CA2459323A1 - G-protein coupled receptors - Google Patents

Abstract

Various embodiments of the invention provide human G-protein coupled receptors (GCREC) and polynucleotides which identify and encode GCREC. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of GCREC.

Description

G-PROTEIN COL~LED RECEPTORS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of G-protein coupled receptors and to the use of these sequences in the diagnosis, treatment, and preventioh of cell proliferative, neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory, and metabolic disorders, and viral infections, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of G-protein coupled receptors. The present invention further relates to the use of specific G-protein coupled receptors to identify molecules that are involved in to modulating taste or olfactory sensation.
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. 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 antiparallel 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) C~rr. 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-urotein Linked Receptor Facts Book, Academic Press, San Diego CA, pp. 2-6;
Bolander, F.F. (1994) Molecular Endocrinoloay, Academic Press, San Diego CA, pp. 162-176;
Baldwin, J.M. (1994) C~rr. 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, CSa anaphylatoxin, endothelia, follicle-stimulating hormone (FSIT), gonadotropic-releasing hormone (GnRl-~, neurokinin, thyrotropin-releasing hormone (TRIG, and oxytocin). GPCRs which act as receptors for stimuli that have yet to be identified are known as orphan receptors.
The diversity of the GPCR family is further increased by alternative splicing.
Many GPCR
2o genes contain introns, and there are currently over 30 such receptors for which spfice variants have been identified. The largest number of variations are at the protein C-terminus. N-terminal and cytoplasmic loop variants are also frequent, while variants in the extracellular loops or transmembrane domains are less common. Some receptors have more than one site at which variance can occur.
The splice variants appear to be functionally distinct, based upon observed differences in distribution, signaling, coupling, regulation, and ligand binding profiles (Kilpatrick, G.J.
et al. (1999) Trends Pharmacol. Sci. 20:294-301).
GPCRs can be divided into three major subfamilies: the rhodopsin-like, secretin-like, and metabotropic glutamate receptor subfamilies. Members of these GPCR subfamilies share similar functions and the characteristic seven transmembrane structure, but have divergent amino acid sequences. The largest family consists of the rhodopsin-like GPCRs, which transmit diverse extracellular signals including hormones, neurotransmitters, and light.
Rhodopsin is a photosensitive GPCR found in animal retinas. In vertebrates, rhodopsin molecules are embedded in membranous stacks found in photoreceptor (rod) cells. 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 are 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 galanin receptors mediate the activity of the neuroendocrine peptide galanin, which inhibits secretion of insulin, acetylcholine, serotonin and noradrenaline, and stimulates prolactin and growth hormone release. Galanin receptors are involved in feeding disorders, pain, depression, and Alzheimer's disease (Kask, K. et al. (1997) Life Sci. 60:1523-1533). Other nervous system rhodopsin-like GPCRs include a growing family of receptors for lysophosphatidic acid and other lysophospholipids, which appear to have roles in development and neuropathology (Chum J. et al.
(1999) Cell Biochem. Biophys. 30:213-242).
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 (Raming, K. et al. (1998) Receptors Channels 6:141-151).
However, the expression of olfactory-like receptors is not confined to olfactory tissues. For example, three rat genes encoding olfactory-like receptors having typical GPCR
characteristics showed expression patterns not only in taste and olfactory tissue, but also in male reproductive tissue (Thomas, M.B. et al. (1996) Gene 178:1-5).
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 (Emrl) and CD97 receptor proteins. 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. 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).
The third GPCR subfamily is the metabotropic glutamate receptor family.
Glutamate is the major excitatory neurotransmitter in the central nervous system. The metabotropic glutamate receptors modulate the activity of intracellular effectors, and are involved in long-term potentiation (Watson, supra, p.130). The Caz+-sensing receptor, which senses changes in the extracellular concentration of calcium ions, has a large extracellular domain including clusters of acidic amino acids which may be involved in calcium binding. The metabotropic glutamate receptor family also includes pheromone receptors, the GABAB receptors, and the taste receptors.
Other subfamilies of GPCRs include two groups of chemoreceptor genes found in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae, which are distantly related to the mammalian olfactory receptor genes. The yeast pheromone receptors STE2 and STE3, involved in the response to mating factors on the cell membrane, have their own seven-transmembrane signature, as do the cAMP receptors from the slime mold Dictyostelium discoideum, which are thought to regulate the aggregation of individual cells and control the expression of numerous developmentally-regulated genes.
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 hyperfunctioniug 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 V2 (X-linked nephrogenic diabetes); glucagon (diabetes and hypertension); calcium (hyperparathyroidism, hypocalcuria, hypercalcemia); parathyroid hormone (short limbed dwarfism); (33-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 Pharmacol. 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 et al., supra; Stadel et al., 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 SHT1D antagonists are used against migraine; and histamine H1 antagonists are used against allergic and anaphylactic reactions, hay fever, itching, and motion sickness (Horn et al., supra).
Recent research suggests potential future therapeutic uses for GPCRs in the treatment of metabolic disorders including diabetes, obesity, and osteoporosis. For example, mutant V2 vasopressin receptors causing nephrogenic diabetes could be functionally rescued in vitro by co-expression of a C-terminal V2 receptor peptide spanning the region containing the mutations.
This result suggests a possible novel strategy for disease treatment (Schoneberg, T. et al. (1996) EMBO J. 15:1283-1291).
Mutations in melanocortin-4 receptor (MC4R) are implicated in human weight regulation and obesity.
As with the vasopressin V2 receptor mutants, these MC4R mutants are defective in trafficking to the plasma membrane (Ho, G. and R.G. MacKenzie (1999) J. Biol. Chem. 274:35816-35822), and thus might be treated with a similar strategy. The type 1 receptor for parathyroid hormone (PTH) is a GPCR that mediates the PTH-dependent regulation of calcium homeostasis in the bloodstream. Study of PTH/receptor interactions may enable the development of novel PTH receptor ligands for the treatment of osteoporosis (Mannstadt, M. et al. (1999) Am. J. Physiol.
277:F665-F675).
The chemokine receptor group of GPCRs have potential therapeutic utility in inflammation and infectious disease. (For review, see Locati, M. and P.M. Murphy (1999) Annu.
Rev. Med. 50:425-440.) Chemokines are small polypeptides that act as intracellular signals in the regulation of leukocyte trafficking, hematopoiesis, and angiogenesis. Targeted disruption of various chemokine receptors in mice indicates that these receptors play roles in pathologic inflammation and in autoimmune disorders such as multiple sclerosis. Chemokine receptors are also exploited by infectious agents, including herpesviruses and the human immunodeficiency virus (HIV-1) to facilitate infection. A truncated version of chemokine receptor CCRS, which acts as a coreceptor for infection of T-cells by HIV-1, results in resistance to AIDS, suggesting that CCRS antagonists could be useful in preventing the development of AIDS.
The involvement of some GPCRs in taste and olfactory sensation has been reported.
Complete or partial sequences of numerous human and other eukaryotic sensory receptors are currently known. (See, e.g., Pilpel, Y. and D. Lancet (1999) Protein Sci.
8:969-977; Mombaerts, P.
(1999) Annu. Rev. Neurosci. 22:487-509. See also, e.g., patents EP 867508A2;
US 5,874,243; WO
92/17585; WO 95/18140; WO 97/17444; and WO 99/67282.) It has been reported that the human genome contains approximately one thousand genes that encode a diverse repertoire of olfactory receptors (Rouquier, S. et al. (1998) Nat. Genet. 18:243-250; Trask, B.J. et al. (1998) Hum. Mol.
Genet.7:2007-2020).
Anatomy and Ph s~gy of the Olfactory BWb The olfactory bulb is an oval anterior outgrowth of brain tissue from the base of the brain ending in a bulbous enlargement which lies over the cribriform plate separating the brain cavity from the upper reaches of the nasal cavity. Its under surface receives the olfactory nerves which pass upward through the cribriform plate from the olfactory region of the nose. The receptor cells for the smell sensation, or olfactory cells, are actually bipolar nerve cells which lie in the olfactory membrane of the nose and connect with globular structures in the olfactory bulb called glomeruli. Each glomerulus is the terminus for about 25,000 axons from olfactory cells, for dendrites from about 25 large mitral cells, and for about 60 smaller tufted cells that send axons through the olfactory tract into the central nervous system. Research suggests that different glomeruli respond to different odors.
Many nerve fibers originating in the olfactory portions of the brain pass backward in the olfactory tract to the olfactory bulb, terminating on a large number of small granule cells located in the center of the bulb. These send short inhibitory dendrites to the mitral and tufted cells.
This inhibitory feedback appears to aid in distinguishing one odor from another. The mitral and tufted cells are continually active, providing a background activity on which is superimposed impulse traffic caused by different odors. Thus, olfactory stimuli modulate the frequency of impulses in the olfactory system leading to transmittal of olfactory information.
The olfactory tract enters the brain at the junction between the mesencephalon and cerebrum.
There the tract divides between two pathways, one to the medial olfactory area, where septal nuclei feed into the hypothalamus and other portions of the limbic system. The other pathway leads to the lateral olfactory area, composed mainly of the prepyriform and pyriform cortex and the cortical portion of the amygdaloid nuclei, where signals pass into the limbic system, especially into the hippocampus.
A third observed olfactory pathway passes through the thalamus, into the dorsomedial thalamic nucleus and then to the lateroposterior quadrant of the orbitofrontal cortex.
Thus, there appears to be an olfactory system that subserves the basic olfactory reflexes, a second that provides automatic but learned control of food intake and aversion to foods, and a third that is comparable to most of the other cortical sensory systems and is used for conscious perception of olfaction.
Some odorants are G-protein coupled receptors, possessing a characteristic seven transmembrane structure. The N-terminus of these seven transmembrane G-protein coupled receptors is extracellular, and is often glycosylated, while the C-terminus is cytoplasmic and is often phosphorylated. Three extracellular loops alternate with three intracellular loops, together forming the seven transmembrane, hydrophobic regions. Sensory transduction in chemoreceptors occurs when l0 odorants activate receptor molecules in olfactory cells, triggering an enzymatic cascade mediated by the G-protein GOLF (G-protein olf). This is similar to the mechanism for sensory transduction in photoreceptors, in which activation by rhodopsin via light triggers an enzymatic cascade mediated by the G-protein transducin (Keio J. (2001) J. Med. 50:13-19).
Anatomy and the Ph s~gy of the Taste System In mammals, there are five primary tastes: salty, sour, bitter, sweet, and umami (the taste of monosodium glutamate). These tastes are believed to be mediated by distinct cell surface receptors on taste receptor cells (TRCs) clustered in taste buds. TRCs are specialized neuroepithelial cells, electrically excitable, and form synapses with afferent gustatory nerve fibers. Taste buds are focal collections of approximately 100 TRCs, clustered within onion-shaped structures. The taste buds of the tongue are found within three types of papillae: fungiform, foliate and vallate, found, respectively, at the front, sides and rear of the tongue. Taste buds are also found in the soft palate, uvula, epiglottis, pharnyx, larnyx, and esophagus.
Taste transduction begins when sapid molecules interact with the receptors and ion channels in the apical microvilli of TRCs that are exposed to the oral cavity. This interaction leads to a change in the membrane conductance, depolarization, and transmitter release onto gustatory afferent neurons.
Taste stimuli vary widely in chemical structure, ranging in size from ions to complex carbohydrates and proteins. Thus, a diversity of mechanisms are required for taste transduction. Ionic stimuli, such as salts and acids (for salty and sour tastes), interact directly with ion channels to depolarize TRCs.
Complex stimuli, such as carbohydrates, alkaloids, and proteins (for sweet, bitter, and umami tastes) activate G-protein coupled receptors (GCRECs) which regulate second-messenger cascades. For example, T1R3, a member of the T1R family of GCRECs is presumed to function as a sweet taste receptor in humans; hT2R4, a member of the T2RlTRB family of GCRECs have been shown to respond to bitter compounds (Gilbertson, T.A. (2000) C~rr. Opinion in Neurobiology 10:519-527).

Expression Information Breast cancer is the most frequently diagnosed type of cancer in American women and the second most frequent cause of cancer death. The lifetime risk of an American woman developing breast cancer is 1 in 8, and one-third of women diagnosed with breast cancer die of the disease. A
number of risk factors have been identified, including hormonal and genetic factors. One genetic defect associated with breast cancer results in a loss of heterozygosity (LOH) at multiple loci such as p53, Rb, BRCA1, and BRCA2. Another genetic defect is gene amplification involving genes such as c-myc and c-erbB2 (Her2-neu gene). Steroid and growth factor pathways are also altered in breast cancer, notably the estrogen, progesterone, and epidermal growth factor (EGF) pathways. Breast cancer evolves through a multi-step process whereby premalignant mammary epithelial cells undergo a relatively defined sequence of events leading to tumor formation. An early event in tumor development is ductal hyperplasia. Cells undergoing rapid neoplastic growth gradually progress to invasive carcinoma and become metastatic to the lung, bone, and potentially other organs. Variables that may influence the process of tumor progression and malignant transformation include genetic factors, environmental factors, growth factors, and hormones.
Prostate cancer develops through a multistage progression ultimately resulting in an aggressive tumor phenotype. The initial step in tumor progression involves the hyperproliferation of normal luminal and/or basal epithelial cells. Androgen responsive cells become hyperplastic and evolve into early-stage tumors. Although early-stage tumors are often androgen sensitive and respond to androgen ablation, a population of androgen independent cells evolve from the hyperplastic population.
These cells represent a more advanced form of prostate tumor that may become invasive and potentially become metastatic to the bone, brain, or lung. A variety of genes may be differentially expressed during tumor progression. For example, loss of heterozygosity (LOH) is frequently observed on chromosome 8p in prostate cancer. Fluorescence in situ hybridization (FISH) revealed a deletion for at least 1 locus on 8p in 29 (69%) tumors, with a significantly higher frequency of the deletion on 8p21.2-p21.1 in advanced prostate cancer than in localized prostate cancer, implying that deletions on 8p22-p21.3 play an important role in tumor differentiation, while 8p21.2-p21.1 deletion plays a role in progression of prostate cancer (Oba, K. et al. (2001) Cancer Genet. Cytogenet. 124:
20-26).
3o Dendritic cells (DCs) are antigen presenting cells that play a crucial role in the initiation of the immune response. DCs can be derived in vitro either from CD34+ bone marrow precursors (IDCs) or from peripheral blood monocytic cells (mDCs). In vivo, DCs reside in two distinct compartments:
the peripheral tissues such as lung, skin, kidney, heart, and intestine; and in secondary lymphoid organs such as lymph node, spleen, and Peyer's patches. In the periphery, DCs are efficient antigen processing cells but are limited in their capacity to activate naive T cells.
Upon activation (injury, inflammation, infection), DCs enter their final stage of maturation during which they downregulate the capacity to process new antigens, migrate out of the periphery into the secondary lymphoid organs, and acquire an extremely potent capacity to activate naive T cells. Although it has been shown that several factors, such as cross-linking the CD40 surface molecules or the presence of TNF-a , can induce this final stage of maturation, little is known about the molecular events that take place during this process.
CD40 is a type I integral membrane glycoprotein belonging to the TNF-receptor family. It is expressed on all mature B lymphocytes, dendritic cells, and some epithelial cells. Antibodies specific for CD40 molecules can induce proliferation of B cells when presented with interleukin-4 (IL-4) or antibodies specific for CD20 molecules. Also, stimulation of B cells with anti-CD40 antibodies and IL-4 can induce the switch of immunoglobulin production to the IgE isotype.
There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of cell proliferative, neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory, and metabolic disorders, and viral infections.
SUMMARY OF THE INVENTION
Various embodiments of the invention provide purified polypeptides, G-protein coupled receptors, referred to collectively as 'GCREC' and individually as 'GCREC-1,' 'GCREC-2,' 'GCREC-3,' 'GCREC-4,' 'GCREC-5,' 'GCREC-6,' 'GCREC-7,' 'GCREC-8,' 'GCREC-9,' 'GCREC-10,' 'GCREC-11,' 'GCREC-12,' 'GCREC-13,' 'GCREC-14,' 'GCREC-15,' 'GCREC-16,' 'GCREC-17,' 'GCREC-18,' 'GCREC-19,' 'GCREC-20,' 'GCREC-21,' and 'GCREC-22' and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utilizing the purified G-protein coupled receptors and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified G-protein coupled receptors and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
The invention additionally provides G-protein coupled receptors that are involved in olfactory and/or taste sensation. The invention further provides polynucleotide sequences that encode said G-protein coupled receptors.
An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ D7 NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID
NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-22. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ m NO:1-22.
Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ )17 NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ B7 NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D NO:1-22. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ )D N0:1-22. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID N0:23-44.
Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ B7 NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ >D NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ D7 NO:l-22.
Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.
Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ B7 NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ Il7 NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 117 NO:1-22, and d) an immunogenic fragment of to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22.
The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ll~ N0:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90%
identical to an amino acid l0 sequence selected from the group consisting of SEQ D7 NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 117 N0:1-22.
Still yet another embodiment provides an isolated polynucleotide selected from the group i5 consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ~ N0:23-44, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ )D N0:23-44, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA
20 equivalent of a)-d). In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44, b) a polynucleotide 25 comprising a naturally occurring polynucleotide sequence at least 90%
identical or at least about 90%
identical to a polynucleotide sequence selected from the group consisting of SEQ >D N0:23-44, 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 30 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. In a related embodiment, the method can include detecting the amount of ii the hybridization complex. In still other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:23-44, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ >D
N0:23-44, 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. In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.
Another embodiment 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 >D NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ >D NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 117 NO:l-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 117 N0:1-22, and a pharmaceutically acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ )D N0:1-22. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional GCREC, comprising administering to a patient in need of such treatment the composition.
Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ )D NO:l-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
)D NO:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-22. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional GCREC, comprising administering to a patient in need of such treatment the composition.
Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ >17 N0:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90%
to identical to an amino acid sequence selected from the group consisting of SEQ 1D NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ Il7 N0:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 117 NO:l-22. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional GCREC, comprising administering to a patient in need of such treatment the composition.
Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ )D N0:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ >D N0:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
)D NO:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 117 N0:1-22. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ~ NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
>D NO:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ >D N0:1-22. 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 l0 polypeptide.
The invention further provides methods of using G-protein coupled receptors of the invention involved in olfactory and/or taste sensation, biologically active fragments thereof (including those having receptor activity), and amino acid sequences having at least 90%
sequence identity therewith, to identify compounds that agonize or antagonize the foregoing receptor polypeptides. These compounds are useful for modulating, blocking and/or mimicking specific tastes and/or odors.
The present invention also relates to the use of olfactory and/or taste receptors of the invention, biologically active fragments thereof (including those having receptor activity), and polypeptides having at least 90% sequence identity therewith, in combination with one or more other olfactory and/or taste receptor polypeptides, to identify a compound or plurality of compounds that modulate, mimic, and/or block a specific olfactory and/or taste sensation.
The invention also relates to cells that express an olfactory or taste receptor polypeptide of the invention, a biologically active fragment thereof (including those having receptor activity), or a polypeptide having at least 90% sequence identity therewith, and the use of such cells in cell-based screens to identify molecules that modulate, mimic, and/or block specific olfactory or taste sensations.
Still further, the invention relates to a cell that co-expresses at least one olfactory or taste G-protein coupled receptor polypeptide of the invention, and a G-protein, and optionally one or more other olfactory and/or taste G-protein coupled receptor polypeptides, and the use of such a cell in screens to identify molecules that modulate, mimic, and/or block specific olfactory and/or taste sensations.
3o Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ m N0:23-44, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
LD N0:23-44, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ >D
N0:23-44, 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 N0:23-44, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ D7 N0:23-44, 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 can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME
database homologs, for polypeptide embodiments of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
Table 5 shows representative cDNA libraries for polynucleotide embodiments.
Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleic acids, and methods are described, it is understood that embodiments of the invention are not limited to the particular machines, instruments, 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 invention.
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 2o 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 various embodiments of 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.
3o DEFINITIONS
"GCREC" refers to the amino acid sequences of substantially purified GCREC
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.

The term "agonist" refers to a molecule which intensifies or mimics the biological activity of GCREC. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of GCREC either by directly interacting with GCREC or by acting on components of the biological pathway in which GCREC
participates.
An "allelic variant" is an alternative form of the gene encoding GCREC.
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 GCREC include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as GCREC or a polypeptide with at least one functional characteristic of GCREC. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding GCREC, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding GCREC. 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 GCREC.
Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of GCREC 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" can refer to an oligopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurnng protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of GCREC. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of GCREC either by directly interacting with GCREC or by acting on components of the biological pathway in which GCREC 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 GCREC 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 Garner protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
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 may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-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 1s cross-linker (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 "spiegeliner" refers to an aptamer which includes L-DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a polynucleotide having a specific nucleic acid sequence.
Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates;
oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation "negative" or "minus" can refer to the antisense strand, and the designation "positive" or "plus" can refer to the sense strand of a reference DNA molecule.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic GCREC, 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, S'-AGT-3' pairs with its complement, 3'-TCA-5'.
A "composition comprising a given polynucleotide" and a "composition comprising a given polypeptide" can refer to any composition containing the given polynucleotide or polypeptide. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotides encoding GCREC or fragments of GCREC 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 GELVIEW fragment assembly system (Accelrys, Burlington MA) 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 Conservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His 2o 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 hefical 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 imrnunological 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 GCREC or a polynucleotide encoding C~CREC
which can be identical in sequence to, but shorter in length than, the parent sequence.
A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from about 5 to about 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. 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 3o encompassed by the present embodiments.
A fragment of SEQ D7 N0:23-44 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ ll~ N0:23-44, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:23-44 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID
N0:23-44 from related polynucleotides. The precise length of a fragment of SEQ ID N0:23-44 and the region of SEQ ID
N0:23-44 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 ID NO:1-22 is encoded by a fragment of SEQ ID N0:23-44. A
fragment of SEQ 117 NO:1-22 can comprise a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-22. For example, a fragment of SEQ ID NO:1-22 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-22.
to The precise length of a fragment of SEQ ID N0:1-22 and the region of SEQ ID
NO:1-22 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
A "full length" polynucleotide is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A "full length"
polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of identical 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 one or more computer algorithms or programs known in the art or described herein. For example, percent identity can 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 3o 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.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms which can be used 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.govBLAST/. 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/bi2.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 l0 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. 50 Expect: 10 Word Size: 11 Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ D7 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 S0, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity' and "% identity," as applied to polypeptide sequences, refer to the percentage of identical residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions.
Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. The phrases "percent similarity" and "% similarity," as applied to polypeptide sequences, refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aligned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=l, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table.
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:
2o Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off.' SO
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 S0, 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 p.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 carned out. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (Tm) 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. and D.W.
Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY, ch. 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°lo 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 acids 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 present in solution and another nucleic acid immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of GCREC
which is capable of eliciting an immune response when introduced into a living organism, for example, a 2o mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of GCREC 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, antibodies, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of GCREC. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of GCREC.
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 which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
"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 GCREC 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 GCREC.
"Probe" refers to nucleic acids encoding GCREC, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acids. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. "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, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 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, for example, Sambrook, J. and D.W. Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY), Ausubel, F.M. et al. (1999; Short Protocols in Molecular Biolo~y, 4''' ed., John Wiley & Sons, New York NY), and 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/MfT
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 nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and Russell (supra). The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. 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 (UTRs). Regulatory elements interact with host or viral proteins which control transcription, l0 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.
15 An "RNA equivalent," in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that 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 GCREC, 20 nucleic acids encoding GCREC, 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 25 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.
30 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 about 60%
free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated.
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. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
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 organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. In another embodiment, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Luis, C. et al. (2002) Science 295:868-872). The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and Russell (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 polynucleotides that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other. A
polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity or sequence similarity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 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 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity or sequence similarity over a certain defined length of one of the polypeptides.
THE INVENTION
Various embodiments of the invention include new human G-protein coupled receptors (GCREC), the polynucleotides encoding GCREC, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory, and metabolic disorders, and viral infections.

Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ll~). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ
ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide B7) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID
NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME
database.
Columns l and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ll~) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank )D
NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID
NO:) of the nearest PROTEOME database homologs. Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s). Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID 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 (Accelrys, Burlington MA). Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are G-protein coupled receptors. For example, SEQ
)D N0:2 is 85% identical, from residue I67 to residue P282, to human olfactory receptor (GenBank )D g2921716) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.) The BLAST probability score is S.Oe-96, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ >17 N0:2 also contains a seven transmembrane receptor (rhodopsin family) domain as determined by searching for statistically significant matches in the hidden Markov model (I~V1NI)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOT1FS, and PROFILESCAN analyses provide further corroborative evidence that SEQ )D N0:2 is an olfactory receptor.
In another example, SEQ ID N0:4 is 48% identical, from residue E2 to residue L330, to mouse olfactory receptor P2 (GenBank 117 87638409) as determined by BLAST. The BLAST
probability score is 4.9e-75. SEQ ID N0:4 also contains a 7 transmembrane receptor (rhodopsin family) domain as determined by searching for statistically significant matches in the hidden Markov model (I~VIM)-based PFAM database. Data from BLI1VIPS, MOTIFS, and PROFILESCAN
analyses provide further corroborative evidence that SEQ ID N0:4 is an olfactory receptor.
In yet another example, SEQ D7 NO:10 is 60% identical, from residue D814 to residue F1004, to human seven transmembrane-domain receptor (GenBank ID 82117161) as determined by BLAST.
The BLAST probability score is 1.6e-70. SEQ ID NO:10 is localized to the plasma membrane, is a receptor involved in G-protein signaling, and is homologous to a member of the secretin family of G-protein coupled receptors with a Latrophilin/CL-1 like GPS domain (PROTEOME

610534~DKFZP564), is homologous to a human G-protein coupled receptor expressed in epithelial cells of the epididymis (PROTEOME ID 342492~GPR64), and is homologous to a human G-protein coupled receptor differentially expressed during metastatic progression of melanomas (PROTEOME >D
342484~GPR56), as determined by BLAST analysis using the PROTEOME database.
SEQ ID
2o NO:10 also contains a 7 transmembrane receptor (secretin family) domain and a CUB domain as determined by searching for statistically significant matches in the hidden Markov model ()-based PFAM database. Data from BL>IVVIPS, BLAST PRODOM, and BLAST DOMO
analyses provide further corroborative evidence that SEQ ll~ N0:10 is a G-protein coupled receptor.
In a further example, SEQ ID N0:16 is 80% identical, from residue M14 to residue D318, to Mus musculus odorant receptor K4h11 (GenBank 117 811692563) as determined by BLAST. The BLAST probability score is 1.4e-127. SEQ )D N0:16 is localized to the plasma membrane, and is a signaling protein, as determined by BLAST analysis using the PROTEOME
database. SEQ D7 N0:16 also contains a 7-transmembrane receptor (rhodopsin family) domain as determined by searching for statistically significant matches in the hidden Markov model (HIVIM)-based PFAM
3o database. Data from BL)IVVIPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ )D N0:16 is an olfactory protein receptor.
In another example, SEQ ID N0:18 is 47% identical, from residue M1 to residue C306, to human breast cancer amplified G-protein coupled receptor 3 (BCA-GPCR-3) (GenBank ID
g15986319) as determined by BLAST. The BLAST probability score is 2.5e-73. SEQ
ID N0:18 is localized to the plasma membrane and is a G-protein coupled receptor, as determined by BLAST
analysis using the PROTEOME database. SEQ ID NO:18 also contains a seven transmembrane receptor (rhodopsin family) domain as determined by searching for statistically significant matches in the hidden Markov model (HIVIM)-based PFAM database. Data from BLIMPS, MOTIFS, and PROF1LESCAN analyses and BLAST analyses of the PRODOM and DOMO databases provide further corroborative evidence that SEQ ID N0:18 is a G-protein coupled receptor.
In yet another example, SEQ ID N0:19 is 98% identical, from residue M1 to residue V281, and 100% from residue A279 to residue T388, to human neurotensin receptor 2 (GenBank ID
g3901028) as determined by BLAST. The BLAST probability score is 3.0e-206. SEQ
ID N0:19 also has homology to proteins that are localized to the plasma membrane and are neurotensin-binding proteins, as determined by BLAST analysis using the PROTEOME database. SEQ ID
N0:19 also contains a 7 transmembrane receptor (rhodopsin family) domain as determined by searching for statistically significant matches in the hidden Markov model (HIVIM)-based PFAM database. Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:19 is a rhodopsin family receptor. SEQ >D N0:1, SEQ ll7 N0:3, SEQ ID
NO:S-9, SEQ
ID N0:11-15, SEQ ID N0:17, and SEQ 117 N0:20-22 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID N0:1-22 are described in Table 7.
As shown in Table 4, the full length polynucleotide embodiments were assembled using cDNA
sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Column 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 embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID N0:23-44 or that distinguish between SEQ ll~ N0:23-44 and related polynucleotides.
The polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA
libraries. Alternatively, the polynucleotide fragments described iu column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides. 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_~:XX~~X N, NZ YYYYY N3 Na represents a "stitched" sequence in which ~:~~~IXXX 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 N1,2,3...~ 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 FZ.~~~KXXX_gAAAAA~BBBBB_1 N is a "stretched" sequence, with 1~'~~KXXX 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") maybe 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 andlor examples of programs GNN, GFG,Exon prediction from genomic sequences using, ENST for example, GENSCAN (Stanford University, CA, USA) or FGENES
(Computer Genomics Group, The Sanger Centre, Cambridge, UK) 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 polynucleotides 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 polynucleotides. The tissues and vectors which were used to to construct the cDNA libraries shown in Table 5 are described in Table 6.
The invention also encompasses GCREC variants. Various embodiments of GCREC
variants can have at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity to the GCREC amino acid sequence, and can contain at least one functional or structural characteristic of GCREC.
Various embodiments also encompass polynucleotides which encode GCREC. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ )D N0:23-44, which encodes GCREC. The polynucleotide sequences of SEQ ID N0:23-44, as presented in the Sequence Listing, embrace the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the 2o sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses variants of a polynucleotide encoding GCREC. In particular, such a variant polynucleotide will have at least about 70%, or alternatively at least about 85 %, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding GCREC. A
particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID N0:23-44 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 N0:23-44. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of GCREC.
In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding GCREC. A splice variant may have portions which have significant sequence identity to a polynucleotide encoding GCREC, 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 a polynucleotide encoding GCREC over its entire length; however, portions of the splice variant will have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding GCREC. For example, a polynucleotide comprising a sequence of SEQ ID N0:44, a polynucleotide comprising a sequence of SEQ 117 N0:30 and a polynucleotide comprising a sequence of SEQ ID
N0:31 are splice variants of each other; and a polynucleotide comprising a sequence of SEQ ID
N0:27, a polynucleotide comprising a sequence of SEQ ID N0:28 and a polynucleotide comprising a sequence of SEQ )D N0:29 are splice variants of each other. Any one of the splice variants described above can encode a polypeptide which contains at least one functional or structural characteristic of GCREC.
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 GCREC, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring 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 standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring GCREC, and all such variations are to be considered as being specifically disclosed.
Although polynucleotides which encode GCREC and its variants are generally capable of hybridizing to polynucleotides encoding naturally occurring GCREC under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding GCREC 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 GCREC 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 polynucleotides which encode GCREC and GCREC derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the synthetic polynucleotide 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 polynucleotide encoding GCREC or any fragment thereof.
Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ D7 N0:23-44 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol.
152:507-S11).
Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad CA). 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).
2o Sequencing is then carried out using either the ABI 373 or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853).
The nucleic acids encoding GCREC 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 (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (Triglia, T. et al. (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 (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 (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 confum 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. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotides or fragments thereof which encode GCREC may be cloned in recombinant DNA molecules that direct expression of GCREC, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionally equivalent polypeptides may be produced and used to express GCREC.
The polynucleotides of the invention can be engineered using methods generally known in the art in order to alter GCREC-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.
l0 Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol.
14:315-319) to alter or improve the biological properties of GCREC, 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 occurnng genes in a directed and controllable manner.
In another embodiment, polynucleotides encoding GCREC may be synthesized, in whole or in part, using one or more chemical methods well known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp.
Ser. 7:225-232).
Alternatively, GCREC itself or a fragment thereof may be synthesized using chemical methods known in the art. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp. 55-60; Roberge, J.Y. et al. (1995) Science 269:202-204). Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the amino acid sequence of GCREC, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.

The peptide may be substantially purified by preparative high performance liquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421). The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
In order to express a biologically active GCREC, the polynucleotides encoding GCREC or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotides encoding GCREC. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding GCREC. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where a polynucleotide sequence encoding GCREC 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 (Scharf, D. et al. (1994) Results Probl.
2o Cell Differ. 20:125-162).
Methods which are well known to those skilled in the art may be used to construct expression vectors containing polynucleotides encoding GCREC and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook and Russell, supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1, 3, and 15).
A variety of expression vector/host systems may be utilized to contain and express polynucleotides encoding GCREC. 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 (Sambrook and Russell, supra; Ausubel et al., 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; 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 polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6340-6344; Buller, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P.
l0 et al. (1994) Mol. Immunol. 31:219-226; 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 polynucleotides encoding GCREC. For example, routine cloning, subcloning, and propagation of polynucleotides encoding GCREC can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Invitrogen).
Ligation of polynucleotides encoding GCREC 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 (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509). When large quantities of GCREC are needed, e.g. for the production of antibodies, vectors which direct high level expression of GCREC 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 GCREC. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. Iu addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184).
Plant systems may also be used for expression of GCREC. Transcription of polynucleotides encoding GCREC 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.
6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984)~EMBO J. 3:1671-1680; Brogue, R. et al. (1984) Science 224:838-843; 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 (The McGraw Hill Yearbook of Science and Technology (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, polynucleotides encoding GCREC 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 GCREC in host cells (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 may 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 (Hartington, J.J. et al. (1997) Nat.
Genet. 15:345-355).
For long term production of recombinant proteins in mammalian systems, stable expression of GCREC in cell lines is preferred. For example, polynucleotides encoding GCREC
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 (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; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Wigler, M. et al.
(1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al.
(1981) J. Mol. Biol.
150:1-14). Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites (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), ~i-glucuronidase and its substrate ~i-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 to transient or stable protein expression attributable to a specific vector system (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 GCREC is inserted within a marker gene sequence, transformed cells containing polynucleotides encoding GCREC can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding GCREC
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 polynucleotide encoding GCREC and that express GCREC 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.
Itnmunological methods for detecting and measuring the expression of GCREC
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 GCREC is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art (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 Itnmunolo~y, Greene Pub.
Associates and Wiley-Interscience, New York NY; Pound, J.D. (1998) hnmunochemical 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 GCREC include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, polynucleotides encoding GCREC, 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 Biosciences, 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 polynucleotides encoding GCREC 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 GCREC may be designed to contain signal sequences which direct secretion of GCREC 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 polynucleotides or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" or "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type C~Iture 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 polynucleotides encoding GCREC 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 GCREC
protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of GCREC activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calinodulin, 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 GCREC encoding sequence and the heterologous protein sequence, so that GCREC may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein expression and purification are discussed in Ausubel et al.
(supra, ch. 10 and 16). A
variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In another embodiment, synthesis of radiolabeled GCREC 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.
GCREC, fragments of GCREC, or variants of GCREC may be used to screen for compounds that specifically bind to GCREC. One or more test compounds may be screened for specific binding to GCREC. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to GCREC. Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.
In related embodiments, variants of GCREC can be used to screen for binding of test compounds, such as antibodies, to GCREC, a variant of GCREC, or a combination of GCREC and/or one or more variants GCREC. In an embodiment, a variant of GCREC can be used to screen for compounds that bind to a variant of GCREC, but not to GCREC having the exact sequence of a sequence of SEQ m NO:1-22. GCREC variants used to perform such screening can have a range of about 50% to about 99% sequence identity to GCREC, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
In an embodiment, a compound identified in a screen for specific binding to GCREC can be closely related to the natural ligand of GCREC, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Coligan, J.E. et al. (1991) Current Protocols in ImmunoloQV 1(2):Chapter 5). In another embodiment, the compound thus identified can be a natural ligand of a receptor GCREC (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132-140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
In other embodiments, a compound identified in a screen for specific binding to GCREC can be closely related to the natural receptor to which GCREC binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the ligand binding site or binding pocket. For example, the compound may be a receptor for GCREC which is capable of propagating a signal, or a decoy receptor for GCREC which is not capable of propagating a signal (Ashkenazi, A. and V.M.
Divit (1999) C~rr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336). The compound can be rationally designed using known techniques. Examples of such techniques include those used to construct the compound etanercept (ENBREL;
Amgen Inc., Thousand Oaks CA), which is efficacious for treating rheumatoid arthritis in humans. Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgGI
(Taylor, P.C. et al. (2001) C~rr. Opin. Immunol. 13:611-616).
In one embodiment, two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to GCREC, fragments of GCREC, or variants of GCREC. The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of GCREC. In one embodiment, an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of GCREC. In another embodiment, an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of GCREC.
In an embodiment, anticalins can be screened for specific binding to GCREC, fragments of GCREC, or variants of GCREC. Anticafins are ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol.
7:8177-8184;
Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities. The amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
In one embodiment, screening for compounds which specifically bind to, stimulate, or inhibit GCREC involves producing appropriate cells which express GCREC, either as a secreted protein or on the cell membrane. Preferred cells can include cells from mammals, yeast, Drosophila, or E. coli.
Cells expressing GCREC or cell membrane fractions which contain GCREC are then contacted with a test compound and binding, stimulation, or inhibition of activity of either GCREC or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide, wherein binding is l0 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 GCREC, either in solution or affixed to a solid support, and detecting the binding of GCREC 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 carned 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.
An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors. Examples of such assays include radio-labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S.
Patent No. 6,372,724.
In a related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands (Matthews, D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30). In another related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (C~nningham, B.C. and J.A. Wells (1991) Proc. Natl. Acad.
Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-10988).
GCREC, fragments of GCREC, or variants of GCREC may be used to screen for compounds that modulate the activity of GCREC. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for GCREC
activity, wherein GCREC is combined with at least one test compound, and the activity of GCREC in the presence of a test compound is compared with the activity of GCREC in the absence of the test compound. A change in the activity of GCREC in the presence of the test compound is indicative of a compound that modulates the activity of GCREC. Alternatively, a test compound is combined with an in vitro or cell-free system comprising GCREC under conditions suitable for GCREC activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of GCREC 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 GCREC 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 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292). 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) Clip. 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 GCREC 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 GCREC 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 GCREC 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 GCREC, e.g., by secreting GCREC 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 GCREC and G-protein coupled receptors. In addition, examples of tissues expressing GCREC can be found in Table 6 and can also be found in Example XI. Therefore, GCREC appears to play a role in cell proliferative, neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory, and metabolic disorders, and viral infections. In the treatment of disorders associated with increased GCREC expression or activity, it is desirable to decrease the expression or activity of GCREC. In the treatment of disorders associated with decreased GCREC expression or activity, it is desirable to increase the expression or activity of GCREC.
Therefore, in one embodiment, GCREC 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 GCREC. 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; 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, priors 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 cardiovascular disorder such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, complications of thrombolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mural annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of cardiac transplantation; a gastrointestinal disorder such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, alphai antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis, verso-occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and carcinomas; 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 heltninthic infections, and trauma; a metabolic disorder such as diabetes, obesity, and osteoporosis; and an infection by a viral agent classified as adenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, l0 paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, and tongavirus.
In another embodiment, a vector capable of expressing GCREC 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 GCREC including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified GCREC 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 GCREC
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of GCREC
may be administered to a subject to treat or prevent a disorder associated with decreased expression or 2o activity of GCREC including, but not limited to, those listed above.
In a further embodiment, an antagonist of GCREC may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of GCREC. Examples of such disorders include, but are not limited to, those cell proliferative, neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory, and metabolic disorders, and viral infections described above. In one aspect, an antibody which specifically binds GCREC 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 GCREC.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding GCREC may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of GCREC including, but not limited to, those described above.
In other embodiments, any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents.

Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of GCREC may be produced using methods which are generally known in the art. In particular, purified GCREC may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind GCREC.
Antibodies to GCREC 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. In an embodiment, neutralizing antibodies (i.e., those which inhibit dimer formation) can be used therapeutically. Single chain antibodies (e.g., from camels or llamas) may be potent enzyme inhibitors and may have application in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S.
(2001) J. Biotechnol.
74:277-302).
For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with GCREC or with any fragment or oligopeptide thereof which has immunogenic 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, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calinette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to GCREC have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are substantially identical to a portion of the amino acid sequence of the natural protein.
Short stretches of GCREC 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 GCREC may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al.
(1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030;
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 (Morrison, S.L. et al. (1984) Proc. Natl. Acad.
Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608;
Takeda, S. et al. (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce GCREC-specific single chain antibodies.
Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton, D.R.
(1991) Proc. Natl. Acad.
Sci. USA 88:10134-10137).
Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al.
(1991) Nature 349:293-299).
Antibody fragments which contain specific binding sites for GCREC 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 (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 GCREC and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering GCREC 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 GCREC. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of GCREC-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 GCREC epitopes, represents the average affinity, or avidity, of the antibodies for GCREC.
The K~ determined for a preparation of monoclonal antibodies, which are monospecific for a particular GCREC epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in which the GCREC-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 GCREC, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRI, 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 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 GCREC-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available (Catty, supra; Coligan et al., supra).
In another embodiment of the invention, polynucleotides encoding GCREC, 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 GCREC. 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 GCREC (Agrawal, S., ed. (1996) Antisense Theraueutics, Humana Press, 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 (Slater, J.E. et al. (1998) J. Allergy Clin. Imtnunol. 102:469-475; Scanlon, K.J. et al. (1995) 9:1288-1296). Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A.D. (1990) Blood 76:271; Ausubel et al., supra; Uckert, W.
and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J.J.
(1995) Br. Med. Bull. 51:217-225; Boado, R.J. et al. (1998) J. Pharm. Sci.
87:1308-1315; Morris, M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736).
In another embodiment of the invention, polynucleotides encoding GCREC may be used for somatic or gertnline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SC117)-Xl disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (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 falciparum and Trypanosoma cruzi). In the case where a genetic deficiency in GCREC expression or regulation causes disease, the expression of GCREC 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 GCREC are treated by constructing mammalian expression vectors encoding GCREC
and introducing these vectors by mechanical means into GCREC-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu.
Rev. Biochem.
62:191-217; 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 GCREC 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).
GCREC
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));
l0 the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding GCREC from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KTT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary 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 GCREC expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding GCREC 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. (1998) 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 an embodiment, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding GCREC to cells which have one or more genetic abnormalities with respect to the expression of GCREC. 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).
In another embodiment, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding GCREC to target cells which have one or more genetic abnormalities with respect to the expression of GCREC. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing GCREC to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. ( 1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S.
Patent 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). The manipulation of cloned herpesvirus sequences, the generation of Ss recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.
In another embodiment, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding GCREC 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) C~rr. 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 GCREC into the alphavirus genome in place of the capsid-coding region results in the production of a large number of GCREC-coding RNAs and the synthesis of high levels of GCREC 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 GCREC 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 2o 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 (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Itnmunolo~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 RNA molecules encoding GCREC.
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 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
molecules encoding GCREC. 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.
In other embodiments of the invention, the expression of one or more selected polynucleotides of the present invention can be altered, inhibited, decreased, or silenced using RNA interference (RNAi) or post-transcriptional gene silencing (PTGS) methods known in the art.
RNAi is a post-transcriptional mode of gene silencing in which double-stranded RNA (dsRNA) introduced into a targeted cell specifically suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantially reduces the expression of the targeted gene. PTGS can also be accomplished by use of DNA
or DNA fragments as well. RNAi methods are described by Fire, A. et al. (1998; Nature 391:806-811) and Gura, T.
(2000; Nature 404:804-808). PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene delivery and/or viral vector delivery methods described herein or known in the art.
RNAi can be induced in mammalian cells by the use of small interfering RNA
also known as siRNA. SiRNA are shorter segments of dsRNA (typically about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease.
SiRNA appear to be the mediators of the RNAi effect in mammals. The most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3' overhangs. The use of siRNA for inducing RNAi in mammalian cells is described by Elbashir, S.M. et al. (2001; Nature 411:494-498).
SiRNA can either be generated indirectly by introduction of dsRNA into the targeted cell, or directly by mammalian transfection methods and agents described herein or known in the art (such as liposome-mediated transfection, viral vector methods, or other polynucleotide delivery/introductory methods). Suitable SiRNAs can be selected by examining a transcript of the target polynucleotide (e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occurrence of each nucleotide and the 3' adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being preferred. Regions to be avoided for target siRNA sites include the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP
endonuclease complex. The selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be eliminated from consideration.
The selected SiRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commercially available methods and kits such as the SILENCER siRNA
construction kit (Ambion, Austin TX).
In alternative embodiments, long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA. This can be accomplished using expression vectors that are engineered to express hairpin RNAs (shRNAs) using methods known in the art (see, e.g., Brummelkamp, T.R. et al. (2002) Science 296:550-553; and Paddison, P.J.
et al. (2002) Genes Dev. 16:948-958). In these and related embodiments, shRNAs can be delivered to target cells using expression vectors known in the art. An example of a suitable expression vector for delivery of siRNA is the PS1LENCER1.0-U6 (circular) plasmid (Ambion). Once delivered to the target tissue, shRNAs are processed in vivo into siRNA-like molecules capable of carrying out gene-specific silencing.
In various embodiments, the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis. Expression levels of the mRNA of a targeted gene, can be deterniined by northern analysis methods using, for example, the NORTHERNMAX-GLY kit (Ambion); by microarray methods; by PCR methods; by real time PCR
methods; and by other RNA/polynucleotide assays known in the art or described herein. Expression l0 levels of the protein encoded by the targeted gene can be determined by Western analysis using standard techniques known in the art.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding GCREC. 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 GCREC
expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding GCREC may be therapeutically useful, and in the treatment of disorders associated with decreased GCREC expression or activity, a compound which specifically promotes expression of the polynucleotide encoding GCREC may be therapeutically useful.
In various embodiments, one or more test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide;
and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding GCREC 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 GCREC 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 GCREC. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system to (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 oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al.
(2000) U.S. Patent No.
6,022,691 ).
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. 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 (Goldman, C.K. et, al. (1997) Nat. Biotechnol. 15:462-466).
Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of GCREC, antibodies to GCREC, and mimetics, agonists, antagonists, or inhibitors of GCREC.
In various embodiments, the compositions described herein, such as pharmaceutical compositions, may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra-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.
to et al., U.S. Patent No. 5,997,848). Pulmonary delivery allows 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 purpose. The determination of an effective dose is well within the capability of those skilled in the art.
Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising GCREC or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, GCREC 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 (Schwarze, 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 GCREC or fragments thereof, antibodies of GCREC, and agonists, antagonists or inhibitors of GCREC, 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 0.1 ~cg to 100,000 /.cg, 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.
2o DIAGNOSTICS
In another embodiment, antibodies which specifically bind GCREC may be used for the diagnosis of disorders characterized by expression of GCREC, or in assays to monitor patients being treated with GCREC or agonists, antagonists, or inhibitors of GCREC.
Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics.
Diagnostic assays for GCREC include methods which utilize the antibody and a label to detect GCREC 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 GCREC, including ELISAs, RIAs, and FAGS, are known in the art and provide a basis for diagnosing altered or abnormal levels of GCREC expression.
Normal or standard values for GCREC expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to GCREC under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of GCREC 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, polynucleotides encoding GCREC may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotides, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of GCREC may be correlated with disease. The l0 diagnostic assay may be used to determine absence, presence, and excess expression of GCREC, and to monitor regulation of GCREC levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding GCREC or closely related molecules may be used to identify nucleic acid sequences which encode GCREC. 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 GCREC, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and may have at least SO%
sequence identity to any of the GCREC encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:23-44 or from genomic sequences including promoters, enhancers, and introns of the GCREC
gene.
Means for producing specific hybridization probes for polynucleotides encoding GCREC
include the cloning of polynucleotides encoding GCREC or GCREC 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
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotides encoding GCREC may be used for the diagnosis of disorders associated with expression of GCREC. 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; 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, priors 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 cardiovascular disorder such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, complications of thrombolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mural annular calcification, mural valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of cardiac transplantation; a gastrointestinal disorder such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired immunodeficiency syndrome (AI17S) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, alphas antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis, verso-occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and carcinomas; 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, irntable 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 helininthic infections, and trauma; a metabolic disorder such as diabetes, obesity, and osteoporosis; and an infection by a viral agent classified as adenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, and tongavirus.
Polynucleotides encoding GCREC 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 GCREC expression.

Such qualitative or quantitative methods are well known in the art.
In a particular embodiment, polynucleotides encoding GCREC may be used in assays that detect the presence of associated disorders, particularly those mentioned above. Polynucleotides complementary to sequences encoding GCREC may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding GCREC 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 GCREC, 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 GCREC, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard values is used to establish the presence of a disorder.
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 GCREC

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 GCREC, or a fragment of a polynucleotide complementary to the polynucleotide encoding GCREC, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from polynucleotides encoding GCREC
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 polynucleotides encoding GCREC 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 pulinonary 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 '70 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;
Kwok, P.-Y. and Z. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) C~rr. Opin.
Neurobiol. 11:637-641).
Methods which may also be used to quantify the expression of GCREC include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves (Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244;
Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236). The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information 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, GCREC, fragments of GCREC, or antibodies specific for GCREC
may be used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S.
Patent No. 5,840,484;

hereby expressly incorporated by reference herein). 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). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data.
The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity (see, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In an embodiment, the toxicity of a test compound can be assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention ~z 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 embodiment relates to the use of the polypeptides disclosed herein to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A
profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type. In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for GCREC
to quantify the levels of GCREC 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 maybe performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol-or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in 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 (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc.
Natl. Acad. Sci. USA
93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalon, D. et al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci.
USA 94:2150-2155;
Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662). Various types of microarrays are well known and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A
Practical Approach, Oxford University Press, London).
In another embodiment of the invention, nucleic acid sequences encoding GCREC
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. 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 Pl constructions, or single chromosome cDNA libraries (Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet.
7:149-154). Once mapped, the nucleic acid sequences may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Larder, E.S. and D. Botstein (1986) Proc. Natl.
Acad. Sci. USA 83:7353-7357).
l0 Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found iu various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding GCREC 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 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 (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, GCREC, 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 3o solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between GCREC 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 (Geysers, 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 GCREC, or fragments thereof, and washed.
Bound GCREC is then detected by methods well known in the art. Purified GCREC
can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-peutralizing 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 GCREC specifically compete with a test compound for binding GCREC.
In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with GCREC.
In additional embodiments, the nucleotide sequences which encode GCREC 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. 60/317,783, U.S. Ser. No. 60/324,079, U.S. Ser. No.
60/329,217, U.S. Ser.
No. 60/348,418, U.S. Ser. No. 60/343,911, U.S. Ser. No. 60/332,362, and U.S.
Ser. No. 60/328,944 are hereby expressly incorporated by reference.
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 (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.

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 plasrnid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al., supra, ch. 5). Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL
S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen, Carlsbad CA), PCDNA2.1 plasmid (Invitrogen), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (lnvitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY
(Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XL1-BlueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX

from Invitrogen.
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 lysis.
Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C.
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 microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences);
the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art.
Reading frames within the cDNA sequences were identified using standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. 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 norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM (Haft, D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864;
Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (HMM is a probabilistic approach which analyzes consensus primary structures of gene families; see, for example, Eddy, S.R. (1996) Curr.
Opin. Struct. Biol.

6:361-365.) The queries were performed using programs based on BLAST, FASTA, BLIIVVIPS, and HIVIIVIER. 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 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, hidden Markov model (HIVIM)-based protein family databases such as PFAM, INCY, and TIGRFAM; and IhVIM-based protein domain databases such as SMART.
Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
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 ID
N0:23-44. 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 G-protein coupled receptors 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 (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94;
Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. 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 G-protein coupled receptors, the encoded polypeptides were analyzed by querying against PFAM models for G-protein coupled receptors. Potential G-protein coupled receptors were also identified by homology to Incyte cDNA sequences that had been annotated as G-protein coupled receptors. 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 so 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 l0 with additional cDNA sequences, or by inspection of genomic DNA, when necessary.
~~Stretched" Seguences 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 GCREC Encoding Polynucleotides The sequences which were used to assemble SEQ m N0:23-44 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 ll7 N0:23-44 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 1D 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 (Sambrook and Russell, supra, ch. 7; Ausubel et al., supra, ch. 4).
Analogous computer techniques applying BLAST were used to search for identical or related molecules in 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 +S 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, polynucleotides encoding GCREC 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 l0 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; heroic 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 is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue-and disease-specific expression of cDNA encoding GCREC. cDNA sequences and cDNA library/tissue information are found in the L1FESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of GCREC Encoding Polynucleotides Fill length polynucleotides are produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in 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 polymerise (Amersham Biosciences), ELONGASE
enzyme (Invitrogen), and Pfu DNA polymerise (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step l: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step S: 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 1: 94°C, 3 min; Step 2:
94 °C, 15 sec; Step 3: 57 °C, 1 min; Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;
l0 Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p,1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 p.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 /.c1 to 10 /.c1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Biosciences). 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 relegated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), 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 Biosciences) 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 Biosciences) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotides are verified using the above procedure or are used to obtain S'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 GCREC Encoding 1o Polynucleotides Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ >D N0:23-44 using the L1FESEQ 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 m N0:23-44 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 /.cCi of [,~ 3zP~ adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a superfine size exclusion dextran bead column (Amersham Biosciences). 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 et al., supra), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena, M., ed.
(1999) DNA Microarrays: A Practical Approach, Oxford University Press, London). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. 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, UV, chemical, or mechanical bonding procedures. A
typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements (Schena, M. et al. (1995) Science 270:467-470;
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 to complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, microarray 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/p.l oligo-(dT) primer (2lmer), 1X first strand buffer, 0.03 units/pl RNase inhibitor, 500 p,M dATP, 500 pM dGTP, 500 pM dTTP, 40 p.M
dCTP, 40 p.M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte Genomics). Specific control poly(A)+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA.
Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech, Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 p1 SX
SSC/0.2% SDS.
Microarray Preparation Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 pg.
s~

Amplified array elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) ate 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 p1 of the array element DNA, at an average concentration of 100 ng/~1, 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 UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 p1 of sample mixture consisting of 0.2 pg each of Cy3 and Cy5 labeled cDNA synthesis products in SX 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 cm2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 ~.1 of SX SSC in a comer of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in a first wash buffer (1X SSC, 0.1%
SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried.
Detection 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 light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm x 1.8 cm array used in the present example is scanned with a ss 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 (Incyte Genomics). Array elements that exhibit at least about a two-fold change in expression, a signal-to-background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentially expressed.
Exuression For example, SEQ ID N0:30 and SEQ ID N0:31 showed differential expression in breast carcinoma cell lines versus primary mammary epithelial cells as determined by microarray analysis.
The breast carcinoma cell lines include BT20, a breast carcinoma cell line derived in vitro from cells emigrating out of thin slices of a tumor mass isolated from a 74-year-old female; BT474, a breast ductal carcinoma cell line isolated from a solid, invasive ductal carcinoma of the breast from a 60-year-old female; BT483, a breast ductal carcinoma cell line isolated from a papillary invasive ductal tumor from a 23-year-old normal, menstruating, parous female; HS578T, a breast ductal carcinoma cell line isolated from a 74-year-old female with breast carcinoma; MCF7, a breast adenocarcinoma cell line derived from the pleural effusion of a 69-year-old female; and MDA-mb-468, a breast adenocarcinoma cell line isolated from the pleural effusion of a 51-year-old female with metastatic adenocarcinoma of the breast. The primary mammary epithelial cell line HMEC
was derived from normal human mammary tissue (Clonetics, San Diego, CA). The microarray experiments showed that the expression of SEQ ID N0:30 and SEQ ID N0:31 were decreased by at least two fold in all six breast carcinoma lines (BT20, BT474, BT483, HS578T, MCF7, and MDA-mb-468) relative to cells from the primary mammary epithelial cell line, HMEC. Therefore, SEQ ID N0:30 and SEQ )D
N0:31 are useful as diagnostic markers or as potential therapeutic targets for breast cancer.
SEQ D7 N0:30 and SEQ B7 N0:31 also showed differential expression in prostate carcinoma cell lines versus normal prostate epithelial cells as determined by microarray analysis. The prostate carcinoma cell lines include DU 145, LNCaP, and PC-3. DU 145 was isolated from a metastatic site in the brain of a 69 year old male with widespread metastatic prostate carcinoma. DU 145 has no detectable sensitivity to hormones; forms colonies in semi-solid medium; is only weakly positive for acid phosphatase; and cells are negative for prostate specific antigen (PSA).
LNCaP is a prostate carcinoma cell line isolated from a lymph node biopsy of a 50 year old male with metastatic prostate carcinoma. LNCaP expresses PSA, produces prostate acid phosphatase, and expresses androgen receptors. PC-3, a prostate adenocarcinoma cell line, was isolated from a metastatic site in the bone of a 62 year old male with grade IV prostate adenocarcinoma. The normal epithelial cell line, PrEC, is a primary prostate epithelial cell line isolated from a normal donor. The microarray experiments showed that the expression of SEQ ID N0:30 and SEQ >D N0:31 were decreased by at least two fold in all three prostate carcinoma lines (DU 145, LNCaP, and PC-3) relative to cells from the normal prostate epithelial cell line, PrEC. Therefore, SEQ ID N0:30 and SEQ >I7 N0:31 are useful as diagnostic markers or as potential therapeutic targets for prostate cancer.
For example, SEQ >D N0:41 showed decreased expression in dendritic cells treated with anti-CD40 versus untreated dendritic cells, as determined by microarray analysis.
Dendritic cells (DCs) are antigen presenting cells that play a crucial role in the initiation of the immune response. Human monocytic dendritic cells (mDCs) were derived in vitro from the adherent cellular fraction of the peripheral blood of 4 healthy volunteer donors. The adherent leukocytes, mostly monocytes, were incubated for 13 days in the presence of recombinant interleukin-4 IL-4 (10 ng/ml) and granulocyte/macrophage colony stimulating factor (10 ng/ ml). Antibodies specific for CD40 molecules can induce proliferation of B cells when presented with IL-4 or antibodies specific for CD20 molecules. The differentiated mDCs were collected after 13 days from the non-adherent cellular fraction and activated in the presence of soluble mouse anti-human CD40 antibodies for 2, 8, and 24 hours. The anti-CD40 treated mDCs were compared to untreated mDCs.
Therefore, SEQ ID
N0:41 is useful in monitoring treatment of, and diagnostic assays for, autoimmune/inflammation disorders.
XII. Complementary Polynucleotides Sequences complementary to the GCREC-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring GCREC.
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 GCREC.
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 GCREC-encoding transcript.
XIII. Expression of GCREC
Expression and purification of GCREC is achieved using bacterial or virus-based expression systems. For expression of GCREC in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the TS 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 GCREC upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of GCREC in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica cali~ornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding GCREC 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 (SP9) insect cells in most cases, or human hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to baculovirus (Engelhard, E.K. et al.
(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.
Gene Ther. 7:1937-1945).
In most expression systems, GCREC 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 to enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences).
Following purification, the GST moiety can be proteolytically cleaved from GCREC 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 et al. (supra, ch. 10 and 16).
Purified GCREC obtained by these methods can be used directly in the assays shown in Examples XVII, XVllI, and XIX where applicable.
XIV. ~nctional Assays GCREC function is assessed by expressing the sequences encoding GCREC at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ~cg 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 pg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP
or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM
detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA
with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY).
The influence of GCREC on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding GCREC and either CD64 or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding GCREC and other genes of interest can be analyzed by northern analysis or microarray techniques.
XV. Production of GCREC Specific Antibodies GCREC substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols.
Alternatively, the GCREC amino acid sequence is analyzed using LASERGENE
software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art (Ausubel et al., supra, ch. 11).
Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-GCREC
activity by, for example, binding the peptide or GCREC 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 GCREC Using Specific Antibodies Naturally occurring or recombinant GCREC is substantially purified by immunoaffinity chromatography using antibodies specific for GCREC. An immunoaffinity column is constructed by covalently coupling anti-GCREC antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing GCREC are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of GCREC (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/GCREC 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 GCREC is collected.
XVII. Identification of Molecules Which Interact with GCREC
Molecules which interact with GCREC may include agonists and antagonists, as well as molecules involved in signal transduction, such as G proteins. GCREC, or a fragment thereof, is labeled with 1~I Bolton-Hunter reagent. (See, e.g., Bolton A.E. and W.M.
Hunter (1973) Biochem. J.
133:529-539.) A fragment of GCREC includes, for example, a fragment comprising one or more of the three extracellular loops, the extracellular N-terminal region, or the third intracellular loop.
Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled GCREC, washed, and any wells with labeled GCREC complex are assayed.
Data obtained using different concentrations of GCREC are used to calculate values for the number, affinity, and association of GCREC with the candidate ligand molecules.
Alternatively, molecules interacting with GCREC 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).
GCREC may also be used in the PATHCALLING process (C~raGen 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).
Potential GCREC agonists or antagonists may be tested for activation or inhibition of GCREC
3o receptor activity using the assays described in sections XVlll and X1X.
Candidate molecules may be selected from known GPCR agonists or antagonists, peptide libraries, or combinatorial chemical libraries.

Methods for detecting interactions of GCREC with intracellular signal transduction molecules such as G proteins are based on the premise that internal segments or cytoplasmic domains from an orphan G protein-coupled seven transmembrane receptor may be exchanged with the analogous domains of a known G protein-coupled seven transmembrane receptor and used to identify the G-proteins and downstream signaling pathways activated by the orphan receptor domains (Kobilka, B.K.
et al. (1988) Science 240:1310-1316). In an analogous fashion, domains of the orphan receptor may be cloned as a portion of a fusion protein and used in binding assays to demonstrate interactions with specific G proteins. Studies have shown that the third intracellular loop of G
protein-coupled seven transmembrane receptors is important for G protein interaction and signal transduction (Conklin, B.R.
1o et al. (1993) Cell 73:631-641). For example, the DNA fragment corresponding to the third intracellular loop of GCREC may be amplified by the polymerase chain reaction (PCR) and subcloned into a fusion vector such as pGEX (Pharmacia Biotech). The construct is transformed into an appropriate bacterial host, induced, and the fusion protein is purified from the cell lysate by glutathione-Sepharose 4B
(Pharmacia Biotech) affinity chromatography.
For in vitro binding assays, cell extracts containing G proteins are prepared by extraction with 50 mM Tris, pH 7.8, 1 mM EGTA, 5 mM MgCl2, 20 mM CHAPS, 20% glycerol, 10 pg of both aprotinin and leupeptin, and 20 p,1 of 50 mM phenylinethylsulfonyl fluoride.
The lysate is incubated on ice for 45 min with constant stirring, centrifuged at 23,000 g for 15 min at 4°C, and the supernatant is collected. 750 p,g of cell extract is incubated with glutathione S-transferase (GST) fusion protein beads for 2 h at 4°C. The GST beads are washed five times with phosphate-buffered saline. Bound G protein subunits are detected by [3zP]ADP-ribosylation with pertussis or cholera toxins. The reactions are terminated by the addition of SDS sample buffer (4.6% (w/v) SDS, 10% (v/v) (3-mercaptoethanol, 20% (w/v) glycerol, 95.2 mM Tris-HCI, pH 6.8, 0.01 % (w/v) bromphenol blue).
The [32P]ADP-labeled proteins are separated on 10% SDS-PAGE gels, and autoradiographed. The separated proteins in these gels are transferred to nitrocellulose paper, blocked with Motto (5% nonfat dried milk, 50 mM Tris-HCl (pH 8.0), 2 mM CaCl2, 80 mM NaCI, 0.02% NaN3, and 0.2% Nonidet P-40) for 1 hour at room temperature, followed by incubation for 1.5 hours with Ga subtype selective antibodies (1:500; Calbiochem-Novabiochem). After three washes, blots are incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit imununoglobulin (1:2000, Cappel, Westchester PA) and visualized by the chemiluminescence-based ECL method (Amersham Corp.).
XVIII. Demonstration of GCREC Activity An assay for GCREC activity measures the expression of GCREC on the cell surface.

cDNA encoding GCREC 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 GCREC-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 GCREC expressed on the cell surface.
In the alternative, an assay for GCREC 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 GCREC is added to to quiescent 3T3 cultured cells using transfection methods well known in the art. The transiently transfected cells are then incubated in the presence of [3H]thymidine, a radioactive DNA precursor molecule. Varying amounts of GCREC 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 GCREC ligand concentration range is indicative of receptor activity. One unit of activity per milliliter is defined as the concentration of GCREC producing a 50% response level, where 100% represents maximal incorporation of [3H]thymidine into acid-precipitable DNA (McKay, I. and I.
Leigh, eds. (1993) Growth Factors: A Practical Approach, Oxford University Press, New York NY, p.
73.) In a further alternative, the assay for GCREC 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 GCREC 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 GCREC
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 AG1-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 GCREC present in the transfected cells.
A yeast two-hybrid system can be used to demonstrate binding and formation of a stable complex between a protein and a specific receptor, in which the yeast (e.g., strain y190 (MATa gal4 l0 ga180 his3 trill-901 ade2-101 ura3-52 leu2-3,-112 + URA3:: GAL_lacZ, LYS2::
GAL_HIS3 cyhr) is grown in yeast extract, peptone, and dextrose (YPD), Synthetic Complete (SC), or drop-out medium (Harper, J.W. et al. (1993) Cell 75:805-816). Affinity chromatography can be used to biochemically characterize the interaction and demonstrate ligand recognition and binding, by mixing purified proteins (GST or GST fusions) with whole-cell protein (e.g., HEK293 human embryonic kidney cells transfected with vector of interest). Bound protein is extracted and fractionated on a 10% SDS-PAGE, and subsequently detected by Western blotting (Tsai, R.Y.L. and R.R.
Reed (1997) J.
Neurosci. 17:4159-4169).
Nerve impulse activity can be assessed directly using a recording pipette, wherein fluoride-sensillum-lymph Ringer (SLR) is applied to single sensilla trichodea (specialized sensory organs). For example, sensilla trichodea from male moths (Bombyx mori) are accessed via the opened tip of a sensory hair, and then used to measure changes in spontaneous nerve impulse activity. This impulse activity can be recorded directly from receptor cells (e.g., bombykol, bombykal ) (Laue, M. et al.
(1997) Cell Tissue Res. 288:149-158).
XIX. Identification of GCREC Ligands GCREC is expressed in a eukaryotic cell fine 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 GCREC 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 3o messengers, such as cyclic AMP or Ca2+. 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 multi-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, GCREC may be coexpressed with the G-proteins Gaisnb 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 GCREC
through a pathway involving phospholipase C and Ca2+ mobilization. Alternatively, GCREC may be expressed in engineered yeast systems which lack endogenous GPCRs, thus providing the advantage of a null background for GCREC 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 (supp.):14-16). The receptors are screened against putative ligands including known GPCR ligands and other naturally occurring bioactive molecules. Biological extracts from tissues, biologtcal fluids and cell supernatants are also screened.
Various modifications and variations of the described compositions, 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. It will be appreciated that the invention provides novel and useful proteins, and their encoding polynucleotides, which can be used in the drug discovery process, as well as methods for using these compositions for the detection, diagnosis, and treatment of diseases and conditions.
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.
Nor should the description of such embodiments be considered exhaustive or limit the invention to the precise forms disclosed. Furthermore, elements from one embodiment can be readily recombined with elements from one or more other embodiments. Such combinations can form a number of embodiments within the scope of the invention. It is intended that the scope of the invention be defined by the following claims and their equivalents.

Table 1 IncytePolypeptideIncyte PolynucleotideIncyte Incyte Full ProjectSEQ ID PolypeptideSEQ ID PolynucleotideLength ID NO: ID NO: ID Clones 724584143 72458414CD125 72458414CB1 3879692CA2, 90110103CA2, CA2, 90110119CA2, 90110127CA2, 90110135CA2, 90110143CA2, 90110203CA2, CA2, 90110227CA2, 90110235CA2, 30350704 3035070CD1 26 3035070CB1 90133367CA2, 90133383CA2, 900234126 90023412CD128 90023412CB1 90008377CA2, 900233208 90023320CD 30 90023320CB 90012132CA2, 90012156CA2, 90012168CA2, 90012184CA2, 900233289 90023328CD131 90023328CB1 90012132CA2, 90012156CA2, 90012168CA2, 90012208CA2, 750432014 7504320CD1 36 7504320CB1 90173722CA2, 90173730CA2, .

90173738CA2, 90173746CA2, 90173814CA2, z ~ .~ o Q ~ ~ c~
°'"~°~NO ~.°~' M ~ r~ _° ~ o ~ a. ~ tx 0.' ~ °'°'~ ~ U~ O~U
N Q, ~ [~ N ! ~" ~'U' V
O O .-r z U ,~-, C i..
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G o ~ W z p; W ~ ~ O o 0 o ., 0.' Q ov U 0 0 ::
~ E'' 0 0 0 ~ ~ U ~ z ~
~ ~ U Q

~~z~. ~~~~3 ~~~ ~AAAA ~~

y N

~, z ~z ~z o zM~

zzz o ~

N O

N
M

s Wn F-a. v~
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.~' N

L~, v~
Pr v~
v~ ~

.b ~

U
Q

N
O

C 'b Q p~., N

A

b U

M
M

C
U O

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z N

N

~ ' ~i , o~ N

~ N N

O ~~ ~ O' N
o0 Ov O~

'r N
~

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~

N
W ~~VN~
O

N ~ ~ ~' ~

N
N N pp' ~ "

, M N O p N

O .w0 ,y0 O N ~ ~i ~ I N

N N "~ v ~ ~ N ~
~

N ~ ~ _ ~

~"i ~~ N ~ .-, ~

W M
O

d0' v N O N
~

l~ ~ d' N
v1 V1 O -~ ~O 00 v~ -~

N M

N
N

O~ ~D ,n N ~O

N N ~O ~
~ ~ p~
N O N
l~ N

~ ~ ~ .-, O
~ M

N v0 , N
M
~

,..., ~ ~ '-' N N
N

Ov o O

p N ~~ ~D ~., ~n ,y0 N
W

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_ ~Nv~'1N $

~p ~mp -~
~.., M N
p~

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v1 O

et ~ 00 N p ~t ~a N t~

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N~

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N N N 00 .,~ ~
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N M O ~ p~
N ~ ~ ~ WO N ~' s ~
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l 0 N
0 N ~j M
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o O
N
N

~ ~ ~ O ~ O ~ ~ v0 Ov N

.:, M M N ~ N
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, V

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N

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M ~ ~O
v0 00 N
M ~ ~ I~ ~ '~ v0 00 ~ N

n op [ ~p ~ 00 N p~
N op N N
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0 yG

p M y " ~ y ~ ~
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. gyp~ .~ p by -r W ~ O , yp 00 f~ t~ N N ~~

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M V1 ~--~ M
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N V1 , M v1 0o O N cn N M ~ ~ M N O
N ~
N

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N

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C
w w w w ~

U U

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oU

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I o N ~ ~ t~ ~ ~ ~ M M v1 O O

~D I~ 00 O M M M M M -~ M
v A M ~ ~n v1 N N N N N O

G A O Ov d' M O O O O O oo O
y ~' G v~ ~ N O O O O O O dw n ~n Y M ~ ~ ~ ~ ~
% ' G ~ ~ ~ ~ ~ O ~ T
V ~ ~

O W M -n v7 v0 ~ 00 01 O N -~ N M
~ ~ M - -~ v1 -~ O --n ~ -~

Q., N -n N N N N N M M M M M M
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N ~ ~ ~ ~~ ~ N
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pp ~O V1 n ~ O~ , 00 ,_, N ~O ~
V1 00 ~D 01 ~' p ~p p 00 M ~
~ 00 l~ ~~ O M

N ~~ N M M
01 O~ O
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7 '-' O~
M G~ ~ ~ ~

~N~ M~ OMO
Ov v0 v0 N -~ _., I~ ~ .~-W O M ~

~ 00 M ~ M M
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.

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V ~O
O O

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Ov ~ ~ ~' ~ ~ ~ o ~ ~ O

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w h N M .~ N ~~ -., v0 v0 ~ ~ v1 ~ ~ $ ,~
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~

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N 00 ~ ,~
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N
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W W W ~ w w ~ W w ~ rr ..w.--r N

_ N O U ~O v7 M ~ O M

z N ~ M N M W N

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~ y ~

C CM v0 v0 v' v~ v~ N C v00 ~t v O W M M _ _ M ~ _ ~ ~ ~
U ~ ~ --n M M O O d' ~~ N
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Table 5 PolynucleotideIncyte ProjectRepresentative Library SEQ ID NO: )D:

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w- w G p ~ ~ v ~

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w ~'U' U ~ irr U ~
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p ~ is 'b ~ G G Wn 7, .~ ~

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U

c~ 0 ' b O
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G vi ~ 'O ~O c0 N j d G ~ ~ G ~ ~O p4 c~ G C p Wn . >, tC cti by w-X ~ N ~
O ~ E ~

p V b cC ee > ' U
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>

G ~ w N

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'O G y,~" G p >, v .G d s.>'.

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U

O
N O .
N
p O eC V
cO !

N _ _ cC cC G ~ .b 'n A"i G ~ E

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G ~ ~ N
O

, ~
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~ >, O C ~' >
s p ~~~~~ ~:~~
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. , ;d ~n G G ' C
i U

. O O v m GD , .b w C .~ j, vj P

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G ~ >

OrUn>,U.~~ A'4.b G

N p ~
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U O 'b O
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N ~ ~ N N ~

v ; ~
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4" ~

V U c~ .b >> O ~ cti ~~ U 'C t-in a .t ~ ~ U C
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p .N ~ ~
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U ~~v0'~ G_ G_ ~ ~
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N "O ~L '~ e ~ Gt O G
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by :~ N G ~ w ~' . m v.N. N eG
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e~c'~bD cd cd :b V ~ ~ CL ~
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p G II ~ c~ ~ o y y II '~.' a, ~. ~ ~' ' E I I
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a~
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t o .-. ~ y w. OD N
E ~ '~~' °~ ~ ~ ~ ~ s x a c~ ~ ~ ~ ai ~° ~ '" ~ i E'oo~~~o.°°c~~oo~ ua.~~~ oa.~~
a. ~ c.~ oo a. ~ W a a ~' w w ~ a, °~ o, E-~ > a Z ~ o. C7 0 0 ~ ,~ w a~ .~ . .E
o ,v, ~
Qv ~ U M ~ G E-H t~1 N N N C. vD .-., O p~ oo Lh ~ ~~, ~ ~, . N p, ~ ~ N
U U U ~ ,r N ~ d~ ~~ E ~ N ~ p ~ O_ ,-~ ~' 'fl ~ ~ b a ~' Y y ~ O ~ G N ~ ~ w ~ N 3 ~ ~ W ~ O ~ ~ S V N
ca ~ O : ~.: ~D ~ P-~ a N O v'i U ~ u.; a. 0. ~ ,>E, ~ ~o~e v~ a E o ~ N ~ ~>_ U ~ y N
fn ina wOVJM ,~N G(/j~, ~N~ C(j~ E yO Co~oCv~ N
0 o U o ~ - ~'; m W N ~ o~ ~ ~ ~ ,~ ~ 00 00 ~Wn'= ~~sN ~~~o~~,~' xy; '~
A a C7 w o °' G :b o~ -o v ~-E E 'd E ~ ,.°~.. ~ -o ~ ~ G '" ,_, ~ o ;' ~ Q' ~ U v ~ . y a :3 :: ~ °~' .r ~ G ~ ~ ~ s .b °~ x oo ° ..-m ~nc~v»Q~, ~ .~..,~~ GR;'~oo,G~,_,U~~dN U
O O 0.' O Ltn p~ 'D ~" UJ p E-~ ~ C/j 'D x N ~ U ~i ~U ~ U ~ ~ ~ ~U
a~ w w c c4 'n ~ Q 3 ~ °' ~_ a. a,.; a ~ ~ -- .~ ° rx - > > _.
C U ~ ' O. w U ~O O ~ a O z ,~ O p O z a~ ~ ~ o .~ o a ~ E a ~ y ~ E ov s ~ ~ ° ~ ~ ~ E ~
c,' G. C. ~ ~O. y'~n' U ~ ~ Qi .d > C U N '~-' O~ V7 ~ ~ z C t G
° c. a ~ a ~ ~., ~ ° ~ yc a~ ~ C7 c . ~ cn ...
xa aa.aaNz a.z3 ~a xz~w ~xN'.r~ ~~c~w.J
.a> w t o c E v,a'~ a~ wG.'~~O ~v 'cdoa t CCj w °~ ~ _E ~ G ~ o i ~ ~ ~ ~ i u" y ~~C~G~~.O U~.G.in y~Ob G~eNO CE
U U G ~OEcd '_'~T"~C Nit UCpp~ GUw.X...NCO~ ~Qir~co ~cOU iat G U C b CC N CG U Cd C 'C O L. .r ~O'w.,~UOO>~cyCNQy E~~dO tUn"O .,Or ~ ~ a. ~ '~ E~ .E ~. ~ .Y ~ ~ ~ .J W ~ o ~ ~ ~ ~ U ::
cUCUU~~O~~W ~~c~~o ~°a.~ c~
c G ~ w c ~ opn ~ ai y v f~ w a~ ~'~~' ~ ~ ~ 'c .. _N C U . eC t~ N >a, e3 td G Pr G G ~ m S N
v~ ~ ~ O t U c~-.C ~ ~ G a, '.' ~ (% U C ~UJ U v ~ ~ w p G
E.°~~~oGn>,~yE~~~ ~s~~~,No~G ~G-o ° ~ a ~, >
~ ~ Q '~ ~ ~ ;.a ~ a~ o o ~, O ~ ~o o E o ~ y ~ E
b 3 s ~~ ~ .c ~1 :_ on w o >, y t ~° w ~~ ~ ~ ~~ ~ Y c a> w c ~ O ' °~ E .~e ~. E
a y~ ~~~~_ac '~ ~~~"~f'y~E~ ~E'~a o E ~ G ~ a~ ~ >~ a~ a~ x y Q. w G .~ w y a~ a~
v O L~ . f.. U U O" .D 0 .''' U > U U U ~i-., O ~ C~ O U U
CO G i~ ~ '~ G N ~- ~ C ~, V O C O C ~~ 00 C G _bA C C
~U O~ ~_ ~ Q O~ ~ = H Q. . _ ~, t-. n.a ~ pp ~ e~ .b ~y '~"~ eti G G
a -° Lr, a a. Pa o- :n ~ 0.~ o- ~ ~ ~ O ~ °'~ c ;c o U G a' c ~a ~a ~aa ~ ~~av ~ ~ ~ a ~Aw~a.~ ~.~ a u.
a W
U
a o ~ x EQ ~ =H a as a,a Q a~ w ~ ~;

U

td i~
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n U :n L~ ~
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m (~ v~ ~ n ~ N
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.

N ~_ G ~ Ow p, ~ U ~
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a o . a 3 c y ~

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C1, VJ~o~o~ ~~ ~~~ ~pv'i Nv ~ ~.C 0.
~
b C~ ~ . '-i a ~n o b ~ . O
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C

N ..~ ~ c ~ ~ C ~

N. . P.~ C d :j e~
'C ~i~ N N Wi-'a oN0 C C
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, _ O
cCaN N d C .--n ~ >~ 'y U U~

u c ~~ p W_ N . G cei a~j ~~ ' U
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'-; w. ~
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U

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~ ~ ~a x a ~ b Vii.a. a. U ran ~ E-' <110> INCYTE GENOMICS, INC.
BAUGHN, Mariah R.
BECHA, Shanya D.
ELLIOTT, Vicki S.
FORSYTHE, Ian J.
GANDHI, Ameena R.
GRIFFIN, Jennifer A.
HONCHELL, Cynthia D.
ISON, Craig H.
JIN, Pei KALLICK, Deborah A.
LEE, Ernestine A.
LEE, Sally RAMKUMAR, Jayalaxmi RICHARDSON, Thomas W.
SPRAGUE, William W.
SWARNAKAR, Anita CHAWLA, Narinder K.
YUE, Henry <120> G-Protein Coupled Receptors <130> PF-1178 PCT
<140> To Be Assigned <141> Herewith <150> US 60/317,783 <151> 2001-09-07 <150> US 60/324,079 <151> 2001-09-21 <150> US 60/328,944 <151> 2001-10-12 <150> US 60/329,217 <151> 2001-10-12 <150> US 60/348,418 <151> 2001-10-26 <150> US 60/343,911 <151> 2001-11-02 <150> US 60/332,362 <151> 2001-11-16 <160> 44 <170> PERL Program <210> 1 <211> 310 <212> PRT
<213> Homo Sapiens <220>
<221> mist feature <223> Incyte ID No: 55036218CD1 <400> 1 Met Gly Gly Asn Gln Thr Ser Ile Thr Glu Phe Leu Leu Leu Gly Phe Pro Ile Gly Pro Arg Ile Gln Met Leu Leu Phe Gly Leu Phe Ser Leu Phe Tyr Ile Phe Ile Leu Leu Gly Asn Gly Thr Ile Leu Gly Leu Ile Ser Leu Asp Ser Arg Leu His Thr Pro Met Tyr Phe Phe Leu Ser His Leu Ala Val Val Asp Ile Ala Cys Ala Cys Ser Thr Val Pro Gln Met Leu Val Asn Leu Leu His Pro Ala Lys Pro Ile Ser Phe Ala Gly Cys Met Thr Gln Met Phe Leu Phe Leu Ser Phe Ala His Thr Glu Cys Leu Leu Leu Val Val Met Ser Tyr Asp Arg Tyr Val Ala Ile Cys His Pro Leu Arg Tyr Ser Thr Ile Met Thr Trp Lys Val Cys Ile Thr Leu Ala Leu Thr Ser Trp Ile Leu Gly Val Leu Leu Ala Leu Val His Leu Val Leu Leu Leu Pro Leu Ser Phe Cys Gly Pro Gln Lys Leu Asn His Phe Phe Cys Glu Ile Met Ala Val Leu Lys Leu Ala Cys Ala Asp Thr His Ile Asn Glu Val Met Val Leu Ala Gly Ala Val Ser Val Leu Val Gly Ala Phe Phe Ser Thr Val Ile Ser Tyr Val His Ile Leu Cys Ala Ile Leu Lys Ile Gln Ser Gly Glu Gly Cys Gln Lys Ala Phe Ser Ile Cys Ser Ser His Leu Cys Val Val Gly Leu Phe Tyr Gly Thr Ala Ile Ile Met Tyr Val Glu Pro Gln Tyr Glu Ser Pro Lys Glu Gln Lys Lys Tyr Leu Leu Leu Phe His Ser Leu Phe Asn Pro Met Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Glu Val Gln Gly Thr Leu Lys Arg Met Leu Glu Lys Lys Arg Thr Ser <210> 2 <211> 310 <212> PRT
<213> Homo Sapiens <220>
<221> mist feature <223> Incyte ID No: 7499741CD1 <400> 2 Met Glu Gly Asn Lys Thr Trp Ile Thr Asp Ile Thr Leu Pro Arg Phe Gln Val Gly Pro Ala Leu Glu Ile Leu Leu Cys Gly Leu Phe Ser Ala Phe Tyr Thr Leu Thr Leu Leu Gly Asn Gly Val Ile Phe Gly Ile Ile Cys Leu Asp Cys Lys Leu His Thr Pro Met Tyr Phe Phe Leu Ser His Leu Ala Ile Val Asp Ile Ser Tyr Ala Ser Asn Tyr Val Pro Lys Met Leu Thr Asn Leu Met Asn Gln Glu Ser Thr Ile Ser Phe Phe Pro Cys Ile Met Gln Thr Phe Leu Tyr Leu Ala Phe Ala His Val Glu Cys Leu Ile Leu Val Val Met Ser Tyr Asp Arg Tyr Ala Asp Ile Cys His Pro Leu Arg Tyr Asn Ser Leu Met Ser Trp Arg Val Cys Thr Val Leu Ala Val Ala Ser Trp Val Phe Ser Phe Leu Leu Ala Leu Val Pro Leu Val Leu Ile Leu Ser Leu Pro Phe Cys Gly Pro His Glu Ile Asn His Phe Phe Cys Glu Ile Leu Ser Val Leu Lys Leu Ala Cys Ala Asp Thr Trp Leu Asn Gln Val Val Ile Phe Ala Ala Cys Val Phe Ile Leu Val Gly Pro Leu Cys Leu Val Leu Val Ser Tyr Leu Arg Ile Leu Ala Ala Ile Leu Arg Ile Gln Ser Gly Glu Gly Arg Arg Lys Ala Phe Ser Thr Cys Ser Ser His Leu Cys Val Val Gly Leu Phe Phe Gly Ser Ala Ile Val Thr Tyr Met Ala Pro Lys Ser Arg His Pro Glu Glu Gln Gln Lys Val Leu Ser Leu Phe Tyr Ser Leu Phe Asn Pro Met Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Ala Glu Val Lys Gly Ala Leu Arg Arg Ala Leu Arg Lys Glu Arg Leu Thr <210> 3 <211> 109 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 72458414CD1 <400> 3 Met Val Ser Tyr Ile Cys Ile Leu Met Thr Ile Leu Lys Ile Gln Thr Ala Asp Gly Lys Gln Lys Ala Phe Phe Thr Cys Phe Ser His Leu Ala Ala Val Ser Ile Leu Tyr Gly Thr Leu Phe Leu Ile Tyr Val Arg Pro Ser Ser Ser Ser Ser Leu Gly Ile Tyr Lys Val Ile Ser Leu Phe Tyr Thr Val Val Ile Pro Met Val Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Glu Val Lys Asp Ala Phe Arg Arg Lys Ile Glu Arg Lys Lys Phe Ile Ile Ala Arg Glu Glu Glu Met Arg Met Lys Asn Gln <210> 4 <211> 301 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3035070CD1 <400> 4 Met Glu Phe Val Leu Leu Gly Phe Ser Asp Ile Pro Asn Leu His Trp Met Leu Phe Ser Ile Phe Leu Leu Met Tyr Leu Met Ile Leu Met Cys Asn Gly Ile Ile Ile Leu Leu Ile Lys Ile His Pro Ala Leu Gln Thr Pro Met Tyr Phe Phe Leu Ser Asn Phe Ser Leu Leu Glu Ile Cys Tyr Val Thr Ile Ile Ile Pro Arg Met Leu Met Asp Ile Trp Thr Gln Lys Gly Asn Ile Ser Leu Phe Ala Cys Ala Thr Gln Met Cys Phe Phe Leu Met Leu Gly Gly Thr Glu Cys Leu Leu Leu Thr Val Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys Lys Pro Leu Gln Tyr Pro Leu Val Met Asn His Lys Val Cys Ile Gln Leu Ile Ile Ala Ser Trp Thr Ile Thr Ile Pro Val Val Ile Gly Glu Thr Cys Gln Ile Phe Leu Leu Pro Phe Cys Gly Thr Asn Thr Ile Asn His Phe Phe Cys Asp Ile Pro Pro Ile Leu Lys Leu Ala Cys Gly Asn Ile Phe Val Asn Glu Ile Thr Val His Val Val Ala Val Val Phe Ile Thr Val Pro Phe Leu Leu Ile Val Val Ser Tyr Gly Lys Ile Ile Ser Asn Ile Leu Lys Leu Ser Ser Ala Arg Gly Lys Ala Lys Ala Phe Ser Thr Cys Ser Ser His Leu Ile Val Val Ile Leu Phe Phe Gly Ala Gly Thr Ile Thr Tyr Leu Gln Pro Lys Pro His Gln Phe Gln Arg Met Gly Lys Leu Ile Ser Leu Phe Tyr Thr Ile Leu Ile Pro Thr Leu Asn Pro Ile Ile Tyr Thr Leu Arg Asn Lys Asp Ile Met Val Ala Leu Arg Lys Leu Leu Ala Lys Leu Leu Thr <210> 5 <211> 596 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90023411CD1 <400> 5 Met Ala Thr Pro Arg Gly Leu Gly Ala Leu Leu Leu Leu Leu Leu Leu Pro Thr Ser Gly Gln Glu Lys Pro Thr Glu Gly Pro Arg Asn Thr Cys Leu Gly Ser Asn Asn Met Tyr Asp Ile Phe Asn Leu Asn Asp Lys Ala Leu Cys Phe Thr Lys Cys Arg Gln Ser Gly Ser Asp Ser Cys Asn Val Glu Asn Leu Gln Arg Tyr Trp Leu Asn Tyr Glu Ala His Leu Met Lys Glu Gly Leu Thr Gln Lys Val Asn Thr Pro Phe Leu Lys Ala Leu Val Gln Asn Leu Ser Thr Asn Thr Ala Glu Asp Phe Tyr Phe Ser Leu Glu Pro Ser Gln Val Pro Arg Gln Val Met Lys Asp Glu Asp Lys Pro Pro Asp Arg Val Arg Leu Pro Lys Ser Leu Phe Arg Ser Leu Pro Gly Asn Arg Ser Val Val Arg Leu Ala Val Thr Ile Leu Asp Ile Gly Pro Gly Thr Leu Phe Lys Gly Pro Arg Leu Gly Leu Gly Asp Gly Ser Gly Val Leu Asn Asn Arg Leu Val Gly Leu Ser Val Gly Gln Met His Val Thr Lys Leu Ala Glu Pro Leu Glu Ile Val Phe Ser His Gln Arg Pro Pro Pro Asn Met Thr Leu Thr Cys Val Phe Trp Asp Val Thr Lys Gly Thr Thr Gly Asp Trp Ser Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu Gly Thr Val Cys Cys Cys Asp His Leu Thr Phe Phe Ala Leu Leu Leu Arg Pro Thr Leu Asp Gln Ser Thr Val His Ile Leu Thr Arg Ile Ser Gln Ala Gly Cys Gly Val Ser Met Ile Phe Leu Ala Phe Thr Ile Ile Leu Tyr Ala Phe Leu Ser Pro Gly Leu Gly His Gly Pro His Pro Gln Gln Ala Glu Cys Asp Leu Pro Ala Gly Arg Ile Ala Gly Pro Cys Glu Arg Gly Leu Glu Arg Gln Glu Lys Leu Pro Pro Ala Pro Gln Gly Gly Ala Trp His Leu Arg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp Ala Pro Lys Ile His Val Ala Leu Gly Gly Ser Leu Phe Leu Leu Asn Leu Ala Phe Leu Val Asn Val Gly Ser Gly Ser Lys Gly Ser Asp Ala Ala Cys Trp Ala Arg Gly Ala Val Phe His Tyr Phe Leu Leu Cys Ala Phe Thr Trp Met Gly Leu 395 ' 400 405 Glu Ala Phe His Leu Tyr Leu Leu Ala Val Arg Val Phe Asn Thr Tyr Phe Gly His Tyr Phe Leu Lys Leu Ser Leu Val Gly Trp Gly Leu Pro Ala Leu Met Val Ile Gly Thr Gly Ser Ala Asn Ser Tyr Gly Leu Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser Leu Glu Leu Cys Trp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr Ile Thr Val His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val Val Leu Ala Leu Val Val Trp Lys Ile Phe Thr Leu Ser Arg Ala Thr Ala Val Lys Glu Arg Gly Lys Asn Arg Lys Lys Val Leu Thr Leu Leu Gly Leu Ser Ser Leu Val Gly Val Thr Trp Gly Leu Ala Ile Phe Thr Pro Leu Gly Leu Ser Thr Val Tyr Ile Phe Ala Leu Phe Asn Ser Leu Gln Gly Val Phe Ile Cys Cys Trp Phe Thr Ile Leu Tyr Leu Pro Ser Gln Ser Thr Thr Val Ser~Ser Ser Thr Ala Arg Leu Asp Gln Ala His Ser Ala Ser Gln Glu <210> 6 <211> 315 <212> PRT

<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 90023412CD1 <400> 6 Met Ala Thr Pro Arg Gly Leu Gly Ala Leu Leu Leu Leu Leu Leu Leu Pro Thr Ser Gly Gln Glu Lys Pro Thr Glu Gly Pro Arg Asn Thr Cys Leu Gly Ser Asn Asn Met Tyr Asp Ile Phe Asn Leu Asn Asp Lys Ala Leu Cys Phe Thr Lys Cys Arg Gln Ser Gly Ser Asp Ser Cys Asn Val Glu Asn Leu Gln Arg Tyr Trp Leu Asn Tyr Glu Ala His Leu Met Lys Glu Gly Leu Thr Gln Lys Val Asn Thr Pro Phe Leu Lys Ala Leu Val Gln Asn Leu Ser Thr Asn Thr Ala Glu Asp Phe Tyr Phe Ser Leu Glu Pro Ser Gln Val Pro Arg Gln Val Met Lys Asp Glu Asp Lys Pro Pro Asp Arg Val Arg Leu Pro Lys Ser Leu Phe Arg Ser Leu Pro Gly Asn Arg Ser Val Val Arg Leu Ala Val Thr Ile Leu Asp Ile Gly Pro Gly Thr Leu Phe Lys Gly Pro Arg Leu Gly Leu Gly Asp Gly Ser Gly Val Leu Asn Asn Arg Leu Val Gly Leu Ser Val Gly Gln Met His Val Thr Lys Leu Ala Glu Pro Leu Glu Ile Val Phe Ser His Gln Arg Pro Pro Pro Asn Met Thr Leu Thr Cys Val Phe Trp Asp Val Thr Lys Gly Thr Thr Gly Asp Trp Ser Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu Gly Thr Val Cys Cys Cys Asp His Leu Thr Phe Phe Ala Leu Leu Leu Arg Pro Thr Leu Asp Gln Ser Thr Val His Ile Leu Thr Arg Ile Ser Gln Ala Gly Cys Gly Val Ser Met Ile Phe Leu Ala Phe Thr Ile Ile Leu Tyr Ala Phe Leu Arg Pro Ala Arg Pro Asp Gly His Arg His Trp Glu Cys Gln Gln Leu Arg Pro Leu His His Pro <210> 7 <211> 424 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 90023428CD1 <400> 7 Met Ala Thr Pro Ser Gly Leu Gly Ala Leu Leu Leu Leu Leu Leu Leu Pro Thr Ser Gly Gln Glu Lys Pro Thr Glu Gly Pro Arg Asn Thr Cys Leu Gly Ser Asn Asn Met Tyr Asp Ile Phe Asn Leu Asn Asp Lys Ala Leu Cys Phe Thr Lys Cys Arg Gln Ser Gly Ser Asp Ser Cys Asn Val Glu Asn Leu Gln Arg Tyr Trp Leu Asn Tyr Glu Ala His Leu Met Lys Glu Gly Leu Thr Gln Lys Val Asn Thr Pro Phe Leu Lys AIa Leu VaI Gln Asn Leu Ser Thr Asn Thr AIa Glu Asp Phe Tyr Phe Ser Leu Glu Pro Ser Gln Val Pro Arg Gln Val Met Lys Asp Glu Asp Lys Pro Pro Asp Arg Val Arg Leu Pro Lys Ser Leu Phe Arg Ser Leu Pro Gly Asn Arg Ser Val Val Arg Leu Ala Val Thr Ile Leu Asp Ile Gly Pro Gly Thr Leu Phe Lys Gly Pro Arg Leu Gly Leu Gly Asp Gly Ser Gly Val Leu Asn Asn Arg Leu Val GIy Leu Ser VaI Gly Gln Met His Val Thr Lys Leu Ala Glu Pro Leu Glu Ile Val Phe Ser His Gln Arg Pro Pro Pro Asn Met Thr Leu Thr Cys Val Phe Trp Asp Val Thr Lys Gly Thr Thr Gly Asp Trp Ser Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu Gly Thr Val Cys Cys Cys Asp His Leu Thr Phe Phe Ala Leu Leu Leu Arg Pro Thr Leu Asp Gln Ser Thr Val His Ile Leu Thr Arg Ile Ser Gln Ala Gly Cys Gly Val Ser Met Ile Phe Leu Ala Phe Thr Ile Ile Leu Tyr Ala Phe Leu Arg Cys Trp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr Ile Thr Val His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val Val Leu Ala Leu Val Val Trp Lys Ile Phe Thr Leu Ser Arg Ala Thr Ala Val Lys Glu Arg Gly Lys Asn Arg Lys Lys Val Leu Thr Leu Leu Gly Leu Ser Ser Leu Val Gly Val Thr Trp Gly Leu Ala Ile Phe Thr Pro Leu Gly Leu Ser Thr Val Tyr Ile Phe Ala Leu Phe Asn Ser Leu Gln Gly Val Phe Ile Cys Cys Trp Phe Thr Ile Leu Tyr Leu Pro Ser Gln Ser Thr Thr Val Ser Ser Ser Thr Ala Arg Leu Asp Gln Ala His Ser Ala Ser Gln Glu <210> 8 <211> 598 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90023320CD1 <400> 8 Met Ile Val Gly Asn Ala Thr Glu Ala Ala Val Ser Ser Phe Val Gln Asn Leu Ser Val Ile Ile Arg Gln Asn Pro Ser Thr Thr Val Gly Asn Leu Ala Ser Val Val Ser Ile Leu Ser Asn Ile Ser Ser Leu Ser Leu Ala Ser His Phe Arg Val Ser Asn Ser Thr Met Glu Asp Val Ile Ser Ile Ala Asp Asn Ile Leu Asn Ser Ala Ser Val Thr Asn Trp Thr Val Leu Leu Arg Glu Glu Lys Tyr Ala Ser Ser Arg Leu Leu Glu Thr Leu Glu Asn Ile Ser Thr Leu Val Pro Pro Thr Ala Leu Pro Leu Asn Phe Ser Arg Lys Phe Ile Asp Trp Lys Gly Ile Pro Val Asn Lys Ser Gln Leu Lys Arg Gly Tyr Ser Tyr Gln Ile Lys Met Cys Pro Gln Asn Thr Ser Ile Pro Ile Arg Gly Arg Val Leu Ile Gly Ser Asp Gln Phe Gln Arg Ser Leu Pro Glu Thr Ile Ile Ser Met Ala Ser Leu Thr Leu Gly Asn Ile Leu Pro Val Ser Lys Asn Gly Asn Ala Gln Val Asn Gly Pro Val Ile Ser Thr Val Ile Gln Asn Tyr Ser Ile Asn Glu Val Phe Leu Phe Phe Ser Lys Ile Glu Ser Asn Leu Ser Gln Pro His Cys Val Phe Trp Asp Phe Ser His Leu Gln Trp Asn Asp Ala Gly Cys His Leu Val Asn Glu Thr Gln Asp Ile Val Thr Cys Gln Cys Thr His Leu Thr Ser Phe Ser Ile Leu Met Ser Pro Phe Val Pro Ser Thr Ile Phe Pro Val Val Lys Trp Ile Thr Tyr Val Gly Leu Gly Ile Ser Ile Gly Ser Leu Ile Leu Cys Leu Ile Ile Glu Ala Leu Phe Trp Lys Gln Ile Lys Lys Ser Gln Thr Ser His Thr Arg Arg Ile Cys Met Val Asn Ile Ala Leu Ser Leu Leu Ile Ala Asp Val Trp Phe Ile Val Gly Ala Thr Val Asp Thr Thr Val Asn Pro Ser Gly Val Cys Thr Ala Ala Val Phe Phe Thr His Phe Phe Tyr Leu Ser Leu Phe Phe Trp Met Leu Met Leu Gly Ile Leu Leu Ala Tyr Arg Ile Ile Leu Val Phe His His Met Ala Gln His Leu Met Met Ala Val Gly Phe Cys Leu Gly Tyr Gly Cys Pro Leu Ile Ile Ser Val Ile Thr Ile Ala Val Thr Gln Pro Ser Asn Thr Tyr Lys Arg Lys Asp Val Cys Trp Leu Asn Trp Ser Asn Gly Ser Lys Pro Leu Leu Ala Phe Val Val Pro Ala Leu Ala Ile Val Ala Val Asn Phe Val Val Val Leu Leu Val Leu Thr Lys Leu Trp Arg Pro Thr Val Gly Glu Arg Leu Ser Arg Asp Asp Lys Ala Thr Ile Ile Arg Val Gly Lys Ser Leu Leu Ile Leu Thr Pro Leu Leu Gly Leu Thr Trp Gly Phe Gly Ile Gly Thr Ile Val Asp Ser Gln Asn Leu Ala Trp His Val Ile Phe Ala Leu Leu Asn Ala Phe Gln Gly Phe Phe Ile Leu Cys Phe Gly Ile Leu Leu Asp Ser Lys Leu Arg Gln Leu Leu Phe Asn Lys Leu Ser Ala Leu Ser Ser Trp Lys Gln Thr Glu Lys Gln Asn Ser Ser Asp Leu Ser Ala Lys Pro Lys Phe Ser Lys Pro Phe Asn Pro Leu Gln Asn Lys Gly His Tyr Ala Phe Ser His Thr Gly Asp Ser Ser Asp Asn Ile Met Leu Thr Gln Phe Val Ser Asn Glu <210> 9 <211> 540 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 90023328CD1 <400> 9 Met Glu Asp Val Ile Ser Ile Ala Asp Asn Ile Leu Asn Ser Ala Ser Val Thr Asn Trp Thr Val Leu Leu Arg Glu Glu Lys Tyr Ala Ser Ser Arg Leu Leu Glu Thr Leu Glu Asn Ile Ser Thr Leu Val Pro Pro Thr Ala Leu Pro Leu Asn Phe Ser Arg Lys Phe Ile Asp Trp Lys Gly Ile Pro Val Asn Lys Ser Gln Leu Lys Arg Gly Tyr Ser Tyr Gln Ile Lys Met Cys Pro Gln Asn Thr Ser Ile Pro Ile Arg Gly Arg Val Leu Ile Gly Ser Asp Gln Phe Gln Arg Ser Leu Pro Glu Thr Ile Ile Ser Met Ala Ser Leu Thr Leu Gly Asn Ile Leu Pro Val Ser Lys Asn Gly Asn Ala Gln Val Asn Gly Pro Val Ile Ser Thr Val Ile Gln Asn Tyr Ser Ile Asn Glu Val Phe Leu Phe Phe Ser Lys Ile Glu Ser Asn Leu Ser Gln Pro His Cys Val Phe Trp Asp Phe Ser His Leu Gln Trp Asn Asp Ala Gly Cys His Leu Val Asn Glu Thr Gln Asp Ile Val Thr Cys Gln Cys Thr His Leu Thr Ser Phe Ser Ile Leu Met Ser Pro Phe Val Pro Ser Thr Ile Phe Pro Val Val Lys Trp Ile Thr Tyr Val Gly Leu Gly Ile Ser Ile Gly Ser Leu Ile Leu Cys Leu Ile Ile Glu Ala Leu Phe Trp Lys Gln Ile Lys Lys Ser Gln Thr Ser His Thr Arg Arg Ile Cys Met Val Asn Ile Ala Leu Ser Leu Leu Ile Ala Asp Val Trp Phe Ile Val Gly Ala Thr Val Asp Thr Thr Val Asn Pro Ser Gly Val Cys Thr Ala Ala Val Phe Phe Thr His Phe Phe Tyr Leu Ser Leu Phe Phe Trp Met Leu Met Leu Gly Ile Leu Leu Ala Tyr Arg Ile Ile Leu Val Phe His His Met Ala Gln His Leu Met Met Ala Val Gly Phe Cys Leu Gly Tyr Gly Cys Pro Leu Ile Ile Ser Val Ile Thr Ile Ala Val Thr Gln Pro Ser Asn Thr Tyr Lys Arg Lys Asp Val Cys Trp Leu Asn Trp Ser Asn Gly Ser Lys Pro Leu Leu Ala Phe Val Val Pro Ala Leu Ala Ile Val Ala Val Asn Phe Val Val Val Leu Leu Val Leu Thr Lys Leu Trp Arg Pro Thr Val Gly Glu Arg Leu Ser Arg Asp Asp Lys Ala Thr Ile Ile Arg Val Gly Lys Ser Leu Leu Ile Leu Thr Pro Leu Leu Gly Leu Thr Trp Gly Phe Gly Ile Gly Thr Ile Val Asp Ser Gln Asn Leu Ala Trp His Val Ile Phe Ala Leu Leu Asn Ala Phe Gln Gly Phe Phe Ile Leu Cys Phe Gly Ile Leu Leu Asp Ser Lys Leu Arg Gln Leu Leu Phe Asn Lys Leu Ser Ala Leu Ser Ser Trp Lys Gln Thr Glu Lys Gln Asn Ser Ser Asp Leu Ser Ala Lys Pro Lys Phe Ser Lys Pro Phe Asn Pro Leu Gln Asn Lys Gly His Tyr Ala Phe Ser His Thr Gly Asp Ser Ser Asp Asn Ile Met Leu Thr Gln Phe Val Ser Asn Glu <210> 10 <211> 1023 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7480155CD1 <400> 10 Met Met Phe Arg Ser Asp Arg Met Trp Ser Cys His Trp Lys Trp Lys Pro Ser Pro Leu Leu Phe Leu Phe Ala Leu Tyr Ile Met Cys Val Pro His Ser Ala Val Trp Gly Cys Ala Asn Cys Arg Val Val Leu Ser Asn Pro Ser Gly Thr Phe Thr Ser Pro Cys Tyr Pro Asn Asp Tyr Pro Asn Ser Gln Ala Cys Met Trp Thr Leu Arg Ala Pro Thr Gly Tyr Ile Ile Gln Ile Thr Phe Asn Asp Phe Asp Ile Glu Glu Ala Pro Asn Cys Ile Tyr Asp Ser Leu Ser Leu Asp Asn Gly Glu Ser Gln Thr Lys Phe Cys Gly Ala Thr Ala Lys Gly Leu Ser Phe Asn Ser Ser Ala Asn Glu Met His Val Ser Phe Ser Ser Asp Phe Ser Ile Gln Lys Lys Gly Phe Asn Ala Ser Tyr Ile Arg Val Ala Val Ser Leu Arg Asn Gln Lys Val Ile Leu Pro Gln Thr Ser Asp Ala Tyr Gln Val Ser Val Ala Lys Ser Ile Ser Ile Pro Glu Leu Ser Ala Phe Thr Leu Cys Phe Glu Ala Thr Lys Val Gly His Glu Asp Ser Asp Trp Thr Ala Phe Ser Tyr Ser Asn Ala Ser Phe Thr Gln Leu Leu Ser Phe Gly Lys Ala Lys Ser Gly Tyr Phe Leu Ser Ile Ser Asp Ser Lys Cys Leu Leu Asn Asn Ala Leu Pro Val Lys Glu Lys Glu Asp Ile Phe Ala Glu Ser Phe Glu Gln Leu Cys Leu Val Trp Asn Asn Ser Leu Gly Ser Ile Gly Val Asn Phe Lys Arg Asn Tyr Glu Thr Val Pro Cys Asp Ser Thr Ile Ser Lys Val Ile Pro Gly Asn Gly Lys Leu Leu Leu Gly Ser Asn Gln Asn Glu Ile Val Ser Leu Lys Gly Asp Ile Tyr Asn Phe Arg Leu Trp Asn Phe Thr Met Asn Ala Lys Ile Leu Ser Asn Leu Ser Cys Asn Val Lys Gly Asn Val Val Asp Trp Gln Asn Asp Phe Trp Asn Ile Pro Asn Leu Ala Leu Lys Ala Glu Ser Asn Leu Ser Cys Gly Ser Tyr Leu Ile Pro Leu Pro Ala Ala Glu Leu Ala Ser Cys Ala Asp Leu Gly Thr Leu Cys Gln Asp Gly Ile Ile Tyr Arg Ile Ser Val Val Ile Gln Asn Ile Leu Arg His Pro Glu Val Lys Val Gln Ser Lys Val Ala Glu Trp Ser Gln Gln Val Met Val Ile Pro Leu His Pro Asn Thr Leu Phe Leu Leu Pro Asn Asp Lys Gln Pro Met Asn Asn Asn Asn Ser Ser Ile Arg Asn Val Ser Leu Val Tyr Asn Ala Thr Asn Asn Thr Asn Leu Glu Gly Lys Ile Ile Gln Gln Lys Leu Leu Lys Asn Asn Glu Ser Leu Asp Glu Gly Leu Arg Leu His Thr Val Asn Val Arg Gln Leu Gly His Cys Leu Ala Met Glu Glu Pro Lys Gly Tyr Tyr Trp Pro Ser Ile Gln Pro Ser Glu Tyr Val Leu Pro Cys Pro Asp Lys Pro Gly Phe Ser Ala Ser Arg Ile Cys Phe Tyr Asn Ala Thr Asn Pro Leu Val Thr Tyr Trp Gly Pro Val Asp Ile Ser Asn Cys Leu Lys Glu Ala Asn Glu Val Ala Asn Gln Ile Leu Asn Leu Thr Ala Asp Gly Gln Asn Leu Thr Ser Ala Asn Ile Thr Asn Ile Val Glu Gln Val Lys Arg Ile Val Asn Lys Glu Glu Asn Ile Asp Ile Thr Leu Gly Ser Thr Leu Met Asn Ile Phe Ser Asn Ile Leu Ser Ser Ser Asp Ser Asp Leu Leu Glu Ser Ser Ser Glu Ala Leu Lys Thr Ile Asp Glu Leu Ala Phe Lys Ile Asp Leu Asn Ser Thr Ser His Val Asn Ile Thr Thr Arg Asn Leu Ala Leu Ser Val Ser Ser Leu Leu Pro Gly Thr Asn Ala Ile Ser Asn Phe Ser Ile Gly Leu Pro Ser Asn Asn Glu Ser Tyr Phe Gln Met Asp Phe Glu Ser Gly Gln Val Asp Pro Leu Ala Ser Val Ile Leu Pro Pro Asn Leu Leu Glu Asn Leu Ser Pro Glu Asp Ser Val Leu Val Arg Arg Ala Gln Phe Thr Phe Phe Asn Lys Thr Gly Leu Phe Gln Ile Leu Glu Gly Pro Leu Pro Ala Asn Thr Gly Asp Leu Cys Leu Pro Leu Gln Val Gln Pro Asp Arg Glu Gln Asp Val Gly Pro Gln Arg Lys Thr Leu Val Ser Tyr Val Met Ala Cys Ser Ile Gly Asn Ile Thr Ile Gln Asn Leu Lys Asp Pro Val Gln Ile Lys Ile Lys His Thr Arg Thr Gln Ala Thr Val Ser Ser Pro Ile Ser Val Glu Ser Pro Gly Gly Phe Asp Leu Gln Val Ile Pro Val Cys Arg Lys Leu Gly Lys Glu Asp Gly Trp Ile Thr Ser Phe Asn Val Asp Gly Leu Cys Ile Ala Val Ala Val Leu Leu His Phe Phe Leu Leu Ala Thr Phe Thr Trp Met Gly Leu Glu Ala Ile His Met Tyr Ile Ala Leu Val Lys Val Phe Asn Thr Tyr Ile Arg Arg Tyr Ile Leu Lys Phe Cys Ile Ile Gly Trp Gly Leu Pro Ala Leu Val Val Ser Val Val Leu Ala Ser Arg Asn Asn Asn Glu Val Tyr Gly Lys Glu Ser Tyr Gly Lys Glu Lys Gly Asp Glu Phe Cys Trp Ile Gln Asp Pro Val Ile Phe Tyr Val Thr Cys Ala Gly Tyr Phe Gly Val Met Phe Phe Leu Asn Ile Ala Met Phe Ile Val Val Met Val Gln Ile Cys Gly Arg Asn Gly Lys Arg Ser Asn Arg Thr Leu Arg Glu Glu Val Leu Arg Asn Leu Arg Ser Val Val Ser Leu Thr Phe Leu Leu Gly Met Thr Trp Gly Phe Ala Phe Phe Ala Trp Gly Pro Leu Asn Ile Pro Phe Met Tyr Leu Phe Ser Ile Phe Asn Ser Leu Gln Glu Phe Pro Gly 5er Phe Ser Asn Leu Gln Gln Leu Gln Leu Ser Val Gln Leu Cys Phe His <210> 11 <211> 312 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504302CD1 <400> 11 Met Ala Asp Asp Asn Phe Thr Val Val Thr Glu Phe Ile Leu Leu Gly Leu Thr Asp His Ala Glu Leu Lys Ala Val Leu Phe Val Val Phe Leu Val Ile Tyr Ala Ile Thr Leu Leu Arg Asn Leu Gly Met Ile Leu Leu Ile Gln Ile Thr Ser Lys Leu His Thr Pro Met Tyr Phe Leu Leu Ser Cys Leu Ser Phe Val Asp Ala Cys Tyr Ser Ser Ala Ile Ala Pro Lys Met Leu Val Asn Leu Leu Val Val Lys Ala Thr Ile Ser Phe Ser Ala Cys Met Val Gln His Leu Cys Phe Gly Val Phe Ile Thr Thr Glu Gly Phe Leu Leu Ser Val Met Ala Tyr Asp Arg Tyr Val Ala Ile Val Ser Pro Leu Leu Tyr Thr Val Ala Met Ser Asp Arg Lys Cys Val Glu Leu Val Thr Gly Ser Trp Ile Gly Gly Ile Val Asn Thr Leu Ile His Thr Ile Ser Leu Arg Arg Leu Ser Phe Cys Arg Leu Asn Ala Val Ser His Phe Phe Cys Asp Ile Pro Ser Leu Leu Lys Leu Ser Cys Ser Asp Thr Ser Met Asn Glu Leu Leu Leu Leu Thr Phe Ser Gly Val Ile Ala Met Ala Thr Phe Leu Thr Val Ile Ile Ser Tyr Ile Phe Ile Ala Phe Ala Ser Leu Arg Ile His Ser Ala Ser Gly Arg Gln Gln Ala Phe Ser Thr Cys Ala Ser His Leu Thr Ala Val Thr Ile Phe Tyr Gly Thr Leu Ile Phe Ser Tyr Ile Gln Pro Ser Ser Gln Tyr Phe Val Glu Gln Glu Lys Val Val Ser Met Phe Tyr Thr Leu Gly Ile Pro Met Leu Asn Leu Leu Ile His Ser Leu Arg Asn Lys Asp Val Lys Glu Ala Val Lys Arg Ala Ile Glu Met Lys His Phe Leu Cys <210> 12 <211> 340 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504308CD1 <400> 12 Met Asp Ser Thr Phe Thr Gly Tyr Asn Leu Tyr Asn Leu Gln Val Lys Thr Glu Met Asp Lys Leu Ser Ser Gly Leu Asp Ile Tyr Arg Asn Pro Leu Lys Asn Lys Thr Glu Val Thr Met Phe Ile Leu Thr Gly Phe Thr Asp Asp Phe Glu Leu Gln Val Phe Leu Phe Leu Leu Phe Phe Ala Ile Tyr Leu Phe Thr Leu Ile Gly Asn Leu Gly Leu Val Val Leu Val Ile Glu Asp Ser Trp Leu His Asn Pro Met Tyr Tyr Phe Leu Ser Val Leu Ser Phe Leu Asp Ala Cys Tyr Ser Thr Val Val Thr Pro Lys Met Leu Val Asn Phe Leu Ala Lys Asn Lys Ser Ile Ser Phe Ile Gly Cys Ala Thr Gln Met Leu Leu Phe Val Thr Phe Gly Thr Thr Glu Cys Phe Leu Leu Ala Ala Met Ala Tyr Asp His Tyr Val Ala Ile Tyr Asn Pro Leu Leu Tyr Ser Val Ser Met Ser Pro Arg Val Tyr Val Pro Leu Ile Thr Ala Ser Tyr Val Ala Gly Ile Leu His Ala Thr Ile His Ile Val Ala Thr Phe Ser Leu Ser Phe Cys Gly Ser Asn Glu Ile Arg His Val Phe Cys Asp Met Pro Pro Leu Leu Ala Ile Ser Cys Ser Asp Thr His Thr Asn Gln Leu Leu Leu Phe Tyr Phe Val Gly Ser Ile Glu Ile Val Thr Ile Leu Ile Val Leu Ile Ser Cys Asp Phe Ile Leu Leu Ser Ile Leu Lys Met His Ser Ala Lys Gly Arg Gln Lys Ala Phe Ser Thr Cys Gly Ser His Leu Thr Gly Val Thr Ile Tyr His Gly Thr Ile Leu Val Ser Tyr Met Arg Pro Ser Ser Ser Tyr Ala Ser Asp His Asp Ile Ile Val Ser Ile Phe Tyr Thr Ile Val Ile Pro Lys Leu Asn Pro Ile Ile Tyr Ser Leu Arg Asn Lys Glu Val Lys Lys Ala Val Lys Lys Met Leu Lys Leu Val Tyr Lys <210> 13 <211> 312 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504312CD1 <400> 13 Met Glu Asn Tyr Asn Gln Thr Ser Thr Asp Phe Ile Leu Leu Gly Leu Phe Pro Gln Ser Arg Ile Gly Leu Phe Val Phe Thr Leu Ile Phe Leu Ile Phe Leu Met Ala Leu Ile Gly Asn Leu Ser Met Ile Leu Leu Ile Phe Leu Asp Ile His Leu His Thr Pro Met Tyr Phe Leu Leu Ser Gln Leu Ser Leu Ile Asp Leu Asn Tyr Ile Ser Thr Ile Val Pro Lys Met Val Tyr Asp Phe Leu Tyr Gly Asn Lys Ser Ile Ser Phe Thr Gly Cys Gly Ile Gln Ser Phe Phe Phe Leu Thr Leu Ala Val Ala Glu Gly Leu Leu Leu Thr Ser Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys Phe Pro Leu His Tyr Pro Ile Arg Ile Ser Lys Arg Val Cys Val Met Met Ile Thr Gly Ser Trp Met Ile Ser Ser Ile Asn Ser Cys Ala His Thr Val Tyr Ala Leu Cys Ile Pro Tyr Cys Lys Ser Arg Ala Ile Asn His Phe Phe Cys Asp Val Pro Ala Met Leu Thr Leu Ala Cys Thr Asp Thr Trp Val Tyr Glu Ser Thr Val Phe Leu Ser Ser Thr Ile Phe Leu Val Leu Pro Phe Thr Gly Ile Ala Cys Ser Tyr Gly Arg Val Leu Leu Ala Val Tyr Arg Met His Ser Ala Glu Gly Arg Lys Lys Ala Tyr Ser Thr Cys Ser Thr His Leu Thr Val Val Ser Phe Tyr Tyr Ala Pro Phe Ala Tyr Thr Tyr Val Arg Pro Arg Ser Leu Arg Ser Pro Thr Glu Asp Lys Ile Leu Ala Val Phe Tyr Thr Ile Leu Thr Pro Met Leu Asn Pro Ile Ile Tyr Ser Leu Arg Asn Lys Glu Val Met Gly Ala Leu Thr Gln Val Ile Gln Lys Ile Phe Ser Val Lys Met <210> 14 <211> 312 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504320CD1 <400> 14 Met Ala Trp Glu Asn Gln Thr Phe Asn Ser Asp Phe Ile Leu Leu Gly Ile Phe Asn His Ser Pro Thr His Thr Phe Leu Phe Phe Leu Val Leu Ala Ile Phe Ser Val Ala Phe Met Gly Asn Ser Val Met Val Leu Leu Ile Tyr Leu Asp Thr Gln Leu His Thr Pro Met Tyr Phe Leu Leu Ser Gln Leu Phe Leu Met Asp Leu Met Leu Ile Cys Ser Thr Val Pro Lys Met Ala Phe Asn Tyr Leu Ser Gly Ser Lys Ser Ile Ser Met Ala Gly Cys Ala Thr Gln Ile Phe Phe Tyr Val Ser Leu Leu Gly Ser Glu Cys Phe Leu Leu Ala Val Met Ser Tyr Asp Arg Tyr Ile Ala Ile Cys His Pro Leu Arg Tyr Thr Asn Leu Met Arg Pro Lys Ile Cys Gly Leu Met Thr Ala Phe Ser Trp Ile Leu Gly Ser Met Asp Ala Ile Ile Asp Ala Val Ala Thr Phe Ser Phe Ser Tyr Cys Gly Ser Arg Glu Ile Ala His Phe Phe Cys Asp Phe Pro Ser Leu Leu Ile Leu Ser Cys Asn Asp Thr Ser Ile Phe Glu Lys Val Leu Phe Ile Cys Cys Ile Val Met Ile Val Phe Pro Val Ala Ile Ile Ile Ala Ser Tyr Ala Arg Val Ile Leu Ala Val Ile His Met Gly Ser Gly Glu Gly Arg Arg Lys Ala Phe Thr Thr Cys Ser Ser His Leu Met Val Val Gly Met Tyr Tyr Gly Ala Gly Leu Phe Met Tyr Ile Arg Pro Thr Ser Asp Arg Ser Pro Met Gln Asp Lys Leu Val Ser Val Phe Tyr Thr Ile Leu Thr Pro Met Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Glu Val Thr Arg Ala Leu Arg Lys Val Leu Gly Lys Gly Lys Cys Gly Glu <210> 15 <211> 311 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504404CD1 <400> 15 Met Ser Gly Glu Asn Asn Ser Ser Val Thr Glu Phe Ile Leu Ala Gly Leu Ser Glu Gln Pro Glu Leu Gln Leu Pro Leu Phe Leu Leu Phe Leu Gly Ile Tyr Val Val Thr Val Val Gly Asn Leu Gly Met Thr Thr Leu Ile Trp Leu Ser Ser His Leu His Thr Pro Met Tyr Tyr Phe Leu Ser Ser Leu Ser Phe Ile Asp Phe Cys His Ser Thr Val Ile Thr Pro Lys Met Leu Val Asn Phe Val Thr Glu Lys Asn Ile Ile Ser Tyr Pro Glu Cys Met Thr Gln Leu Tyr Phe Phe Leu Val Phe Ala Ile Ala Glu Cys His Met Leu Ala Ala Met Ala Tyr Asp Arg Tyr Met Ala Ile Cys Ser Pro Leu Leu Tyr Ser Val Ile Ile Ser Asn Lys Ala Cys Phe Ser Leu Ile Leu Gly Val Tyr Ile 140 . 145 150 Ile Gly Leu Val Cys Ala Ser Val His Thr Gly Cys Met Phe Arg Val Gln Phe Cys Lys Phe Asp Leu Ile Asn His Tyr Phe Cys Asp Leu Leu Pro Leu Leu Lys Leu Ser Cys Ser Ser Ile Tyr Val Asn Lys Leu Leu Ile Leu Cys Val Gly Ala Phe Asn Ile Leu Val Pro Ser Leu Thr Ile Leu Cys Ser Tyr Ile Phe Ile Ile Ala Ser Ile Leu His Ile Arg Ser Thr Glu Gly Arg Ser Lys Ala Phe Ser Thr Cys Ser Ser His Met Leu Ala Val Val Ile Phe Phe Gly Ser Ala Ala Phe Met Tyr Leu Gln Pro Ser Ser Ile Ser Ser Met Asp Gln Gly Lys Val Ser Ser Val Phe Tyr Thr Ile Ile Val Pro Met Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Asp Val His Val Ser Leu Lys Lys Met Leu Gln Arg Arg Thr Leu Leu <210> 16 <211> 323 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504536CD1 <400> 16 Met Val Phe Leu Ser Ser Val Glu Thr Asp Gln Arg Lys Met Ser Ala Gly Asn His Ser Ser Val Thr Glu Phe Ile Leu Ala Gly Leu Ser Glu Gln Pro Glu Leu Gln Leu Arg Leu Phe Leu Leu Phe Leu Gly Ile Tyr Val Val Thr Val Val Gly Asn Leu Ser Met Ile Thr Leu Ile Gly Leu Ser Ser His Leu His Thr Pro Met Tyr Tyr Phe Leu Ser Gly Leu Ser Phe Ile Asp Ile Cys His Ser Thr Ile Ile Thr Pro Lys Met Leu Val Asn Phe Val Thr Glu Lys Asn Ile Ile Ser Tyr Pro Glu Cys Met Thr Gln Leu Tyr Phe Phe Leu Ile Phe Ala Ile Ala Glu Cys His Met Leu Ala Val Thr Ala Tyr Asp Arg Tyr Val Ala Ile Cys Ser Pro Leu Leu Tyr Asn Val Ile Met Ser Tyr His His Cys Phe Trp Leu Thr Val Gly Val Tyr Ile Leu Gly Ile Leu Gly Ser Thr Ile His Thr Gly Phe Met Leu Arg Leu Phe Leu Cys Lys Thr Asn Val Ile Asn His Tyr Phe Cys Asp Leu Phe Pro Leu Leu Gly Leu Ser Cys Ser Ser Thr Tyr Ile Asn Glu Leu Leu Val Leu Val Leu Ser Ala Phe Asn Ile Leu Thr Pro Ala Leu Thr Ile Leu Ala Ser Tyr Ile Phe Ile Ile Ala Ser Ile Leu Arg Ile Arg Ser Thr Glu Gly Arg Ser Lys Ala Phe Ser Thr Cys Ser Ser His Ile Leu Ala Val Ala Val Phe Phe Gly Ser Ala Ala Phe Met Tyr Leu Gln Pro Ser Ser Val Ser Ser Met Asp Gln Gly Lys Val Ser Ser Val Phe Tyr Thr Ile Val Val Pro Met Leu Asn Pro Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Phe Ala Leu Lys Lys Asn Leu Asp Ser Lys Ala Cys Ser <210> 17 <211> 320 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7505015CD1 <400> 17 Met Glu Met Arg Asn Thr Thr Pro Asp Phe Ile Leu Leu Gly Leu Phe Asn His Thr Arg Ala His Gln Val Leu Phe Met Met Leu Leu Ala Thr Val Leu Thr Ser Leu Phe Ser Asn Ala Leu Met Ile Leu Leu Ile His Trp Asp His Arg Leu His Arg Pro Met Tyr Phe Leu Leu Ser Gln Leu Ser Leu Met Asp Met Met Leu Val Ser Thr Thr Val Pro Lys Met Ala Ala Asp Tyr Leu Thr Gly Asn Lys Ala Ile Ser Arg Ala Gly Cys Gly Val Gln Ile Phe Phe Leu Pro Thr Leu Gly Gly Gly Glu Cys Phe Leu Leu Ala Ala Met Ala Tyr Asp Arg Tyr Ala Ala Val Cys His Pro Leu Arg Tyr Pro Thr Leu Met Ser Trp Gln Leu Cys Leu Arg Met Thr Met Ser Ser Trp Leu Leu Gly Ala Ala Asp Gly Leu Leu Gln Ala Val Ala Thr Leu Ser Phe Pro Tyr Cys Gly Ala His Glu Ile Asp His Phe Phe Cys Glu Ala Pro Val Leu Val Arg Leu Ala Cys Ala Asp Thr Ser Val Phe Glu Asn Ala Met Tyr Ile Cys Cys Val Leu Met Leu Leu Val Pro Phe Ser Leu Ile Leu Ser Ser Tyr Gly Leu Ile Leu Ala Ala Val Leu Leu Met Arg Ser Thr Glu Ala Arg Lys Lys Ala Phe Ala Thr Cys Ser Ser His Val Ala Val Val Gly Leu Phe Tyr Gly Ala Gly Ile Phe Thr Tyr Met Arg Pro Lys Ser His Arg Ser Thr Asn His Asp Lys Val Val Ser Ala Phe Tyr Thr Met Phe Thr Pro Leu Leu Asn Pro Leu Ile Tyr Ser Val Arg Asn Ser Glu Val Lys Glu Ala Leu Lys Arg Trp Leu Gly Thr Cys Val Asn Leu Lys His Gln Gln Asn Glu Ala His Arg Ser Arg <210> 18 <211> 320 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7505023CD1 <400> 18 Met Glu Met Arg Asn Thr Thr Pro Asp Phe Ile Leu Leu Gly Leu Phe Asn His Thr Arg Ala His Gln Val Leu Phe Met Met Val Leu Ser Ile Val Leu Thr Ser Leu Phe Gly Asn Ser Leu Met Ile Leu Leu Ile His Trp Asp His Arg Leu His Thr Pro Met Tyr Phe Leu Leu Ser Gln Leu Ser Leu Met Asp Met Met Leu Val Ser Thr Thr Val Pro Lys Met Ala Ala Asp Tyr Leu Thr Gly Ser Lys Ala Ile Ser Arg Ala Gly Cys Gly Val Gln Ile Phe Phe Leu Pro Thr Leu Gly Gly Gly Glu Cys Phe Leu Leu Ala Ala Met Ala Tyr Asp Arg Tyr Ala Ala Val Cys His Pro Leu Arg Tyr Pro Thr Leu Met Ser Trp Gln Leu Cys Leu Arg Met Thr Met Ser Cys Trp Leu Leu Gly Ala Ala Asp Gly Leu Leu Gln Ala Val Val Thr Leu Ser Phe Pro Tyr Cys Gly Ala His Glu Ile Asp His Phe Phe Cys Glu Thr Pro Val Leu Val Arg Leu Ala Cys Ala Asp Thr Ser Val Phe Glu Asn Ala Met Tyr Ile Cys Cys Val Leu Met Leu Leu Val Pro Phe Ser Leu Ile Leu Ser Ser Tyr Gly Leu Ile Leu Ala Ala Val Leu His Met Arg Ser Thr Glu Ala Arg Lys Lys Ala Phe Ala Thr Cys Ser Ser His Val Ala Val Val Gly Leu Phe Tyr Gly Ala Ala Ile Phe Thr Tyr Met Arg Pro Lys Ser His Arg Ser Thr Asn His Asp Lys Val Val Ser Ala Phe Tyr Thr Met Phe Thr Pro Leu Leu Asn Pro Leu Ile Tyr Ser Val Lys Asn Ser Glu Val Lys Gly Ala Leu Lys Arg Trp Leu Gly Thr Cys Val Asn Ile Lys His Gln Gln Asn Glu Ala His Arg Ser Arg <210> 19 <211> 388 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 70490395CD1 <400> 19 Met Glu Thr Ser Ser Pro Arg Pro Pro Arg Pro Ser Ser Asn Pro Gly Leu Ser Leu Asp Ala Arg Leu Gly Val Asp Thr Arg Leu Trp Ala Lys Val Leu Phe Thr Ala Leu Tyr Ala Leu Ile Trp Ala Leu Gly Ala Ala Gly Asn Ala Leu Ser Val His Val Val Leu Lys Ala Arg Ala Gly Arg Ala Gly Arg Leu Arg His His Val Leu Ser Leu Ala Leu Ala Gly Leu Leu Leu Leu Leu Val Gly Val Pro Val Glu Leu Tyr Ser Phe Val Trp Phe His Tyr Pro Trp Val Phe Gly Asp Leu Gly Cys Arg Gly Tyr Tyr Phe Val His Glu Leu Cys Ala Tyr Ala Thr Val Leu Ser Val Ala Gly Leu Ser Ala Glu Arg Cys Leu Ala Val Cys Gln Pro Leu Arg Ala Arg Ser Leu Leu Thr Pro Arg Arg Thr Arg Trp Leu Val Ala Leu Ser Trp Ala Ala Ser Leu Gly Leu Ala Leu Pro Met Ala Val Ile Met Gly Gln Lys His Glu Leu Glu Thr Ala Asp Gly Glu Pro Glu Pro Ala Ser Arg Val Cys Thr Val Leu Val Ser Arg Thr Ala Leu Gln Val Phe Ile Gln Val Asn Val Leu Val Ser Phe Val Leu Pro Leu Ala Leu Thr Ala Phe Leu Asn Gly Val Thr Val Ser His Leu Leu Ala Leu Cys Ser Gln Val Pro Ser Thr Ser Thr Pro Gly Ser Ser Thr Pro Ser Arg Leu Glu Leu Leu Ser Glu Glu Gly Leu Leu Ser Phe Ile Val Trp Lys Lys Thr Phe Ile Gln Gly Gly Pro Gly Ala Ile Val Val Met Tyr Val Ile Cys Trp Leu Pro Tyr His Ala Arg Arg Leu Met Tyr Cys Tyr Val Pro Asp Asp Ala Trp Thr Asp Pro Leu Tyr Asn Phe Tyr His Tyr Phe Tyr Met Val Thr Asn Thr Leu Phe Tyr Val Ser Ser Ala Val Thr Pro Leu Leu Tyr Asn Ala Val Ser Ser Ser Phe Arg Lys Leu Phe Leu Glu Ala Val Ser Ser Leu Cys Gly Glu His His Pro Met Lys Arg Leu Pro Pro Lys Pro Gln Ser Pro Thr Leu Met Asp Thr Ala Ser Gly Phe Gly Asp Pro Pro Glu Thr Arg Thr <210> 20 <211> 587 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503250CD1 <400> 20 Met Leu Leu Cys Thr Ala Arg Leu Val Gly Leu Gln Leu Leu Ile Ser Cys Cys Trp Ala Phe Ala Cys His Ser Thr Glu Ser Ser Pro Asp Phe Thr Leu Pro Gly Asp Tyr Leu Leu Ala Gly Leu Phe Pro Leu His Ser Gly Cys Leu Gln Val Arg His Arg Pro Glu Val Thr Leu Cys Asp Arg Ser Cys Ser Phe Asn Glu His Gly Tyr His Leu Phe Gln Ala Met Arg Leu Gly Val Glu Glu Ile Asn Asn Ser Thr Ala Leu Leu Pro Asn Ile Thr Leu Gly Tyr Gln Leu Tyr Asp Val Cys Ser Asp Ser Ala Asn Val Tyr Ala Thr Leu Arg Val Leu Ser Leu Pro Gly Gln His His Ile Glu Leu Gln Gly Asp Leu Leu His Tyr Ser Pro Thr Val Leu Ala Val Ile Gly Pro Asp Ser Thr Asn Arg Ala Ala Thr Thr Ala Ala Leu Leu Ser Pro Phe Leu Val Pro Met Leu Leu Glu Gln Ile His Lys Val His Phe Leu Leu His Lys Asp Thr Val Ala Phe Asn Asp Asn Arg Asp Pro Leu Ser Ser Tyr Asn Ile Ile Ala Trp Asp Trp Asn Gly Pro Lys Trp Thr Phe Thr Val Leu Gly Ser Ser Thr Trp Ser Pro Val Gln Leu Asn Ile Asn Glu Thr Lys Ile Gln Trp His Gly Lys Asp Asn Gln Val Pro Lys Ser Val Cys Ser Ser Asp Cys Leu Glu Gly His Gln Arg Val Val Thr Gly Phe His His Cys Cys Phe Glu Cys Val Pro Cys Gly Ala Gly Thr Phe Leu Asn Lys Ser Asp Leu Tyr Arg Cys Gln Pro Cys Gly Lys Glu Glu Trp Ala His Glu Gly Ser Gln Thr Cys Phe Pro Arg Thr Val Val Phe Leu Ala Leu Arg Glu His Thr Ser Trp Val Leu Leu Ala Ala Asn Thr Leu Leu Leu Leu Leu Leu Leu Gly Thr Ala Gly Leu Phe Ala Trp His Leu Asp Thr Pro Val Val Arg Ser Ala Gly Gly Arg Leu Cys Phe Leu Met Leu Gly Ser Leu Ala Ala Gly Ser Gly Ser Leu Tyr Gly Phe Phe Gly Glu Pro Thr Arg Pro Ala Cys Leu Leu Arg Gln Ala Leu Phe Ala Leu Gly Phe Thr Ile Phe Leu Ser Cys Leu Thr Val Arg Ser Phe Gln Leu Ile Ile Ile 395 . 400 405 Phe Lys Phe Ser Thr Lys Val Pro Thr Phe Tyr His Ala Trp Val Gln Asn His Gly Ala Gly Leu Phe Val Met Ile Ser Ser Ala Ala Gln Leu Leu Ile Cys Leu Thr Trp Leu Val Val Trp Thr Pro Leu Pro Ala Arg Glu Tyr Gln Arg Phe Pro His Leu Val Met Leu Glu Cys Thr Glu Thr Asn Ser Leu Gly Phe Ile Leu Ala Phe Leu Tyr Asn Gly Leu Leu Ser Ile Ser Ala Phe Ala Cys Ser Tyr Leu Gly Lys Asp Leu Pro Glu Asn Tyr Asn Glu Ala Lys Cys Val Thr Phe Ser Leu Leu Phe Asn Phe Val Ser Trp Ile Ala Phe Phe Thr Thr Ala Ser Val Tyr Asp Gly Lys Tyr Leu Pro Ala Ala Asn Met Met Ala Gly Leu Ser Ser Leu Ser Ser Gly Phe Gly Gly Tyr Phe Leu Pro Lys Cys Tyr Val Ile Leu Cys Arg Pro Asp Leu Asn Ser Thr Glu His Phe Gln Ala Ser Ile Gln Asp Tyr Thr Arg Arg Cys Gly Ser Thr <210> 21 <211> 926 <212> PRT
<213> Homo Sapiens <220>
<221> mist feature <223> Incyte ID No: 7505723CD1 <400> 21 Met Ala Phe Leu Ile Ile Leu Ile Thr Cys Phe Val Ile Ile Leu Ala Thr Ser Gln Pro Cys Gln Thr Pro Asp Asp Phe Val Ala Ala Thr Ser Pro Gly His Ile Ile Ile Gly Gly Leu Phe Ala Ile His Glu Lys Met Leu Ser Ser Glu Asp Ser Pro Arg Arg Pro Gln Ile Gln Glu Cys Val Gly Phe Glu Ile Ser Val Phe Leu Gln Thr Leu Ala Met Ile His Ser Ile Glu Met Ile Asn Asn Ser Thr Leu Leu Pro Gly Val Lys Leu Gly Tyr Glu Ile Tyr Asp Thr Cys Thr Glu Val Thr Val Ala Met Ala Ala Thr Leu Arg Phe Leu Ser Lys Phe Asn Cys Ser Arg Glu Thr Val Glu Phe Lys Cys Asp Tyr Ser Ser Tyr Met Pro Arg Val Lys Ala Val Ile Gly Ser Gly Tyr Ser Glu Ile Thr Met Ala Val Ser Arg Met Leu Asn Leu Gln Leu Met Pro Gln Val Gly Tyr Glu Ser Thr Ala Glu Ile Leu Ser Asp Lys Ile Arg Phe Pro Ser Phe Leu Arg Thr Val Pro Ser Asp Phe His Gln Ile Lys Ala Met Ala His Leu Ile Gln Lys Ser Gly Trp Asn Trp Ile Gly Ile Ile Thr Thr Asp Asp Asp Tyr Gly Arg Leu Ala Leu Asn Thr Phe Ile Ile Gln Ala Glu Ala Asn Asn Val Cys Ile Ala Phe Lys Glu Val Leu Pro Ala Phe Leu Ser Asp Asn Thr Ile Glu Val Arg Ile Asn Arg Thr Leu Lys Lys Ile Ile Leu Glu Ala Gln Val Asn Val Ile Val Val Phe Leu Arg Gln Phe His Val Phe Asp Leu Phe Asn Lys Ala Ile Glu Met Asn Ile Asn Lys Met Trp Ile Ala Ser Asp Asn Trp Ser Thr Ala Thr Lys Ile Thr Thr Ile Pro Asn Val Lys Lys Ile Gly Lys Val Val Gly Phe Ala Phe Arg Arg Gly Asn Ile Ser Ser Phe His Ser Phe Leu Gln Asn Leu His Leu Leu Pro Ser Asp Ser His Lys Leu Leu His Glu Tyr Ala Met His Leu Ser Ala Cys Ala Tyr Val Lys Asp Thr Asp Leu Ser Gln Cys Ile Phe Asn His Ser Gln Arg Thr Leu Ala Tyr Lys Ala Asn Lys Ala Ile Glu Arg Asn Phe Val Met Arg Asn Asp Phe Leu Trp Asp Tyr Ala Glu Pro Gly Leu Ile His Ser Ile Gln Leu Ala Val Phe Ala Leu Gly Tyr Ala Ile Arg Asp Leu Cys Gln Ala Arg Asp Cys Gln Asn Pro Asn Ala Phe Gln Pro Trp Glu Leu Leu Gly Val Leu Lys Asn Val Thr Phe Thr Asp Gly Trp Asn Ser Phe His Phe Asp Ala His Gly Asp Leu Asn Thr Gly Tyr Asp Val Val Leu Trp Lys Glu Ile Asn Gly His Met Thr Val Thr Lys Met Ala Glu Tyr Asp Leu Gln Asn Asp Val Phe Ile Ile Pro Asp Gln Glu Thr Lys Asn Glu Phe Arg Asn Leu Lys Gln Ile Gln Ser Lys Cys Ser Lys Glu Cys Ser Pro Gly Gln Met Lys Lys Thr Thr Arg Ser Gln His Ile Cys Cys Tyr Glu Cys Gln Asn Cys Pro Glu Asn His Tyr Thr Asn Gln Thr Asp Met Pro His Cys Leu Leu Cys Asn Asn Lys Thr His Trp Ala Pro Val Arg Ser Thr Met Cys Phe Glu Lys Glu Val Glu Tyr Leu Asn Trp Asn Asp Ser Leu Ala Ile Leu Leu Leu Ile Leu Ser Leu Leu Gly Ile Ile Phe Val Leu Val Val Gly Ile Ile Phe Thr Arg Asn Leu Asn Thr Pro Val Val Lys Ser Ser Gly Gly Leu Arg Val Cys Tyr Val Ile Leu Leu Cys His Phe Leu Asn Phe Ala Ser Thr Ser Phe Phe Ile Gly Glu Pro Gln Asp Phe Thr Cys Lys Thr Arg Gln Thr Met Phe Gly Val Ser Phe Thr Leu Cys Ile Ser Cys Ile Leu Thr Lys Ser Leu Lys Ile Leu Leu Ala Phe Ser Phe Asp Pro Lys Leu Gln Lys Phe Leu Lys Cys Leu Tyr Arg Pro Ile Leu Ile Ile Phe Thr Cys Thr Gly Ile Gln Val Val Ile Cys Thr Leu Trp Leu Ile Phe Ala Ala Pro Thr Val Glu Val Asn Val Ser Leu Pro Arg Val Ile Ile Leu Glu Cys Glu Glu Gly Ser Ile Leu Ala Phe Gly Thr Met Leu Gly Tyr Ile Ala Ile Leu Ala Phe Ile Cys Phe Ile Phe Ala Phe Lys Gly Lys Tyr Glu Asn Tyr Asn Glu Ala Lys Phe Ile Thr Phe Gly Met Leu Ile Tyr Phe Ile Ala Trp Ile Thr Phe Ile Pro Ile Tyr Ala Thr Thr Phe Gly Lys Tyr Val Pro Ala Val Glu Ile Ile Val Ile Leu Ile Ser Asn Tyr Gly Ile Leu Tyr Cys Thr Phe Ile Pro Lys Cys Tyr Val Ile Ile Cys Lys Gln Glu Ile Asn Thr Lys Ser Ala Phe Leu Lys Met Ile Tyr Ser Tyr Ser Ser His Ser Val Ser Ser Ile Ala Leu Ser Pro Ala Ser Leu Asp Ser Met Ser Gly Asn Val Thr Met Thr Asn Pro Ser Ser Ser Gly Lys Ser Ala Thr Trp Gln Lys Ser Lys Asp Leu Gln Ala Gln Ala Phe Ala His Ile Cys Arg Glu Asn Ala Thr Ser Val Ser Lys Thr Leu Pro Arg Lys Arg Met Ser Ser Ile <210> 22 <211> 256 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90023339CD1 <400> 22 Met Gln Met Glu Leu Lys Phe Lys Asn Gly Ser Ile Val Ala Gly Tyr Glu Val Val Gly Ser Ser Ser Ala Ser Glu Leu Leu Ser Ala Ile Glu His Val Ala Glu Lys Ala Lys Thr Ala Leu His Lys Leu Phe Pro Leu Glu Asp Gly Ser Phe Arg Val Phe Gly Lys Ala Gln Cys Asn Asp Ile Val Phe Gly Phe Gly Ser Lys Asp Asp Glu Tyr Thr Leu Pro Cys Ser Ser Gly Tyr Arg Gly Asn Ile Thr Ala Lys Cys Glu Ser Ser Gly Trp Gln Val Ile Arg Glu Thr Cys Val Leu Ser Leu Leu Glu Glu Leu Asn Lys Asn Phe Ser Met Ile Val Gly Asn Ala Thr Glu Ala Ala Val Ser Ser Phe Val Gln Asn Leu Ser Val Ile Ile Arg Gln Asn Pro Ser Thr Thr Val Gly Asn Leu Ala Ser Val Val Ser Ile Leu Ser Asn Ile Ser Ser Leu Ser Leu Ala Ser His Phe Arg Val Ser Asn Ser Thr Met Glu Gly Phe Phe Ile Leu Cys Phe Gly Ile Leu Leu Asp Ser Lys Leu Arg Gln Leu Leu Phe Asn Lys Leu Ser Ala Leu Ser Ser Trp Lys Gln Thr Glu Lys Gln Asn Ser Ser Asp Leu Ser Ala Lys Pro Lys Phe Ser Lys Pro Phe Asn Pro Leu Gln Asn Lys Gly His Tyr Ala Phe Ser His Thr Gly Asp Ser Ser Asp Asn Ile Met Leu Thr Gln Phe Val Ser Asn Glu <210> 23 <211> 1138 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 55036218CB1 <400> 23 ttttcttaga aaacactcaa acaataatat gtacagttgc agcatgctta aacatttgcc 60 ttagacattc agacttggtg agctccttgt ttcttctaca gggaaatggg gggaaatcag 120 acttccatca cagagttcct cctactggga tttcccattg gcccaaggat tcagatgctc 180 ctctttgggc tcttctccct gttctacatc ttcattctgt tggggaacgg gacaatcctg 240 gggctcatct cactggactc cagactccac acccccatgt acttcttcct ctcacacctg 300 gcggtcgtcg acatcgcctg tgcttgcagc acggtgcccc agatgctggt gaacctcctg 360 catccagcca agcccatctc ctttgctggc tgcatgaccc agatgtttct gtttttgagt 420 tttgcacata cagaatgtct cctcctggtg gtgatgtcct atgatcggta cgtggccatc 480 tgccaccctc tccgatattc taccatcatg acctggaaag tctgcatcac tttggcattg 540 acttcctgga ttttaggagt cttattggcc cttgtccatc tagtgttact gctaccactg 600 tccttctgtg gaccccagaa acttaatcac tttttctgtg aaattatggc tgttctcaaa 660 cttgcctgtg cggataccca cattaatgag gtaatggttt tggcaggggc agtgtctgtg 720 ctggtgggag ccttcttttc cactgtaata tcttatgttc atattctatg tgccattcta 780 aagatccagt caggagaggg gtgccagaaa gccttctcca tctgctcctc ccacctctgt 840 gtggttggac tcttttatgg cacagccatc atcatgtatg ttgagcccca gtatgagagc 900 cccaaggagc agaagaaata tctcctgctg tttcacagcc tcttcaatcc catgcttaat 960 cccctaattt atagtcttag gaacaaggaa gtccaaggta ctctaaagag gatgcttgaa 1020 aagaagagaa cttcatgaaa gcctgaaaga atagtaaaat agctggcttc aaagggcttt 1080 gagattacat ctgaacccat ccctactcag gatacataat cacactctag agaaccct 1138 <210> 24 <211> 1351 <212> DNA
<213> Homo Sapiens <220>
<221> mist feature <223> Incyte ID No: 7499741CB1 <400> 24 ccccttatgt agggtatatt ataatagatc ctgttttgtt atattttatt aaatgttata 60 tacatgtaca catacacaca caaatatata tacacatata catgtatgta ttcttattat 120 tccattgata caatgcgatt ttttctcatt tttatgagca cacttggtta gttttggcaa 180 catatctgag aataaattct agaaaattca ttcaaagtta taatttctaa gtgctccctc 240 tgtgcatctt tgactggccc acagctctga ccttcctgtc ctagatgtcc acaagagcat 300 ggaaggcaac aagacatgga tcacagacat caccttgccg cgattccagg ttggtccagc 360 actggagatt ctcctctgtg gacttttctc tgccttctat acactcaccc tgctggggaa 420 tggggtcatc tttgggatta tctgcctgga ctgtaagctt cacacaccca tgtacttctt 480 cctctcacac ctggccattg ttgacatatc ctatgcttcc aactatgtcc ccaagatgct 540 gacgaatctt atgaaccagg aaagcaccat ctcctttttt ccatgcataa tgcagacatt 600 cttgtatttg gcttttgctc acgtagagtg tctgattttg gtggtgatgt cctatgatcg 660 ctatgcggac atctgccacc ccttacgtta caatagcctc atgagctgga gagtgtgcac 720 tgtcctggct gtggcttcct gggtgttcag cttcctcctg gctctggtcc ctttagttct 780 catcctgagc ctgcccttct gcgggcctca tgaaatcaac cacttcttct gtgaaatcct 840 gtctgtcctc aagttggcct gtgctgacac ctggctcaac caggtggtca tctttgcagc 900 ctgcgtgttc atcctggtgg ggccactctg cctggtgctg gtctcctact tgcgcatcct 960 ggccgccatc ttgaggatcc agtctgggga gggccgcaga aaggccttct ccacctgctc 1020 ctcccacctt tgcgtggtgg gactcttctt tggcagcgcc attgtcacgt acatggcccc 1080 caagtcccgc catcctgagg agcagcagaa agttctttcc ctgttttaca gccttttcaa 1140 tccaatgctg aaccccctga tatatagcct aaggaatgca gaggtcaagg gcgccctgag 1200 gagggcactg aggaaggaga ggctgacgtg agacatctca aagggaacca tggggaggga 1260 gccttgctcc ctgcaaaata tagaagttgg cttttttttt ttgtcttctg ctagaataaa 1320 tgctacctta aactggaata ctatagacct a 1351 <210> 25 <211> 1156 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 72458414CB1 <400> 25 atcacttttc tgggttgtgc tgcacagatg tggttctttg gtctctttga ggcagctgag 60 tgttttctcc tggctgccat ggcatatgac cggtatgtgg ccatctgcaa gcccttgttg 120 tatacgctca ttatgtctca gcaggtctgt atgcagctgg tggtagggcc ttatgccatg 180 gctcttataa gcaccatgac tcatacaatt ttcacttttg cttacccttt tgtggttcaa 240 atattatcaa tcactttttc tgtgatattt ttccactgct ttccctagca tgtgcagaca 300 cctgggtgaa taaatttgtg ctgtttgtct tggctggagc tataggagta ctcagtggtc 360 tgatcatcat ggtctcctat atttgcatcc tgatgaccat cttgaagatc cagactgctg 420 atgggaagca aaaagctttc ttcacctgtt tttctcacct tgcggctgtc tccatcctgt 480 atgggactct tttcttgatt tatgttcggc caagttcaag ttcctccctg ggtatctata 540 aagtgatttc tctattttat actgtggtaa tccccatggt taaccccctt atttacagct 600 tgaggaataa ggaggtgaaa gatgcattca gaagaaaaat tgagaggaaa aaatttatta 660 tagcaagaga agaggagatg aggatgaaga accagtagtg acttacagtg cacatgtgtt 720 gtgatgatga ttaagtttca gcatacatca ttccacattt atagatgaat gtgccctgaa 780 caggagagag gttccgcacc cattctggga gtggtgtcat cagtatggaa cactgcatca 840 ccatgaaggc atgaggtgtg aaagtgttgg gacactcttc ttttcctgct cttggacatc 900 agaactccag gctctccagc gtttggactc caggatttgc accagtggtc ccaggttctc 960 agtcctcaag cctcagactt agagttacaa catcagcttc cctgtttttg aggcctttag 1020 gcttggactg agccatgcta ctgtcatcct aaggtctcta gcttgcagat tatctgcctg 1080 ggacttaaac ttcgtaatca tgaaccagtt cccctaataa attccctctt aggcatctaa 1140 aaaaaaaaaa aaaggg 1156 <210> 26 <211> 1108 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3035070CB1 <400> 26 gttaacccaa gaggaaaata ttgttcaaaa tatggatgaa ttaactgtct tgaatttgct 60 atatctgcag atggagagca aactaaaaga gaaaaatcaa atgtgactac aataatggaa 120 tttgttcttt tggggttttc tgatattccc aatctccact ggatgctttt tagtatattt 180 ttacttatgt atttgatgat cctgatgtgc aatggcatca taatactact aataaaaatt 240 caccccgctc tccagactcc catgtatttt tttcttagca atttttccct tttggaaatc 300 tgttatgtaa caatcattat cccaagaatg ctcatggaca tttggactca gaaaggaaat 360 atttctttgt ttgcttgtgc tacacaaatg tgtttttttc ttatgcttgg aggcacggag 420 tgtctccttc tgacagtgat ggcctatgac cgctacgtgg ctatttgtaa gcctttgcag 480 tatcctctag tgatgaacca caaagtctgc attcagctga taatagcttc ctggaccatc 540 acaattcctg tagtaattgg ggaaacatgc caaattttcc ttttgccctt ttgcggaact 600 aacacaatta atcatttctt ttgtgacatc ccgccaatac tcaagcttgc ttgtggaaac 660 atatttgtga atgagataac agtccatgta gtagcggtgg tgtttatcac ggtgccattt 720 ctgttgattg ttgtctctta tggcaaaatt atctccaaca ttttgaaatt gtcatcagcc 780 agaggaaagg ctaaagcctt ctccacctgc tcatctcacc taatagttgt aatcttattc 840 tttggagcag gtactatcac ttatttacag cccaaaccac atcagtttca aaggatgggg 900 aaactgattt ctcttttcta caccattctg attccaactt tgaatcctat tatatatacc 960 ctgaggaaca aagatatcat ggtggcattg agaaaattac tagctaagtt attaacatga 1020 gatgaagact tgaaattaca gaaataattt ctttataggt ttgtcatgtg gcttcagtga 1080 attttttttt aactttgtcc ctatggta 1108 <210> 27 <211> 2596 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 90023411CB1 <400> 27 cgttgctgtc gagagctcct ggcgtgggca aggctggcca aggatggcga cgcccagggg 60 cctgggggcc ctgctcctgc tcctcctgct cccgacctca ggtcaggaaa agcccaccga 120 agggccaaga aacacctgcc tggggagcaa caacatgtac gacatcttca acttgaatga 180 caaggctttg tgcttcacca agtgtaggca gtcgggcagc gactcctgca atgtggaaaa 240 cttgcagaga tactggctaa actacgaggc ccatctgatg aaggaaggtt tgacgcagaa 300 ggtgaacacg cctttcctga aggctttggt ccagaacctc agcaccaaca ctgcagaaga 360 cttctatttc tctctggagc cctctcaggt tccgaggcag gtgatgaagg acgaggacaa 420 gccccctgac agagtgcgac ttcccaagag cctttttcga tccctgccag gcaacaggtc 480 tgtggtccgc ttggccgtca ccattctgga cattggtcca gggactctct tcaagggccc 540 ccggctcggc ctgggagatg gcagcggcgt gttgaacaat cgcctggtgg gtttgagtgt 600 gggacaaatg catgtcacca agctggctga gcctctggag atcgtcttct ctcaccagcg 660 accgccccct aacatgaccc tcacctgtgt attctgggat gtgactaaag ggaccactgg 720 agactggtct tctgagggct gctccacgga ggtcagacct gaggggaccg tgtgctgctg 780 tgaccacctg acctttttcg ccctgctcct gagacccacc ttggaccagt ccacggtgca 840 tatcctcaca cgcatctccc aggcgggctg tggggtctcc atgatcttcc tggccttcac 900 cattattctt tatgcctttc tgagccctgg cctaggacat ggtccccatc cccaacaagc 960 cgagtgtgac ttgccagctg gccgcattgc tggaccctgt gagcggggct tggagcgcca 1020 ggagaaatta ccaccagctc cccagggtgg ggcctggcat ctcaggcttt cccgggagag 1080 gttcaagtca gaagatgccc caaagatcca cgtggccctg ggtggcagcc tgttcctcct 1140 gaatctggcc ttcttggtca atgtggggag tggctcaaag gggtctgatg ctgcctgctg 1200 ggcccggggg gctgtcttcc actacttcct gctctgtgcc ttcacctgga tgggccttga 1260 agccttccac ctctacctgc tcgctgtcag ggtcttcaac acctacttcg ggcactactt 1320 cctgaagctg agcctggtgg gctggggcct gcccgccctg atggtcatcg gcactgggag 1380 tgccaacagc tacggcctct acaccatccg tgatagggag aaccgcacct ctctggagct 1440 atgctggttc cgtgaaggga caaccatgta cgccctctat atcaccgtcc acggctactt 1500 cctcatcacc ttcctctttg gcatggtggt cctggccctg gtggtctgga agatcttcac 1560 cctgtcccgt gctacagcgg tcaaggagcg ggggaagaac cggaagaagg tgctcaccct 1620 gctgggcctc tcgagcctgg tgggtgtgac atgggggttg gccatcttca ccccgttggg 1680 cctctccacc gtctacatct ttgcactttt caactccttg caaggtgtct tcatctgctg 1740 ctggttcacc atcctttacc tcccaagtca gagcaccaca gtctcctcct ctactgcaag 1800 attggaccag gcccactccg catctcaaga ataggaaggc acggccctgc aatatggact 1860 cagctctggc tctctgtgtg accttgggcg gctccgtgcc tctctctgta ctccctcagt 1920 ttccttctct gtacaatgtg gctggggagg gagaggatgg gaccaggttg gaccacgtgg 1980 catcagaggt cccatccaga tccaactata ggtccaagag tccacgtaag caggtttgca 2040 aggctctaaa gttcctatag tcctgagacc ccctgccagc aaagagtgac agtcacctcc 2100 atgccctgcc ctcattgcaa agccctcact caccttctgg tctcagcaag ggaggagagt 2160 ctgttgctgg catagccctg gaaggagccc ccagcctctc ccctcctcct ccttgtcact 2220 ggcctcccac aactcccctt ctggctgcct gtaaccttga ggggcattca ggaggccagc 2280 gttccctcag gcactggggg tttgttttgg ggggtgggag ttgatcctcc cacccagtct 2340 gcccctggtc tctgcccatc caatcagagc ccaccctcct ggaagagacc cccgtgttca 2400 gagtgctggc agccctgcac gtgtccaggg acactgcatt tcaaagaacc actgagtggg 2460 tgagctacct tgggcaaacc ccccactcct gactctgact gccacgtggg tggcccgacc 2520 tctgacctgc tgtcatcgta gaggtagaaa gcaaacaatc tggggctcag cacacctggg 2580 ggtgctccca ctcatt 2596 <210> 28 <211> 2174 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90023412CB1 <400> 28 cgttgctgtc gagagctcct ggcgtgggca aggctggcca aggatggcga cgcccagggg 60 cctgggggcc ctgctcctgc tcctcctgct cccgacctca ggtcaggaaa agcccaccga 120 agggccaaga aacacctgcc tggggagcaa caacatgtac gacatcttca acttgaatga 180 caaggctttg tgcttcacca agtgcaggca gtcgggcagc gactcctgca atgtggaaaa 240 cttgcagaga tactggctaa actacgaggc ccatctgatg aaggaaggtt tgacgcagaa 300 ggtgaacacg cctttcctga aggctttggt ccagaacctc agcaccaaca ctgcagaaga 360 cttctatttc tctctggagc cctctcaggt tccgaggcag gtgatgaagg acgaggacaa 420 gccccctgac agagtgcgac ttcccaagag cctttttcga tccctgccag gcaacaggtc 480 tgtggtccgc ttggccgtca ccattctgga cattggtcca gggactctct tcaagggccc 540 ccggctcggc ctgggagatg gcagcggcgt gttgaacaat cgcctggtgg gtttgagtgt 600 gggacaaatg catgtcacca agctggctga gcctctggag atcgtcttct ctcaccagcg 660 accgccccct aacatgaccc tcacctgtgt attctgggat gtgactaaag ggaccactgg 720 agactggtct tctgagggct gctccacgga ggtcagacct gaggggaccg tgtgctgctg 780 tgaccacctg acctttttcg ccctgctcct gagacccacc ttggaccagt ccacggtgca 840 tatcctcaca cgcatctccc aggcgggctg tggggtctcc atgatcttcc tggccttcac 900 cattattctt tatgcctttc tgaggcctgc ccgccctgat ggtcatcggc actgggagtg 960 ccaacagcta cggcctctac accatccgtg atagggagaa ccgcacctct ctggagctat 1020 gctggttccg tgaagggaca gccatgtacg ccctctatat caccgtccac ggctacttcc 1080 tcatcacctt cctctttggc atggtggtcc tggccctggt ggtctggaag atcttcaccc 1140 tgtcccgtgc tacagcggtc aaggagcggg ggaagaaccg gaagaaggtg ctcaccctgc 1200 tgggcctctc gagcctggtg ggtgtgacat gggggttggc catcttcacc ccgttgggcc 1260 tctccaccgt ctacatcttt gcacttttca actccttgca aggtgtcttc atctgctgct 1320 ggttcaccat cctttacctc ccaagtcaga gcaccacagt ctcctcctct actgcaagat 1380 tggaccaggc ccactccgca tctcaagaat aggaaggcac ggccctgcaa tatggactca 1440 gctctggctc tctgtgtgac cttgggcagc tccgtgcctc tctctgtact ccctcagttt 1500 ccttctctgt acaatgtggc tggggaggga gaggatggga ccaggttgga ccacgtggca 1560 tcagaggtcc catccagatc caactatagg tccaagagtc cacgtaagca ggtttgcaag 1620 gctctaaagt tcctatagtc ctgagacccc ctgccagcaa agagtgacag tcacctccat 1680 gccctgccct cattgcaaag ccctcactca ccttctggtc tcagcaaggg aggagagtct 1740 gttgctggca tagccctgga aggagccccc agcctctccc ctcctcctcc ttgtcactgg 1800 cctcccacaa ctccccttct ggctgcctgt aaccttgagg ggcattcagg aggccagcgt 1860 tccctcaggc actgggggtt tgttttgggg ggtgggagtt gatcctccca cccagtctgc 1920 ccctggtctc tgcccatcca atcagagccc accctcctgg aagagacccc cgtgttcaga 1980 gtgctggcag ccctgcacgt gtccagggac actgcatttc aaagaaccac tgagtgggtg 2040 agctaccttg ggcaaacccc ccactcctga ctctgactgc cacgtgggtg gcccgacctc 2100 tgacctgctg tcatcgtaga ggtagaaagc aaacaatctg gggctcagca cacctggggg 2160 tgctcccact catt 2174 <210> 29 <211> 2079 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 90023428CB1 <400> 29 cgttgctgtc gagagctcct ggcgtgggca aggctggcca aggatggcga cgcccagtgg 60 cctgggggcc ctgctcctgc tcctcctgct cccgacctca ggtcaggaaa agcccaccga 120 agggccaaga aacacctgcc tggggagcaa caacatgtac gacatcttca acttgaatga 180 caaggctttg tgcttcacca agtgcaggca gtcgggcagc gactcctgca atgtggaaaa 240 cttgcagaga tactggctaa actacgaggc ccatctgatg aaggaaggtt tgacgcagaa 300 ggtgaacacg cctttcctga aggctttggt ccagaacctc agcaccaaca ctgcagaaga 360 cttctatttc tctctggagc cctctcaggt tccgaggcag gtgatgaagg acgaggacaa 420 gccccctgac agagtgcgac ttcccaagag cctttttcga tccctgccag gcaacaggtc 480 tgtggtccgc ttggccgtca ccattctgga cattggtcca gggactctct tcaagggccc 540 ccggctcggc ctgggagatg gcagcggcgt gttgaacaat cgcctggtgg gtttgagtgt 600 gggacaaatg catgtcacca agctggctga gcctctggag atcgtcttct ctcaccagcg 660 accgccccct aacatgaccc tcacctgtgt attctgggat gtgactaaag ggaccactgg 720 agactggtct tctgagggct gctccacgga ggtcagacct gaggggaccg tgtgctgctg 780 tgaccacctg acctttttcg ccctgctcct gagacccacc ttggaccagt ccacggtgca 840 tatcctcaca cgcatctccc aggcgggctg tggggtctcc atgatcttcc tggccttcac 900 cattattctt tatgcctttc tgagatgctg gttccgtgaa gggacaacca tgtacgccct 960 ctatatcacc gtccacggct acttcctcat caccttcctc tttggcatgg tggtcctggc 1020 cctggtggtc tggaagatct tcaccctgtc ccgtgctaca gcggtcaagg agcgggggaa 1080 gaaccggaag aaggtgctca ccctgctggg cctctcgagc ctggtgggtg tgacatgggg 1140 gttggccatc ttcaccccgt tgggcctctc caccgtctac atctttgcac ttttcaactc 1200 cttgcaaggt gtcttcatct gctgctggtt caccatcctt tacctcccaa gtcagagcac 1260 cacagtctcc tcctctactg caagattgga ccaggcccac tccgcatctc aagaatagga 1320 aggcacggcc ctgcaatatg gactcagctc tggctctctg tgtgaccttg ggcagctccg 1380 tgcctctctc tgtactccct cagtttcctt ctctgtacaa tgtggctggg gagggagagg 1440 atgggaccag gttggaccac gtggcatcag aggtcccatc cagatccaac tataggtcca 1500 agagtccacg taagcaggtt tgcaaggctc taaagttcct atagtcctga gaccccctgc 1560 cagcaaagag tgacagtcac ctccatgccc tgccctcatt gcaaagccct cactcacctt 1620 ctggtctcag caagggagga gagtctgttg ctggcatagc cctggaagga gcccccagcc 1680 tctcccctcc tcctccttgt cactggcctc ccacaactcc ccttctggct gcctgtaacc 1740 ttgaggggca ttcaggaggc cagcgttccc tcaggcactg ggggtttgtt ttggggggtg 1800 ggagttgatc ctcccaccca gtctgcccct ggtctctgcc catccaatca gagcccaccc 1860 tcctggaaga gacccccgtg ttcagagtgc tggcagccct gcacgtgtcc agggacactg 1920 catttcaaag aaccactgag tgggtgagct accttgggca aaccccccac tcctgactct 1980 gactgccacg tgggtggccc gacctctgac ctgctgtcat cgtagaggta gaaagcaaac 2040 aatctggggc tcagcacacc tgggggtgct cccactcat 2079 <210> 30 <211> 3296 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90023320CB1 <400> 30 gtggctcaga tactgatact ttctttccaa acagcataag aagtgattga gccacaagta 60 tactgaagga agggctccct cgagttgtgg tgtgaagaga taaatcacca ggcccagtcg 120 aagaatatca gctgctgctt caggtgacct atagagattc caaggagaaa agagatttga 180 gaaattttct gaagctcttg aagcctccat tattatggtc acatgggcta attagaatta 240 tcagagcaaa ggctaccaca gactgcaaca gcctgaatgg agtcctgcag tgtacctgtg 300 aagacagcta cacctggttt cctccctcat gccttgatcc ccagaactgc taccttcaca 360 cggctggagc actcccaagc tgtgaatgtc atctcaacaa cctcagccag agtgtcaatt 420 tctgtgagag aacaaagatt tggggcactt tcaaaattaa tgaaaggttt acaaatgacc 480 ttttgaattc atcttctgct atatactcca aatatgcaaa tggaattgaa attcaactta 540 aaaaagcata tgaaagaatt caaggttttg agtcggttca ggtcacccaa tttcgaaatg 600 gaagcatcgt tgctgggtat gaagttgttg gctccagcag tgcatctgaa ctgctgtcag 660 ccattgaaca tgttgccgag aaggctaaga cagcccttca caagctgttt ccattagaag 720 acggctcttt cagagtgttc ggaaaagccc agtgtaatga cattgtcttt ggatttgggt 780 ccaaggatga tgaatatacc ctgccctgca gcagtggcta caggggaaac atcacagcca 840 agtgtgagtc ctctgggtgg caggtcatca gggagacttg tgtgctctct ctgcttgaag 900 aactgaacaa gaatttcagt atgattgtag gcaatgccac tgaggcagct gtgtcatcct 960 tcgtgcaaaa tctttctgtc atcattcggc aaaacccatc aaccacagtg gggaatctgg 1020 cttcggtggt gtcgattctg agcaatattt catctctgtc actggccagc catttcaggg 1080 tgtccaattc aacaatggag gatgtcatca gtatagctga caatatcctt aattcagcct 1140 cagtaaccaa ctggacagtc ttactgcggg aagaaaagta tgccagctca cggttactag 1200 agacattaga aaacatcagc actctggtgc ctccgacagc tcttcctctg aatttttctc 1260 ggaaattcat tgactggaaa gggattccag tgaacaaaag ccsaactcaaa aggggttaca 1320 gctatcagat taaaatgtgt ccccaaaata catctattcc catcagaggc cgtgtgttaa 1380 ttgggtcaga ccaattccag agatcccttc cagaaactat tatcagcatg gcctcgttga 1440 ctctggggaa cattctaccc gtttccaaaa atggaaatgc tcaggtcaat ggacctgtga 1500 tatccacggt tattcaaaac tattccataa atgaagtttt cctatttttt tccaagatag 1560 agtcaaacct gagccagcct cattgtgtgt tttgggattt cagtcatttg cagtggaacg 1620 atgcaggctg ccacctagtg aatgaaactc aagacatcgt gacgtgccaa tgtactcact 1680 tgacctcctt ctccatattg atgtcacctt ttgtcccctc tacaatcttc cccgttgtaa 1740 aatggatcac ctatgtggga ctgggtatct ccattggaag tctcatttta tgcctgatca 1800 tcgaggcttt gttttggaag cagattaaaa aaagccaaac ctctcacaca cgtcgtattt 1860 gcatggtgaa catagccctg tccctcttga ttgctgatgt ctggtttatt gttggtgcca 1920 cagtggacac cacggtgaac ccttctggag tctgcacagc tgctgtgttc tttacacact 1980 tcttctacct ctctttgttc ttctggatgc tcatgcttgg catcctgctg gcttaccgga 2040 tcatcctcgt gttccatcac atggcccagc atttgatgat ggctgttgga ttttgcctgg 2100 gttatgggtg ccctctcatt atatctgtca ttaccattgc tgtcacgcaa cctagcaata 2160 cctacaaaag gaaagatgtg tgttggctta actggtccaa tggaagcaaa ccactcctgg 2220 cttttgttgt ccctgcactg gctattgtgg ctgtgaactt cgttgtggtg ctgctagttc 2280 tcacaaagct ctggaggccg actgttgggg aaagactgag tcgggatgac aaggccacca 2340 tcatccgcgt ggggaagagc ctcctcattc tgacccctct gctagggctc acctggggct 2400 ttggaatagg aacaatagtg gacagccaga atctggcttg gcatgttatt tttgctttac 2460 tcaatgcatt ccagggattt tttatcttat gctttggaat actcttggac agtaagctgc 2520 gacaacttct gttcaacaag ttgtctgcct taagttcttg gaagcaaaca gaaaagcaaa 2580 actcatcaga tttatctgcc aaacccaaat tctcaaagcc tttcaaccca ctgcaaaaca 2640 aaggccatta tgcattttct catactggag attcctccga caacatcatg ctaactcagt 2700 ttgtctcaaa tgaataaggc aaggaatcat aaaatcaaga aaaaatttcc agaacaactt 2760 gacatttaga gacaaatgtc aatgaagaaa ttatgctcag tattcgatcg ggttttctga 2820 tttaggggtc tgggaataaa acaagaatgt ctcagtggct tcattactgc tcccttttgt 2880 cttcaattaa atgaaaagaa gatttatttc catgtgattt gattcaaaga aagtgctcca 2940 taaatgcaga agagtaggtt ttgttggaaa tcgtgtcagt tgtaccctga ccataaaata 3000 tggtttctat tttcataaaa cagcattatt cacatggcat ttccaataat ctggattgaa 3060 ggaagaaaat tttatgaaat agctttagat aaattaatag gccacgttca ttttcttgtc 3120 aaaaagttac tggtgggggg atggtgggaa aaagttatta gtgcaaattt cctagagaaa 3180 aaaccatttc tctttcaaat tttccagttg aattttatgt tcgcttttgc ttcttaggtt 3240 ctatcactta atattgaaag ttaatcagaa ataaaatgta aacttctatt taaaaa 3296 <210> 31 <211> 3112 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 90023328CB1 <400> 31 gtggctcaga tactgatact ttctttccaa acagcataag aagtgattga gccacaagta 60 tactgaagga agggctccct cgagttctgg tgtgaagaga taaatcacca gaaaaatgat 120 ggcatcaaaa caaaaaaaga actcattgtg aataagaaaa aacatctagg cccagtcgaa 180 gaatatcagc tgctgcttca ggtgacctat agagattcca aggagaaaag agatttgaga 240 aattttctga agctcttgaa gcctccatta ttatggtcac atgggctaat tagaattatc 300 agagcaaagg ctaccacaga ctgcaacagc ctgaatggag tcctgcagtg tacctgtgaa 360 gacagctaca cctggtttcc tccctcatgc cttgatcccc agaactgcta ccttcacacg 420 gctggagcac tcccaagctg tgaatgtcat ctcaacaacc tcagccagag tgtcaatttc 480 tgtgagagaa caaagatttg gggcactttc aaaattaatg aaaggtttac aaatgacctt 540 ttgaattcat cttctgctat atactccaaa tatgcaaatg gaattgaaat tcaaaaatgg 600 aagcatcgtt gctgggtatg aagttgttgg ctccagcagt gcatctgaac tgctgtcagc 660 cattgaacat gttgccgaga aggctaagac agcccttcac aagctgtttc cattagaaga 720 cggctctttc agagtgttcg gaaaaggcaa tgccactgag gcagctgtgt catccttcgt 780 gtaaaatctt tctgtcatca ttcggcaaaa cccatcaacc acagtgggga atctggcttc 840 ggtggtgtcg attctgagca atatttcatc tctgtcactg gccagccatt tcagggtgtc 900 caattcaaca atggaggatg tcatcagtat agctgacaat atccttaatt cagcctcagt 960 aaccaactgg acagtcttac tgcgggaaga aaagtatgcc agctcacggt tactagagac 1020 attagaaaac atcagcactc tggtgcctcc gacagctctt cctctgaatt tttctcggaa 1080 attcattgac tggaaaggga ttccagtgaa caaaagccaa ctcaaaaggg gttacagcta 1140 tcagattaaa atgtgtcccc aaaatacatc tattcccatc agaggccgtg tgttaattgg 1200 gtcagaccaa ttccagagat cccttccaga aactattatc agcatggcct cgttgactct 1260 ggggaacatt ctacccgttt ccaaaaatgg aaatgctcag gtcaatggac ctgtgatatc 1320 cacggttatt caaaactatt ccataaatga agttttccta tttttttcca agatagagtc 1380 aaacctgagc cagcctcatt gtgtgttttg ggatttcagt catttgcagt ggaacgatgc 1440 aggctgccac ctagtgaatg aaactcaaga catcgtgacg tgccaatgta ctcacttgac 1500 ctccttctcc atattgatgt caccttttgt cccctctaca atcttccccg ttgtaaaatg 1560 gatcacctat gtgggactgg gtatctccat tggaagtctc attttatgcc tgatcatcga 1620 ggctttgttt tggaagcaga ttaaaaaaag ccaaacctct cacacacgtc gtatttgcat 1680 ggtgaacata gccctgtccc tcttgattgc tgatgtctgg tttattgttg gtgccacagt 1740 ggacaccacg gtgaaccctt ctggagtctg cacagctgct gtgttcttta cacacttctt 1800 ctacctctct ttgttcttct ggatgctcat gcttggcatc ctgctggctt accggatcat 1860 cctcgtgttc catcacatgg cccagcattt gatgatggct gttggatttt gcctgggtta 1920 tgggtgccct ctcattatat ctgtcattac cattgctgtc acgcaaccta gcaataccta 1980 caaaaggaaa gatgtgtgtt ggcttaactg gtccaatgga agcaaaccac tcctggcttt 2040 tgttgtccct gcactggcta ttgtggctgt gaacttcgtt gtggtgctgc tagttctcac 2100 aaagctctgg aggccgactg ttggggaaag actgagtcgg gatgacaagg ccaccatcat 2160 ccgcgtgggg aagagcctcc tcattctgac ccctctgcta gggctcacct ggggctttgg 2220 aataggaaca atagtggaca gccagaatct ggcttggcat gttatttttg ctttactcaa 2280 tgcattccag ggatttttta tcttatgctt tggaatactc ttggacagta agctgcgaca 2340 acttctgttc aacaagttgt ctgccttaag ttcttggaag caaacagaaa agcaaaactc 2400 atcagattta tctgccaaac ccaaattctc aaagcctttc aacccactgc aaaacaaagg 2460 ccattatgca ttttctcata ctggagattc ctccgacaac atcatgctaa ctcagtttgt 2520 ctcaaatgaa taaggcaagg aatcataaaa tcaagaaaaa atttccagaa caacttgaca 2580 tttagagaca aatgtcaatg aagaaattat gctcagtatt cgatcgggtt ttctgattta 2640 ggggtctggg aataaaacaa gaatgtctca gtggcttcat tactgctccc ttttgtcttc 2700 aattaaatga aaagaagatt tatttccatg tgatttgatt caaagaaagt gctccataaa 2760 tgcagaagag taggttttgt tggaaatcgt gtcagttgta ccctgaccat aaaatatggt 2820 ttctattttc ataaaacagc attattcaca tggcatttcc aataatctgg attgaaggaa 2880 gaaaatttta tgaaatagct ttagataaat taataggcca cgttcatttt cttgtcaaaa 2940 agttactggt ggggggatgg tgggaaaaag ttattagtgc aaatttccta gagaaaaaac 3000 catttctctt tcaaattttc cagttgaatt ttatgttcgc ttttgcttct taggttctat 3060 cacttaatat tgaaagttaa tcagaaataa aatgtaaact tctatttaaa as 3112 <210> 32 <211> 3455 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7480155CB1 <400> 32 ggccgagtcc cgcctcccgc cagcgggggc gaggacctgc gacgcgcacc cctgcctggc 60 ccggtctcct cagcaccagc cccacgcaca ccctacttcc tcagcttctc gccctcaccc 120 tgccaacttc cctgcgagga gggacctgcc gccagcctgc ttcctcgtcc gcaggccctg 180 cgctgaacgc tgccgcgccc agggttcacc ttgcgccgtc gggaaagccc atgaactctc 240 cagaaacggc gtaaaggagg gtcccgccgc ggcgcagggc tggggcgcct gggttccccc 300 tgggtggagc agcggcagca gagcgggaaa gtggtggagg atgatcttgc ggccaaaggg 360 gacctcggcg cagtaatgtc aacatgatgt ttcgctcaga tcgaatgtgg agctgccatt 420 ggaaatggaa gcccagtcct ctcctgttct tatttgcttt atatatcatg tgtgttcctc 480 actcagcagt gtggggatgt gccaactgcc gagtggtttt gtccaaccct tctgggacct 540 ttacttctcc atgctaccct aacgactacc caaacagcca ggcttgcatg tggacgctcc 600 gagcccccac cggttatatc attcagataa catttaacga ctttgacatt gaagaagctc 660 ccaattgcat ttatgactca ttatcccttg ataatggaga gagccagact aaattttgtg 720 gagcaactgc caaaggccta tcatttaact caagtgcgaa tgagatgcat gtgtcctttt 780 caagtgactt tagcatccag aagaaaggtt tcaatgccag ctacatcaga gttgccgtgt 840 ccttaaggaa tcaaaaggtc attttacccc agacatcaga tgcttaccag gtatctgttg 900 caaaaagcat ctctattcca gagctcagtg ctttcacact ctgctttgaa gcaaccaaag 960 ttggccatga agacagtgat tggacagctt tctcctactc aaatgcatcc ttcacacaat 1020 tgctcagttt tggaaaggcc aagagtggct actttctatc catttctgat tcaaaatgtt 1080 tgttgaataa tgcattacct gtcaaagaaa aagaagacat ttttgcagaa agctttgaac 1140 agctctgcct tgtttggaat aattctttgg gctctattgg tgtaaatttc aaaagaaact 1200 atgaaacagt tccatgtgat tctaccatta gtaaagttat tcctgggaat gggaaattgt 1260 tgttgggctc caatcaaaat gaaattgtct ctctaaaagg ggacatttat aactttcgac 1320 tttggaattt taccatgaat gccaaaatcc tctccaacct cagctgtaat gtgaaaggga 1380 atgtagtcga ctggcaaaat gacttctgga atatcccaaa cctagctctg aaagctgaaa 1440 gcaacctaag ctgtggttcc tacctgatcc cgctcccagc agcagaactg gccagctgtg 1500 cagacctggg gaccctctgt caagatggaa ttatctatag aatatccgta gtgattcaga 1560 acatccttcg tcaccctgag gtaaaagtac agagcaaggt ggcagaatgg tctcaacagg 1620 tgatggtcat ccctctacat ccaaatactc tgtttctcct accaaatgac aagcaaccaa 1680 tgaacaacaa caacagttct atccgaaatg tttccctagt ttacaatgct accaacaata 1740 ctaatttgga aggaaaaatc attcagcaga agctcctaaa aaataatgag tccttggatg 1800 aaggcttgag gctacataca gtgaatgtga gacaactggg tcattgtctt gccatggagg 1860 aacccaaagg ctactactgg ccatctatcc aaccttctga atacgttctt ccttgtccag 1920 acaagcctgg cttttctgct tctcggatat gtttttacaa tgctaccaac ccattggtaa 1980 cctactgggg acctgttgat atctccaact gtttaaaaga agcaaatgaa gttgctaacc 2040 agattttaaa tttaactgct gatgggcaga acttaacctc agccaatatt accaacattg 2100 tggaacaggt caaaagaatt gtgaataaag aagaaaacat tgatataaca cttggctcaa 2160 ctctaatgaa tatattttct aatatcttaa gcagttcaga cagtgacttg cttgagtcat 2220 cttctgaagc tttaaaaaca attgatgaat tggccttcaa gatagaccta aatagcacat 2280 cacatgtgaa tattacaact cggaacttgg ctctcagcgt atcatccctg ttaccaggga 2340 caaatgcaat ttcaaatttt agcattggtc ttccaagcaa taatgaatcg tatttccaga 2400 tggattttga gagtggacaa gtggatccac tggcatctgt aattttgcct ccaaacttac 2460 ttgagaattt aagtccagaa gattctgtat tagttagaag agcacagttt actttcttca 2520 acaaaactgg acttttccag attctagaag gcccactgcc agcaaacact ggtgacctct 2580 gtttgccttt acaagtacaa cctgatcgag agcaggatgt aggaccccaa agaaaaactt 2640 tagtgagtta tgtgatggcg tgcagtattg gaaacattac tatccagaat ctgaaggatc 2700 ctgttcaaat aaaaatcaaa catacaagaa ctcaggcaac cgtctcctct ccaatttctg 2760 ttgagagtcc tggtggtttt gatcttcagg ttataccggt ttgcagaaag cttgggaaag 2820 aagatggctg gatcacctcc ttcaatgtgg atggactttg cattgctgtt gcagtcctgt 2880 tgcatttctt ccttctggca acctttacct ggatggggct agaagcaatt cacatgtaca 2940 ttgctctagt taaagtattt aacacttaca ttcgccgata cattctaaaa ttctgcatca 3000 ttggctgggg tttgcctgcc ttagtggtgt cagttgttct agcgagcaga aacaacaatg 3060 aagtctatgg aaaagaaagt tatgggaaag aaaaaggtga tgaattctgt tggattcaag 3120 atccagtcat attttatgtg acctgtgctg ggtattttgg agtcatgttt tttctgaaca 3180 ttgccatgtt cattgtggta atggtgcaga tctgtgggag gaatggcaag agaagcaacc 3240 ggaccctgag agaagaagtg ttaaggaacc tgcgcagtgt ggttagcttg acctttctgt 3300 tgggcatgac atggggtttt gcattctttg cctggggacc cttaaatatc cccttcatgt 3360 acctcttctc catcttcaat tcattacaag aatttcctgg cagtttcagt aaccttcaac 3420 agctacagtt gtctgttcag ctctgtttcc attaa 3455 <210> 33 <211> 1100 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504302CB1 <400> 33 tatgtatttt ccccctgaca aattatgagt ctatcttttt ttcacagtag attttgtgaa 60 aaagaaagaa acatatggct gatgataatt ttacagttgt cactgagttt attcttttgg 120 gattgacaga tcatgctgaa ctaaaagctg tgctttttgt ggtgttcctg gtgatttacg 180 ccattacctt gttgaggaat ctgggcatga tcctcttaat ccaaatcacc tccaaactcc 240 acacacccat gtacttttta ctcagctgtc tttcatttgt ggatgcctgc tattcatctg 300 caattgcacc caaaatgctg gtgaacctcc tggttgtgaa ggcaacaatt tctttctctg 360 cttgcatggt acagcatttg tgtttcggag tgttcatcac cacagaaggc ttcttactgt 420 cagtgatggc ctatgaccgc tatgtggcca ttgtgagtcc cttgctttac actgtagcca 480 tgtctgatag aaagtgtgtg gagcttgtca caggatcatg gataggtgga atagttaaca 540 cattaatcca cacaatcagc ttgaggagac tgtccttttg taggctaaat gctgtcagcc 600 acttcttctg tgacattcct tcactgctaa agctgtcatg ttctgacacc tccatgaatg 660 agttgttgct gttaaccttc tccggagtca ttgccatggc caccttcttg actgtgatca 720 tttcctacat cttcattgct tttgctagcc taaggatcca ctcagcatca ggcagacagc 780 aagccttctc cacctgtgcc tctcacctga ctgctgtgac catattctat ggtaccttaa 840 tctttagcta cattcagcca agctcccagt attttgtgga acaagagaaa gtggtttcta 900 tgttctatac gctagggatt cccatgttaa acctgttgat acacagtttg agaaacaagg 960 acgtaaagga ggcagtgaaa agggccatag aaatgaaaca tttcctctgt taatttcaag 1020 tcactattaa tcctacaaca ttctgtcatt cagttctcac ttccaagaat tccgtgaatg 1080 ccaaagcttc tcaagacttt 1100 <210> 34 <211> 1150 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504308CB1 <400> 34 tatgccccaa gaaaaaaaca aggatacaat tgaatggact ccactttcac aggctataac 60 ctttataacc tgcaagtaaa aactgaaatg gacaagttgt catcaggttt ggatatatac 120 aggaatccac tgaagaacaa gactgaagtc accatgttta tattgacagg cttcacagat 180 gattttgagc tgcaagtctt cctattttta ctattttttg caatctatct ctttaccttg 240 ataggcaatt tagggctggt tgtgttggtc attgaggatt cctggctcca caaccccatg 300 tattattttc ttagtgtttt atcattcttg gatgcttgct attctacagt tgtcactcca 360 aaaatgttgg tcaatttcct ggcaaaaaat aaatccattt catttatcgg atgtgcaaca 420 cagatgcttc tttttgttac ttttggaact acagaatgtt ttctcttggc tgcaatggct 480 tatgatcact atgtagccat ctacaaccct ctcctgtatt cagtgagcat gtcacccaga 540 gtctatgtgc cactcatcac tgcttcctac gttgctggca ttttacatgc tactatacat 600 atagtggcta catttagcct gtccttctgt ggatccaatg aaattaggca tgtcttttgt 660 gatatgcctc ctctccttgc tatttcttgt tctgacactc acacaaacca gcttctactc 720 ttctactttg tgggttctat tgagatagtc actatcctga ttgtcctcat ttcctgtgat 780 ttcattctgt tgtccattct gaagatgcat tctgctaagg gaaggcaaaa ggccttctct 840 acatgtggct ctcacctaac tggagtgaca atttatcatg gaacaattct cgtcagttat 900 atgagaccaa gttccagcta tgcttcagac catgacatca tagtgtcaat attttacaca 960 attgtgattc ccaagttgaa tcccatcatc tatagtttga ggaacaaaga agtaaaaaag 1020 gcagtgaaga aaatgttgaa attggtttac aaatgaagaa tatatttaaa attgagtaaa 1080 cctgaaaaaa atgttgagtg tcagagttca catctctata ttttagttaa agtatttgca 1140 tatcaaagaa 1150 <210> 35 <211> 1034 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7504312CB1 <400> 35 atggaaaatt acaatcaaac atcaactgat ttcatcttat tggggctgtt cccacaatca 60 agaattggcc ttttcgtatt caccctcatt tttctcattt tcctaatggc tctaattgga 120 aatctatcca tgattcttct catctttttg gacatccatc tccacacacc tatgtatttc 180 ctacttagtc agctctccct cattgaccta aattacatct ccaccattgt tccaaagatg 240 gtttatgatt ttctgtatgg aaacaagtct atctccttca ctggatgtgg gattcagagt 300 ttcttcttct tgactttagc agttgcagaa gggctgctcc tgacatcaat ggcctatgat 360 cgttatgtgg ccatttgctt tcctctccac tatcccatcc gtataagcaa aagagtgtgt 420 gtgatgatga taacaggatc ttggatgata agctctatca actcttgtgc tcacacagta 480 tatgcactct gtatcccata ttgcaagtcc agagccatca atcatttttt ctgtgatgtt 540 ccagctatgt tgacgctagc ctgcacagac acttgggtct atgagagcac agtgtttttg 600 agcagcacca tctttcttgt gcttcctttc actggtattg catgttccta tggccgggtt 660 ctccttgctg tctaccgcat gcactctgca gaagggagga agaaggccta ttcaacctgt 720 agcacccacc tcactgtagt gtccttctac tatgcaccct ttgcttatac ctatgtacgt 780 ccaagatccc tgcgatctcc aacagaggac aagattctgg ctgttttcta caccatcctc 840 accccaatgc tcaaccccat catctacagc ctgagaaaca aggaggtgat gggggccctg 900 acacaagtga ttcagaaaat cttctcagtg aaaatgtaga catacgttct gtgttagagt 960 caaagcgcta ggttcatatc aacttagtag tgtacagcag tgaagaaaaa cattattaca 1020 tgcccagtgt gtca 1034 <210> 36 <211> 1100 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504320CB1 <400> 36 ttataaggta ctgggtaaag ttttaccaaa tcaatgaact ggttttgtgt tactaggtaa 60 aaaacatatt catcatggca tgggagaatc agaccttcaa ctctgacttc atcctcctgg 120 gaatcttcaa tcacagcccc acccacacct tcctcttctt tctggtcctg gccatctttt 180 cagtggcctt catgggaaac tctgtcatgg ttctcctcat ctacctggac acccagctcc 240 acacccccat gtacttcctc ctcagtcaac tgttcctcat ggacctcatg ctcatctgct 300 ctaccgtacc caagatggcc ttcaactact tgtctggcag caagtccatt tctatggctg 360 gttgtgccac acaaattttc ttctatgtat cactgcttgg ctccgaatgc tttctgttgg 420 ctgttatgtc ttatgaccgc tatattgcca tttgccaccc tctaagatac accaatctca 480 tgagacccaa aatttgtgga cttatgactg ccttctcctg gatcctgggc tctatggatg 540 caatcattga tgctgtagcg acattttcct tctcctactg tgggtctcgg gaaatagccc 600 acttcttctg tgacttccct tccctactaa tcctctcatg caatgacaca tcaatatttg 660 aaaaggttct tttcatctgc tgtatagtaa tgattgtttt tcctgttgca atcatcatcg 720 cttcctatgc tcgagttatt ctggctgtca ttcacatggg atctggagag ggtcgtcgca 780 aagcttttac tacctgttcc tctcacctca tggtggtggg aatgtactat ggagcaggtt 840 tgttcatgta catacggccc acatctgatc gctcccctat gcaggacaag ctggtgtctg 900 tattctacac catcctcact cccatgctga atcccctcat ctacagcctc cgcaacaagg 960 aggtgaccag agcactcagg aaagtgttag gaaagggcaa gtgtggagag tgagtacttt 1020 gtaaacttta tgctttgctg tctgctaaat tcttttaact gtcccttttt ttccattaag 1080 tcctcaaaat agctttcatt 1100 <210> 37 <211> 1100 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7504404CB1 <400> 37 gaatatctgg gaaccttttc tcaatgaaga ataataaagt ccctcccaca accatggggt 60 ttttttgttt tgttttttct cttcctgcag aaactgactg gaggagatgt caggagaaaa 120 taattcctca gtgactgagt tcattctggc tgggctctca gaacagccag agctccagct 180 gcccctcttc ctcctgttct taggaatcta tgtggtcaca gtggtgggca acctgggcat 240 gaccacactg atttggctca gttctcacct gcacacccct atgtactatt tcctcagcag 300 tctgtccttc attgacttct gccattccac tgtcattacc cctaagatgc tggtgaactt 360 tgtgacagag aagaacatca tctcctaccc tgaatgcatg actcagctct acttcttcct 420 cgtttttgct attgcagagt gtcacatgtt ggctgcaatg gcgtatgacc gttacatggc 480 catctgtagc cccttgctgt acagtgtcat catatccaat aaggcttgct tttctctgat 540 tttaggggtg tatataatag gcctggtttg tgcatcagtt catacaggct gtatgtttag 600 ggttcaattc tgcaaatttg atttgattaa ccattatttc tgtgatcttc ttcccctcct 660 aaagctctct tgctctagta tctatgtcaa caaactactt attctatgtg ttggtgcatt 720 taacatcctt gtccccagcc tgaccatcct ttgctcttac atctttatta ttgccagcat 780 cctccacatt cgctccactg agggcaggtc caaagccttc agcacttgta gctcccacat 840 gttggcggtt gtaatctttt ttggatctgc agcattcatg tacttgcagc catcttcaat 900 cagctccatg gaccagggga aagtatcctc tgtgttttat actattattg tgcccatgtt 960 gaaccctctg atttatagcc tgaggaataa agatgtccat gtttccctga agaaaatgct 1020 acagagaaga acattattgt aaacagtaat aataagaaga tgatatatta atcctaagta 1080 gtggactgtt acattgtatg 1100 <210> 38 <211> 1148 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504536CB1 <400> 38 tgaatcctca gcatgggata ccctcctcca aaagtagaat agcaactcaa tccctacaaa 60 caccatggtt tttctttcct ccgtagaaac tgaccaaagg aaaatgtcag caggaaacca 120 ttcctcagtg actgagttca ttctggctgg gctctcagaa cagccagagc tccagctgcg 180 cctcttcctc ctgttcttag gaatctatgt ggtcacagtg gtgggcaact tgagcatgat 240 cacactgatt gggctcagtt ctcacctgca tacccccatg tactatttcc tcagtggtct 300 gtccttcatt gatatctgcc attccactat cattaccccc aaaatgctgg tgaactttgt 360 gacagagaag aacatcatct cctaccctga atgcatgact cagctttact tcttcctcat 420 ttttgctatt gcagagtgtc acatgttggc tgtaacggca tatgaccgct atgttgccat 480 ctgcagcccc ttgctgtaca atgtcatcat gtcctatcac cactgcttct ggctcacagt 540 gggagtttac attttaggca tccttggatc tacaattcac accggcttta tgttgagact 600 ctttttgtgc aagactaatg tgattaacca ttatttttgt gatctcttcc ctctcttggg 660 gctctcctgc tccagcacct acatcaatga attactggtt ctggtcttga gtgcatttaa 720 catcctgacg cctgccttaa ccatccttgc ttcttacatc tttatcattg ccagcatcct 780 ccgcattcgc tccactgagg gcaggtccaa agccttcagc acttgcagct cccacatctt 840 ggctgttgct gttttctttg ggtctgcagc attcatgtac ctgcagccat catctgtcag 900 ctccatggac caggggaaag tgtcctctgt gttttatact attgttgtgc ccatgctgaa 960 cccaatctat agcctaagaa ataaggatgt caaatttgcc ctgaagaaaa atctggacag 1020 caaagcatgt tcatgaatcc aattattgcc atgttgtcat atcaaaacag gctttcagtt 1080 tgattagata tgtaatatgt tagatttatt gtgcaaattt atcaaaatat agtgatgctc 1140 ttttatgt 1148 <210> 39 <211> 1097 <212> DNA

<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7505015CB1 <400> 39 agaaactgtg cagtcaaaca gttattcaga ccataaaagc ctcttctttc tctatttttc 60 catttgcacc atcacaccag gggaaattat ggagatgaga aatactaccc cagattttat 120 tctcctagga ctctttaacc acaccagagc ccaccaagtc ctcttcatga tgcttctggc 180 caccgttttg acctccctgt ttagcaatgc cctcatgatt ctcctgattc actgggacca 240 ccggctccac aggcccatgt acttcctcct gagccaactt tccctcatgg acatgatgct 300 ggtttccacc actgtgccca aaatggcggc tgactacttg accggaaata aggccatctc 360 ccgcgctggc tgtggtgtgc agatcttctt cctccccaca ctgggtggtg gagagtgctt 420 cctcttagca gccatggcct atgaccgcta tgcggctgtc tgccacccac tccgatatcc 480 cactctcatg agctggcagc tgtgcctgag gatgaccatg tcgtcctggc tcctgggtgc 540 agctgacggc ctcctgcagg ctgttgctac cctgagcttc ccatattgcg gtgcacacga 600 gatcgatcac ttcttctgcg aggcccccgt gttggtgcgt ttggcttgtg ctgacacttc 660 agtcttcgaa aacgccatgt acatctgctg tgtgttaatg ctcctggtcc ccttttccct 720 catcctgtcc tcctatggtc tcatcctcgc tgctgttctg ctcatgcgct ctacagaagc 780 ccgcaagaag gcctttgcca cctgctcttc acatgtggct gtggtgggac tcttttatgg 840 agctggcatt tttacctata tgagacccaa atcccacagg tccactaacc acgataaggt 900 tgtgtcagcc ttctatacta tgttcacccc tttactaaat cccctcatct acagtgtgag 960 gaacagtgag gtcaaggaag ccctgaaacg gtggctgggg acgtgtgtaa acctaaaaca 1020 ccagcaaaat gaggcccaca ggtcaagatg atctagtgtc agatgagtct aagttcctga 1080 atttattaac attttaa 1097 <210> 40 <211> 1038 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7505023CB1 <400> 40 ttccttctct atttttccat ttgcaccatc acaccagggg aaattatgga gatgagaaat 60 actaccccag attttattct cctaggactc tttaaccaca ccagagccca ccaagtcctc 120 ttcatgatgg ttctgagtat cgttttgacc tccctgtttg gcaattccct catgattctc 180 ctgattcact gggaccaccg gctccacacg cccatgtact tcctcctgag ccaactttcc 240 ctcatggaca tgatgctggt ttccaccact gtgcccaaaa tggcggctga ctacttgacc 300 ggaagtaagg ccatctcccg cgctggctgt ggtgtgcaga tcttcttcct ccccacactg 360 ggtggtggag agtgcttcct cttagcagcc atggcctatg accgctatgc ggctgtctgc 420 cacccactcc gatatcccac tctcatgagc tggcagctgt gcctgaggat gaccatgtcg 480 tgttggctcc tgggtgcagc tgacgggctc ctgcaggctg ttgttaccct gagcttccca 540 tattgtggtg cacacgagat cgatcacttc ttctgcgaga cccccgtgct ggtgcgtttg 600 gcttgtgctg acacttcagt cttcgaaaac gccatgtaca tctgctgtgt gttaatgctc 660 ctggtcccct tttccctcat cctgtcctcc tatggtctca tcctcgctgc tgttctgcac 720 atgcgctcta cagaagcccg caagaaggcc tttgccacct gctcttcaca tgtggctgtg 780 gtgggactct tttatggagc tgccattttt acctatatga gacccaaatc ccataggtcc 840 actaaccatg acaaggttgt gtcagccttc tatactatgt tcaccccttt actaaacccc 900 ctcatctaca gtgtgaagaa cagtgaggtg aagggagccc tgaaacggtg gctggggacg 960 tgtgtaaaca taaaacacca gcaaaatgag gcccacaggt caagatgatc taatgtcaaa 1020 tgagtctaag ttcctgaa 1038 <210> 41 <211> 1522 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 70490395CB1 <400> 41 agcggagtga gagggaggga gcgccggccg cgggagcggg atggaaacca gcagcccgcg 60 gcccccgcgg cccagctcca acccggggct gagcctggac gcccggctgg gcgtggacac 120 tcgcctctgg gccaaggtgc tgttcaccgc gctctacgca ctcatctggg cgctgggcgc 180 ggcgggcaat gcgctgtccg tgcacgtggt gctgaaggcg cgggccgggc gcgcggggcg 240 cctgcgccac cacgtgctca gcctggcgct cgcgggcctg ctgctgctgc tggtcggcgt 300 gccggtggag ctctacagct tcgtgtggtt ccactacccc tgggtcttcg gcgacctggg 360 ctgccgcggc tactacttcg tgcacgagct gtgcgcctac gccacggtgc tgagcgtggc 420 aggcctgagc gccgagcgct gcctagccgt gtgccagccc ctgcgtgccc gcagcctgct 480 gacgccacgc cggacccggt ggctggtggc gctctcgtgg gccgcctcgc tcggcctcgc 540 cctgcccatg gccgtcatca tggggcagaa gcacgaactc gagacggcgg acggggagcc 600 ggagcccgcc tcgcgagtgt gcacggtgct ggtgagccgc accgcgctcc aagtctttat 660 ccaggtgaat gtgctggtgt ccttcgtgct ccccttggca ctaactgctt tcctgaatgg 720 ggtcacagtg agccacctgc tggccctctg ctcccaagtg ccgtccactt ctaccccggg 780 cagctccacc cccagccgcc tggagctgct gagtgaggag ggtctcctca gcttcatcgt 840 atggaagaag acctttatcc agggaggccc aggagccatc gtggtcatgt atgtcatctg 900 ctggctgccg taccatgccc gcaggctcat gtactgctac gtacctgatg acgcgtggac 960 tgacccactg tacaatttct accactactt ctacatggtg accaacacac ttttctacgt 1020 cagctcagct gtgactcctc ttctctacaa cgccgtgtcc tcctccttca gaaaactctt 1080 cctggaagcc gtcagctccc tgtgtggaga gcaccacccc atgaagcggt tacccccgaa 1140 gccccagagt cccaccctaa tggatacagc ttcaggcttt ggggatcccc cagaaacccg 1200 gacctgaatg taatgcaaga atgaacagaa caagcaaaat gaccagctgc ttagtcacct 1260 ggcaaagcag gtgagcaacc tcatcactaa tcattcaagc ttcgcagcca gggcgacttc 1320 tatcaacccc tgctctgctg agaaccatca agcgcaggga agccacgtga cccctcctag 1380 cctcaggctc cctcgtctgt gtagtggaga taaagaacag cacccatctc ttagtgttgc 1440 ctgagactta agtgcttagc acagaacctg gtgcgtagta gatgctccaa taaatttttg 1500 ctggcacgaa aaaaaaaaaa gg 1522 <210> 42 <211> 1964 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503250CB1 <400> 42 cgcgggcatc tggccagcat gctgctctgc acggctcgcc tggtcggcct gcagcttctc 60 atttcctgct gctgggcctt tgcctgccat agcacggagt cttctcctga cttcaccctc 120 cccggagatt acctcctggc aggcctgttc cctctccatt ctggctgtct gcaggtgagg 180 cacagacccg aggtgaccct gtgtgacagg tcttgtagct tcaatgagca tggctaccac 240 ctcttccagg ctatgcggct tggggttgag gagataaaca actccacggc cctgctgccc 300 aacatcaccc tggggtacca gctgtatgat gtgtgttctg actctgccaa tgtgtatgcc 360 acgctgagag tgctctccct gccagggcaa caccacatag agctccaagg agaccttctc 420 cactattccc ctacggtgct ggcagtgatt gggcctgaca gcaccaaccg tgctgccacc 480 acagccgccc tgctgagccc tttcctggtg cccatgcttt tggagcagat ccacaaggtg 540 catttccttc tacacaagga cactgtggcg tttaatgaca acagagatcc cctcagtagc 600 tataacataa ttgcctggga ctggaatgga cccaagtgga ccttcacggt cctcggttcc 660 tccacatggt ctccagttca gctaaacata aatgagacca aaatccagtg gcacggaaag 720 gacaaccagg tgcctaagtc tgtgtgttcc agcgactgtc ttgaagggca ccagcgagtg 780 gttacgggtt tccatcactg ctgctttgag tgtgtgccct gtggggctgg gaccttcctc 840 aacaagagtg acctctacag atgccagcct tgtgggaaag aagagtgggc acatgaggga 900 agccagacct gcttcccgcg cactgtggtg tttttggctt tgcgtgagca cacctcttgg 960 gtgctgctgg cagctaacac gctgctgctg ctgctgctgc ttgggactgc tggcctgttt 1020 gcctggcacc tagacacccc tgtggtgagg tcagcagggg gccgcctgtg ctttcttatg 1080 ctgggctccc tggcagcagg tagtggcagc ctctatggct tctttgggga acccacaagg 1140 cctgcgtgct tgctacgcca ggccctcttt gcccttggtt tcaccatctt cctgtcctgc 1200 ctgacagttc gctcattcca actaatcatc atcttcaagt tttccaccaa ggtacctaca 1260 ttctaccacg cctgggtcca aaaccacggt gctggcctgt ttgtgatgat cagctcagcg 1320 gcccagctgc ttatctgtct aacttggctg gtggtgtgga ccccactgcc tgctagggaa 1380 taccagcgct tcccccatct ggtgatgctt gagtgcacag agaccaactc cctgggcttc 1440 atactggcct tcctctacaa tggcctcctc tccatcagtg cctttgcctg cagctacctg 1500 ggtaaggact tgccagagaa ctacaacgag gccaaatgtg tcaccttcag cctgctcttc 1560 aacttcgtgt cctggatcgc cttcttcacc acggccagcg tctacgacgg caagtacctg 1620 cctgcggcca acatgatggc tgggctgagc agcctgagca gcggcttcgg tgggtatttt 1680 ctgcctaagt gctacgtgat cctctgccgc ccagacctca acagcacaga gcacttccag 1740 gcctccattc aggactacac gaggcgctgc ggctccacct gaccagtggg tcagcaggca 1800 cggctggcag ccttctctgc cctgagggtc gaaggtcgag caggccgggg gtgtccggga 1860 ggtctttggg catcgcggtc tggggttggg acgtgtaagc gcctgggaga gcctagacca 1920 ggctccgggc tgccaataaa gaagtgaaat gcgaaaaaaa aaaa 1964 <210> 43 <211> 2781 <212> DNA
<213> Homo sapiens <220>
<221> mist feature <223> Incyte ID No: 7505723CB1 <400> 43 atggcattct taattatact aattacctgc tttgtgatta ttcttgctac ttcacagcct 60 tgccagaccc ctgatgactt tgtggctgcc acttctccgg gacatatcat aattggaggt 120 ttgtttgcta ttcatgaaaa aatgttgtcc tcagaagact ctcccagacg accacaaatc 180 caggagtgtg ttggctttga aatatcagtt tttcttcaaa ctcttgccat gatacacagc 240 attgagatga tcaacaattc aacactctta cctggagtca aactggggta tgaaatctat 300 gacacttgta cagaagtcac agtggcaatg gcagccactc tgaggtttct ttctaaattc 360 aactgctcca gagaaactgt ggagtttaag tgtgactatt ccagctacat gccaagagtt 420 aaggctgtca taggttctgg gtactcagaa ataactatgg ctgtctccag gatgttgaat 480 ttacagctca tgccacaggt gggttatgaa tcaactgcag aaatcctgag tgacaaaatt 540 cgctttcctt catttttacg gactgtgccc agtgacttcc atcaaattaa agcaatggct 600 cacctgattc agaaatctgg ttggaactgg attggcatca taaccacaga tgatgactat 660 ggacgattgg ctcttaacac ttttataatt caggctgaag caaataacgt gtgcatagcc 720 ttcaaagagg ttcttccagc ctttctttca gataatacca ttgaagtcag aatcaatcgg 780 acactgaaga aaatcatttt agaagcccag gttaatgtca ttgtggtatt tctgaggcaa 840 ttccatgttt ttgatctctt caataaagcc attgaaatga atataaataa gatgtggatt 900 gctagtgata attggtcaac tgccaccaag attaccacca ttcctaatgt taaaaagatt 960 ggcaaagttg tagggtttgc ctttagaaga gggaatatat cctctttcca ttcctttctt 1020 caaaatctgc acttgcttcc cagtgacagt cacaaactct tacatgaata tgccatgcat 1080 ttatctgcct gcgcatatgt caaggacact gatttgagtc aatgcatatt caatcattct 1140 caaaggactt tggcctacaa ggctaacaag gctatagaaa ggaacttcgt catgagaaat 1200 gacttcctct gggactatgc tgagccagga ctcattcata gtattcagct tgcagtgttt 1260 gcccttggtt atgccattcg ggatctgtgt caagctcgtg actgtcagaa ccccaacgcc 1320 tttcaaccat gggagttact tggtgtgcta aaaaatgtga cattcactga tggatggaat 1380 tcatttcatt ttgatgctca cggggattta aatactggat atgatgttgt gctctggaag 1440 gagatcaatg gacacatgac tgtcactaag atggcagaat atgacctaca gaatgatgtc 1500 ttcatcatcc cagatcagga aacaaaaaat gagttcagga atcttaagca aattcaatct 1560 aaatgctcca aggaatgcag tcctgggcaa atgaagaaaa ctacaagaag tcaacacatc 1620 tgttgctatg aatgtcagaa ctgtcctgaa aatcattaca ctaatcagac agatatgcct 1680 cactgccttt tatgcaacaa caaaactcac tgggcccctg ttaggagcac tatgtgcttt 1740 gaaaaggaag tggaatatct caactggaat gactccttgg ccatcctact cctgattctc 1800 tccctactgg gaatcatatt tgttctggtt gttggcataa tatttacaag aaacctgaac 1860 acacctgttg tgaaatcatc cgggggatta agagtctgct atgtgatcct tctctgtcat 1920 ttcctcaatt ttgccagcac gagctttttc attggagaac cacaagactt cacatgtaaa 1980 accaggcaga caatgtttgg agtgagcttt actctttgca tctcctgcat tttgacgaag 2040 tctctgaaaa ttttgctagc cttcagcttt gatcccaaat tacagaaatt tctgaagtgc 2100 ctctatagac cgatccttat tatcttcact tgcacgggca tccaggttgt catttgcaca 2160 ctctggctaa tctttgcagc acctactgta gaggtgaatg tctccttgcc cagagtcatc 2220 atcctggagt gtgaggaggg atccatactt gcatttggca ccatgctggg ctacattgcc 2280 atcctggcct tcatttgctt catatttgct ttcaaaggca aatatgagaa ttacaatgaa 2340 gccaaattca ttacatttgg catgctcatt tacttcatag cttggatcac attcatccct 2400 atctatgcta ccacatttgg caaatatgta ccagctgtgg agattattgt catattaata 2460 tctaactatg gaatcctgta ttgcacattc atccccaaat gctatgttat tatttgtaag 2520 caagagatta acacaaagtc tgcctttctc aagatgatct acagttattc ttcccatagt 2580 gtgagcagca ttgccctgag tcctgcttca ctggactcca tgagcggcaa tgtcacaatg 2640 accaatccca gctctagtgg caagtctgca acctggcaga aaagcaaaga tcttcaggca 2700 caagcatttg cacacatatg cagggaaaat gccacaagtg tatctaaaac tttgcctcga 2760 aaaagaatgt caagtatatg a 2781 <210> 44 <211> 1863 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 90023339CB1 <400> 44 gtggctcaga tactgatact ttctttccaa acagcataag aagtgattga gccacaagta 60 tactgaagga agggctccct cgagttgtgg tgtgaagaga taaatcacca ggcccagtcg 120 aagaatatca gctgctgctt caggtgacct atagagattc caaggagaaa agagatttga 180 gaaattttct gaagctcttg aagcctccat tattatggtc acatgggcta attagaatta 240 tcagagcaaa ggctaccaca gactgcaaca gcctgaatgg agtcctgcag tgtacctgtg 300 aagacagcta cacctggttt cctccctcat gccttgatcc ccagaactgc taccttcaca 360 cggctggagc actcccaagc tgtgaatgtc atctcaacaa cctcagccag agtgtcaatt 420 tctgtgagag aacaaggatt tggggcactt tcaaaattaa tgaaaggttt acaaatgacc 480 ttttgaattc atcttctgct atatactcca aatatgcaaa tggaattgaa attcaaaaat 540 ggaagcatcg ttgctgggta tgaagttgtt ggctccagca gtgcatctga actgctgtca 600 gccattgaac atgttgccga gaaggctaag acagcccttc acaagctgtt tccattagaa 660 gacggctctt tcagagtgtt cggaaaagcc cagtgtaatg acattgtctt tggatttggg 720 tccaaggatg atgaatatac cctgccctgc agcagtggct acaggggaaa catcacagcc 780 aagtgtgagt cctctgggtg gcaggtcatc agggagactt gtgtgctctc tctgcttgaa 840 gaactgaaca agaatttcag tatgattgta ggcaatgcca ctgaggcagc tgtgtcatcc 900 ttcgtgcaaa atctttctgt catcattcgg caaaacccat caaccacagt ggggaatctg 960 gcttcggtgg tgtcgattct gagcaatatt tcatctctgt cactggccag ccacttcagg 1020 gtgtccaatt caacaatgga gggatttttt atcttatgct ttggaatact cttggacagt 1080 aagctgcgac aacttctgtt caacaagttg tctgccttaa gttcttggaa gcaaacagaa 1140 aagcaaaact catcagattt atctgccaaa cccaaattct caaagccttt caacccactg 1200 caaaacaaag gccattatgc attttctcat actggagatt cctccgacaa catcatgcta 1260 actcagtttg tctcaaatga ataaggcaag gaatcataaa atcaagaaaa aatttccaga 1320 acaacttgac atttagagac aaatgtcaat gaagaaatta tgctcagtat tcgatcgggt 1380 tttctgattt aggggtctgg gaataaaaca agaatgtctc agtggcttca ttactgctcc 1440 cttttgtctt caattaaatg aaaagaagat ttatttccat gtgatttgat tcaaagaaag 1500 tgctccataa atgcagaaga gtaggttttg ttggaaatcg tgtcagttgt accctgacca 1560 taaaatatgg tttctatttt cataaaacag cattattcac atggcatttc caataatctg 1620 gattgaagga agaaaatttt atgaaatagc tttagataaa ttaataggcc acgttcattt 1680 tcttgtcaaa aagttactgg tggggggatg gtgggaaaaa gttattagtg caaatttcct 1740 agagaaaaaa ccatttctct ttcaaatttt ccagttgaat tttatgttcg cttttgcttc 1800 ttaggttcta tcacttaata ttgaaagtta atcagaaata aaatgtaaac ttctatttaa 1860 aaa 1863

Claims (105)

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:1-22, 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:1-18 and SEQ ID NO:20-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-22.

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:23-44.

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:1-22.

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:23-44, 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:23-44, 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:1-22.

19. A method for treating a disease or condition associated with decreased expression of functional GCREC, 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 GCREC, 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 GCREC, 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 method for a diagnostic test for a condition or disease associated with the expression of GCREC 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 Flab' )Z 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 GCREC
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 GCREC
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:1-22, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from the animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-22.

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:1-22, 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 specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-22.

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:1-22 in a sample, the method comprising:
a) incubating the antibody of claim 11 with the 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:1-22 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-22 from a sample, the method comprising:
a) incubating the antibody of claim 11 with the 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:1-22.

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 method of identifying a compound that modulates, mimics and/or blocks an olfactory and/or taste sensation, the method comprising:
a) contacting the compound with an olfactory and/or taste receptor polypeptide selected from the group consisting of:
i) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, ii) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, and iii) an olfactory and/or taste receptor having an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-22.
b) identifying whether the compound specifically binds to and/or affects the activity of said receptor polypeptide.

57. The method of claim 56, wherein said receptor polypeptide is expressed on the surface of a mammalian cell.

58. The method of claim 57, wherein said mammalian cell expresses a G-protein.

59. The method of claim 58, wherein said mammalian cell expresses a plurality of G-protein coupled receptors.

60. The method of claim 59, wherein said mammalian cell expresses another olfactory and/or taste receptor polypeptide.

61. The method of claim 56, wherein said receptor polypeptide is fused to another polypeptide.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

78. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:17.

79. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:18.

80. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:19.

81. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:20.

82. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:21.

83. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:22.

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

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

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

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

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

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

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

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

92. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:31.

93. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:32.

94. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:33.

95. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:34.

96. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:35.

97. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:36.

98. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:37.

99. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:38.

100. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:39.

101. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:40.

102. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:41.

103. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:42.

104. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:43.

105. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:44.