttg ccagtttttc 1560 cacatggatt tttggcttct tatcattatt tccagcagca ttcttacctc ~tcttcaggtt 1620 ctgggaacac tttttattta tgtcttattt atggttgagg aattcagaaa agagccagtg 1680 gaaaacatgg atgatgtcat ctactatgtg aatggcactt accgcctgct ggagtttctt 1740 gtggccctct gtgtggtggc ctatggcgtc tcagagacca tctttggaga atggacagtg 1800 atgggctcaa tgatcatctt cattcattcc tactataacg tgtggcttcg ggcccagctg 1860 gggtggaaga gctttcttct ccgcagggat gctgtgaata agattaaatc gttacccatt 1920 gctacgaaag agcagcttga gaaacacaat gatatttgtg ccatctgtta tcaggacatg 1980 aaatctgctg tgatcacgcc ttgcagtcat tttttccatg caggctgtct taagaaatgg 2040 ctgtatgtcc aggagacctg ccctctgtgc cactgccatc tgaaaaactc ctcccagett 2100 ccaggattag gaactgagcc agttctacag cctcatgctg gagctgagca aaacgtcatg 2160 tttcaggaag gtactgaacc cccaggccag gagcatactc cagggaccag gatacaggaa 2220 ggttccaggg acaataatga gtacattgcc agacgaccag ataaccagga aggggctttt 2280 gaccccaaag aatatcctca cagtgcgaaa gatgaagcac atcctgttga atcagcctag 2340 aggagaagca gcaggaatga tgctttgata ctctggagga gaagttaact caagatggaa 2400 ttcatgttct gatttgagga atgaaaatga gatgatcagg caggaaactg acattccaag 2460 gatctaatcc aggaagtact ctcagtgggg accacctgct ttcatcccct gacattgtgg 2520 gagaaatttt gcaatgtatg ctaatcaaaa tgtatttata tgttctctgc tgatgtttta 2580 tagaggtttg tgaagaaaat tcaacctcag caacttcaga aactgcccct gatacgtgtg 2640 agagagaaat aaaatcagat tttgagtgtt gaagggactg aggaagtgag gataaagagc 2700 atgaggacag catggaaaga aggaggcaga agtggaactg aactttcact ctccatggga 2760 cagatcaatc tcattatcaa gtctgaatag caaccagccc tctcctccac cccgtttctc 2820 ctcagttaat tggagctcag tcaggtgatt attgagtctt gtacagcact gaaatgaaat 2880 caaagatgaa gaagcattga ttgtattcaa agattgaagc acgctcatac tttgtatgtg 2940 ctttagggaa ggggtgggtg ggcacttggg ccttgcgggt gcattcatgt aatctgagac 3000 tcttgaactt tatgacggag tcttcaatat tttgatgtat atgaaacttt tgttaaatat 3060 gttgtatact tcgctggctg tgtgaagtaa actaaaactc tgatgaacac tttggagtct 3120 gctttagtga aggagaccaa agtgggaagg gctttagggc actgatagag gccctgggtg 3180 tacttttcaa tcctgtgtaa tgtttaattc ttgcaactga atcaaaacag tgttaaatta 3240 tggcaatatt tgcactttgg gaatgagtac ataactgtat gatcacactc tgcaaatgcc 3300 acttttaaag ctgttaatag actttgcacc ttttctttga caaggatgtg tcatatttaa 3360 atttttacat tcatcatggc tacaggtaga actggggagg ggggaatgta attttttatg 3420 ggaattttga tatgaaaaga aactagtcat ttatttatac aataggcttg gctcaaaaag 3480 tgtttttcag acctcggtat tcctaatgtg ggatgtgact ttattttatt tttagtagca 3540 aatttggatg tagactgaca gacatagctg aatgtcttaa taaatttaaa tttgaagata 3600 aaaaaaaaaa aaaa 3614 <210> 18 <211> 1585 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7291877C81 <400> 18 cgcgcaccaa caacagcaac aactccactg cgccgggctg aggagcagga attaggagct 60 cgcgaataat atgaaaggga tccgcaaagg ggaaagccga gcaaaggaat ccaaaccctg 120 ggagcctggc aagcgaagat gcgctaaatg tggccgccta gacttcatcc tgatgaagaa 180 aatggggatt aaaagtggat ttacgttttg gaacctcgtc tttttattga cggtgtcttg 240 tgtgaaagga tttatttata catgtggtgg aactttaaaa ggacttaatg gcactataga 300 aagccctggt tttccatatg gatatccaaa tggtgcaaac tgcacatggg taataatagc 360 agaagaacga aatagaatac aaattgtttt tcagtcattt gctctagaag aagaatacga 420 ctacttatca ttatatgatg gacatcctca tcctacaaac tttaggacaa ggttaacagg 480 attccatctg ccacctccag tgacaagtac caaatctgtg ttctcactac gtttgaccag 540 tgattttgca gttagtgctc atggatttaa ggtatattac gaagaattgc agagtagctc 600 ttgtggaaat cctggtgttc cacccaaagg tgtattatat ggcacaagat tcgacgtcgg 660 ggacaagatc cgctacagct gtgtaactgg atacatcctt gatggccacc ctcagctcac 720 ctgcatagcc aattcagtta atacagcttc gtgggatttt cctgttccta tctgtagagc 780 tgaagatgct tgtggaggaa caatgagagg atccagtggc atcatatcca gccctagttt 840 tcctaatgag taccataaca atgctgattg cacttggacc attgtagcag agcctgggga 900 cacaatttca ctcatattta ctgattttca aatggaagag aaatatgatt acttagaaat 960 agaaggttct gagccaccta ccatatggtt atctggaatg aatataccac caccaattat 1020 cagcaacaaa aactggctca gactgcattt tgttacagac agcaatcatc gataccgtgg 1080 atttagtgct ccctatcaag gttcttctac attgacccac actacctcca ctggtgagtt 1140 agaggagcat aacaggacta ccactggtgc tattgctgtt gctagcacac ctgcagatgt 1200 tactgtatcc agtgttacag ctgtcaccat ccatagactt tccgaggaac agcgagtgca 1260 agttacgagt ctcagaaatt caggtctgga ccccaacacg tccaaggacg ggctctctcc 1320 tcatccagca gatacacaaa gtaccaggag aagaccaaga catgctgaac agatagaaag 1380 aactaaagag cttgcagttg ttactcatag aggacattgc aatagagtcg aggacataga 1440 aaaacccatt ctagtggtac aagatagatt ttgtaaaatg aattctgatc aaagtactaa 1500 agaagttaca gtgtgtatgc agagagtgag tcttttaagt tactttttca atgagttggt 1560 aaacaaccga aaaccaattg cttaa 1585 <210> 19 <211> 5618 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1218126CB1 <400> 19 gatatgactg aggcgcccat gggggtggcg gggcggctgt aggagcaggg gcctagcaag 60 cgcccagcgg agcgacccct gcctggccgt ggctagcatg gcccctacgc tgttccagaa 120 gctcttcagc aagaggaccg ggctgggcgc gcccggccgc gacgcccggg acccagattg 180 cgggttcagt tggcctttac cagagtttga tccaagccag attcgactga ttgtatatca 240 agactgtgaa agacgaggga gaaatgtttt gtttgactcc agtgttaaga gaagaaatga 300 ggacatatca gtatcgaaac tctgcagtga tgctcaagtt aaagtctttg ggaaatgctg 360 ccaactgaaa cctggaggag acagttcttc ctctttagat agttctgtga cttcatcttc 420 tgatataaaa gaccagtgtc ttaagtacca gggttctcgg tgctcttctg atgccaatat 480 gcttggagag atgatgtttg gctcagtagc aatgagctac aaaggatcca ccttaaaaat 540 tcatcagatt cgttcccctc cacagctcat gctcagcaaa gtgtttactg ctcggactgg 600 cagcagtatt tgtgggagtc tcaatacgct acaagatagt cttgaattca tcaatcagga 660 caacaataca ttaaaggctg ataataacac agttattaat ggactgcttg gaaatatagc 720 atctctcagc agcttgctga tcactccatt tccttcccca aactcctcac ttacccgaag 780 ttgtgccagc agctaccagc gacgttggcg acgcagccaa acaacaagtt tggaaaatgg 840 ggtatttcct agatggtcta tagaagaaag ctttaatctc tcagatgaaa gctgtggccc 900 taacccagga attgtgcgga aaaagaagat tgcaattggg gtaatctttt cattgtccaa 960 agatgaagat gaaaataaca aatttaatga attctttttt tcacattttc ctctctttga 1020 aagctacatg aacaaattaa agagtgcaat agaacaggct atgaaaatga gccggagatc 1080 agctgatgcc agtcagagaa gtttggcata taatcgaata gttgatgccc taaatgaatt 1140 cagaacaaca atttgtaatc tttacacgat gccacgaatt ggagaacctg tctggcttac 1200 aatgatgtcg gggactccag aaaagaacca cctttgctat cgtttcatga aggagttcac 1260 ctttctaatg gaaaatgctt ccaaaaatca attcttgcca gctctcatta ctgcagttct 1320 gaccaatcat cttgcctggg ttccaacagt catgccaaat ggacaaccac ctataaaaat 1380 atttttagaa aagcattcct ctcagagtgt ggacatgttg gcaaagactc atccatataa 1440 cccactttgg gcacaactgg gagacttgta tggcgctatt ggctctcccg tacggttagc 1500 aaggactgtg gtagttggca aacgacaaga catggtccag aggctacttt attttcttac 1560 ttattttata agatgctctg aacttcaaga aa.cgcatctt ttagaaaatg gagaagatga 1620 agccatcgtt atgccaggca cagtaattac taccacttta gagaaaggtg aaatagaaga 1680 atcagagtat gtccttgtca caatgcatag aaacaaaagc agtttgctct ttaaagagtc 1740 agaagaaatt agaactccca attgtaactg taaatattgc agtcatccac tccttgggca 1800 aaatgtagag aacatttcac aacaagagag agaagatatt caaaacagct ctaaggagct 1860 gctaggaatt tcagatgagt gccggatgat ttctccttct gactgccaag aagaaaatgc 1920 tgttgatgtt aaacagtaca gagataaatt aagaacttgc tttgacgcca agttagagac 1980 agttgtttgc acaggatctg ttccagtaga caaatgtgca ttgtcagagt caggcttaga 2040 gtcaacagag gaaacatggc agagtgagaa gttgctggat tcagacagtc acacaggcaa 2100 agcaatgaga tccacaggaa tggttgtgga aaaaaaacct ccagataaga ttgtgcctgc 2160 ttcattttct tgtgaggctg cccagacaaa ggttactttc ctgattgggg attctatgtc 2220 acctgattca gatactgagc ttcgaagtca ggcagtggtg gatcagatta ccagacatca 2280 caccaaacca ttgaaggaag aaagaggggc tattgatcag catcaagaaa ctaaacaaac 2340 aaccaaggac caatctggag agtctgatac acagaacatg gtttctgaag agccctgtga 2400 acttccctgt tggaatcatt cagacccaga aagcatgagc ttattcgacg aatattttaa 2460 tgatgattca atcgaaacca ggactattga tgatgttcca tttaaaacaa gtacagatag 2520 taaagaccat tgctgtatgt tagagttttc aaaaatattg tgtacaaaaa ataacaagca 2580 gaacaatgaa ttttgtaaat gtatagaaac agttccccaa gattcatgta aaacctgctt 2640 tcctcagcag gaccaaagag atacactctc cattcttgtc ccccatgggg ataaagagag 2700 ttcagataaa aaaattgctg taggaactga atgggacatt ccaagaaatg aaagttcaga 2760 cagtgccctt ggggatagtg aaagtgaaga tacaggtcat gatatgacta gacaagttag 2820 cagttattat ggaggagagc aagaagattg ggcagaagag gatgagatac cttttcctgg 2880 gtcaaagtta atcgaagtga gtgctgttca gcccaacatt gccaacttcg ggaggtcctt 2940 gctgggtggc tactgctcat cttatgtgcc tgactttgtt cttcaaggaa ttgggagtga 3000 tgagaggttc cgtcagtgtc tgatgtcaga tttatctcat gctgtgcagc atccagtttt 3060 ggatgaacca atagcagaag ctgtctgtat tatagctgac atggataaat ggactgttca 3120 agtggccagt agccagagac gagtgacaga taataaattg ggaaaggaag tattggtttc 3180 cagtcttgtt tccaatctgc ttcattccac acttcagctt tataagcata acttgtctcc 3240 aaatttttgt gtaatgcatc ttgaagaccg gttgcaggag ctatacttca aaagtaaaat 3300 gctgtctgaa tacctgaggg ggcagatgcg tgttcatgtc aaggagctgg gagtggttct 3360 ggggattgaa tccagtgatc ttccacttct ggctgctgta gcaagcactc actctccata 3420 tgttgcacaa atactccttt aatataccta aaaattgtta gaaattggtg ggaaaatagg 3480 tagaaaccaa ggaagcagac acaacatgca tttatggaga ttctttttcc cttttagact 3540 tccatctgaa tgagtcagtc accagggtat tctgcatagc attgtatatt ctgtgtatgt 3600 cagatggctt tttctttttg actggacttt tgggtggtgg tagattttta aacaaatgaa 3660 attaaagcaa caataatttt gaagcatttg aaaaagccaa agtgtacggt agaaatttct 3720 acaaaatgaa tattatcaag agtttcatgt gatcactgca gtgttgtcac agctcataaa 3780 tagcaacagt gtttcatgat ttaatggctc agaaatagtt attcattagt ttttaatttt 3840 taatttctaa ggtacagaga tctataaaac cttgattatt tgttagtttt gcaattcaaa 3900 acagctaatg tctggttatt tctcaaagta agtattttaa acagcctgtt aattataaga 3960 aactcagaat aatgagtgta aatgtgttat gttatccacc caagtgtaca tatgtaccta 4020 ttttttttta aaaagcagaa atagaaatac aagactggta aacatgcctt taaaaatata 4080 tatattttca actagtattg tctataatgc tgaaatatta cttattggtg atttttctgt 4140 ttcacacact ctaaaatata agtaaagcca accttttttt taaggctgag attcccaaaa 4200 tgagaatact actttatacc atttgtttat aagtatgaac tgttcttata aatattaata 4260 tttacatatt cactaattta acataaatga aaattaggat taaaaattgc accaaagcat 4320 cggcaaaaac aatactatat tctttaaaag tgctcaggta gccaaggccc ttgcttttgg 4380 tatcaaccct catgaaccca taggagctga tatttgtttc actgcttaat aatcctcaat 4440 ttacactatt cataactctt aaaattattc tctttttttc taagagcccc tcccttccaa 4500 aagtgtattt ttttcaaaga ttttcacttc tcaattgttg cctttgtaca tactatagag 4560 tgttgcttgt aagaaaggct aatatggaac caaactcttg taagtaatgt aaatagaaag 4620 gtgggtggat aaagttttca atactttcta ctacctcagt ttacttgagt actacattat 4680 agtttattct ttgcttatct ggtctaagag acttttaatg ctagtagtaa agttggtttc 4740 tgctttcatt gactattttc atcataattt catcattgat taaaaaaaga aaaccacttg 4800 tttattcagt tattaaatat atttactata taacacatcc attcttgctg tttaaatttt 4860 caatagttaa tggaaagttg tctttgacct tgaatttaca gcattgggtc acattttgcc 4920 ttgctgtgta tgtattcaag agacttccaa ctagacaaag aaaaaattgt tgttttaatg 4980 gaatgtaaac ctgaaattgg,~gtgtctgca atctgtttgg cccatgacct tttacctagt 5040 cccagttatt acctgagtct cccatggatg acttgctgcc aaggagtgtt tgtggatata 5100 ttttctttgg cttaattttc ttattctgtg cattaacaaa attatccagt tgtctgattt 5160 tggaattcta tgagtcaatc tttttggcag aattcagaat attaaaaagt tcatacattt 5220 gcgggccatt gtaccttttt tttttttttt ttttttttga cggagtttca ctcttgttac 5280 ccaggctgga gttgcaatgg tgcgatctca gctcactgca acctccgcct cccagttcaa 5340 gtgattctcc gtgcctcagc ccccaagtta gctgggatta caatttgtgc gccaccacac 5400 ccagctaatt ttttattttt agtagagatg agatttcacc atgttttggt caggctggtc 5460 ttgaactcct gaactcaagt gatccaccgt gcctcgacct cccaaagtac tgggattaca 5520 ggcgtgagcc atgtgtgccc agccttgtac tttttttttt tttaatggta gctctgttta 5580 gcattggggc atatgtcggg gtgtctcttt aaccttaa 5618 <210> 20 <211> 1641 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7479161CB1 <400> 20 ccgacaggag gccgggagcc cccacctacc ccttgtggag ctgcaggagc aagggcatgc 60 agccagtcat gctggccctg tggtccctgc ttctgctctg gggcctggcg actccatgcc 120 aggagctgct agagacggtg ggcacgctcg ctcggattga caaggatgaa ctcggcaaag 180 ccatccagaa ctcactggtt ggggagccca ttctgcagaa tgtgctggga tcggtcacag 240 ctgtgaaccg gggcctcttg ggctcaggag ggctgcttgg aggaggcggc ttgctgggcc 300 acggaggggt ttttggcgtt gtcgaggagc tctctggtct gaagattgag gagctcacgc 360 tgccaaaggt gttgctgaag ctgctgccgg gatttggggt gcagctgagc ctgcacacca 420 aagtgggcat gcattgctct ggcccccttg gtggccttct gcagctggct gcggaggtga 480 acgtgacatc gcgggtggcg ctggccgtga gctcaagggg cacacccatc cttatcctca 540 agcgctgcag cacgctcctg ggccacatca gcctgttctc agggctgctg cccacaccac 600 tctttggggt cgtggaacag atgctcttca aggtgcttcc gggactgctg tgccccgtgg 660 tggacagtgt gctgggtgtg gtgaatgagc tcctgggggc tgtgctgggc ctggtgtccc 720 ttggggctct tgggtccgtg gaattctctc tggccacatt gcctctcatc tccaaccagt 780 acatagaact ggacatcaac cctatcgtga agagtgtagc tggtgatatc attgacttcc 840 ccaagtcccg tgccccagcc aaggtgcccc ccaagaagga ccacacatcc caggtgatgg 900 tgccactgta cctcttcaac accacgtttg gactcctgca gaccaacggc gccctcgaca 960 tggacatcac ccctgagctg gttcccagcg atgtcccact gacaactaca gacctggcag 1020 ctttgctccc tgaggccctg gggaagctgc ccctgcacca gcaactccta ctgttcctgc 1080 gggtgaggga agctcccacg gtcacactcc acaacaagaa ggccttggtc tccctcccag 1140 ccaacatcca tgtgctgttc tatgtcccta aggggacccc tgaatccctc tttgagctga 1200 actccgtcat gactgtgcgt gcccagctgg ctccctcggc taccaagctg cacatctccc 1260 tgtccctgga acggctcagt gtcaaggtgg cctcctcctt tacccatgcc tttgacggat 1320 cgcgtttaga agaatggctc agccatgtgg tcggggcagt gtatgcacca aagcttaacg 1380 tggccctgga tgttggaatt cccctgccta aggttcttaa tatcaatttt tccaattcag 1440 ttctggagat cgtagagaat gctgtggcag ctctctatgt ccttgtagta gcatagaaga 1500 tggtgttctt ctcagatcag tggactatgc catgttattt tgttcttgga ctaaggccct 1560 gtgaggtgca actggtccac tttcattttt ggtcagagat ggagaataag gaattatatg 2620 ttggtactag cactggaata g 1641 <210> 21 <211> 6056 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7722591CB1 <400> 21 gactgacctg tgaggactgc ctggccaact ctagccagtg cgcctggtgc cagtccaccc 60 acacctgctt cctgtttgct gcctacttgg cccggtaccc acacgggggc tgtcgaggct 120 gggacgacag gtatggtccc tggggcaggg ctaacagagg aagattcccc accggcaagg 180 ggctggggct ctgaccccca cccctgccat cctgcagtgt acactcggag ccacggtgcc 240 ggagctgcga tggcttcctg acctgccatg agtgtctgca gagccacgag tgtggctggt 300 gtggcaatga ggacaacccc acactgggac ggtgagcccg ggcaggtggg tgggcagggt 360 gcccggctgt gtccttcctc catgaccggt cattctaatg gcctctttgc ttctctgccc 420 tcttgcccat cccatggcca ccttcccttt tccgtactgt ttttctgttg tttattttac 480 CCtCCCgCtC ttttttCtCC atttttCCtC CttttCCggC tCtCtgCgat tcgttttctt 540 tctccttgtc tgtttctgtc tctccgctct ccctttcact gcatctctgt ctatgtctct 600 cttctgtctt ccaaaattgt ttttgtctgc gacttctcct ggtttctctg tctctctttc 660 caaatttgta tttgcatccc tctccttcca attggcctcc tctctccctg tcattgtttc 720 tatgtatggc tcctgtttct atgttgtCCC CCgCttCttC aCtCtCCCaC CCtgCaggtg 78O
cctacagggg gacttctcag ggcccctcgg tgggggtaac tgctccctgt ggtgtggggg 840 agggcctggg tgcttcccgt ggccctgccc tgcccgtctg ggcatacgcc cgctgtcctg 900 acgtggatga gtgtcgcctg ggcctggccc ggtgccaccc gcgggcgacc tgcctgaaca 960 cgcccctcag ctacgagtgt cactgccagc ggggctacca gggtgatggc atctcacact 1020 gcaaccgcac gtgcttggag gactgtggcc atggtgtgtg cagtggcccc ccggacttta 1080 cctgcgtgtg tgacctaggc tggacatcag acctgccccc tcccacacct gccccgggtc 1140 cgccagcccc ccgctgctcc cgggactgtg gctgcagctt ccacagccac tgccgcaagc 1200 ggggccctgg cttctgcgac gagtgccagg actggacatg gggggagcac tgcgaacgat 1260 gccggcccgg cagcttcggc aacgccacag gctctagggg ctgccggccc tgccagtgca 1320 acgggcacgg ggacccacgc cgtggccact gcgacaacct cagtgggctc tgcttctgcc 1380 aggaccacac cgagggtgcc cactgccagc tctgctcccc aggctattat ggggatccca 1440 gggccggtgg ttcctgcttt cgggagtgtg ggggtcgcgc cctcctcacc aacgtgtcct 1500 cagtggcact gggctcacgc cgggtcgggg ggctgctgcc tccaggtggc ggggctgcaa 1560 gagccgggcc tggcctgtcc tactgtgtgt gggttgtctc ggccactgag gagctacagc 1620 cctgtgctcc cgggaccctc tgtcccccac tcaccctcac cttctccccc gacagcagca 1680 CCCCCtgCaC gctgagctac gtcctggcgt ttgatggatt cccacgcttc ctggacactg 1740 gtgttgtcca gtcggaccgc agcctcatag ctgccttctg cggccagcga cgggacaggc 1800 ccctcactgt tcaggccctg tctgggctgc tcgtgctgca ctgggaggcc aatggctcct 1860 catcctgggg cttcaatgct tcggtgggct ctgcccgctg tgggtcaggg ggccccggga 1920 gctgtcccgt cccccaggaa tgcgtgcccc aggacggtgc tgcaggtgcg gggctctgcc 1980 gatgtcctca gggctgggct ggcccacact gccgcatggc tctgtgtcct gagaactgca 2040 atgcccacac tggggcagga acttgtaacc agagcctggg tgtgtgcatc tgtgccgagg 2100 gcttcggggg ccccgactgc gccaccaagc tggatggcgg gcagctggtc tgggagaccc 2160 tcatggacag ccgcctctca gccgacactg ccagccgctt cctgcaccgc ctgggccaca 2220 ccatggtgga tggacccgat gccaccttgt ggatgtttgg gggcctgggc ctgccccagg 2280 ggctgctggg aaacctgtac aggtactcag tgagtgagcg gcggtggaca cagatgctgg 2340 cgggagccga ggacgggggc ccaggcccat cgccccgctc cttccatgca gctgcatatg 2400 tgcccgctgg ccgtggtgcc atgtatctgc tggggggact taccgctgga ggcgtcaccc 2460 gtgatttctg ggtcctcaac ctcaccaccc tgcaatggcg gcaggagaag gccccccaga 2520 ccgtggagct gccagccgtt gctggtcaca cccttactgc ccgccgaggc ctgtctctgc 2580 tcctggtggg cggttactcc ccggaaaatg gcttcaacca gcagctgctg gagtaccagc 2640 tggcaaccgg cacctgggtg tcaggagccc agagtgggac accccccaca ggtctctatg 2700 gtcactctgc tgtctaccac gaggccaccg actccctcta cgtgtttggg gggttccgat 2760 tccatgtgga gctggcggcc CCatCCCCCg agctctactc cctgcactgt cctgaccgca 2820 cctggagtct gctggcccct tctcaggggg caaagccccg cccccggctt ttccacgcct 2880 cagccctgtt aggggacacc atggtggttc ttggggggcg ctcggaccct gacgagttca 2940 gcagcgacgt tctgctctac caggtcaact gcaatgcctg gcttctgccc gacctcaccc 3000 gctcggcctc tgtggggccc ccaatggagg agtctgtggc ccatgctgtg gcagcagtcg 3060 ggagccgcct gtatatctct gggggtttcg ggggagtggc cctgggccgc ctgctggcac 3120 tgaCCCtgCC CCCtgaCCCC tgccgcctgc tgtcctcacc tgaagcttgt aaccagtctg 3180 gggcctgcac ctggtgccat ggggcctgct tgtccgggga tcaggcccac aggctgggct 3240 gcgggggctc cccctgctcc ccaatgcctc gctccccgga ggaatgtcga cgtctccgga 3300 cctgcagtga gtgCCtggCC CgCCatCCtC ggaccctgca acctggagat ggagaggcgt 3360 ccaccccccg ctgtaagtgg tgtaccaact gccccgaagg tgcttgcatt ggacgcaatg 3420 ggtcctgcac ctctgagaat gactgtcgga tcaaccagcg agaggtcttc tgggcaggga 3480 actgctccga ggctgcgtgc ggggctgctg actgcgagca gtgcacgcgg gagggcaagt 3540 gcatgtggac gcggcagttc aagaggacag gggagacccg ccgcatcctc tccgtgcagc 3600 ccacctatga ctggacgtgc ttcagccact ctctgctgaa tgtgtccccc atgccggtgg 3660 aatcatcacc cccactgccc tgCCCCaCCC CttgtCaCCt cctacccaac tgtacctcct 3720 gcctggactc taagggagca gatgggggct ggcagcactg tgtttggagc agcagcctgc 3780 agcagtgtct gagcccttcc tacctgcccc tgcgatgtat ggccggaggc tgtgggcggc 3840 tgctccgggg acctgagagc tgctccctgg gctgtgctca ggcaactcag tgcgccttgt 3900 gcctgcggcg cccccattgc ggctggtgtg cctggggggg ccaggatggg ggtggccgct 3960 gcatggaggg tggactcagc ggcccccgtg atgggctgac atgtgggcgt ccgggggcct 4020 cctgggcctt cctgtcctgc ccccctgagg acgagtgtgc aaacgggcac cacgactgca 4080 acgagacgca gaattgccac gaccagcccc acggctatga gtgcagctgc aagaccggct 4140 ataccatgga caacatgaca gggctgtgcc gccctgtgtg cgcccagggc tgcgtgaacg 4200 gctcatgtgt ggagcccgac cactgccgct gccactttgg ctttgtgggc cgcaactgct 4260 ccacggaatg ccgctgcaac cgccacagtg aatgcgctgg tgttggggcg cgtgaccact 4320 gcttgctctg ccgcaaccac accaagggca gccactgtga gcagtgcctc ccgctgtttg 4380 tgggttcagc tgtcggaggc gggacctgcc ggccctgcca cgccttttgt cgtggaaata 4440 gccacatctg catctccagg aaggagttac aaatgtccaa gggagagcca aagaagtact 4500 cactggaccc agaggagatt gaaaactggg tgacagaggg tcctagtgaa gacgaggccg 4560 tgtgcgtgaa ctgccagaat aacagctatg gggagaaatg cgagagctgc ctgcagggct 4620 acttcctcct ggacgggaag tgcaccaaat gccagtgtaa tggccacgcg gacacatgta 4&80 acgagcagga tgggacgggc tgtccatgtc agaataacac agagacgggc acatgccagg 4740 gcagctcccc cagtgaccgt cgagactgct acaagtacca gtgcgccaag tgccgggaat 4800 catttcacgg gagtccgctg ggcggccagc agtgctaccg cctcatctcg gtggagcagg 4860 agtgctgcct ggaccccacg tcccagacca actgcttcca tgagcccaaa cgccgggcgc 4920 taggccccgg ccgcactgtc ctctttggcg tgcagcccaa attcaccaac gtggacatcc 4980 gcctgacgct ggacgtgacc ttcggggccg tggacctcta tgtctccacc tcctatgaca 5040 CCttCgtggt ccgtgtggcc cctgacactg gcgtccatac tgtacacatc cagccacccc 5100 cagccccacc acctccacca ccccctgcag atggtgggcc ccggggggct ggggatccag 5160 gaggagcagg ggccagcagt gggccgggcg ccccagcaga gccacgggta cgggaggtat 5220 ggccgcgggg cctgattacc tacgtgacgg tgacggagcc gtcggcagtg ctggtggtcc 5280 gcggcgtgcg ggaccggctg gtcatcacct acccacacga gcaccatgcc ctcaagtcga 5340 gccgcttcta cctgctgctg ctgggcgtgg gagacccaag~tgggcccggc gccaacggct 5400 cagccgactc gcagggcctg ctcttcttcc ggcaggacca ggcccacatt gacctgtttg 5460 tCttCttCtC CgtCttCttC tCCtgCttCt tCCtCttCCt Ct CdCtCtgt gtgctcctct 5520 ggaaggccaa gcaggctctg gaccagcggc aggagcagcg ccggcacttg caggagatga 5580 ccaagatggc cagccgcccc ttcgccaagg tcaccgtctg cttcccacct gaccctactg 5640 ccccggcctc cgcctggaag ccggctgggc tcccacctcc cgccttccgc cgctctgagc 5700 ccttcctggc acccctgctg ctgacagggg ccggtgggcc ctggggaccc atggg~gggg 5760 gctgctgccc accagccatc cccgccacca ctgctgggct gcgagctggg cccatcactc 5820 tcgagcccac agaagatggc atggctggcg tggccacact gctgctccag ctgcctggcg 5880 ggccccatgc acccaacggc gcctgcctgg ggtcagccct cgtcacactg cggcacaggc 5940 tgcacgagta ctgtgggggt ggtgggggtg ctgggggcag tgggcatggg actggtgcgg 6000 gccggaaggg actgttgagc caggacaacc tcaccagcat gtccctctga catgcc 6056 <210> 22 <211> 1699 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2173285CB1 <400> 22 ggggcgctgg gggtgacggt gcggagccgc tgccagcgct gggcgagagt cggcggccgg 60 atccgaggag caggcgggcc tgaggccgag tcagctgcgc gggcccccgg atcccccgac 120 agagcggcgg cggtgtctgg ccaggcggta ggcgctgcct ggccgcggcg gggaagatgt 180 tcagcgtaga gtcgctggag cgggcggagc tgtgcgagag cctcctcact tggatccaga 240 catttaatgt ggatgcacca tgccagaccg tggaagattt aacgaatggg gttgtgatgg 300 cccaggttct tcaaaagata gatcctgcat attttgatga aaattggcta aacagaatca 360 aaactgaagt aggagataat tggaggctaa agataagcaa tttaaagaaa attttaaaag 420 gaatcttgga ttataatcat gagattttag gacagcaaat taatgacttt acccttcctg 480 atgtgaacct tattggggag cattctgatg cagcagagct tggaaggatg cttcagctca 540 tcttaggctg tgctgtgaac tgtgaacaga agcaagagta catccaagcc attatgatga 600 tggaggaatc tgttcaacat gttgtcatga cagccattca agagctgatg agtaaagaat 660 CtCCtgtCtC tgctggaaat gatgcctatg ttgaccttga tcgtcagctg aagaaaacta 720 cagaggaact aaatgaagct ttgtcagcaa aggaagaaat tgctcaaaga tgccatgaac 780 tggatatgca ggttgcagca ttgcaggaag agaaaagtag tttgttggca gagaatcagg 840 tattaatgga aagactcaat caatctgatt ctatagaaga ccctaacagt ccagcaggaa 900 gaaggcattt gcagctccag actcaattag aacagctcca agaagaaaca ttcagactag 960 aagcagccaa agatgattat cgaatacgtt gtgaagagtt agaaaaggag atctctgaac 1020 ttcggcaaca gaatgatgaa ctgaccactt tggcagatga agctcagtct ctgaaagatg 1080 agatcgacgt gctgagacat tcttctgata aagtatctaa actagaaggt caagtagaat 1140 cttataaaaa gaagctagaa gaccttggtg atttaaggcg gcaggttaaa ctcttagaag 1200 agaagaatac catgtatatg cagaatactg tcagtctaga ggaagagtta agaaaggcca 2260 acgcagcgcg aagtcaactt gaaacctaca agagacaggt aaaagaaaca cagcatcttg 1320 atgatggttt caggcaagct ctcagttatg acatgtagct taccaaaatt actaatttgt 1380 tttcatggta ttctgttttt taccttttct ttattgtatt gattcattta ggagactgag 1440 tctcactctg tcacccagcc tggagtgcag tggcatgatc tcagctcact gcaacctcca 1500 cctcccaggt tcaagctatt CttCtgCCCC agCCtCCtga gtagctggaa ctacagacgc 1560 atgctgccac acctggctaa ttttttgtat tttggtaaag acagggtttc actgtgttgc 1620 ccaggctggt cttgtactcc tgagctcaag tgatccacca gcctcagcct tccaaagtgc 1680 taggattaca agcgtgagc 1699 <210> 23 <211> 1661 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7487619CB1 <400> 23 ctatttggtc cagccttatc gccccggact cgtaaccttc ggccatgccg tattataggc 60 tcccccatcc agtagtacag acattgcaat taactggcag agtcttttcc ctgattgaga 120 agtaaccctg tcatgtcatg cgaaggccag tgaaggagga ttttaggacc ataggtgacc 180 tctgtaaaat gagattgata tgtcctgcca aaaccagcaa aagacagaga cctctgccca 240 aaactgcaag aatgggatac tgccaacacc tgcaatagac ttggaagagg actgtggtcc 300 tcccatgaga tttcagcttc tggggacaca ttgattgaga cctgttgaga ctctcagcag 360 agggtccagc tgacctggcc cacagagagg gtgagataaa aattttacac gttttatggc 420 actaagtttg tgttattttt ttaatatatc aatggaaatc taatatagtg tttctctact 480 ttcttctgca tgtgtgtctc tgtgtgtgtg cacctgtgtg catgtgtgtg agagaggctg 540 aaataatttc atcatcatct ctgtgaggga agctttgtaa caagcgaagt gcaggataac 600 tccagaatta tctacctggt tgatgcagtt tccacataga gaatggattc tcatttctca 660 attaagtgct aaatgctggg tgctctttat atccccagag ggagagagac caagggtgag 720 aagaaatgtc caacgccagc ctcgtgacag cgttcatcct cacgggcctt ccccatgccc 780 cagggctgga cgcccccctc tttggaatct tcctggtggt ttacgtgctc actgtgctgg 840 ggaacctcct catcctgctg gtgatcaggg tggattctca cctccacacc cccatgtact 900 acttcctcac caacctgtcc ttcattgaca tgtggttctc cactgtcacg gtgcccaaaa 960 tgctgatgac ettggtgtcc ccaagcggca gggetatctc cttccacagc tgcgtggctc 2020 agctctattt tttccacttc ctggggagca ccgagtgttt cctctacaca gtcatggcct 1080 acgatcgcta cctggccatc agttacccgc tcaggtacac cagcatgatg actgggcgct 1140 cgtgtactct tctggccacc agcacttggc tcagtggctc tctgcactct gctgtccagg 1200 ccatattgac tttccatttg ccctactgtg gacccaactg gatccagcac tatttgtgtg 1260 atgcaccgcc catcctgaaa ctggcctgtg cagacacctc agccatagag actgtcattt 1320 ttgtgactgt tggaatagtg gcctcgggct gctttgtcct gatagtgctg tcctatgtgt 1380 CCatCgtCtg ttCCatCCtg cggatccgca cctcagaggg gaagcacaga gcctttcaga 1440 cctgtgcctc ccactgtatc gtggtccttt gcttctttgg ccctggtctt ttcatttacc 1500 tgaggccagg ctccaggaaa gctgtggatg gagttgtggc cgttttctac actgtgctga 1560 cgccccttct caaccctgtt gtgtacaccc tgaggaacaa ggaggtgaag aaagctctgt 1620 tgaagctgaa agacaaagta gcacattctc agagcaaata g 1661 <210> 24 <211> 2033 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7487607CB1 <400> 24 caatttccgg acctcggtgg tctgcccgcc tcgccctccc aaagtactgg gatttacagg 60 tcacctgccc tgctgggtag tatagaaaag gacatgattc aggtggggat gtcgaggata 120 caaagatgaa ggaagaagaa tggctgtctt tctttctatt cagaaaagat cccagaattc 180 tacatttatt cagcagaaac tatgccatgt atactatgtg agaggaagat cagcagaaca 240 gctgttcgag cagaggaagc tgacttctct gcaggaagaa ggcaatatgt attttctcat 300 acttagagat aatataccct gtgtcatcat ggcttccaaa cagtaatccc ttcttcagaa 360 attcatcagc ttcacagaaa attttgttga acagcctaaa aacagaagat caaggcaaaa 420 caatctgctg tgtattgcaa cctaagaagt gagctgacct tccatttaga ggttaaatag 480 agagtaaaat ggaatgggaa aaccacacca ttctggtgga attttttctg aagggacttt 540 ctggtcaccc aagacttgag ttactctttt ttgtgctcat cttcataatg tatgtggtca 600 tccttctggg gaatggtact ctcattttaa tcagcatctt ggaccctcac ettcacaccc 660 ctatgtactt ctttctgggg aacctctcct tcttggacat ctgctacacc accacctcta 720 ttCCCtCCaC gctagtgagc ttcctttcag aaagaaagac catttccctt tctggctgtg 780 cagtgcagat gttcctcggc ttggccatgg ggacaacaga gtgtgtgctt ctgggcatga 840 tggcctatga cegctatgtg gctatctgca accctctgag atatcccatc atcatgagta 900 aggatgccta tgtacccatg gcagctgggt cctggatcat aggagctgtc aattctgcag 960 tacaatcagt gtttgtggta caattgcctt tctgcaggaa taacatcatc aatcatttca 1020 cctgtgaaat tctggctgtc atgaaactgg cctgtgctga catctcagac aatgagttca 1080 tcatgcttgt ggccacaaca ttgttcatat tgacaccttt gttattaatc attgtctctt 1140 acacgttaat cattgtgagc atcttcaaaa ttagctcttc cgaggggaga agcaaagctt 1200 cctctacctg ttcagcccat ctgactgtgg tcataatatt ctatgggacc atcctcttca 1260 tgtacatgaa gcccaagtct aaagagacac ttaattcgga tgacttggat gctaccgaca 1320 aaattatatc catgttctat ggggtgatga ctcccatgat gaatccttta atctacagtc 1380 ttagaaacaa ggatgtgaaa gaggcagtaa aacacctact gaacagaagg ttctttagca 1440 agtgagtgca aaatgtactg gaatatgaac acacttgata ttgttgaaac ttcagaatta 1500 tgttagaatt ttgggtactt ttactatttt tatgcatttt catatattat gttaaaataa 1560 tgagatacag catttcaaaa ttattgcatg tccactctag agaatttgca agatacaggg 1620 cagtaggatg aagaagaaag aggggttacc tattactcta ctagtgggaa atggccccgt 1680 ttcaacattt tgaacagtaa ctttcatatt atgggttttt ttttctgcat tggaattggg 1740 tgtgatgtgc ctttttatgt tcactttttt ccataatgtt atttcatagg caacatttca 1800 tagaatcttt caaaataaat aaagccctct gttgtagaaa aagcaaaaca gaaaaacccc 1860 aacatagtgt actcacattt tccagggaca agcctgtgtt atagtttcac attaatctcc 1920 agatcctgtt aaagccacta aataaccagt ttctttttct gtatttaaat tttggtgtcg 1980 ggtgtccagc ctccaggttt ctcgggacca tcccaaaggg gcgggaataa atg 2033 <210> 25 <211> 1659 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7487616CB1 <400> 25 ctattcaccc tgagcaaata ctgacccatt tttcagcttc aacagagctt tctttacctc 60 cttgtttctc agggtgtaca caacagggtt gaaaagagga gtcagcgtgg tgtagaaaac 120 ggccacaacc ccatgcaagg cgtccctgga gcctggcctc aggtaaatga aaagaccagg 180 gccaaagaag caaaggacca cgatacagtg ggaggcacag gtctgaaagg ctctgtgcct 240 cccctctgag gtgcggatcc gcaggatgga acagacgatg gacacatagg acagcactat 300 caggacaaag cagcccgagg ccactagccc aatattcaca aagatgacca tctcgttggc 360 tgaggtgtct gcacaggcca gtttcaggat gggcggtgcg tcacagaagt agtgctggat 420 ctggttgggt ccacagtagg gcaaatggaa agtcaatatg gtctggacag cagagtgcag 480 agagccactg agccaagtgc cggtggccag gagggcacac gagcgcccag tcatcatgtt 540 ggtgtacctg agcgggtaac tgatggccag gtagcgatca taggacatga ctgtgtagag 600 gaaacactcg gtgctcccca ggaagtggaa aaaatagagc tgagccacgc agctgtggaa 660 ggagatagtc ctgccgcttg gggacaccaa ggtcatcagc attttgggca ccgtgacagt 720 ggagaaccac atgtcaatga aggacaggtt ggtgaggaag tagtacatgg gggtgtggag 780 gtgagaatcc accctgatca ccagcaggat gaggaggttc cccagcacag tgagcacgta 840 aaccaccagg aagattccaa agaggggggc gtccagccct ggggcatggg gaaggcccgt 900 gaggatgaac gctgtcacga ggctggcgtt ggacatttct tctcaccctt ggtctctctc 960 cctctgggga tataaagagc acccagcatt tagcacttaa ttgagaaatg agaatccatt 1020 ctctatgtgg aaactgcatc aaccaggtag ataattctgg agttatcctg cacttcgctt 1080 gttacaaagc ttccctcaca gagatgatga tgaaattatt tcagcctctc tcacacacat 1140 gcacacaggt gcacacacac agagacacac atgcagaaga aagtagagaa acactatatt 1200 agatttccat tgatatatta aaaaaataac acaaacttag tgccataaaa cgtgtaaaat 1260 ttttatctca ccctctctgt gggccaggtc agctggaccc tctgctgaga gtctcaacag 1320 gtctcaatca atgtgtcccc agaagctgaa atctcatggg aggaccacag tcctcttcca 1380 agtctattgc aggtgttggc agtatcccat tcttgcagtt ttgggcagag gtctctgttt 1440 tgctggtttt ggcaggacat atcaatctca ttttacagag gtcacctatg gtcctaaaat 1500 cctccttcac tggccttcgc atgacatgac agggttactt ctcaatcagg gaaaagactc 1560 tgccagttaa ttgcaatgtc tgtactactg gatgggggag cctataatac ggcatggccg 1620 aaggttacga gtccggggcg ataaggctgg accaaatag 1659 <210> 26 <211> 1175 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7483204CB1 <400> 26 cccttttagg gttttttttt tttaatacag taataatgcc tctttgaaag tgacactcac 60 ctgggatact ttttgagggt aaagaagata atttacataa accaatcttg ttctacttta 120 cagataattt tttttttgat atcttggaaa gtcaagttct agcctgtcat tctcgtaatg 180 atttctgtag cagtttgaaa caagaacaag gaagaatgga ctgggaaaat tgctcctcat 240 taactgattt ttttctcttg ggaattacca ataacccaga gatgaaagtg accctatttg 300 ctgtattctt ggctgtttat atcattaatt tctcagcaaa tcttggaatg atagttttaa 360 tcagaatgga ttaccaactt cacacaccaa tgtatttctt cctcagtcat ctgtctttct 420 gtgatctctg ctattctact gcaactgggc ccaagatgct ggtagatcta cttgccaaga 480 acaagtcaat acccttctat ggctgtgctc tgcaattctt ggtcttctgt atctttgcag 540 attctgagtg tctactgctg tcagtgatgg cctttgatcg gtacaaggcc atcatcaacc 600 ccctgctcta tacagtcaac atgtctagca gagtgtgcta tctactcttg actggggttt 660 atctggtggg aatagcagat gctttgatac atatgacact ggccttccgc ctatgcttct 720 gtgggtctaa tgagattaat catttcttct gtgatatccc tcctctctta ttactctctt 780 gctcagatac acaggtcaat gagttagtgt tattcaccgt ctttggtttt attgaactga 840 gtaccatttc aggagttttc atttcttatt gttatatcat cctatcagtc ttggagatac 900 actctgctga ggggaggttc aaagctctct ctacatgcac ttcccactta tctgcggttg 960 caattttcca gggaactctg ctctttatgt atttccggcc aagttcttcc tattctctag 1020 atcaagataa aatgacctca ttgttttaca cccttgtggt tcccatgttg aaccccctga 1080 tttatagcct gaggaacaag gatgtgaaag aggccctgaa aaaactgaaa aatgaaattt 1140 tattttaagg aaatagtaaa aatacatgtt tatac 1175 <210> 27 <211> 1737 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472099CB1 <400> 27 tcagagttga gctgggggag gcttcagagg ctctgcatgc ccaggaggct tctctgagac 60 tgggaaggga gtggaaactc agtagcccgt ggtcctggtt gcgcgctccc tcccatgtct 120 tcattatctg atttggtaaa atatctacaa gacgggggag tccgggctgc tttctttgac 180 actggtcatc actccacacc tagacgctat gtccatgtct ggaagatgct tttaaggaag 240 ttttgatgca gagaagagtg gccggtgctc ttCCtCgCgt gCCaCCatCt gCttaCttCC 300 agaagctaat tattttcatg tgactgatgt gactaatatt ttttaacgcc tgcaggttcg 360 ttatgtacca gcaaatgttc atggtactga agatatacca gttgaagaga aagaatgtct 420 ggttgtcata gtacctattt gctacgaggt caaatcttgt ccctggaaga aatgtacaaa 480 tattaatact taaaacagta ttttccatta agaagagaat tttattctga taaggtgaag 540 gagcctatga agatcaacaa ggagaatttc caagagtcat gtcagcctcc agtatcacct 600 caacacatcc aacttccttc ttgttgatgg ggattccagg cctggagcac ctgcacatct 660 ggatctccat ccccttctca gcatatacac tggccctgct tggaaactgc accctccttc 720 tcatcatcca ggctgatgca gccctccatg agcccatata cctctttetg gccatgttgg 780 cagccatcga cctggtcctt tcctcctcag cattgcccaa aatgcttgcc atattctggt 840 tcagggatcg ggagatcaac ttttttgcct gtctggtcca gatgttcttc cttcactcct 900 tctccatcat ggagtcagca gtgctgctgg ccatggcctt tgaccgctat gtggccatct 960 gcaagccact gcactacacc acggtcctga ctgggtccct catcaccaag attggcatgg 1020 ctgctgtggc ccgggctgtg acactaatga ctccactccc cttcctgctg agatgtttcc 1080 actactgccg aggcccagtg attgcccgct gctactgtga acacatggct gtggtcaggc 1140 tggctgtggg aacactaggc ttcaacaata tctatggcat tgctgtggcc atgtttattg 1200 gagtgttgga tctattcttt atcatcctat cttatatctt tatccttcag gcagttctac 1260 aactctcctc tcaggaggcc cgctacaaag catttgggac atgtgtctct cacataggtg 1320 ccatcttagc cttctacaca ccttcagtca tctcttcagt catgcaccgt gtggcccgct 2380 gtgctgtgcc acacgtccac attctcctcg ccaatttcta tCtgCtCttC ccacccatgg 1440 tcaatcccat catctacggc gttaagacca agcagatccg tgacagtctt gggagtattc 1500 ccgagaaagg atgtgtgaat agagagtgag gaataagtgg aaaaagagtg gggcacagtg 1560 aatgctgtag tgggccaggg ctgtgctgag agtagatggg tgctagactc cacgtttagt 1620 tcttttcttg tattatgaaa agaataaatg atgtcctgaa gctcagtgcc acagtctgtt 1680 aagaattgtg ggtctttgcc ctcggtacct ctggattgaa ctggtgactg tgcggtc 1737 <210> 28 <211> 972 .
<212> DNA
<213> Homo Sapiens <220>

<221> misc_feature <223> Incyte ID No: 7485443CB1 <400> 28 taaatagaga gtaaaatgga atgggaaaac cacaccattc tggtggaatt ttttctgaag 60 ggactttctg gtcacccaag acttgagtta ctcttttttg tgctcatctt cataatgtat 120 gtggtcatcc ttctggggaa tggtactctc attttaatca gcatcttgga ccctcacctt 180 cacaceccga tgtacttctt tctggggaac ctetccttct tggacatctg ctacaccacc 240 acctctattc cctccacact agtgagcttc ctttcagaaa gaaagaccat ttccttttct 300 ggctgtgcag tgcagatgtt ccttggcttg gccatgggga caacagagtg tgtgcttctg 360 ggcatgatgg cctttgaccg ctatgtggct atctgcaacc ctctgagata tcccatcatc 420 atgagcaaga atgcctatgt acccatggct gttgggtcct ggtttgcagg gattgtcaac 480 tctgcagtac aaactacatt tgtagtacaa ttgcctttct gcaggaagaa tgtcatcaat 540 catttctcat gtgaaattct agctgtcatg aagttggcct gtgctgacat ctcaggcaat 600 gagttcctca tgcttgtggc cacaatattg ttcacattga tgccactgct cttgatagtt 660 atctcttact cattaatcat ttccagcatc ctcaagattc actcctctga ggggagaagc 720 aaagctttct ctacctgctc agcccatctg actgtggtca taatattcta tgggaccatc 780 ctcttcatgt atatgaagcc caagtctaaa gagacactta attcagatga cttggatgct 840 accgacaaaa ttatatccat gttctatggg gtgatgactc ccatgatgaa tcctttaatc 900 tacagtctta gaaacaagga tgtgaaagag gcagtaaaac acctaccgaa cagaaggttc 960 tttagcaagt ga 972 <210> 29 <211> 1592 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3090414CB1 <400> 29 tagactacag gcccagagac tggaaacttt tccacgctag gtcccagctt gagctgtgtc 60 ataaccaagt tctctaagat tcaactataa aaacttacta aggtatggag aagaagaaaa 120 cattaatctg cagaaagccc agacaaattt tgagctattt cataacctac cagacttatc 180 atgctaacac tgaataaaac agacctaata ccagcttcat ttattctgaa tggagtccca 240 ggactggaag acacacaact ctggatttcc ttcccattct gctctatgta tgttgtggct 300 atggtaggga attgtggact cctctacctc attcactatg aggatgccct gcacaaaccc 360 atgtactact tcttggccat gctttccttt actgaccttg ttatgtgctc tagtacaatc 420 cctaaagccc tctgcatctt ctggtttcat ctcaaggaca ttggatttga tgaatgcctt 480 gtccagatgt tcttcatcca caccttcaca gggatggagt ctggggtgct tatgcttatg 540 gccctggatc gctatgtggc catctgctac cccttacgct attcaactat cctcaccaat 600 cctgtaattg caaaggttgg gactgccacc ttcctgagag gggtattact cattattccc 660 tttactttcc tcaccaagcg cctgccctac tgcagaggca atatacttcc ccatacctac 720 tgtgaccaca tgtatgtagc caaattgtcc tgtggtaatg tcaaggtcaa tgccatctat 780 ggtctgatgg ttgccctcct gatttggggc tttgacatac tgtgtatcac catctcctat 840 accatgattc tccgggcagt ggtcagcctc tcctcagcag atgctcggca gaaggccttt 900 aatacctgca ctgcccacat ttgtgccatt gttttctcct atactccagc tttcttctcc 960 ttcttttccc accgctttgg ggaacacata atcccccctt cttgccacat cattgtagcc 1020 aatatttatc tgctcctacc acccactatg aaccctattg tctatggggt gaaaaccaaa 1080 cagatacgag actgtgtcat aaggatcctt tcaggttcta aggataccaa atcctacagc 1140 atgtgaatga acacttgcca ggagtgagaa gagaaggaaa gaattacttc tatttgcctc 1200 ttatgcagga gttcataaaa tctttctgga agtactgtat tgatcacaaa atggagtttg 1260 ttgactggtg cattctcaat aagtaccttg ggaatctcaa catcattgga aggcccacca 1320 acatttctat aaatttttta ccttctcact catgtgaagg accagtctaa taattaaacc 1380 atattttatt cgacaaaaaa aaaaaaaaaa aaaaaacggg ggggggcccg caactatgac 1440 gcccgcaacc ccggaatata ctccggcacg ggaaacaaca gggcgtaatt ctcgcacaaa 1500 ttttggcccc taaatggggc ccccgcgtgg gcgtccacct tgtactccca tcttgtgggg 1560 cgcccacggc ggggaaacct ccggccagga tg 1592 <210> 30 <211> 1480 <212> DNA

<213> Homo Sapiens ~220>
<221> misc_feature <223> Incyte ID No: 7503710CB1 <400> 30 ggtggaggaa ccgacaggag gccgggagcc cccacctacc ccttgtggag ctgcaggagc 60 aagggcatgc agccagtcat gctggccctg tggtccctgc ttctgctctg gggcctggcg 120 actccatgcc aggagctgct agagacggtg ggcacgctcg ctcggattga caaggatgaa 180 ctcggcaaag ccatccagaa ctcactggtt ggggagccca ttctgcagaa tgtgctggga 240 tcggtcacag ctgtgaaccg gggcctcttg ggctcaggag ggctgcttgg aggaggcggc 300 ttgctgggcc acggaggggt ttttggcgtt gtcgaggagc tctctggtct gaagattgag 360 gagctcacgc tgccaaaggt gttgctgaag ctgctgccgg gatttggggt gcagctgagc 420 ctgcacacca aagtgggcat gcattgctct ggcccccttg gtggccttct gcagctggct 480 gcggaggtga acgtgacatc gcgggtggcg ctggccgtga gctcaagggg cacacccatc 540 cttatcctca agcgctgcag cacgctcctg ggccacatca gcctgttctc agggctgctg 600 cccacaccac tctttggggt cgtggaacag atgctcttca aggtgcttcc gggactgctg 660 tgccccgtgg tggacagtgt gctgggtgtg gtgaatgagc tcctgggggc tgtgctgggc 720 ctggtgtccc ttggggctct tgggtccgtg gaattctctc tggccacatt gcctctcatc 780 tccaaccagt acatagaact ggacatcaac cctatcgtga agagtgtagc tggtgatatc 840 attgacttcc ccaagtcccg tgccccagcc aaggtgcccc ccaagaagga ccacacatcc 900 caggtgatgg tgccactgta cctcttcaac accacgtttg gactcctgca gaccaacggc 960 gccctcgaca tggacatcac ccctgagctg gttcccagcg atgtcccact gacaactaca 1020 gacctggcag ctttgctccc tgaggtcatg actgtgcgtg cccagctggc tccctcggct 1080 accaagctgc acatctccct gtccctggaa cggctcagtg tcaaggtggc ctcctccttt 1140 acccatgcct ttgacggatc gcgtttagaa gaatggctca gccatgtggt cggggcagtg 1200 tatgcaccaa agcttaacgt ggccctggat gttggaattc ccctgcctaa ggttcttaat 1260 atcaattttt ccaattcagt tctggagatc gtagagaatg ctgtggcagc tctctatgtc 1320 cttgtagtag catagaagat ggtgttcttc tcagatcagt ggactatgcc atgttatttt 1380 gttcttggac taaggccctg tgaggtgcaa ctggtccact ttcatttttg gtcagagatg 1440 gagaataagg aattatatgt tggtactagc actggaatag ' 1480

Claims (85)

What is claimed is:

1. 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:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90%
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:1-14, c) a polypeptide comprising a naturally occurring amino acid sequence at least 98%
identical to the amino acid sequence of SEQ ID NO:15, d) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, and e) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.

3. An isolated polynucleotide encoding a polypeptide of claim 1.

4. An isolated polynucleotide encoding a polypeptide of claim 2.

5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:16-30.

6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3.

7. A cell transformed with a recombinant polynucleotide of claim 6.

8. A transgenic organism comprising a recombinant polynucleotide of claim 6.

9. A method of producing a polypeptide of claim 1, the method comprising:
a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.

10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15.

11. An isolated antibody which specifically binds to a polypeptide of claim 1.

12. An isolated polynucleotide selected from the group consisting of:
a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:16-30, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:16-30, c) a polynucleotide complementary to a polynucleotide of a), d) a polynucleotide complementary to a polynucleotide of b), and e) an RNA equivalent of a)-d).

13. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 12.

14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:
a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.

15. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides.

16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:
a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.

17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.

18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.

19. A method for treating a disease or condition associated with decreased expression of functional REMAP, comprising administering to a patient in need of such treatment the composition of claim 17.

20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.

21. A composition comprising an agonist compound identified by a method of claim 20 and a pharmaceutically acceptable excipient.

22. A method for treating a disease or condition associated with decreased expression of functional REMAP, comprising administering to a patient in need of such treatment a composition of claim 21.

23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.

24. A composition comprising an antagonist compound identified by a method of claim 23 and a pharmaceutically acceptable excipient.

25. A method for treating a disease or condition associated with overexpression of functional REMAP, comprising administering to a patient in need of such treatment a composition of claim 24.

26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1.

27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, the method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1.

28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising:
a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.

29. A method of assessing toxicity of a test compound, the method comprising:
a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.

30. A diagnostic test for a condition or disease associated with the expression of REMAP in a biological sample, the method comprising:
a) combining the biological sample with an antibody of claim 11, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex, and b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.

31. The antibody of claim 11, wherein the antibody is:
a) a chimeric antibody, b) a single chain antibody, c) a Fab fragment, d) a F(ab')2 fragment, or e) a humanized antibody.

32. A composition comprising an antibody of claim 11 and an acceptable excipient.

33. A method of diagnosing a condition or disease associated with the expression of REMAP
in a subject, comprising administering to said subject an effective amount of the composition of claim 32.

34. A composition of claim 32, wherein the antibody is labeled.

35. A method of diagnosing a condition or disease associated with the expression of REMAP
in a subject, comprising administering to said subject an effective amount of the composition of claim 34.

36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 11, the method comprising:
a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from said animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which binds specifically to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.

37. A polyclonal antibody produced by a method of claim 36.

38. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier.

39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising:
a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.

40. A monoclonal antibody produced by a method of claim 39.

41. A composition comprising the monoclonal antibody of claim 40 and a suitable carrier.

42. The antibody of claim 11, wherein the antibody is produced by screening a Fab expression library.

43. The antibody of claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library.

44. A method of detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15 in a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15 from a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:1-15.

46. A microarray wherein at least one element of the microarray is a polynucleotide of claim 13.

47. A method of generating an expression profile of a sample which contains polynucleotides, the method comprising:
a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.

48. An array comprising different nucleotide molecules affixed in distinct physical locations on a solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, and wherein said target polynucleotide is a polynucleotide of claim 12.

49. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.

50. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide.

51. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to said target polynucleotide.

52. An array of claim 48, which is a microarray.

53. An array of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule comprising said first oligonucleotide or polynucleotide sequence.

54. An array of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.

55. An array of claim 48, wherein each distinct physical location on the substrate contains multiple nucleotide molecules, and the multiple nucleotide molecules at any single distinct physical location have the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another distinct physical location on the substrate.

56. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:1.

57. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:2.

58. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:3.

59. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:4.

60. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:5.

61. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:6.

62. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:7.

63. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:8.

64. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:9.

65. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:10.

66. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:11.

67. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:12.

68. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:13.

69. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:14.

70. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:15.

71. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:16.

72. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:17.

73. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:18.

74. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:19.

75. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:20.

76. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:21.

77. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:22.

78. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:23.

79. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:24.

80. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:25.

81. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:26.

82. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:27.

83. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:28.

84. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:29.

85. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:30.