CA2437717A1 - G-protein coupled receptors - Google Patents

Abstract

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

Description

G-PROTEIN COUPLED 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 prevention 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 and odorant receptors. The present invention further relates to the use of specific G-protein coupled receptors to identify l0 molecules that are involved in 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) Curr. Opin. Cell Biol. 6:191-197). The amino-terminus of a GPCR is extracellular, is of variable length, and is often glycosylated. The carboxy-terminus is cytoplasmic and generally phosphorylated. Extracellular loops alternate with intracellular loops and link the transmembrane domains. Cysteine disulfide bridges linking the second and third extracellular loops may interact with agonists and antagonists. The most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops. The transmembrane domains account, in part, for structural and functional features of the receptor. In most cases, the bundle of a helices forms a ligand-binding pocket. The extracellular N-terminal segment, or one or more of the three extracellular loops, may also participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor. In turn, the large, third intracellular loop of the activated receptor interacts with a heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, including the activation of second messengers such as cyclic AMP (CAMP), phospholipase C, and inositol triphosphate, and the interaction of the activated GPCR with ion channel proteins. (See, e.g., Watson, S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego CA, pp. 2-G;
Bolander, F.F. (1994) Molecular EndocrinoloQV, Academic Press, San Diego CA, pp. 1G2-17G;
Baldwin, J.M. (1994) Curr. Opin. Cell Biol. 6:180-190.) GPCRs include receptors for sensory signal mediators (e.g., light and olfactory stimulatory l0 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, endothelin, follicle-stimulating hormone (FSH), gonadotropic-releasing hormone (GnRH), neurokinin, thyrotropin-releasing hormone (TRH), and oxytocin). GPCRs which act as receptors for stimuli that have yet to be identified are known as orphan receptors.
The diversity of the GPCR family is further increased by alternative splicing.
Many GPCR
genes contain introns, and there are currently over 30 such receptors for which splice 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

2 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.

3

4 PCT/US02/03635 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). Another subfamily of the secretin-like GPCRs was recently defined by the Ig-Hepta protein. Ig-Hepta contains a seven transmembrane domain characteristic of secretin-like GPCRs, as well as a large extracellular domain containing two immunoglobulin-like repeats. Ig-Hepta expression is localized to the aveolar walls of the lung and the intercalated cells in the collecting duct of the kidney, suggesting a role for Ig-Hepta in pH sensing or regulation (Abe, J. et al. (1999) J. Biol.
Chem. 274:19957-19964).
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 Ca2+-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 brig sae, 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 hyperfunctioning thyroid adenomas, suggesting that certain GPCRs susceptible to constitutive activation may behave as protooncogenes (Parma, J. et al.
(1993) Nature 365:649-651). GPCR receptors for the following ligands also contain mutations associated with human disease: luteinizing hormone (precocious puberty);
vasopressin Vz (X-linked nephrogenic diabetes); glucagon (diabetes and hypertension); calcium (hyperparathyroidism, hypocalcuria, hypercalcemia); parathyroid hormone (short limbed dwarfism); (33-adrenoceptor (obesity, non-insulin-dependent diabetes mellitus); growth hormone releasing hormone (dwarfism); and adrenocorticotropin (glucocorticoid deficiency) (Wilson, S. et al. (1998) Br.
J. Phannocol. 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, supra;
Stadel, supra). For example, the dopamine agonist L-dopa is used to treat Parkinson's disease, while a dopamine antagonist is used to treat schizophrenia and the early stages of Huntington's disease. Agonists and antagonists of adrenoceptors have been used for the treatment of asthma, high blood pressure, other cardiovascular disorders, and anxiety; muscarinic agonists are used in the treatment of glaucoma and tachycardia; serotonin SHT1D antagonists are used against migraine; and histamine H1 antagonists are used against allergic and anaphylactic reactions, hay fever, itching, and motion sickness (Horn, 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

5 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).
IL-5 treatment and Immune Response Cells undergoing neoplastic growth gradually progress to invasive carcinoma and become metastatic. Factors involved in tumor progression and malignant transformation include genetic factors, environmental factors, growth factors, and hormones. Histological and molecular evaluation of breast tumors has revealed that the development of breast cancer evolves through a multi-step process whereby pre-malignant mammary epithelial cells undergo a relatively defined sequence of events leading to tumor formation.
Neoplastic growth is mediated by a variety of factors such as Interleukin 5 (IL-5), a T cell-derived factor that promotes the proliferation, differentiation, and activation of eosinophils. IL-5 has also been known as T cell replacing factor (TRF), B cell growth factor II
(BCGFII), B cell differentiation factor m (BCDF m), eosinophil differentiation factor (EDF), and eosinophil colony-stimulating factor (Eo-CSF). IL-5 exerts its activity on target cells by binding to specific cell surface receptors. The effect of IL-5 may be observed in human peripheral blood mononuclear cells (PBMCs), which contain about 52% lymphocytes (12% B lymphocytes, 40% T
lymphocytes {25%
CD4+ and 15% CD8+}), 20% NK cells, 25% monocytes, and 3% various cells that include dendritic cells and progenitor cells.
The discovery of new G-protein coupled receptors and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention,

6 and treatment 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.
SUMMARY OF THE INVENTION
The invention features 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," "GCREC-22," "GCREC-23," "GCREC-24,"
"GCREC-25," "GCREC-26," "GCREC-27," "GCREC-28," "GCREC-29," "GCREC-30," "GCREC-31," "GCREC-32," "GCREC-33," "GCREC-34," "GCREC-35," "GCREC-36," "GCREC-37,"
"GCREC-38," "GCREC-39," "GCREC-40," "GCREC-41," "GCREC-42," "GCREC-43," "GCREC-44," "GCREC-45," "GCREC-46," "GCREC-47," and "GCREC-48." In one aspect, the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ 1D NO:1-48, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90°Io identical to an amino acid sequence selected from the group consisting of SEQ >D NO:1-48, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-48. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-48.
The invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90°lo identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-48, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-48. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ
ID NO:1-48. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID N0:49-96.

7 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.
Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90°lo identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-48, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-48. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ >D NO:1-48, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90°Io identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ >D NO:l-48. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ >D NO:1-48, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90°Io identical to an amino acid sequence selected from the group consisting of SEQ >D NO:1-48, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
ID NO:1-48.
The invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ

ID N0:49-96, b) a polynucleotide comprising a naturally occurnng polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:49-96, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:49-96, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ 1D N0:49-96, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID
N0:49-96, 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 N0:49-96, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ >D NO:1-48, 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:1-48, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-48, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ >D NO:1-48. The invention additionally 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.
The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, 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:1-48, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D
NO:1-48, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ >D NO:1-48. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional GCREC, comprising administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, b) a polypeptide comprising a naturally occurnng amino acid sequence at least 90°Io identical to an amino acid sequence selected from the group consisting of SEQ m NO:1-48, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 1D NO:1-48, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-48. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional GCREC, comprising administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-48, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m NO:1-48, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-48, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ >D NO:1-48. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-48, 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 B7 NO:1-48, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D
NO:1-48, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D NO:1-48. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
The invention further provides 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.
The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID N0:49-96, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:49-96, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ )D N0:49-96, 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 1D N0:49-96, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ m N0:49-96, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences ofthe presentinvention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
Table 5 shows the representative cDNA library for polynucleotides of the invention.
Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"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 mare 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 polynucIeotide encoding GCREC, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence 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 similarity 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" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) 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, 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')z, 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 Garners 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 cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.) The term "intramer" refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA
aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci.
USA 96:3606-3610).
The term "spiegelmer" refers to an aptamer 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 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, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotide sequences encoding 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 (GCG, Madison WI) 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 Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gtn, His Gly Ala His Asn, Arg, Gln, Glu Iie Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or polypeptide.
Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A
derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
A "fragment" is a unique portion of GCREC or the polynucleotide encoding GCREC
which is identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, I00, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ ID N0:49-96 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:49-96, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:49-96 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ
ID N0:49-96 from related polynucleotide sequences. The precise length of a fragment of SEQ ID
N0:49-96 and the region of SEQ ID N0:49-96 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:1-48 is encoded by a fragment of SEQ ID N0:49-96. A
fragment of SEQ ID NO:1-48 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-48. For example, a fragment of SEQ ID NO:1-48 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-48.
The precise length of a fragment of SEQ ID NO:1-48 and the region of SEQ ID
NO:1-48 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a translation initiation 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, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS

8:189-191. For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off. 50 Expect: 10 Word Size: 1l Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over 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 "°lo identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and-hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 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 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1 % (w/v) SDS, and about 100 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 carried 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. et al. (1989) Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ~g/ml. Organic solvent, such as formamide at a concentration of about 35-50°Io v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases., A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"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 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, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of 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 rnay 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.
"OperabIy 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 S nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers soiubility 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 acid sequences encoding GCREC, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. "Primers"
are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the 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 the references, for example Sambrook, J. et al. (I989) Molecular Cloning: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring Harbor Fress, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Bi-olo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Inriis, 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 l0 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/MIT
Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, su ra. 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, translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of containing GCREC, 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 synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90°!o 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. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA
molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation.
Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%~at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length. A variant may be described as, for example, an "allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human 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 sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ~). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ m NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide 1D) 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. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog.
Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s).
Column 5 shows the annotation of the GenBank homolog(s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ 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 (Genetics Computer Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains, and motifs. Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are G-protein coupled receptors. For example, SEQ
)D NO:1 is 28% identical, from residue I370 to residue K680, to chicken ovarian follicle-stimulating hormone receptor (GenBank ID g1256414) and 51% identical, from residue L136 to residue E702, to human leucine-rich repeat-containing G protein-coupled receptor 7 (GenBank ID
g10441730) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability scores are 7.5e-41 and 1.4e-153, respectively, indicating the probabilities of obtaining the observed polypeptide sequence alignments by chance. SEQ ID NO:1 also contains a rhodopsin family 7-transmembrane receptor domain, 9 leucine rich repeats, and a low-density lipoprotein receptor domain, as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ
ID NO:1 is a G-protein coupled hormone receptor with leucine-rich repeats. In a further example, SEQ ID N0:2 is 29% identical, from residue V203 to residue 5871, to rat seven transmembrane receptor Ig-Hepta (GenBank ID g5525078) with a BLAST probability score of 6.7e-67. (See Table 2.) SEQ ID N0:2 also contains a 7 transmembrane receptor (secretin family) domain and a latrophilin/CL-1-like GPS domain as determined by searching for statistically significant matches in the HMM-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses provide further corroborative evidence that SEQ ID
N0:2 is a secretin-like GPCR. In a further example, SEQ ID N0:3 is 36% identical, from residue L56 to residue T243, to human small cell vasopressin subtype 1b receptor (GenBank ID
g2613125) with a BLAST probability score of 9.7e-41. (See Table 2.) SEQ ID N0:3 also contains a 7 transmembrane receptor (rhodopsin family) domain as determined by searching for statistically significant matches in the HMM-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ
ID N0:3 is a vasopressin receptor. In a further example, SEQ ID N0:4 is 23%
identical, from residue S30 to residue Q301, to human cysteinyl leukotriene receptor (GenBank ID g5359718) with a BLAST probability score of 4.5e-21. (See Table 2.) Data from BLIMPS and additional BLAST
analyses provide corroborative evidence that SEQ ID N0:4 is a G-protein coupled receptor. In a further example, SEQ ID N0:8 is 99% identical, from residue M1 to residue V345, to human orphan G-protein coupled receptor (GenBank ID g8118040) with a BLAST probability score of 2.9e-186.
(See Table 2.) SEQ >Z7 N0:8 also contains a 7 transmembrane receptor metabotropic glutamate family domain as determined by searching for statistically significant matches in the HMM-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLAST analysis provide further corroborative evidence that SEQ ID N0:8 is a G-protein coupled receptor. In a further example, example, SEQ ID NO:10 is 64% identical, from residue N5 to residue I307, to Mus musculus odorant receptor S46 (GenBank Ip g4680268)with a BLAST probability score of 6.2e-111.
(See Table 2.) SEQ ID NO:10 also contains a 7-transmembrane receptor (rhodopsin family) domain as determined by searching for statistically significant matches in the HMM-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ID NO:10 is an olfactory GPCR. SEQ ID NO:S-7, SEQ >D N0:9, SEQ ID NO:11-44, and SEQ ID N0:45-48 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO:I-48 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte 1D) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs. Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the full length polynucleotide sequences of the invention, and of fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ID
N0:49-96 or that distinguish between SEQ ID N0:49-96 and related polynucleotide sequences.
The polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotide sequences. In addition, the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST"). Alternatively, the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP"). Alternatively, the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. For example, a polynucleotide sequence identified as FL_h~~:~XXX N~ NZ_YYYYY N3 N4 represents a "stitched" sequence in which XXXXXX
is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and N,,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.~~XX gAAAAA_gBBBBB_I IV is a "stretched" sequence, with x~~XXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB
being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V). In instances where a RefSeq sequence was used as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier (denoted by "NM,"
"NP," or "NT") may be used in place of the GenBank identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs GNN, GFG,Exon prediction from genomic sequences using, for example, ENST GENSCAN (Stanford University, CA, USA) or FGENES

(Computer Genomics Group, The Sanger Centre, Cambridge, 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 polynucleotide sequences which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
The invention also encompasses GCREC variants. A preferred GCREC variant is one which has at least about 80%, or alternatively at least about 90%, or alternatively at least about 95%, or even at least about 99% amino acid sequence identity to the GCREC amino acid sequence, and which contains at least one functional or structural characteristic of GCREC.
The invention also encompasses polynucleotides which encode GCREC. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:49-96, which encodes GCREC. The polynucleotide sequences of SEQ n7 N0:49-96, as presented in the Sequence Listing, embrace the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding GCREC. In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or even at least about 99%
polynucleotide sequence identity to the polynucleotide sequence encoding GCREC. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:49-96 which has at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or even at least about 99% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID N0:49-96.
Any one of the polynucleotide variants described above can encode an amino acid sequence 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 sequence encoding GCREC. A splice variant may have portions which have significant sequence identity to the polynucleotide sequence encoding GCREC, but will generally have a greater l0 or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing. A splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50%
polynucleotide sequence identity to the polynucleotide sequence encoding 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 sequence encoding GCREC. Any one of the splice variants described above can encode an amino acid sequence 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 nucleotide sequences which encode GCREC and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring GCREC
under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences 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 DNA sequences which encode GCREC
and GCREC derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding GCREC or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID
N0:49-96 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise (Applied Biosystems), thermostable T7 polymerise (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerises and proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular BioloQV, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp.
856-853.) The nucleic acid sequences 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. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the' emitted wavelengths. Outpudlight intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode 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 DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express GCREC.
The nucleotide sequences of the present 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 MOLECLJLARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of 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 occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding GCREC may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively, GCREC itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, the amino acid sequence of 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. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing.
(See, e.g., Creighton, supra, pp. 28-53.) In order to express a biologically active GCREC, the nucleotide sequences 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 polynucleotide sequences encoding GCREC. Such elements may vary in their strength and specificity.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding GCREC. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences 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. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding GCREC and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A
Laboratorv Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular BioloQV, John Wiley & Sons, New York NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences 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. (See, e.g., Sambrook, supra; Ausubel, su ra; 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 TechnoloQV (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc.
Natl. Acad. Sci. USA
90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al. (1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding GCREC. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding GCREC can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding 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. (See, e.g., 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. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, 1995, supra;
Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et al. (1994) Bio/Technology 12:181-184.) Plant systems may also be used for expression of GCREC. Transcription of sequences encoding GCREC may be driven by viral promoters, e.g., the 355 and 195 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. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp. 191-196.) _ In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding 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. (See, e.g., Logan, J. and T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. 5V40 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. (See, e.g., Harnngton, 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, sequences 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.
(See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic. or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), f3 glucuronidase and its substrate f3-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding GCREC is inserted within a marker gene sequence, transformed cells containing sequences 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 nucleic acid sequence 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.
Immunological 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. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in ImmunoloQV, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.) A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding GCREC include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, the sequences 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 Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding 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 sequences or to process the expressed protein in the desired fashion.
Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" or "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding 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, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the 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 (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled 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 of the present invention or fragments thereof may be used to screen for compounds that specifically bind to GCREC. At least one and up to a plurality of test compounds may be screened for specific binding to GCREC. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the natural ligand of GCREC, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current Protocols in ImmunoloQV 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which GCREC
binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express GCREC, either as a secreted protein or on the cell membrane. Preferred cells 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 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 carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compounds) may be free in solution or affixed to a solid support.
GCREC of the present invention or fragments thereof 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) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BLJ6 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 are peripheral blood cells, and human mammary epithelial cells, and also can be found in Table 6. 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, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a 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, mitral 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, alpha,-antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis, veno-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 extracoiporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic 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.
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 Garner 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 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 of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of 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. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use. Single chain antibodies (e.g., from camels or llamas) may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J.
Biotechnol. 74:277-302).
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 Calmette-Guerin) and Corvnebacterium 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 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. (See, e.g., 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; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.) In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., MoiTison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (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. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites for GCREC may also be generated.
For example, such fragments include, but are not limited to, F(ab')z fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between 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, su ra).
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, K~, 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 K
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 10'2 L/mole are preferred for use in immunoassays in which the GCREC-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with I~
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, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For 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.
(See, e.g., Catty, su ra, and Coligan et al. supra.) In another embodiment of the invention, the 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. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.) In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g., Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon, K.J. et al. (1995)

9(13):1288-1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Moms, M.C. et al. (1997) Nucleic Acids Res.
25(14):2730-2736.) In another embodiment of the invention, polynucleotides encoding GCREC may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), 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 Tr'panosoma 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 expressedwsing (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ~i-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci.
USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V.
and H.M. Blau, su ra)), or (iii) a tissue-sped ific 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 KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and 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 the alternative, 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, both incorporated by reference herein.
In another alternative, 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, hereby incorporated by reference. The manipulation of cloned hetpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver 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) Curr. Opin. Biotechnol.
9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for 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 manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunolog_ic Approaches, Futura Publishing, Mt. Kisco NY, pp.
163-177.) A
complementary sequence or antisense molecule may also be designed to block translation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding 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 of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA
sequences encoding 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.
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.
At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide;
and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding 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~ombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem.
Biophys. Res. Commun.

268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified 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. (See, e.g., Goldman, C.K. et al. (1997) Nat.
Biotechno1.15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remin. t Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of GCREC, antibodies to GCREC, and mimetics, agonists, antagonists, or inhibitors of GCREC.
The compositions utilized in this invention 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.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
Compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended 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 l0 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 ~g 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.
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 l0 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 FACS, 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, the polynucleotides encoding GCREC may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of GCREC
may be correlated with disease. The 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 polynucleotide sequences, 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 occurnng sequences encoding GCREC, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and may have at least 50%
sequence identity to any of the 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
N0:49-9G or from genomic sequences including promoters, enhancers, and introns of the GCREC
gene.
Means for producing specific hybridization probes for DNAs encoding GCREC
include the cloning of polynucleotide sequences 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.
Polynucleotide sequences 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, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a 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, mitral 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 (A)DS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, alpha,-antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis,.liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis, veno-occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic chotestasis of pregnancy, and hepatic tumors including nodular hypeiplasias, 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 helminthic 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. The polynucleotide sequences 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 aspect, the nucleotide sequences encoding GCREC may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide 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 nucleotide sequences 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 the polynucleotide sequences 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 the polynucleotide sequences 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 pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOXS gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well as for tracing the origins of populations and their migrations. (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu .(1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin.
Neurobiol. 11:637-641.) Methods which may also be used to quantify the expression of GCREC include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al. (1993) J. 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 polynucleotide sequences described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
In another embodiment, 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. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurnng environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity. (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type. In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for 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 microanay to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol-or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample.
A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116; Shalom D. et al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA
94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types of microarrays are well known and thoroughly described in DNA Microarrays: A
Practical Approach, M. Schena, ed. (1999) Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding 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 mufti-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harnngton, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.) Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding 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.
(See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, 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 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. (See, e.g., Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with 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-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding 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. 60/267,322, U.S. Ser. No. 60/271,215, U.S. Ser. No.
60/274,551, U.S. Ser.

No. 60/278,507, U.S. Ser. No. 60/280,597, U.S. Ser. No. 60/281,107, and No.
U.S. Ser. No.
60/282,121, are expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL 51000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE
(Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones Plasniids 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 Pharmacia Biotech or supplied in ABI
sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI
protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, su ra, unit 7.7). 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 norve~icus, Mus musculus, Caenorhabditis ele~ans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM; and HMM-based protein domain databases such as SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA
95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (HMM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R.
(1996) Curr. Opin.
Struct. Biol. 6:361-365.) The queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV
and V) were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences.
Alternatively, a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide.
Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM; and HMM-based protein domain databases such as SMART. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ ID
N0:49-96. 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 l0 a general-purpose gene identification program which analyzes genomic DNA
sequences from a variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop 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.
3o V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals thus identified were then "stitched" together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.
"Stretched" 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 1V. 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 ID N0:49-96 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID N0:49-96 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI "GeneMap'99" World Wide Web site (http://www.ncbi.nlm.nih.gov/genemapn, can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity 5 x minimum { length(Seq. 1 ), length(Seq. 2) }
The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding 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 derived from a cDNA library constructed from a human tissue. Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system;
nervous system;
pancreas; respiratory system; sense organs; skin; stomatognathic system;
unclassified/mixed; or urinary tract. The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, each human tissue 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 LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of GCREC Encoding Polynucleotides Full length polynucleotide sequences were also produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg'-+, (NH,,),SOa, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 60°C, 1 min;
Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C. In the alternative, the parameters for primer pair T7 and SK+
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; Step 6: 68 °C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~,1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 ~.1 of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 ,u1 to 10 ~1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37°C in 384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7:
storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with 20% dimethysulfoxide ( 1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the above procedure or are used to obtain 5'regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in GCREC Encoding Polynucleotides Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ ID N0:49-96 using the LIFESEQ database (Incyte Genomics).
Sequences from the same gene were clustered together and assembled as described in Example III, allowing the identification of all sequence variants in the gene. An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecall errors by requiring a minimum Phred quality score of 15, and removed sequence alignment errors and errors resulting from improper trimming of vector sequences, chimeras, and splice variants.
An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP. Clone error filters used statistically generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering error filters used statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed duplicates and SNPs found in immunoglobulins or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations. The Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezualan, and two Amish individuals. The African population comprised 194 individuals (97 male, 97 female), all African Americans. The Hispanic population comprised 324 individuals (162 male, 162 female), all Mexican Hispanic. The Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
Allele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no allelic variance in this population were not further tested in the other three populations.
X. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ 1D N0:49-96 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 ,uCi of [Y-3zP] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
XI. Microarrays The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), supra).
Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, 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. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470; Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.) Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, 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/~,1 oligo-(dT) primer (2lmer), 1X first strand buffer, 0.03 units/~l RNase inhibitor, 500 ~.M dATP, 500 p.M dGTP, 500 ~,M dTTP, 40 ~M
dCTP, 40 p.M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 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 N,1 5X 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 p,g.

Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~.l of the array element DNA, at an average concentration of 100 ng/~l, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATAL1NKER 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 ~tl of sample mixture consisting of 0.2 ,ug each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65°C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 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 corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60°C. The arrays are washed far 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 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).
Expression For example, for component 2112194 of SEQ ID N0:64, peripheral blood cells (PBMCs) are collected from the blood of 6 donors using standard gradient separation. The PBMCs from each donor are placed in culture for 2 hours in the presence or absence of recombinant interleukin-5 (IL-5).
IL-5 treated PBMCs and untreated control PBMCs from the different donors are pooled according to their respective treatments. In this manner, it was demonstrated that treatment with IL-5 alters the expression of component 2112194 of SEQ >D N0:64 in PBMCs by a factor of at least 2.

Alternatively, for component 2112194 of SEQ >D N0:64, a normal human mammary epithelial cell (HMEC) population is compared to breast carcinoma lines at various stages of tumor progression.
Samples are lysed in Trizol and the total RNA fraction is recovered. Poly-A
mRNA is purified using a standard oligo-dT selection method. Gene expression profiles of HMEC cells are compared to those of the breast carcinoma lines. In this manner, it was demonstrated that the expression of component 2112194 of SEQ m N0:64 is altered by a factor of at least 2 during breast tumor progression.
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 Autograuhica californica 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 (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther.
7:1937-1945.) In most expression systems, 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 enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from 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 (1995, supra, ch. 10 and 16). Purified GCREC obtained by these methods can be used directly in the assays shown in Examples XVII, XVIII, and XIX, where applicable.
XIV. Functional 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 (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ,ug of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 ~cg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide;
changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cvtometry, Oxford, New York NY.
The influence of 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., Harnngton, 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. (See, e.g., Ausubel, 1995, su ra, ch. 1 I.) Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 43IA
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. (See, e.g., Ausubel, 1995, su ra.) 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 I% 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 Pharmacia Biotech). 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'z5I 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 mufti-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 (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
Potential GCREC agonists or antagonists may be tested for activation or inhibition of GCREC
receptor activity using the assays described in sections XVII and XVIII.
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.
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 MgClz, 20 mM CHAPS, 20% glycerol, 10 ~g of both aprotinin and leupeptin, and 20 ~,1 of 50 mM phenylmethylsulfonyl fluoride.
The lysate is incubated on ice for 45 min with constant stirnng, centrifuged at 23,000 g for 15 min at 4°C, and the supernatant is collected. 750 ~,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 [32P]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) ~i-mercaptoethanol, 20% (w/v) glycerol, 95.2 mM Tris-HCI, pH 6.8, 0.01% (w/v) bromphenol blue).
The [3zP]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 blotto (S% 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 immunoglobulin (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 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 pCi/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.
XIX. Identification of GCREC Ligands GCREC is expressed in a eukaryotic cell line such as CHO (Chinese Hamster Ovary) or HEK (Human Embryonic Kidney) 293 which have a good history of GPCR expression and which contain a wide range of G-proteins allowing for functional coupling of the expressed 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 messengers, such as cyclic AMP or CaZ+. These may be measured directly using standard methods well known in the art, or by the use of reporter gene assays in which a luminescent protein (e.g. firefly luciferase or green fluorescent protein) is under the transcriptional control of a promoter responsive to the stimulation of protein kinase C by the activated receptor (Milligan, G. et al. (1996) Trends Pharmacol. Sci. 17:235-237). Assay technologies are available for both of these second messenger systems to allow high throughput readout in mufti-well plate format, such as the adenylyl cyclase activation FlashPlate Assay (NEN Life Sciences Products), or fluorescent Caz+
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 Ga,sn6 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 occurnng bioactive molecules.
Biological extracts from tissues, biological fluids and cell supernatants are also screened.
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Table 1 Incyte ProjectPolypeptideIncyte PolynucleotideIncyte ID SEQ ID PolypeptideSEQ ID NO: Polynucleotide NO: ID ID

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Table 5 Polynucleotide Representative Library SEQ
Incyte Project ID:
ID NO:

49 ' 7485090CB ~ ADRETUT07 50 ( 7474890CB ( PROSTMY01 52 ( 90012430CB ~ MONOTXNOS

55 2880041CB1 ~MIXDUNBO1 57 190012163CB1 ~LUNGTUT09 66 ~7476781CB1 GPCRGSV02 74 ~ 7475057CB ~ SINITMRO1 95 ~ 55036208CB ~ GPCRDPV02 v .

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<110> INCYTE GENOMICS, INC.
BAUGHN, Mariah R.
TRIBOULEY,Catherine M.
NGUYEN,Danniel B.
THORNTON,Michael YAO,Monique G.
KALLICK,Deborah A.
GANDHI,Ameena R.
WALIA,Narinder K.
ARVIZU,Chandra ELLIOTT,Vicki S.
HAFALIA,April J.A.
RAMKUMAR, Jayalaaani JIN,Pei TANG,Y.Tom YUE,Henry REDDY,ROOpa BURFORD,Neil LU,Dyung Aina M.
GRAUL,Richard C.
KAHN,Farrah A.
WALSH,ROderick T.
ISON,Craig H.
RICHARDSON,Thomas W.
GRIFFIN,Jennifer A.
WARREN,Bridget A.
YANG,Junming LEE,Ernestine A.
HARLAND,Lee <120> G-PROTEIN COUPLED RECEIPTORS
<130> PI-0357 PCT
<140> To Be Assigned <141> Herewith <150> 60/267,322; 60/271,215; 60/274,551; 60/278,507;
60/280,597; 60/281,107; 60/282,121 <151> 2001-02-07; 2001-02-23; 2001-03-08; 2001-03-23;
2001-03-30; 2001-04-02; 2001-04-06 <160> 96 <170> PERL Program <210> 1 <211> 726 <212> PRT
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 7485090CD1 <400> 1 Met Gln Lys Thr Lys Gln Asp Glu Asp Tyr Glu Arg Ala Ile Gly Phe Ser Val Lys Met Asp Asp Ser Asp Ser Asp Phe Ala Leu~Thr Gln Gly Ser Met Ile Thr Pro Ser Cys Gln Lys Gly Tyr Phe Pro Cys Gly Asn Leu Thr Lys Cys Leu Pro Arg Ala Phe His Cys Asp Gly Lys Asp Asp Cys Gly Asn Gly Ala Asp Glu Glu Asn Cys Gly Asp Thr Ser Gly Trp Ala Thr Ile Phe Gly Thr Val His Gly Asn Ala Asn Ser Val Ala Leu Thr Gln Glu Cys Phe Leu Lys Gln Tyr Pro Gln Cys Cys Asp Cys Lys Glu Thr Glu Leu Glu Cys Val Asn Gly Asp Leu Lys Ser Val Pro Met Ile Ser Asn Asn Val Thr Leu Leu Ser Leu Lys Lys Asn Lys Ile His Ser Leu Pro Asp Lys Val Phe Ile Lys Tyr Thr Lys Leu Lys Lys Ile Phe Leu Gln His Asn Cys Ile Arg His Ile Ser Arg Lys Ala Phe Phe Gly Leu Cys Asn Leu Gln Ile Leu Tyr Leu Asn His Asn Cys Ile Thr Thr Leu Arg Pro Gly Ile Phe Lys Asp Leu His Gln Leu Thr Trp Leu Ile Leu Asp Asp Asn Pro Ile Thr Arg Ile Ser Gln Arg Leu Phe Thr Gly Leu Asn Ser Leu Phe Phe Leu Ser Met Val Asn Asn Tyr Leu Glu Ala Leu Pro Lys Gln Met Cys Ala Gln Met Pro Gln Leu Asn Trp Val Asp Leu Glu Gly Asn Arg Ile Lys Tyr Leu Thr Asn Ser Thr Phe Leu Ser Cys Asp Ser Leu Thr Val Leu Asp Leu Ser Ser Asn Thr Ile Thr Glu Leu Ser Pro His Leu Phe Lys Asp Leu Lys Leu Leu Gln Lys Leu Asn Leu Ser Ser Asn Pro Leu Met Tyr Leu His Lys Asn Gln Phe Glu Ser Leu Lys Gln Leu Gln Ser Leu Asp Leu Glu Arg Ile Glu Ile Pro Asn Ile Asn Thr Arg Met Phe Gln Pro Met Lys Asn Leu Ser His Ile Pro Cys Tyr Phe Lys Asn Phe Arg Tyr Cys Ser Tyr Ala Pro His Val Arg Ile Cys Met Pro Leu Thr Asp Gly Ile Ser Ser Phe Glu Asp Leu Leu Ala Asn Asn Ile Leu Arg Ile Phe Val Trp Val Ile Ala Phe Ile Thr Cys Phe Gly Asn Leu Phe Val Ile Gly Met Arg Ser Phe Ile Lys Ala Glu Asn Thr Thr His Ala Met Ser Ile Lys Ile Leu Cys Cys Ala Asp Cys Leu Met Gly Val Tyr Leu Phe Phe Val Gly Ile Phe Asp Ile Lys Tyr Arg Gly Gln Tyr Gln Lys Tyr Ala Leu Leu Trp Met Glu Ser Val Gln Cys Arg Leu Met Gly Phe Leu Ala Met Leu Ser Thr Glu Val Ser Val Leu Leu Leu Thr Tyr Leu Thr Leu Glu Lys Phe Leu Val Ile Val Phe Pro Phe Ser Asn Ile Arg Pro Gly Lys Arg Gln Thr Ser Val Ile Leu Ile Cys Ile Trp Met Ala Gly Phe Leu Ile Ala Val Ile Pro Phe Trp Asn Lys Asp Tyr Phe Gly Asn Phe Tyr Gly Lys Asn Gly Val Cys Phe Pro Leu Tyr Tyr Asp Gln Thr Glu Asp Ile Gly Ser Lys Gly Tyr Ser Leu Gly Ile Phe Leu Gly Val Asn Leu Leu Ala Phe Leu Ile Ile Val Phe Ser Tyr Ile Thr Met Phe Cys Ser Ile Gln Lys Thr Ala Leu Gln Thr Thr Glu Val Arg Asn Cys Phe Gly Arg Glu Val Ala Val Ala Asn Arg Phe Phe Phe Ile Val Phe Ser Asp Ala Ile Cys Trp Ile Pro Val Phe Val Val Lys Ile Leu Ser Leu Phe Arg Val Glu Ile Pro Asp Thr Met Thr Ser Trp Ile Val Ile Phe Phe Leu Pro Val Asn Ser Ala Leu Asn Pro Ile Leu Tyr Thr Leu Thr Thr Asn Phe Phe Lys Asp Lys Leu Lys Gln Leu Leu His Lys His Gln Arg Lys Ser Ile Phe Lys Ile Lys Lys Lys Ser Leu Ser Thr Ser Ile Val Trp Ile Glu Asp Ser Ser Ser Leu Lys Leu Gly Val Leu Asn Lys Ile Thr Leu Gly Asp Ser Ile Met Lys Pro Val Ser <210> 2 <211> 924 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474890CD1 <400> 2 Met Val Cys Ser Ala Ala Pro Leu Leu Leu Leu Ala Thr Thr Leu Pro Leu Leu Gly Ser Pro Val Ala Gln Ala Ser Gln Pro Gly Gln Ser Gln Ala Gly Gly Glu Ser Gly Ser Gly Gln Leu Leu Asp Gln Glu Asn Gly Ala Gly Glu Cys Asn Val Asn His Lys Gly Asn Phe Tyr Cys Ala Cys Leu Ser Gly Tyr Gln Trp Asn Thr Ser Ile Cys Leu His Tyr Pro Pro Cys Gln Ser Leu His Asn His Gln,Pro Cys Gly Cys Leu Val Phe Ser His Pro Glu Pro Gly Tyr Cys Gln Leu Leu Pro Pro Val Pro Gly Ile Leu Asn Leu Asn Ser Gln Leu Gln Met Pro Gly Asp Thr Leu Ser Leu Thr Leu His Leu Ser Gln Glu Ala Thr Asn Leu Ser Trp Phe Leu Arg His Pro Gly Ser Pro Ser Pro Ile Leu Leu Gln Pro Gly Thr Gln Val Ser Val Thr Ser Ser His Gly Gln Ala Ala Leu Ser Val Ser Asn Met Ser His His Trp Ala Gly Glu Tyr Met Ser Cys Phe Glu Ala Gln Gly Phe Lys Trp Asn Leu Tyr Glu Val Val Arg Val Pro Leu Lys Ala Thr Asp Val Ala Arg Leu Pro Tyr Gln Leu Ser Ile Ser Cys Ala Thr Ser Pro Gly Phe Gln Leu Ser Cys Cys Ile Pro Ser Thr Asn Leu Ala Tyr Thr Ala Ala Trp Ser Pro Gly Glu Gly Ser Lys Ala Ser Ser Phe Asn Glu Ser Gly Ser Gln Cys Phe Val Leu Ala Val Gln Arg Cys Pro Met Ala Asp Thr Thr Tyr Ala Cys Asp Leu Gln Ser Leu Gly Leu Ala Pro Leu Arg Val Pro Ile Ser Ile Thr Ile Ile Gln Asp Gly Asp Ile Thr Cys Pro Glu Asp Ala Ser Val Leu Thr Trp Asn Val Thr Lys Ala Gly His Val Ala Gln Ala Pro Cys Pro Glu Ser Lys Arg Gly Ile Val Arg Arg Leu Cys Gly Ala Asp Gly Val Trp Gly Pro Val His Ser Ser Cys Thr Asp Ala Arg Leu Leu Ala Leu Phe Thr Arg Thr Lys Leu Leu Gln Ala Gly Gln Gly Ser Pro Ala Glu Glu Val Pro Gln Ile Leu Ala Gln Leu Pro Gly Gln Ala Ala Glu Ala Ser Ser Pro Ser Asp Leu Leu Thr Leu Leu Ser Thr Met Lys Tyr Val Ala Lys Val Val Ala Glu Ala Arg Ile Gln Leu Asp Arg Arg Ala Leu Lys Asn Leu Leu Ile Ala Thr Asp Lys Val Leu Asp Met Asp Thr Arg Ser Leu Trp Thr Leu Ala Gln Ala Arg Lys Pro Trp Ala Gly Ser Thr Leu Leu Leu Ala Val Glu Thr Leu Ala Cys Ser Leu Cys Pro Gln Asp His Pro Phe Ala Phe Ser Leu Pro Asn Val Leu Leu Gln Ser Gln Leu Phe Gly Pro Thr Phe Pro Ala Asp Tyr Ser Ile Ser Phe Pro Thr Arg Pro Pro Leu Gln Ala Gln Ile Pro Arg His Ser Leu Ala Pro Leu Val Arg Asn Gly Thr Glu Ile Ser Ile Thr Ser Leu Val Leu Arg Lys Leu Asp His Leu Leu Pro Ser Asn Tyr Gly Gln Gly Leu Gly Asp Ser Leu Tyr Ala Thr Pro Gly Leu Val Leu Val Ile Ser Ile Met Ala Gly Asp Arg Ala Phe Ser Gln Gly Glu Val Ile Met Asp Phe Gly Asn Thr Asp Gly Ser Pro His Cys Val Phe Trp Asp His Ser Leu Phe Gln Gly Arg Gly Gly Trp Ser Lys Glu Gly Cys Gln Ala Gln Val Ala Ser Ala Ser Pro Thr Ala Gln Cys Leu Cys Gln His Leu Thr Ala Phe Ser Val Leu Met Ser Pro His Thr Val Pro Glu Glu Pro Ala Leu Ala Leu Leu Thr Gln Val Gly Leu Gly Ala Ser Ile Leu Ala Leu Leu Val Cys Leu Gly Val Tyr Trp Leu Val Trp Arg Val Val Val Arg Asn Lys Ile Ser Tyr Phe Arg His Ala Ala Leu Leu Asn Met Val Phe Cys Leu Leu Ala Ala Asp Thr Cys Phe Leu Gly Ala Pro Phe Leu Ser Pro Gly Pro Arg Ser Pro Leu Cys Leu Ala Ala Ala Phe Leu Cys His Phe Leu Tyr Leu Ala Thr Phe Phe Trp Met Leu Ala Gln Ala Leu Val Leu Ala His Gln Leu Leu Phe Val Phe His Gln Leu Ala Lys His Arg Val Leu Pro Leu Met Val Leu Leu Gly Tyr Leu Cys Pro Leu Gly Leu Ala Gly Val Thr Leu Gly Leu Tyr Leu Pro Gln Gly Gln Tyr Leu Arg Glu Gly Glu Cys Trp Leu Asp Gly Lys Gly Gly Ala Leu Tyr Thr Phe Val Gly Pro Val Leu Ala Ile Ile Gly Val Asn Gly Leu Val Leu Ala Met Ala Met Leu Lys Leu Leu Arg Pro Ser Leu Ser Glu Gly Pro Pro Ala Glu Lys Arg Gln Ala Leu Leu Gly Val Ile Lys Ala Leu Leu Ile Leu Thr Pro Ile Phe Gly Leu Thr Trp Gly Ala Gly Pro Gly His Ser Val Arg Gly Ser Leu His Gly Pro Ser Leu His Leu His His Ser Gln His Pro Pro Gly Arg Leu His Pro Ile Val Trp Leu Pro His Gly Gln Glu Asp Thr Arg Ser Phe Ala Gln Thr Leu Leu Pro Arg Pro Ser Pro Gln Leu His His Leu Pro Gly His Lys <210> 3 <211> 371 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474936CD1 <400> 3 Met Pro Ala Asn Phe Thr Glu Gly Ser Phe Asp Ser Ser Gly Thr Gly Gln Thr Leu Asp Ser Ser Pro Val Ala Cys Thr Glu Thr Val Thr Phe Thr Glu Val Val Glu Gly Lys Glu Trp Gly Ser Phe Tyr Tyr Ser Phe Lys Thr Glu Gln Leu Ile Thr Leu Trp Val Leu Phe Val Phe Thr Ile Val Gly Asn Ser Val Val Leu Phe Ser Thr Trp Arg Arg Lys Lys Lys Ser Arg Met Thr Phe Phe Val Thr Gln Leu Ala Ile Thr Asp Ser Phe Thr Gly Leu Val Asn Ile Leu Thr Asp Ile Ile Trp Arg Phe Thr Gly Asp Phe Thr Ala Pro Asp Leu Val Cys Arg Val Val Arg Tyr Leu Gln Val Val Leu Leu Tyr Ala Ser Thr Tyr Val Leu Val Ser Leu Ser Ile Asp Arg Tyr His Ala Ile Val Tyr Pro Met Lys Phe Leu Gln Gly Glu Lys Gln Ala Arg Val Leu Ile Val Ile Ala Trp Ser Leu Ser Phe Leu Phe Ser Ile Pro Thr Leu Ile Ile Phe Gly Lys Arg Thr Leu Ser Asn Gly Glu Val Gln Cys Trp Ala Leu Trp Pro Asp Asp Ser Tyr Trp Thr Pro Tyr Met Thr Ile Val Ala Phe Leu Val Tyr Phe Ile Pro Leu Thr Ile Ile Ser Ile Met Tyr Gly Ile Val Ile Arg Thr Ile Trp Ile Lys Ser Lys Thr Tyr Glu Thr Val Ile Ser Asn Cys Ser Asp Gly Lys Leu Cys Ser Ser Tyr Asn Arg Gly Leu Ile Ser Lys Ala Lys Ile Lys Ala Ile Lys Tyr Ser Ile Ile Ile Ile Leu Ala Phe Ile Cys Cys Trp Ser Pro Tyr Phe Leu Phe Asp Ile Leu Asp Asn Phe Asn Leu Leu Pro Asp Thr Gln Glu Arg Phe Tyr Ala Ser Val Ile Ile Gln Asn Leu Pro Ala Leu Asn Ser Ala Ile Asn Pro Leu Ile Tyr Cys Val Phe Ser Ser Ser Ile Ser Phe Pro Cys Arg Glu Arg Arg Ser Gln Asp Ser Arg Met Thr Phe Arg Glu Arg Thr Glu Arg His Glu Met Gln Ile Leu Ser Lys Pro Glu Phe Ile <210> 4 <211> 313 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 90012430CD1 <400> 4 Met Gln Lys Cys Asp Phe Pro Ser Met Pro Gly His Asn Thr Ser Arg Asn Ser Ser Cys Asp Pro Ile Val Thr Pro His Leu Ile Ser Leu Tyr Phe Ile Val Leu Ile Gly Gly Leu Val Gly Val Ile Ser Ile Leu Phe Leu Leu Val Lys Met Asn Thr Arg Ser Val Thr Thr Met Ala Val Ile Asn Leu Val Val Val His Ser Val Phe Leu Leu Thr Val Pro Phe Arg Leu Thr Tyr Leu Ile Lys Lys Thr Trp Met Phe Gly Leu Pro Phe Cys Lys Phe Val Ser Ala Met Leu His Ile His Met Tyr Leu Thr Phe Leu Phe Tyr Val Val Ile Leu Val Thr Arg Tyr Leu Ile Phe Phe Lys Cys Lys Asp Lys Val Glu Phe Tyr Arg Lys Leu His Ala Val Ala Ala Ser Ala Gly Met Trp Thr Leu Val Ile Val Ile Val Val Pro Leu Val Val Ser Arg Tyr Gly Ile His Glu Glu Tyr Asn Glu Glu His Cys Phe Lys Phe His Lys Glu Leu Ala Tyr Thr Tyr Val Lys Ile Ile Asn Tyr Met Ile Val Ile Phe Val Ile Ala Val Ala Val Ile Leu Leu Val Phe Gln Val Phe Ile Ile Met Leu Met Val Gln Lys Leu Arg His Ser Leu Leu Ser His Gln Glu Phe Trp Ala Gln Leu Lys Asn Leu Phe Phe Ile Gly Val Ile Leu Val Cys Phe Leu Pro Tyr Gln Phe Phe Arg Ile Tyr Tyr Leu Asn Val Val Thr His Ser Asn Ala Cys Asn Ser Lys Val Ala Phe Tyr Asn Glu Ile Phe Leu Ser Val Thr Ala Ile Ser Cys Tyr Asp Leu Leu Leu Phe Val Phe Gly Gly Ser His Trp Phe Lys 290 ~ 295 300 Gln Lys Ile Ile Gly Leu Trp Asn Cys Val Leu Cys Arg <210> 5 <211> 305 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 90012586CD1 <400> 5 Met Pro Gly His Asn Thr Ser Arg Asn Ser Ser Cys Asp Pro Ile Val Thr Pro His Leu Ile Ser Leu Tyr Phe Ile Val Leu Ile Gly Gly Leu Val Gly Val Ile Ser Ile Leu Phe Leu Leu Val Lys Met Asn Thr Arg Ser Val Thr Thr Met Ala Val Ile Asn Leu Val Val Val His Ser Val Phe Leu Leu Thr Val Pro Phe Arg Leu Thr Tyr Leu Ile Lys Lys Thr Trp Met Phe Gly Leu Pro Phe Cys Lys Phe Val Ser Ala Met Leu His Ile His Met Tyr Leu Thr Phe Leu Phe Tyr Val Val Ile Leu Val Thr Arg Tyr Leu Ile Phe Phe Lys Cys Lys Asp Lys Val Glu Phe Tyr Arg Lys Leu His Ala Val Ala Ala Ser Ala Gly Met Trp Thr Leu Val Ile Val Ile Val Val Pro Leu Val Val Ser Arg Tyr Gly Ile His Glu Glu Tyr Asn Glu Glu His Cys Phe Lys Phe His Lys Glu Leu Ala Tyr Thr Tyr Val Lys Ile Ile Asn Tyr Met Ile Val Ile Phe Val Ile Ala Val Ala Val Ile Leu Leu Val Phe Gln Val Phe Ile Ile Met Leu Met Val Gln Lys Leu Arg His Ser Leu Leu Ser His Gln Glu Phe Trp Ala Gln Leu Lys Asn Leu Phe Phe Ile Gly Val Ile Leu Val Cys Phe Leu Pro Tyr Gln Phe Phe Arg Ile Tyr Tyr Leu Asn Val Val Thr His Ser Asn Ala Cys Asn Ser Lys Val Ala Phe Tyr Asn Glu Ile Phe Leu Ser Val Thr Ala Ile Ser Cys Tyr Asp Leu Leu Leu Phe Val Phe Gly Gly Ser His Trp Phe Lys Gln Lys Ile Ile Gly Leu Trp Asn Cys Val Leu Cys Arg <210> 6 <211> 367 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 90012670CD1 <400> 6 Met Val Phe Arg Leu Ile His Gln Ser Ser Glu Ile Ala Cys Lys Asn Gly Leu Ser Leu Cys Cys Pro Gly Trp Ser Leu Ala Thr Gln Ser Trp Leu Thr Asp Cys Ser Ile Asp Leu Arg Gly Ser Ser Asp Pro Phe Ile Ser Ala Ser Ser Val Ala Glu Thr Thr Gly Asp Phe Pro'Ser Met Pro Gly His Asn Thr Ser Arg Asn Ser Ser Cys Asp Pro Ile Val Thr Pro His Leu Ile Ser Leu Tyr Phe Ile Val Leu Ile Gly Gly Leu Val Gly Val Ile Ser Ile Leu Phe Leu Leu Val Lys Met Asn Thr Arg Ser Val Thr Thr Met Ala Val Ile Asn Leu Val Val Val His Ser Val Phe Leu Leu Thr Val Pro Phe Arg Leu Thr Tyr Leu Ile Lys Lys Thr Trp Met Phe Gly Leu Pro Phe Cys Lys Phe Val Ser Ala Met Leu His Ile His Met Tyr Leu Thr Phe Leu Phe Tyr Val Val Ile Leu Val Thr Arg Tyr Leu Ile Phe Phe Lys Cys Lys Asp Lys Val Glu Phe Tyr Arg Lys Leu His Ala Val Ala Ala Ser Ala Gly Met Trp Thr Leu Val Ile Val Ile Val Val Pro Leu Val Val Ser Arg Tyr Gly Ile His Glu Glu Tyr Asn Glu Glu His Cys Phe Lys Phe His Lys Glu Leu Ala Tyr Thr Tyr Val Lys Ile Ile Asn Tyr Met Ile Val Ile Phe Val Ile Ala Val Ala Val Ile Leu Leu Val Phe Gln Val Phe Ile Ile Met Leu Met Val Gln Lys Leu Arg His Ser Leu Leu Ser His Gln Glu Phe Trp Ala Gln Leu Lys Asn Leu Phe Phe Ile Gly Val Ile Leu Val Cys Phe Leu Pro Tyr Gln Phe Phe Arg Ile Tyr Tyr Leu Asn Val Val Thr His Ser Asn Ala Cys Asn Ser Lys Val Ala Phe Tyr Asn Glu Ile Phe Leu Ser Val Thr Ala Ile Ser Cys Tyr Asp Leu Leu Leu Phe Val Phe Gly Gly Ser His Trp Phe Lys Gln Lys Ile Ile Gly Leu Trp Asn Cys Val Leu Cys Arg <210> 7 <211> 1124 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2880041CD1 <400> 7 Met His Arg Trp Val Lys Glu Lys Asn Ile Thr Val Arg Asp Thr Arg Cys Val Tyr Pro Lys Ser Leu Gln Ala Gln Pro Val Thr Gly Val Lys Gln Glu Leu Leu Thr Cys Asp Pro Pro Leu Glu Leu Pro Ser Phe Tyr Met Thr Pro Ser His Arg Gln Val Val Phe Glu Gly Asp Ser Leu Pro Phe Gln Cys Met Ala Ser Tyr Ile Asp Gln Asp Met Gln Val Leu Trp Tyr Gln Asp Gly Arg Ile Val Glu Thr Asp Glu Ser Gln Gly Ile Phe Val Glu Lys Asn Met Ile His Asn Cys g5 100 105 Ser Leu Ile Ala Ser Ala Leu Thr Ile Ser Asn Ile Gln Ala Gly Ser Thr Gly Asn Trp Gly Cys His Val Gln Thr Lys Arg Gly Asn Asn Thr.Arg Thr Val Asp Ile Val Val Leu Glu Ser Ser Ala Gln Tyr Cys Pro Pro Glu Arg Val Val Asn Asn Lys Gly Asp Phe Arg Trp Pro Arg Thr Leu Ala Gly Ile Thr Ala Tyr Leu Gln Cys Thr Arg Asn Thr His Gly Ser Gly Ile Tyr Pro Gly Asn Pro Gln Asp Glu Arg Lys Ala Trp Arg Arg Cys Asp Arg Gly Gly Phe Trp Ala Asp Asp Asp Tyr Ser Arg Cys Gln Tyr Ala Asn Asp Val Thr Arg Val Leu Tyr Met Phe Asn Gln Met Pro Leu Asn Leu Thr Asn Ala Val Ala Thr Ala Arg Gln Leu Leu Ala Tyr Thr Val Glu Ala Ala Asn Phe Ser Asp Lys Met Asp Val Ile Phe Val Ala Glu Met Ile Glu Lys Phe Gly Arg Phe Thr Lys Glu Glu Lys Ser Lys Glu Leu Gly Asp Val Met Val Asp Ile Ala Ser Asn Ile Met Leu Ala Asp Glu Arg Val Leu Trp Leu Ala Gln Arg Glu Ala Lys Ala Cys Ser Arg Ile Val Gln Cys Leu Gln Arg Ile Ala Thr Tyr Arg Leu Ala Gly Gly Ala His Val Tyr Ser Thr Tyr Ser Pro Asn Ile Ala Leu Glu Ala Tyr Val Ile Lys Ser Thr Gly Phe Thr Gly Met Thr Cys Thr Val Phe Gln Lys Val Ala Ala Ser Asp Arg Thr Gly Leu Ser Asp Tyr Gly Arg Arg Asp Pro Glu Gly Asn Leu Asp Lys Gln Leu Ser Phe Lys Cys Asn Val Ser Asn Thr Phe Ser Ser Leu Ala Leu Lys Asn Thr Ile Val Glu Ala Ser Ile Gln Leu Pro Pro Ser Leu Phe Ser Pro Lys Gln Lys Arg Glu Leu Arg Pro Thr Asp Asp Ser Leu Tyr Lys Leu Gln Leu Ile Ala Phe Arg Asn Gly Lys Leu Phe Pro Ala Thr Gly Asn Ser Thr Asn Leu Ala Asp Asp Gly Lys Arg Arg Thr Val Val Thr Pro Val Ile Leu Thr Lys Ile Asp Gly Val Asn Val Asp Thr His His Ile Pro Val Asn Val Thr Leu Arg Arg Ile Ala His Gly Ala Asp Ala Val Ala Ala Arg Trp Asp Phe Asp Leu Leu Asn Gly Gln Gly Gly Trp Lys Ser Asp Gly Cys His Ile Leu Tyr Ser Asp Glu Asn Ile Thr Thr Ile Gln Cys Tyr Ser Leu Ser Asn Tyr Ala Val Leu Met Asp Leu Thr Gly Ser Glu Leu Tyr Thr Gln Ala Ala Ser Leu Leu His Pro Val Val Tyr Thr Thr Ala Ile Ile Leu Leu Leu Cys Leu Leu Ala Val Ile Val Ser Tyr Ile Tyr His His Ser Leu Ile Arg Ile Ser Leu Lys Ser Trp His Met Leu Val Asn Leu Cys Phe His Ile Phe Leu Thr Cys Val Val Phe Val Gly Gly Ile Thr Gln Thr Arg Asn Ala Ser Ile Cys Gln Ala Val Gly Ile Ile Leu His Tyr Ser Thr Leu Ala Thr Val Leu Trp Val Gly Val Thr Ala Arg Asn Ile Tyr Lys Gln Val Thr Lys Lys Ala Lys Arg Cys Gln Asp Pro Asp Glu Pro Pro Pro Pro Pro Arg Pro Met Leu Arg Phe Tyr Leu Ile Gly Gly Gly Ile Pro Ile Ile Val Cys Gly Ile Thr Ala Ala Ala Asn Ile Lys Asn Tyr Gly Ser Arg Pro Asn Ala Pro Tyr Cys Trp Met Ala Trp Glu Pro Ser Leu Gly Ala Phe Tyr Gly Pro Ala Ser Phe Ile Thr Phe Val Asn Cys Met Tyr Phe Leu Ser Ile Phe Ile Gln Leu Lys Arg His Pro Glu Arg Lys Tyr Glu Leu Lys Glu Pro Thr Glu Glu Gln Gln Arg Leu Ala Ala Asn Glu Asn Gly Glu Ile Asn His Gln Asp Ser Met Ser Leu Ser Leu Ile Ser Thr Ser Ala Leu Glu Asn Glu His Thr Phe His Ser Gln Leu Leu Gly Ala Ser Leu Thr Leu Leu Leu Tyr Val Ala Leu Trp Met Phe Gly Ala Leu Ala Val Ser Leu Tyr Tyr Pro Leu Asp Leu Val Phe Ser Phe Val Phe Gly Ala Thr Ser Leu Ser Phe Ser Ala Phe Phe Met Val His His Cys Val Asn Arg Glu Asp Val Arg Leu Ala Trp Ile Met Thr Cys Cys Pro Gly Arg Ser Ser Tyr Ser Val Gln Val Asn Val Gln Pro Pro Asn Ser Asn Gly Thr Asn Gly Glu Ala Pro Lys Cys Pro Asn Ser Ser Ala Glu Ser Ser Cys Thr Asn Lys Ser Ala Ser Ser Phe Lys Asn Ser Ser Gln Gly Cys Lys Leu Thr Asn Leu Gln Ala Ala Ala Ala Gln Cys His Ala Asn Ser Leu Pro Leu Asn Ser Thr Pro Gln Leu Asp Asn Ser Leu Thr Glu His Ser Met Asp Asn Asp Ile Lys Met His Val Ala Pro Leu Glu Val Gln Phe Arg Thr Asn Val His Ser Ser Arg His His Lys Asn Arg Ser Lys Gly His Arg Ala Ser Arg Leu Thr Val Leu Arg Glu Tyr Ala Tyr Asp Val Pro Thr Ser Val Glu Gly Ser Val Gln Asn Gly Leu Pro Lys Ser Arg Leu Gly Asn Asn Glu Gly His Ser Arg Ser Arg Arg Ala Tyr Leu Ala Tyr Arg Glu Arg Gln Tyr Asn Pro Pro Gln Gln Asp Ser Ser Asp Ala Cys Ser Thr Leu Pro Lys Ser Ser Arg Asn Phe Glu Lys Pro Val Ser Thr Thr Ser Lys Lys Asp Ala Leu Arg Lys Pro Ala Val Val Glu Leu Glu Asn Gln Gln Lys Ser Tyr Gly Leu Asn Leu Ala Ile Gln Asn Gly Pro Ile

12/82 Lys Ser Asn Gly Gln Glu Gly Pro Leu Leu Gly Thr Asp Ser Thr Gly Asn Val Arg Thr Gly Leu Trp Lys His Glu Thr Thr Val <210> 8 <211> 345 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90012123CD1 <400> 8 Met Tyr Lys Asp Cys Ile Glu Ser Thr Gly Asp Tyr Phe Leu Leu Cys Asp Ala Glu Gly Pro Trp Gly Ile Ile Leu Glu Ser Leu Ala Ile Leu Gly Ile Val Val Thr Ile Leu Leu Leu Leu Ala Phe Leu Phe Leu Met Arg Lys Ile Gln Asp Cys Ser Gln Trp Asn Val Leu Pro Thr Gln Leu Leu Phe Leu Leu Ser Val Leu Gly Leu Phe Gly Leu Ala Phe Ala Phe Ile Ile Glu Leu Asn Gln Gln Thr Ala Pro Val Arg Tyr Phe Leu Phe Gly Val Leu Phe Ala Leu Cys Phe Ser Cys Leu Leu Ala His Ala Ser Asn Leu Val Lys Leu Val Arg Gly Cys Val Ser Phe Ser Trp Thr Thr Ile Leu Cys Ile Ala Ile Gly Cys Ser Leu Leu Gln Ile Ile Ile Ala Thr Glu Tyr Val Thr Leu Ile Met Thr Arg Gly Met Met Phe Val Asn Met Thr Pro Cys Gln .

Leu Asn Val Asp Phe Val Val Leu Leu Val Tyr Val Leu Phe Leu Met Ala Leu Thr Phe Phe Val Ser Lys Ala Thr Phe Cys Gly Pro Cys Glu Asn Trp Lys Gln His Gly Arg Leu Ile Phe Ile Thr Val Leu Phe Ser Ile Ile Ile Trp Val Val Trp Ile Ser Met Leu Leu Arg Gly Asn Pro Gln Phe Gln Arg Gln Pro Gln Trp Asp Asp Pro Val Val Cys Ile Ala Leu Val Thr Asn Ala Trp Val Phe Leu Leu Leu Tyr Ile Val Pro Glu Leu Cys Ile Pro Tyr Arg Ser Cys Arg Gln Glu Cys Pro Leu Gln Gly Asn Ala Cys Pro Val Thr Ala Tyr Gln His Ser Phe Gln Val Glu Asn Gln Glu Leu Ser Arg Ala Arg

13/82 Asp Ser Asp Gly Ala Glu Glu Asp Val Ala Leu Thr Ser Tyr Gly Thr Pro Ile Gln Pro Gln Thr Val Asp Pro Thr Gln Glu Cys Phe Ile Pro Gln Ala Lys Leu Ser Pro Gin Gln Asp Ala Gly Gly Val <210> 9 <211> 300 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90012163CD1 <400> 9 Met Tyr Lys Asp Cys Ile Glu Ser Thr Gly Asp Tyr Phe Leu Leu Cys Asp Ala Glu Gly Pro Trp Gly Ile Ile Leu Glu Ser Leu Ala Ile Leu Gly Ile Val Val Thr Ile~Leu Leu Leu Leu Ala Phe Leu Phe Leu Met Arg Lys Ile Gln Asp Cys Ser Gln Trp Asn Val Leu Pro Thr Gln Leu Leu Phe Leu Leu Ser Val Leu Gly Leu Phe Gly 65 70 ' 75 Leu Ala Phe Ala Phe Ile Ile Glu Leu Asn Gln Gln Thr Ala Pro Val Arg Tyr Phe Leu Phe Gly Val Leu Phe Ala Leu Cys Phe Ser Cys Leu Leu Ala His Ala Ser Asn Leu Val Lys Leu Val Arg Gly Cys Val Ser Phe Ser Trp Thr Thr Ile Leu Cys Ile Ala Ile Gly Cys Ser Leu Leu Gln Ile Ile Ile Ala Thr Glu Tyr Val Thr Leu Ile Met Thr Arg Gly Met Met Phe Val Asn Met Thr Pro Cys Gln Leu Asn Val Asp Phe Val Val Leu Leu Val Tyr Val Leu Phe Leu Met Ala Leu Thr Phe Phe Val Ser Lys Ala Thr Phe Cys Gly Pro Cys Glu Asn Trp Lys Gln His Gly Arg Leu Ile Phe Ile Thr Val Leu Phe Ser Ile Ile Ile Trp Val Val Trp Ile Ser Met Leu Leu Arg Gly Asn Pro Gln Phe Gln Arg Gln Pro Gln Trp Asp Asp Pro Val Val Cys Ile Ala Leu Val Thr Asn Ala Trp Val Phe Leu Leu Leu Tyr Ile Val Pro Glu Leu Cys Ile Leu Tyr Arg Ser Cys Arg Gln Glu Cys Pro Leu Gln Gly Asn Ala Cys Pro Val Thr Ala Tyr

14/82 Gln His Ser Phe Gln Val Glu Asn Gln Glu Leu Ser Arg Asp Cys <210> 10 <211> 312 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472462CD1 <400> 10 Met Pro Ser Ile Asn Asp Thr His Phe Tyr Pro Pro Phe Phe Leu Leu Leu Gly Ile Pro Gly Leu Asp Thr Leu His Ile Trp Ile Ser Phe Pro Phe Cys Ile Val Tyr Leu Ile Ala Ile Val Gly Asn Met Thr Ile Leu Phe Val Ile Lys Thr Glu His Ser Leu His Gln Pro Met Phe Tyr Phe Leu Ala Met Leu Ser Met Ile Asp Leu Gly Leu Ser Thr Ser Thr Ile Pro Lys Met Leu Gly Ile Phe Trp Phe Asn Leu Gln Glu Ile Ser Phe Gly Gly Cys Leu Leu Gln Met Phe Phe Ile His Met Phe Thr Gly Met Glu Thr Val Leu Leu Val Val Met Ala Tyr Asp Arg Phe Val Ala Ile Cys Asn Pro Leu Gln Tyr Thr Met Ile Leu Thr Asn Lys Thr Ile Ser Ile Leu Ala Ser Val Val Val Gly Arg Asn Leu Val Leu Val Thr Pro Phe Val Phe Leu Ile Leu Arg Leu Pro Phe Cys Gly His Asn Ile Val Pro His Thr Tyr Cys Glu His Arg Gly Leu Ala Gly Leu Ala Cys Ala Pro Ile Lys Ile Asn Ile Ile Tyr Gly Leu Met Val Ile Ser Tyr Ile Ile Val Asp Val Ile Leu Ile Ala Ser Ser Tyr Val Leu Ile Leu Arg Ala Val Phe Arg Leu Pro Ser Gln Asp Val Arg Leu Lys Ala Phe Asn Thr Cys Gly Ser His Val Cys Val Met Leu Cys Phe Tyr Thr Pro Ala Phe Phe Ser Phe Met Thr His Arg Phe Gly Gln Asn Ile Pro His Tyr Ile His Ile Leu Leu Ala Asn Leu Tyr Val Val Val Pro Pro Ala Leu Asn Pro Val Ile Tyr Gly Val Arg Thr Lys Gln Ile Arg Glu Gln Ile Val Lys Ile Phe Val Gln Lys Glu

15/82 <210> 11 <211> 317 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474873CD1 <400> 11 Met Gly Met Val Arg His Thr Asn Glu Ser Asn Leu Ala Gly Phe Ile Leu Leu Gly Phe Ser Asp Tyr Pro Gln Leu Gln Lys Val Leu Phe Val Leu Ile Leu Ile Leu Tyr Leu Leu Thr Ile Leu Gly Asn Thr Thr Ile Ile Leu Val Ser Arg Leu Glu Pro Lys Leu His Met Pro Met Tyr Phe Phe Leu Ser His Leu Ser Phe Leu Tyr Arg Cys Phe Thr Ser Ser Val Ile Pro Gln Leu Leu Val Asn Leu Trp Glu Pro Met Lys Thr Ile Ala Tyr Gly Gly Cys Leu Val His Leu Tyr Asn Ser His Ala Leu Gly Ser Thr Glu Cys Val Leu Pro Ala Val Met Ser Cys Asp Arg Tyr Val Ala Val Cys Arg Pro Leu His Tyr Thr Val Leu Met His Ile His Leu Cys Met Ala Leu Ala Ser Met ,Ala Trp Leu Ser Gly Ile Ala Thr Thr Leu Val Gln Ser Thr Leu Thr Leu Gln Leu Pro Phe Cys Gly His Arg Gln Val Asp His Phe Ile Cys Glu Val Pro Val Leu Ile Lys Leu Ala Cys Val Gly Thr Thr Phe Asn Glu Ala Glu Leu Phe Val Ala Ser Ile Leu Phe Leu Ile Val Pro Val Ser Phe Ile Leu Val Ser Ser Gly Tyr Ile Ala His Ala Val Leu Arg Ile Lys Ser Ala Thr Arg Arg Gln Lys Ala Phe Gly Thr Cys Phe Ser His Leu Thr Val Val Thr Ile Phe Tyr Gly Thr Ile Ile Phe Met Tyr Leu Gln Pro Ala Lys Ser Arg Ser Arg Asp Gln Gly Lys Phe Val Ser Leu Phe Tyr Thr Val Val Thr Arg Met Leu Asn Pro Leu Ile Tyr Thr Leu Arg Ile Lys Glu Val Lys Gly Ala Leu Lys Lys Val Leu Ala Lys Ala Leu Gly Val Asn Ile Leu <210> 12

16/82 <211> 309 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475172CD1 <400> 12 Met Ala Ala Gly Asn His Ser Thr Val Thr Glu Phe Ile Leu Lys Gly Leu Thr Lys Arg Ala Asp Leu Gln Leu Pro Leu Phe Leu Leu Phe Leu Gly Ile Tyr Leu Val Thr Ile Val Gly Asn Leu Gly Met Ile Thr Leu Ile Cys Leu Asn Ser Gln Leu His Thr Pro Met Tyr Tyr Phe Leu Ser Asn Leu Ser Leu Met Asp Leu Cys Tyr Ser Ser Val Ile Thr Pro Lys Met Leu Val Asn Phe Val Ser Glu Lys Asn Ile Ile Ser Tyr Ala Gly Cys Met,Ser Gln Leu Tyr Phe Phe Leu Val Phe Val Ile Ala Glu Cys Tyr Met Leu Thr Val Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys His Pro Leu Leu Tyr Asn Ile Ile Met Ser His His Thr Cys Leu Leu Leu Val Ala Val Val Tyr Ala Ile Gly Leu Ile Gly Ser Thr Ile Glu Thr Gly Leu Met Leu Lys Leu Pro Tyr Cys Glu His Leu Ile Ser His Tyr Phe Cys Asp Ile Leu Pro Leu Met Lys Leu Ser Cys Ser Ser Thr Tyr Asp Val Glu Met Thr Val Phe Phe Ser Ala Gly Phe Asn Ile Ile Val Thr Ser Leu Thr Val Leu Val Ser Tyr Thr Phe Ile Leu Ser Ser Ile Leu Gly Ile Ser Thr Thr Glu Gly Arg Ser Lys Ala Phe Ser Thr Cys Ser Ser His Leu Ala Ala Val Gly Met Phe Tyr Gly Ser Thr Ala Phe Met Tyr Leu Lys Pro Ser Thr Ile Ser Ser Leu Thr Gln Glu Asn Val Ala Ser Val Phe Tyr Thr Thr Val Ile Pro Met Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Glu Val Lys Ala Ala Val Gln Lys Thr Leu Arg Gly Lys Leu Phe <210> 13 <211> 343 <212> PRT
<213> Homo Sapiens

17/82 <220>
<221> misc_feature <223> Incyte ID No: 7475259CD1 <400> 13 Met Lys Gln Tyr Ser Val Gly Asn Gln His Ser Asn Tyr Arg Ser Leu Leu Phe Pro Phe Leu Cys Ser Gln Met Thr Gln Leu Thr Ala Ser Gly Asn Gln Thr Met Val Thr Glu Phe Leu Phe Ser Met Phe Pro His Ala His Arg Gly Gly Leu Leu Phe Phe Ile Pro Leu Leu Leu Ile Tyr Gly Phe Ile Leu Thr Gly Asn Leu Ile Met Phe Ile Val Ile Gln Val Gly Met Ala Leu His Thr Pro Leu Tyr Phe Phe Ile Ser Val Leu Ser Phe Leu Glu Ile Cys Tyr Thr Thr Thr Thr Ile Pro Lys Met Leu Ser Cys Leu Ile Ser Glu Gln Lys Ser Ile Ser Val Ala Gly Cys Leu Leu Gln Met Tyr Phe Phe His Ser Leu Gly Ile Thr Glu Ser Cys Val Leu Thr Ala Met Ala Ile Asp Arg Tyr Ile Ala Ile Cys Asn Pro Leu Arg Tyr Pro Thr Ile Met Ile Pro Lys Leu Cys Ile Gln Leu Thr Val Gly Ser Cys Phe Cys Gly Phe Leu Leu Val Leu Pro Glu Ile Ala Trp Ile Ser Thr Leu Pro Phe Cys Gly Ser Asn Gln Ile His Gln Ile Phe.Cys Asp Phe Thr Pro Val Leu Ser Leu Ala Cys Thr Asp Thr Phe Leu Val Val Ile Val Asp Ala Ile His Ala Ala Glu Ile Val Ala Ser Phe Leu Val Ile Ala Leu Ser Tyr Ile Arg Ile Ile Ile Val Ile Leu Gly Met His Ser Ala Glu Gly His His Lys Ala Phe Ser Thr Cys Ala Ala His Leu Ala Val Phe Leu Leu Phe Phe Gly Ser Val Ala Val Met Tyr Leu Arg Phe Ser Ala Thr Tyr Ser Val Phe Trp Asp Thr Ala Ile Ala Val Thr Phe Val Ile Leu Ala Pro Phe Phe Asn Pro Ile Ile Tyr Ser Leu Lys Asn Lys Asp Met Lys Glu Ala Ile Gly Arg Leu Phe His Tyr Gln Lys Arg Ala Gly Trp Ala Gly Lys <210> 14 <211> 311 <212> PRT
<213> Homo Sapiens

18/82 <220>
<221> misc_feature <223> Incyte ID No: 7475267CD1 <400> 14 Met Asp His His Met Pro Pro Asn Asn Val Thr Glu Phe Ile Leu Leu Gly Leu Thr Gln Asn Pro His Leu Gln Lys Ile Leu Phe Ile Val Phe Leu Phe Ile Phe Leu Phe Thr Met Leu Ala Asn Leu Phe Ile Val Ile Thr Ile Ser Cys Ser Pro Thr Leu Ser Ser Pro Met Tyr Phe Phe Leu Thr Tyr Leu Ser Phe Ile Asp Ala Ser Tyr Thr Ser Val Thr Thr Pro Lys Met Ile Thr Asp Leu Leu Tyr Gln Arg Arg Thr Ile Ser Leu Ala Gly Cys Leu Thr Gln Leu Phe Val Glu His Leu Leu Gly Gly Ser Glu Ile Ile Leu Leu Ile Val Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys Lys Pro Leu His Tyr Thr Thr Ile Met Gln Gln Gly Ile Cys His Leu Leu Val Val Ile Ala Trp Ile Gly Gly Ile Leu His Ala Thr Val Gln Ile Leu Phe Met Thr Asp Leu Pro Phe Cys Gly Pro Asn Val Ile Asp His Phe Met Cys Asp Leu Phe Pro Leu Leu Lys Leu Ala Cys Arg Asp Thr Tyr Arg Leu Gly Met Leu Val Ala Ala Asn Ser Gly Ala Met Cys Leu Leu Ile Phe Ser Leu Leu Val Ile Ser Tyr Ile Val Ile Leu Ser Ser Leu Lys Ser Tyr Ser Ser Glu Gly Gln His Lys Ala Leu Ser Thr Cys Gly Ser His Phe Thr Val Val Val Leu Phe Phe Val Pro Cys Ile Phe Thr Tyr Met His Pro Val Val Thr Tyr Ser Val Asp Lys Leu Val Thr Val Phe Phe Ala Ile Leu Thr Pro Met Leu Asn Pro Ile Ile Tyr Thr Val Arg Asn Thr Glu Val Lys Asn Ala Val Arg Ser Leu Leu Arg Lys Arg Val Thr Val Tyr Ala <210> 15 <211> 307 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475271CD1

19/82 <400> 15 Met Asn His Ser Val Val Thr Glu Phe Ile Ile Leu Gly Leu Thr Lys Lys Pro Glu Leu Gln Gly Ile Ile Phe Leu Phe Phe Leu Ile

20 25 30 Val Tyr Leu Val Ala Phe Leu Gly Asn Met Leu Ile Ile Ile Ala Lys Ile Tyr Asn Asn Thr Leu His Thr Pro Met Tyr Val Phe Leu Leu Thr Leu Ala Val Val Asp Ile Ile Cys Thr Thr Ser Ile Ile Pro Lys Met Leu Gly Thr Met Leu Thr Ser Glu Asn Thr Ile Ser Tyr Ala Gly Cys Met Ser Gln Leu Phe Leu Phe Thr Trp Ser Leu Gly Ala Glu Met Val Leu Phe Thr Thr Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys Phe Pro Leu His Tyr Ser Thr Ile Met Asn His His Met Cys Val Ala Leu Leu Ser Met Val Met Ala Ile Ala Val Thr Asn Ser Trp Val His Thr Ala Leu Ile Met Arg Leu Thr Phe Cys Gly Pro Asn Thr Ile Asp His Phe Phe Cys Glu Ile Pro Pro Leu Leu Ala Leu Ser Cys Ser Pro Val Arg Ile Asn Glu Val Met Val Tyr Val Ala Asp Ile Thr Leu Ala Ile Gly Asp Phe Ile Leu Thr Cys Ile Ser Tyr Gly Phe Ile Ile Val Ala Ile Leu Arg Ile Arg Thr Val Glu Gly Lys Arg Lys Ala Phe Ser Thr Cys Ser Ser His Leu Thr Val Val Thr Leu Tyr Tyr Ser Pro Val Ile Tyr Thr Tyr Ile Arg Pro Ala Ser Ser Tyr Thr Phe Glu Arg Asp Lys Val Val Ala Ala Leu Tyr Thr Leu Val Thr Pro Thr Leu Asn Pro Met Val Tyr Ser Phe Gln Asn Arg Glu Met Gln Ala Gly Ile Arg Lys Val Phe Ala Phe Leu Lys His .305 <210> 16 <211> 316 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475305CD1 <400> 16 Met Lys Ile Ala Asn Asn Thr Val Val Thr Glu Phe Ile Leu Leu Gly Leu Thr Gln Ser Gln Asp Ile Gln Leu Leu Val Phe Val Leu Ile Leu Ile Phe Tyr Leu Ile Ile Leu Pro Gly Asn Phe Leu Ile Ile Phe Thr Ile Arg Ser Asp Pro Gly Leu Thr Ala Pro Leu Tyr Leu Phe Leu Gly Asn Leu Ala Phe Leu Asp Ala Ser Tyr Ser Phe Ile Val Ala Pro Arg Met Leu Val Asp Phe Leu Ser Glu Lys Lys Val Ile Ser Tyr Arg Gly Cys Ile Thr Gln Leu Phe Phe Leu His Phe Leu Gly Gly Gly Glu Gly Leu Leu Leu Va1 Val Met Ala Phe Asp Arg Tyr Ile Ala Ile Cys Arg Pro Leu His Cys Ser Thr Val Met Asn Pro Arg Ala Cys Tyr Ala Met Met Leu Ala Leu Trp Leu Gly Gly Phe Val His Ser Ile Ile Gln Val Val Leu Ile Leu Arg Leu Pro Phe Cys,Gly Pro Asn Gln Leu Asp Asn Phe Phe Cys Asp Val Arg Gln Val Ile Lys Leu Ala Cys Thr Asp Met Phe Val Val Glu Leu Leu Met Val Phe Asn Ser Gly Leu Met Thr Leu Leu Cys Phe Leu Gly Leu Leu Ala Ser Tyr Ala Val Ile Leu Cys His Val Arg Arg Ala Ala Ser Glu Gly Lys Asn Lys Ala Met Ser Thr Cys Thr Thr Arg Val Ile Ile Ile Leu Leu Met Phe Gly Pro Ala Ile Phe Ile Tyr Met Cys Pro Phe Arg Ala Leu Pro Ala Asp Lys Met Val Ser Leu Phe His Thr Val Ile Phe Pro Leu Met Asn Pro Met Ile Tyr Thr Leu Arg Asn Gln Glu Val Lys Thr Ser Met Lys Arg Leu Leu Ser Arg His Val Val Cys Gln Val Asp Phe Ile IIe Arg Asn <210> 17 <211> 317 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7476160CD1 <400> 17 Met Met Arg Leu Met Lys Glu Val Arg Gly Arg Asn Gln Thr Glu Val Thr Glu Phe Leu Leu Leu Gly Leu Ser Asp Asn Pro Asp Leu

21/82 Gln Gly Val Leu Phe Ala Leu Phe Leu Leu Ile Tyr Met Ala Asn Met Val Gly Asn Leu Gly Met Ile Val Leu Ile Lys Ile Asp Leu Cys Leu His Thr Pro Met Tyr Phe Phe Leu Ser Ser Leu Ser Phe Val Asp Ala Ser Tyr Ser Ser Ser Val Thr Pro Lys Met Leu Val Asn Leu Met Ala Glu Asn Lys Ala Ile Ser Phe His Gly Cys Ala Ala Gln Phe Tyr Phe Phe Gly Ser Phe Leu Gly Thr Glu Cys Phe Leu Leu Ala Met Met Ala Tyr Asp Arg Tyr Ala Ala Ile Trp Asn Pro Leu Leu Tyr Pro Val Leu Val Ser Gly Arg Ile Cys Phe Leu Leu Ile Ala Thr Ser Phe Leu Ala Gly Cys Gly Asn Ala Ala Ile His Thr Gly Met Thr Phe Arg Leu Ser Phe Cys Gly Ser Asn Arg Ile Asn His Phe Tyr Cys Asp Thr Pro Pro Leu Leu Lys Leu Ser Cys Ser Asp Thr His Phe Asn Gly Ile Val Ile Met Ala Phe Ser Ser Phe Ile Val Ile Ser Cys Val Met Ile Val Leu Ile Ser Tyr Leu Cys Ile Phe Ile Ala Val Leu Lys Met Pro Ser Leu Glu Gly Arg His Lys Ala Phe Ser Thr Cys Ala Ser Tyr Leu Met Ala Val Thr Ile Phe Phe Gly Thr Ile Leu Phe Met Tyr Leu Arg Pro Thr Ser Ser Tyr Ser Met Glu Gln Asp Lys Val Val Ser Val Phe Tyr Thr Val Ile Ile Pro Val Leu Asn Pro Leu Ile Tyr Ser Leu Lys Asn Lys Asp Val Lys Lys Ala Leu Lys Lys Ile Leu Trp Lys His Ile Leu <210> 18 <211> 317 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7476781CD1 <400> 18 Met Gly Asp Asn Gln Ser Arg Val Thr Glu Phe Ile Leu Val Gly Phe Gln Leu Ser Val Glu Met Glu Val Leu Leu Phe Trp Ile Phe

22/82 Ser Leu Leu Tyr Leu Phe Ser Leu Leu Gly Asn Gly Val Ile Phe Gly Leu Ile Cys Leu Asp Ser Lys Leu His Thr Pro Met Tyr Phe Phe Leu Ser His Leu Ala Val Ile Asp Met Ser Tyr Ala Ser Asn Asn Val Pro Lys Met Leu Ala Asn Leu Val Asn Gln Lys Arg Thr Ile Ser Phe Ile Ser Cys Ile Met Gln Thr Phe Leu Tyr Leu Ala Phe Ala Val Thr Val Cys Leu Ile Leu Val Val Met Ser Tyr Asp Arg Phe Val Ala Ile Cys His Pro Leu His Tyr Thr Val Ile Met Ser Trp Arg Val Cys Thr Val Leu Ala Val Ala Ser Trp Val Phe Ser Phe Leu Leu Ala Leu Val His Leu Val Leu Ile Leu Arg Leu Pro Phe Cys Gly Pro Gln Glu Val Asn His Phe Phe Gly Glu Ile Leu Ser Val Leu Lys Leu Ala Cys Ala Asp Thr Trp Leu Asn Gln Val Val Ile Phe Ala Ala Cys Met Phe Ile Leu Val Gly Pro Leu Cys Leu Val Leu Val Ser Tyr Leu His 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 Met Tyr Met Ala Pro Lys Ser Ser His Ser Gln Glu Arg Arg Lys Ile Leu Ser Leu Phe Tyr Ser Leu Phe Asn Pro Ile Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Ala Glu Val Lys Gly Ala Leu Lys Arg Val Leu Trp Lys Gln Arg Ser Ile Glu Glu Ser Phe Glu Ile Ser <210> 19 <211> 319 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7487603CD1 <400> 19 Met Phe Pro Ala Asn Trp Thr Ser Val Lys Val Phe Phe Phe Leu Gly Phe Phe His Tyr Pro Lys Val Gln Val Ile Ile Phe Ala Val Cys Leu Leu Met Tyr Leu Ile Thr Leu Leu Gly Asn Ile Phe Leu

23/82 Ile Ser Ile Thr Ile Leu Asp Ser His Leu His Thr Pro Met Tyr Leu Phe Leu Ser Asn Leu Ser Phe Leu Asp Ile Trp Tyr Ser Ser Ser Ala Leu Ser Pro Met Leu Ala Asn Phe Val Ser Gly Arg Asn Thr Ile Ser Phe Ser Gly Cys Ala Thr Gln Met Tyr Leu Ser Leu Ala Met Gly Ser Thr Glu Cys Val Leu Leu Pro Met Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys Asn Pro Leu Arg Tyr Pro Val Ile Met Asn Arg Arg Thr Cys Val Gln Ile Ala Ala Gly Ser Trp Met Thr Gly Cys Leu Thr Ala Met Val Glu Met Met Ser Val Leu Pro Leu Ser Leu Cys Gly Asn Ser Ile Ile Asn His Phe Thr Cys Glu Ile Leu Ala Ile Leu Lys Leu Val Cys Val Asp Thr Ser Leu Val Gln Leu Ile Met Leu Val Ile Ser Val Leu Leu Leu Pro Met Pro Met Leu Leu Ile Cys Ile Ser Tyr Ala Phe Ile Leu Ala Ser Ile Leu Arg Ile Ser Ser Val Glu Gly Arg Ser Lys Ala Phe Ser Thr Cys Thr Ala His Leu Met Val Val Val Leu Phe Tyr Gly Thr Ala Leu Ser Met His Leu Lys Pro Ser Ala Val Asp Ser Gln Glu Ile Asp Lys Phe Met Ala Leu Val Tyr Ala Gly Gln Thr Pro Met Leu Asn Pro Ile Ile Tyr Ser Leu Arg Asn Lys Glu Val Lys Val Ala Leu Lys Lys Leu Leu Ile Arg Asn His Phe Asn Thr Ala Phe Ile Ser Ile Leu Lys <210> 20 <211> 318 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No. 58015601CD1 <400> 20 Met Glu Gly Asn Gln Thr Trp Ile Thr Asp Ile Thr Leu Leu Gly Phe Gln Ala Gly Pro Ala Leu Ala Ile Leu Leu Cys Gly Leu Phe Ser Val Phe Tyr Thr Leu Thr Leu Leu Gly Asn Gly Val Ile Phe

24/82 Gly Ile Ile Cys Leu Asp Ser Lys Leu His Thr Pro Met Tyr Phe Phe Leu Ser His Leu Ala Ile Ile Asp Met Ser Tyr Ala Ser Asn Asn Val Pro Lys Met Leu Ala Asn Leu Met Asn Gln Lys Arg Thr Ile Ser Phe Val Pro Cys Ile Met Gln Thr Phe Leu Tyr Leu Ala Phe Ala Val Thr Glu Cys Leu Ile Leu Val Val Met Ser Tyr Asp Arg Tyr Val Ala Ile Cys His Pro Phe Gln Tyr Thr Val Ile Met Ser Trp Arg Val Cys Thr Ile Leu Val Leu Thr Ser Trp Ser Cys Gly Phe Ala Leu Ser Leu Val His Glu Ile Leu Leu Leu Arg Leu Pro Phe Cys Gly Pro Arg Asp Val Asn His Leu Phe Cys Glu Ile Leu Ser Val Leu Lys Leu Ala Cys Ala Asp Thr Trp Val Asn Gln Val Val Ile Phe Ala Thr Cys Val Phe Val Leu Val Gly Pro Leu Ser Leu Ile Leu Val Ser Tyr Met His Ile Leu Gly Ala Ile Leu Lys Ile Gln Thr Lys Glu Gly Arg Ile Lys Ala Phe Ser Thr Cys Ser Ser His Leu Cys Val Val Gly Leu Phe Phe Gly Ile Ala Met Val Val'Tyr Met Val Pro Asp Ser Asn Gln Arg Glu Glu Gln Glu Lys Met Leu Ser Leu Phe His Ser Val Leu Asn Pro Met Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Ala Gln Leu Lys Gly Ala Leu His Arg Ala Leu Gln Arg Lys Arg Ser Met Arg Thr Val Tyr Gly Leu Cys Leu <210> 21 <211> 351 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 6541249CD1 <400> 21 Met Ser Gly Asp Asn Ser Ser Ser Leu Thr Pro Gly Phe Phe Ile Leu Asn Gly Val Pro Gly Leu Glu Ala Thr His Ile Trp Ile Ser Leu Pro Phe Cys Phe Met Tyr Ile Ile Ala Val Val Gly Asn Cys Gly Leu Ile Cys Leu Ile Ser His Glu Glu Ala Leu His Arg Pro

25/82 Met Tyr Tyr Phe Leu Ala Leu Leu Ser Phe Thr Asp Val Thr Leu Cys Thr Thr Met Val Pro Asn Met Leu Cys Ile Phe Trp Phe Asn Leu Lys Glu Ile Asp Phe Asn Ala Cys Leu Ala Gln Met Phe Phe Val His Met Leu Thr Gly Met Glu Ser Gly Val Leu Met Leu Met Ala Leu Asp Arg Tyr Val Ala Ile Cys Tyr Pro Leu Arg Tyr Ala Thr Ile Leu Thr Asn Pro Val Ile Ala Lys Ala Gly Leu Ala Thr Phe Leu Arg Asn Val Met Leu Ile Ile Pro Phe Thr Leu Leu Thr Lys Arg Leu Pro Tyr Cys Arg Gly Asn Phe Ile Pro His Thr Tyr Cys Asp His Met Ser Val Ala Lys Val Ser Cys Gly Asn Phe Lys Val Asn Ala Ile Tyr Gly Leu Met Val Ala Leu Leu Ile Gly Val Phe Asp Ile Cys Cys Ile Ser Val Ser Tyr Thr Met Ile Leu Gln Ala Val Met Ser Leu Ser Ser Ala Asp Ala Arg His Lys Ala Phe Ser Thr Cys Thr Ser His Met Cys Ser Ile Val Ile Thr Tyr Val Ala Ala Phe Phe Thr Phe Phe Thr His Arg Phe Val Gly His Asn Ile Pro Asn His Ile His Ile Ile Val Ala Asn Leu Tyr Leu Leu Leu Pro Pro Thr Met Asn Pro Ile Val Tyr Gly Val Lys Thr Lys Gln Ile Gln Glu Gly Val Ile Lys Phe Leu Leu Gly Asp Lys Lys Asn Val Gln Gly Phe Cys Phe Ser Gln Val Ile Ser Leu Gly Ser Pro Phe Lys Met Asp Leu Asn Gly Asn Asn Arg Leu Gln Val Leu Arg Lys Glu Arg Glu Glu <210> 22 <211> 315 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472078CD1 <400> 22 Met Glu Gly Gln Ser Thr Val Thr Glu Phe Ser Asn Phe Ile Thr Gly Phe Gln Gln Asp Gly Ser Leu Leu Phe Pro Leu Tyr Phe Pro

26/82 Leu Leu Phe Ile Tyr Thr Phe Ile Ile Ile Asp Asn Leu Leu Ile Phe Ser Ala Val Arg Leu Asp Thr His Leu His Asn Pro Met Tyr Asn Phe Ile Ser Ile Phe Ser Phe Leu Glu Ile Trp Tyr Thr Thr Ala Thr Ile Pro Lys Met Leu Ser Asn Leu Ile Ser Glu Lys Lys Ala Ile Ser Met Thr Gly Cys Ile Leu Gln Met Tyr Phe Phe His Ser Leu Glu Asn Ser Glu Gly Ile Leu Leu Thr Thr Met Ala Ile Asp Arg Tyr Val Ala Ile Cys Asn Pro Leu Arg Tyr Gln Met Ile Met Thr Pro Arg Leu Cys Ala Gln Leu Ser Ala Gly Ser Cys Leu Phe Gly Phe Leu Ile Leu Leu Pro Glu Ile Val Met Ile Ser Thr Leu Pro Phe Cys Gly Pro Asn Gln Ile His Gln Ile Phe Cys Asp Leu Val Pro Val Leu Ser Leu Ala Cys Thr Asp Thr Ser Met Ile Leu Ile Glu Asp Val Ile His Ala Val Thr Ile Ile Ile Thr Phe Leu Ile Ile Ala Leu Ser Tyr Val Arg Ile Val Thr Val Ile Leu Arg Ile Ser Ser Ser Glu Gly Arg Gln Lys Ala Phe Ser Thr Cys Ala Gly His Leu Met Val Phe Leu Ile Phe Phe Gly Ser Val Ser Leu Met Tyr Leu Arg Phe Ser Asp Thr Tyr Pro Pro Val Leu Asp Thr Ala Ile Ala Leu Met Phe Thr Val Leu Ala Pro Phe Phe Asn Pro Ile Ile Tyr Ser Leu Arg Asn Lys Asp Met Asn Asn Ala Ile Lys Lys Leu Phe Cys Leu Gln Lys Val Leu Asn Lys Pro Gly Gly <210> 23 <211> 312 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472087CD1 <400> 23 Met Ser Val Leu Asn Asn Ser Glu Val Lys Leu Phe Leu Leu Ile Gly Ile Pro Gly Leu Glu His Ala His Ile Trp Phe Ser Ile Pro Ile Cys Leu Met Tyr Leu Leu Ala Ile Met Gly Asn Cys Thr Ile

27/82 Leu Phe Ile Ile Lys Thr Glu Pro Ser Leu His Glu Pro Met Tyr Tyr Phe Leu Ala Met Leu Ala Val Ser Asp Met Gly Leu Ser Leu Ser Ser Leu Pro Thr Met Leu Arg Val Phe Leu Phe Asn Ala Met Gly Ile Ser Pro Asn Ala Cys Phe Ala Gln Glu Phe Phe Ile His Gly Phe Thr Val Met Glu Ser Ser Val Leu Leu Ile Met Ser Leu Asp Arg Phe Leu Ala Ile His Asn Pro Leu Arg Tyr Ser Ser Ile Leu Thr Ser Asn Arg Val Ala Lys Met Gly Leu Ile Leu Ala Ile Arg Ser Ile Leu Leu Val Ile Pro Phe Pro Phe Thr Leu Arg Arg Leu Lys Tyr Cys Gln Lys Asn Leu Leu Ser His Ser Tyr Cys Leu His Gln Asp Thr Met Lys Leu Ala Cys Ser Asp Asn Lys Thr Asn Val Ile Tyr Gly Phe Phe Ile Ala Leu Cys Thr Met Leu Asp Leu Ala Leu Ile Val Leu Ser Tyr Val Leu Ile Leu Lys Thr Ile Leu Ser Ile Ala Ser Leu Ala Glu Arg Leu Lys Ala Leu Asn Thr Cys Val Ser His Ile Cys Ala Val Leu Thr Phe Tyr Val Pro Ile Ile Thr Leu Ala Ala Met His His Phe Ala Lys His Lys Ser Pro Leu Val Val Ile Leu Ile Ala Asp Met Phe Leu Leu Val Pro Pro Leu Met Asn Pro Ile Val Tyr Cys Val Lys Thr Arg Gln Ile Trp Glu Lys Ile Leu Gly Lys Leu Leu Asn Val Cys Gly Arg <210> 24 <211> 330 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472089CD1 <400> 24 Met Ser Val Leu Asn Asn Thr Ile Ala Glu Pro Leu Ile Phe Leu Leu Met Gly Ile Pro Gly Leu Lys Ala Thr Gln Tyr Trp Ile Ser Ile Pro Phe Cys Leu Leu Tyr Val Val Ala Val Ser Gly Asn Ser Met Ile Leu Phe Val Val Leu Cys Glu Arg Ser Leu His Lys Pro Met Tyr Tyr Phe Leu Ser Met Leu Ser Ala Thr Asp Leu Ser Leu

28/82 Ser Leu Cys Thr Leu Ser Thr Thr Leu Gly Val Phe Trp Phe Glu Ala Arg Glu Ile Asn Leu Asn Ala Cys Ile Ala Gln Met Phe Phe Leu His Gly Phe Thr Phe Met Glu Ser Gly Val Leu Leu Ala Met Ala Phe Asp Arg Phe Val Ala Ile Cys Tyr Pro Leu Arg Tyr Thr Thr Ile Leu Thr Asn Ala Arg Ile Ala Lys Ile Gly Met Ser Met Leu Ile Arg Asn Val Ala Val Met Leu Pro Val Met Leu Phe Val Lys Arg Leu Ser Phe Cys Ser Ser Met Val Leu Ser His Ser Tyr Cys Tyr His Val Asp Leu Ile Gln Leu Ser Cys Thr Asp Asn Arg Ile Asn Ser Ile Leu Gly Leu Phe Ala Leu Leu Ser Thr Thr Gly Phe Asp Cys Pro Cys Ile Leu Leu Ser Tyr Ile Leu Ile Ile Arg Ser Val Leu Ser Ile Ala Ser Ser Glu Glu Arg Arg Lys Ala Phe Asn Thr Cys Thr Ser His Ile Ser Ala Val Ser Ile Phe Tyr Leu Pro Leu Ile Ser Leu Ser Leu Val His Arg Tyr Gly His Ser Ala Pro Pro Phe Val His Ile Ile Met Ala Asn Val Phe Leu Leu Ile Pro Pro Val Leu Asn Pro Ile Ile Tyr Ser Val Lys Ile Lys Gln Ile Gln Lys Ala Ile Ile Lys Val Leu Ile Gln Lys His Ser Lys Ser Asn His Gln Leu Phe Leu Ile Arg Asp Lys Ala Ile Tyr Glu <210> 25 <211> 314 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474902CD1 <400> 25 Met Thr Leu Gly Ser Leu Gly Asn Ser Ser Ser Ser Val Ser Ala Thr Phe Leu Leu Ser Gly Ile Pro Gly Leu Glu Arg Met His Ile Trp Ile Ser Ile Pro Leu Cys Phe Met Tyr Leu Val Ser Ile Pro Gly Asn Cys Thr Ile Leu Phe Ile Ile Lys Thr Glu Arg Ser Leu His Glu Pro Met Tyr Leu Phe Leu Ser Met Leu Ala Leu Ile Asp

29/82 Leu Gly Leu Ser Leu Cys Thr Leu Pro Thr Val Leu Gly Ile Phe Trp Val Gly Ala Arg Glu Ile Ser His Asp Ala Cys Phe Ala Gln Leu Phe Phe Ile His Cys Phe Ser Phe Leu Glu Ser Ser Val Leu Leu Ser Met Ala Phe Asp Arg Phe Val Ala Ile Cys His Pro Leu His Tyr Val Ser Ile Leu Thr Asn Thr Val Ile Gly Arg Ile Gly Leu Val Ser Leu Gly Arg Ser Val Ala Leu Ile Phe Pro Leu Pro Phe Met Leu Lys Arg Phe Pro Tyr Cys Gly Ser Pro Val Leu Ser His Ser Tyr Cys Leu His Gln Glu Val Met Lys Leu Ala Cys Ala Asp Met Lys Ala Asn Ser Ile Tyr Gly Met Phe Val Ile Val Ser Thr Val Gly Ile Asp Ser Leu Leu Ile Leu Phe Ser Tyr Ala Leu Ile Leu Arg Thr Val Leu Ser Ile Ala Ser Arg Ala Glu Arg Phe Lys Ala Leu Asn Thr Cys Val Ser His Ile Cys Ala Val Leu Leu Phe Tyr Thr Pro Met Ile Gly Leu Ser Val Ile His Arg Phe Gly Lys Gln Ala Pro His Leu Val Gln Val Val Met Gly Phe Met Tyr Leu Leu Phe Pro Pro Val Met Asn Pro Ile Val Tyr Ser Val Lys Thr Lys Gln Ile Arg Asp Arg Val Thr His Ala Phe Cys Tyr <210> 26 <211> 320 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475057CD1 <400> 26 Met Ala Glu Thr Leu Gln Leu Asn Ser Thr Phe Leu His Pro Asn Phe Phe Ile Leu Thr Gly Phe Pro Gly Leu Gly Ser Ala Gln Thr Trp Leu Thr Leu Val Phe Gly Pro Ile Tyr Leu Leu Ala Leu Leu Gly Asn Gly Ala Leu Pro Ala Val Val Trp Ile Asp Ser Thr Leu His Gln Pro Met Phe Leu Leu Leu Ala Ile Leu Ala Ala Thr Asp Leu Gly Leu Ala Thr Ser Ile Ala Pro Gly Leu Leu Ala Val Leu

30/82 Trp Leu Gly Pro Arg Ser Val Pro Tyr Ala Val Cys Leu Val Gln Met Phe Phe Val. His Ala Leu Thr Ala Met Glu Ser Gly Val Leu Leu Ala Met Ala Cys Asp Arg Ala Ala Ala Ile Gly Arg Pro Leu His Tyr Pro Val Leu Val Thr Lys Ala Cys Val Gly Tyr Ala Ala Leu Ala Leu Ala Leu Lys Ala Val Ala Ile Val Val Pro Phe Pro Leu Leu Val Ala Lys Phe Glu His Phe Gln Ala Lys Thr Ile Gly His Thr Tyr Cys Ala His Met Ala Val Val Glu Leu Val Val Gly Asn Thr Gln Ala Thr Asn Leu Tyr Gly Leu Ala Leu Ser Leu Ala Ile Ser Gly Met Asp Ile Leu Gly Ile Thr Gly Ser Tyr Gly Leu Ile Ala His Ala Val Leu Gln Leu Pro Thr Arg Glu Ala His Ala Lys Ala Phe Gly Thr Cys Ser Ser His Ile Cys Val Ile Leu Ala Phe Tyr Ile Pro Gly Leu Phe Ser Tyr Leu Thr His Arg Phe Gly His His Thr Val Pro Lys Pro Val His Ile Leu Leu Ser Asn Ile Tyr Leu Leu Leu Pro Pro Ala Leu Asn Pro Leu Ile Tyr Gly Ala Arg Thr Lys Gln Ile Arg Asp Arg Leu Leu Glu Thr Phe Thr Phe Arg Lys Ser Pro Leu <210> 27 <211> 331 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475261CD1 <400> 27 Met Ser Gly Glu Asn Val Thr Arg Val Gly Thr Phe Ile Leu Val Gly Phe Pro Thr Ala Pro Gly Leu Gln Tyr Leu Leu Phe Leu Leu Phe Leu Leu Thr Tyr Leu Phe Val Leu Val Glu Asn Leu Ala Ile Ile Leu Thr Val Trp Ser Ser Thr Ser Leu His Arg Pro Met Tyr Tyr Phe Leu Ser Ser Met Ser Phe Leu Glu Ile Trp Tyr Val Ser Asp Ile Thr Pro Lys Met Leu Glu Gly Phe Leu Leu Gln Gln Lys Arg Ile Ser Phe Val Gly Cys Met Thr Gln Leu Tyr Phe Phe Ser

31/82 Ser Leu Val Cys Thr Glu Cys Val Leu Leu Ala Ser Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys His Pro Leu Arg Tyr His Val Leu Val Thr Pro Gly Leu Cys Leu Gln Leu Val Gly Phe Ser Phe Val Ser Gly Phe Thr Ile Ser Met Ile Lys Val Cys Phe Ile Ser Ser Val Thr Phe Cys Gly Ser Asn Val Leu Asn His Phe Phe Cys Asp Ile Ser Pro Ile Leu Lys Leu Ala Cys Thr Asp Phe Ser Thr Ala Glu Leu Val Asp Phe Ile Leu Ala Phe Ile Ile Leu Val Phe Pro Leu Leu Ala Thr Met Leu Ser Tyr Ala His Ile Thr Leu Ala Val Leu Arg Ile Pro Ser Ala Thr Gly Cys Trp Arg Ala Phe Phe Thr Cys Ala Ser His Leu Thr Val Val Thr Val Phe Tyr Thr Ala Leu Leu Phe Met Tyr Val Arg Pro Gln Ala Ile Asp Ser Arg Ser Ser Asn Lys Leu Ile Ser Val Leu Tyr Thr Val Ile Thr Pro Ile Leu Asn Pro Leu Ile Tyr Cys Leu Arg Asn Lys Glu Phe Lys Asn Ala 290 295. 300 Leu Lys Lys Ala Phe Gly Leu Thr Ser Cys Ala Val Glu Gly Arg Leu Ser Ser Leu Leu Glu Leu His Leu Gln Ile His Ser Gln Pro Leu <210> 28 <211> 311 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475262CD1 <400> 28 Met Lys Gly Ala Asn Leu Ser Gln Gly Met Glu Phe Glu Leu Leu Gly Leu Thr Thr Asp Pro Gln Leu Gln Arg Leu Leu Phe Val Val Phe Leu Gly Met Tyr Thr Ala Thr Leu Leu Gly Asn Leu Val Met Phe Leu Leu Ile His Val Ser Ala Thr Leu His Thr Pro Met Tyr Ser Leu Leu Lys Ser Leu Ser Phe Leu Asp Phe Cys Tyr Ser Ser Thr Val Val Pro Gln Thr Leu Val Asn Phe Leu Ala Lys Arg Lys

32/82 Val Ile Ser Tyr Phe Gly Cys Met Thr Gln Met Phe Phe Tyr Ala Gly Phe Ala Thr Ser Glu Cys Tyr Leu Ile Ala Ala Met Ala Tyr Asp Arg Tyr Ala Ala Ile Cys Asn Pro Leu Leu Tyr Ser Thr Ile Met Ser Pro Glu Val Cys Ala Ser Leu Ile Val Gly Ser Tyr Ser Ala Gly Phe Leu Asn Ser Leu Ile His Thr Gly Cys Ile Phe Ser Leu Lys Phe Cys Gly Ala His Val Val Thr His Phe Phe Cys Asp Gly Pro Pro Ile Leu Ser Leu Ser Cys Val Asp Thr Ser Leu Cys Glu Ile Leu Leu Phe Ile Phe Ala Gly Phe Asn Leu Leu Ser Cys Thr Leu Thr Ile Leu Ile Ser Tyr Phe Leu Ile Leu Asn Thr Ile Leu Lys Met Ser Ser Ala Gln Gly Arg Phe Lys Ala Phe Ser Thr Cys Ala Ser His Leu Thr Ala Ile Cys Leu Phe Phe Gly Thr Thr Leu Phe Met Tyr Leu Arg Pro Arg Ser Ser Tyr Ser Leu Thr Gln Asp Arg Thr Val Ala Val Ile Tyr Thr Val Val Ile Pro Val Leu Asn Pro Leu Met Tyr Ser Leu Arg Asn Lys Asp Val Lys Lys Ala Leu Ile Lys Val Trp Gly Arg Lys Thr Met Glu <210> 29 <211> 308 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475266CD1 <400> 29 Met Thr Met Glu Asn Tyr Ser Met Ala Ala Gln Phe Val Leu Asp Gly Leu Thr Gln Gln Ala Glu Leu Gln Leu Pro Leu Phe Leu Leu Phe Leu Gly Ile Tyr Val Val Thr Val Val Gly Asn Leu Gly Met Ile Leu Leu Ile Ala Val Ser Pro Leu Leu His Thr Pro Met Tyr Tyr Phe Leu Ser Ser Leu Ser Phe Val Asp Phe Cys Tyr Ser Ser Val Ile Thr Pro Lys Met Leu Val Asn Phe Leu Gly Lys Lys Asn Thr Ile Leu Tyr Ser Glu Cys Met Val Gln Leu Val Phe Phe Val Val Phe Val Val Ala Glu Gly Tyr Leu Leu Thr Ala Met Ala Tyr

33/82 Asp Arg Tyr Val Ala Ile Cys Ser Pro Leu Leu Tyr Asn Ala Ile Met Ser Ser Trp Val Cys Ser Leu Leu Val Leu Ala Ala Phe Phe Leu Gly Phe Leu Ser Ala Leu Thr His Thr Ser Ala Met Met Lys Leu Ser Phe Cys Lys Ser His Ile Ile Asn His Tyr Phe Cys Asp Val Leu Pro Leu Leu Asn Leu Ser Cys Ser Asn Thr His Leu Asn 185 . 190 195 Glu Leu Leu Leu Phe Ile Ile Ala Gly Phe Asn Thr Leu Val Pro Thr Leu Ala Val Ala Val Ser Tyr Ala Phe Ile Leu Tyr Ser Ile Leu His Ile Arg Ser Ser Glu Gly Arg Ser Lys Ala Phe Gly Thr Cys Ser Ser His Leu Met Ala Val Val Ile Phe Phe Gly Ser Ile Thr Phe Met Tyr Phe Lys Pro Pro Ser Ser Asn Ser Leu Asp Gln Glu Lys Val Ser Ser Val Phe Tyr Thr Thr Val Ile Pro Met Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Lys Ala Leu Arg Lys Val Leu Val Gly Lys <210> 30 <211> 298 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475284CD1 <400> 30 Met Arg Asn His Thr Met Val Thr Glu Phe Ile Leu Leu Gly Ile Pro Glu Thr Glu Gly Leu Glu Thr Ala Leu Leu Phe Leu Phe Ser Ser Phe Tyr Leu Cys Thr Leu Leu Gly Asn Val Leu Ile Leu Thr Ala Ile Ile Ser Ser Thr Arg Leu His Thr Pro Met Tyr Phe Phe Leu Gly Asn Leu Ser Ile Phe Asp Leu Gly Phe Ser Ser Thr Thr Val Pro Lys Met Leu Phe Tyr Leu Ser Gly Asn Ser His Ala Ile Ser Tyr Ala Gly Cys Val Ser Gln Leu Phe Phe Tyr His Phe Leu Gly Cys Thr Glu Cys Phe Leu Tyr Thr Val Met Ala Cys Asp Arg Phe Val Ala Ile Cys Phe Pro Leu Arg Tyr Thr Val Ile Met Asn

34/82 His Arg Val Cys Phe Met Leu Ala Thr Gly Thr Trp Met Ile Gly Cys Val His Ala Met Ile Leu Thr Pro Leu Thr Phe Gln Leu Pro Tyr Cys Gly Pro Asn Lys Val Gly Tyr Tyr Phe Cys Asp Ile Pro Ala Val Leu Pro Leu Ala Cys Lys Asp Thr Ser Leu Ala Gln Arg Val Gly Phe Thr Asn Val Gly Leu Leu Ser Leu Ile Cys Phe Phe Leu Ile Leu Val Ser Tyr Thr Cys Ile Gly Ile Ser Ile Ser Lys Ile Arg Ser Ala Glu Gly Arg Gln Arg Ala Phe Ser Thr Cys Ser Ala His Leu Thr Ala Ile Leu Cys Ala Tyr Gly Pro Val Ile Val Ile Tyr Leu Gln Pro Asn Pro Ser Ala Leu Leu Gly Ser Ile Ile Gln Ile Leu Asn Asn Leu Val Thr Pro Met Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Ser Asp Gln Pro <210> 31 <211> 317 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475309CD1 <400> 31 .
Met Ser Gln Val Thr Asn Thr Thr Gln Glu Gly Ile Tyr Phe Ile Leu Thr Asp Ile Pro Gly Phe Glu Ala Ser His Ile Trp Ile Ser Ile Pro Val Cys Cys Leu Tyr Thr Ile Ser Ile Met Gly Asn Thr

35 40 45 Thr Ile Leu Thr Val Ile Arg Thr Glu Pro Ser Val His Gln Arg Met Tyr Leu Phe Leu Ser Met Leu Ala Leu Thr Asp Leu Gly Leu Thr Leu Thr Thr Leu Pro Thr Val Met Gln Leu Leu Trp Phe Asn Val Arg Arg Ile Ser Ser Glu Ala Cys Phe Ala Gln Phe Phe Phe Leu His Gly Phe Ser Phe Met Glu Ser Ser Val Leu Leu Ala Met Ser Val Asp Cys Tyr Val Ala Ile Cys Cys Pro Leu His Tyr Ala Ser Ile Leu Thr Asn Glu Val Ile Gly Arg Thr Gly Leu Ala Ile Ile Cys Cys Cys Val Leu Ala Val Leu Pro Ser Leu Phe Leu Leu Lys Arg Leu Pro Phe Cys His Ser His Leu Leu Ser Arg Ser Tyr Cys Leu His Gln Asp Met Ile Arg Leu Val Cys Ala Asp Ile Arg Leu Asn Ser Trp Tyr Gly Phe Ala Leu Ala Leu Leu Ile Ile Ile Val Asp Pro Leu Leu Ile Val Ile Ser Tyr Thr Leu Ile Leu Lys Asn Ile Leu Gly Thr Ala Thr Trp Ala Glu Arg Leu Arg Ala Leu Asn Asn Cys Leu Ser His Ile Leu Ala Val Leu Val Leu Tyr Ile Pro Met Val Gly Val Ser Met Thr His Arg Phe Ala Lys His Ala Ser Pro Leu Val His Val Ile Met Ala Asn Ile Tyr Leu Leu Ala Pro Pro Val Met Asn Pro Ile Ile Tyr Ser Val Lys Asn Lys Gln Ile Gln Trp Gly Met Leu Asn Phe Leu Ser Leu Lys Asn Met His Ser Arg <210> 32 <211> 309 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477359CD1 <400> 32 Met Gly Leu Gly Asn Glu Ser Ser Leu Met Asp Phe Ile Leu Leu Gly Phe Ser Asp His Pro Arg Leu Glu Ala Val Leu Phe Val Phe Val Leu Phe Phe Tyr Leu Leu Thr Leu Val Gly Asn Phe Thr Ile Ile Ile Ile Ser Tyr Leu Asp Pro Pro Leu His Thr Pro Met Tyr Phe Phe Leu Ser Asn Leu Ser Leu Leu Asp Ile Cys Phe Thr Thr Ser Leu Ala Pro Gln Thr Leu Val Asn Leu Gln Arg Pro Lys Lys Thr Ile Thr Tyr Gly Gly Cys Val Ala Gln Leu Tyr Ile Ser Leu Ala Leu Gly Ser Thr Glu Cys Ile Leu Leu Ala Asp Met Ala Leu Asp Arg Tyr Ile Ala Val Cys Lys Pro Leu His Tyr Val Val Ile Met Asn Pro Arg Leu Cys Gln Gln Leu Ala Ser Ile Ser Trp Leu Ser Gly Leu Ala Ser Ser Leu Ile His Ala Thr Phe Thr Leu Gln Leu Pro Leu Cys Gly Asn His Arg Leu Asp His Phe Ile Cys Glu

36/82 Val Pro Ala Leu Leu Lys Leu Ala Cys Val Asp Thr Thr Val Asn Glu Leu Val Leu Phe Val Val Ser Val Leu Phe Val Val Ile Pro Pro Ala Leu Ile Ser Ile Ser Tyr Gly Phe Ile Thr Gln Ala Val Leu Arg Ile Lys Ser Val Glu Ala Arg His Lys Ala Phe Ser Thr Cys Ser Ser His Leu Thr Val Val Ile Ile Phe Tyr Gly Thr Ile Ile Tyr Val Tyr Leu Gln Pro Ser Asp Ser Tyr Ala Gln Asp Gln Gly Lys Phe Ile Ser Leu Phe Tyr Thr Met Val Thr Pro Thr Leu Asn Pro Ile Ile Tyr Thr Leu Arg Asn Lys Asp Met Lys Glu Ala Leu Arg Lys Leu Leu Ser Gly Lys Leu <210> 33 <211> 312 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 58004547CD1 <400> 33 Met Ser Gly Glu Asn Val Thr Lys Val Ser Thr Phe Ile Leu Val Gly Leu Pro Thr Ala Pro Gly Leu Gln Tyr Leu Leu Phe Leu Leu Phe Leu Leu Thr Tyr Leu Phe Val Leu Val Glu Asn Leu Ala Ile Ile Leu Ile Val Trp Ser Ser Thr Ser Leu His Arg Pro Met Tyr Tyr Phe Leu Ser Ser Met Ser Phe Leu Glu Ile Trp Tyr Val Ser Asp Ile Thr Pro Lys Met Leu Glu Gly Phe Leu Leu Gln Gln Lys Arg Ile Ser Phe Val Gly Cys Met Thr Gln Leu Tyr Phe Phe Ser Ser Leu Val Cys Thr Glu Cys Val Leu Leu Ala Ser Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys His Pro Leu Arg Tyr His Val Leu Val Thr Pro Gly Leu Cys Leu Gln Leu Val Gly Phe Ser Phe Val Ser Gly Phe Thr Ile Ser Met Ile Lys Val Cys Phe Ile Ser Ser Val Thr Phe Cys Gly Ser Asn Val Leu Asn His Phe Phe Cys Asp Ile Ser Pro Ile Leu Lys Leu Ala Cys Thr Asp Phe Ser Thr Ala Glu Leu Val Asp Phe Ile Leu Ala Phe Ile Ile Leu Val Phe Pro

37/82 Leu Leu Ala Thr Ile Leu Ser Tyr Trp His Ile Thr Leu Ala Val Leu Arg Ile Pro Ser Ala Thr Gly Cys Trp Arg Ala Phe Ser Thr Cys Ala Ser His Leu Thr Val Val Thr Val Phe Tyr Thr Ala Leu Leu Phe Met Tyr Val Arg Pro Gln Ala Ile Asp Ser Gln Ser Ser Asn Lys Leu Ile Ser Ala Val Tyr Thr Val Val Thr Pro Ile Ile Asn Pro Leu Ile Tyr Cys Leu Arg Asn Lys Glu Phe Lys Asp Ala Leu Lys Lys Ala Leu Gly Leu Gly Gln Thr Ser His <210> 34 <211> 310 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7476156CD1 <400> 34 Met Asp Lys Glu Asn Ser Ser Met Val Thr Glu Phe Ile Phe Met Gly Ile Thr Gln Asp Pro Gln Met Glu Ile Ile Phe Phe Val Val Phe Leu Ile Val Tyr Leu Val Asn Val Val Gly Asn Ile Gly Met Ile Ile Leu Ile Thr Thr Asp Thr Gln Leu His Thr Pro Met Tyr Phe Phe Leu Cys Asn Leu Ser Phe Val Asp Leu Gly Tyr Ser Ser Ala Ile Ala Pro Arg Met Leu Ala Asp Phe Leu Thr Asn His Lys Val Ile Ser Phe Ser Ser Cys Ala Thr Gln Phe Ala Phe Phe Val Gly Phe Val Asp Ala Glu Cys Tyr Val Leu Ala Ala Met Ala Tyr Gly Arg Phe Val Ala Ile Cys Arg Pro Leu His Tyr Ser Thr Phe Met Ser Lys Gln Val Cys Leu Ala Leu Met Leu Gly Ser Tyr Leu Ala Gly Leu Val Ser Leu Val Ala His Thr Thr Leu Thr Phe Ser Leu Ser Tyr Cys Gly Ser Asn Ile Ile Asn His Phe Phe Cys Glu Ile Pro Pro Leu Leu Ala Leu Ser Cys Ser Asp Thr Tyr Ile Ser Glu Ile Leu Leu Phe Ser Leu Cys Gly Phe Ile Glu Phe Ser Thr Ile Leu Ile Ile Phe Ile Ser Tyr Thr Phe Ile Leu Val Ala Ile

38/82 Ile Arg Met Arg Ser Ala Glu Gly Arg Leu Lys Ala Phe Ser Thr Cys Gly Ser His Leu Thr Gly Ile Thr Leu Phe Tyr Gly Thr Val Met Phe Met Tyr Leu Arg Pro Thr Ser Ser Tyr Ser Leu Asp Gln Asp Lys Trp Ala Ser Val Phe Tyr Thr Val Ile Ile Pro Met Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Ala Ala Phe Lys Lys Leu Ile Gly Lys Lys Ser Gln <210> 35 <211> 314 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475114CD1 <400> 35 Met Ala Asn Val Thr Leu Val Thr Gly Phe Leu Leu Met Gly Phe Ser Asn Ile Gln Lys Leu Arg Ile Leu Tyr Gly Val Leu Phe Leu Leu Ile Tyr Leu Ala Ala Leu Met Ser Asn Leu Leu Ile Ile Thr Leu Ile Thr Leu Asp Val Lys Leu Gln Thr Pro Met Tyr Phe Phe Leu Lys Asn Leu Ser Phe Leu Asp Val Phe Leu Val Ser Val Pro Ile Pro Lys Phe Ile Val Asn Asn Leu Thr His Asn Asn Ser Ile Ser Ile Leu Gly Cys Ala Phe Gln Leu Leu Leu Met Thr Ser Phe Ser Ala Gly Glu Ile Phe Ile Leu Thr Ala Met Ser Tyr Asp Arg Tyr Val Ala Ile Cys Cys Pro Leu Asn Tyr Glu Val Ile Met Asn Thr Gly Val Cys Val Leu Met Ala Ser Val Ser Trp Ala Ile Gly Gly Leu Phe Gly Thr Ala Tyr Thr Ala Gly Thr Phe Ser Met Pro Phe Cys Gly Ser Ser Val Ile Pro Gln Phe Phe Cys Asp Val Pro Ser Leu Leu Arg Ile Ser Cys Ser Glu Thr Leu Met Val Ile Tyr Ala Gly Ile Gly Val Gly Ala Cys Leu Ser Ile Ser Cys Phe Ile Cys Ile Val Ile Ser Tyr Ile Tyr Ile Phe Ser Thr Val Leu Lys Ile Pro Thr Thr Lys Gly Gln Ser Lys Ala Phe Ser Thr Cys Phe Pro His Leu Thr Val Phe Thr Val Phe Ile Ile Thr Ala Tyr Phe

39/82 Val Tyr Leu Lys Pro Pro Ser Asn Ser Pro Ser Val Ile Asp Arg Leu Leu Ser Val Ile Tyr Thr Val Met Pro Pro Val Phe Asn Pro Val Thr Tyr Ser Leu Arg Asn Asn Asp Met Lys Cys Ala Leu Ile Arg Leu Leu Gln Lys Thr Tyr Gly Gln Glu Ala Tyr Phe Ile <210> 36 <211> 311 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 55003505CD1 <400> 36 Met Ser Asn Ala Ser Leu Val Thr Ala Phe Ile Leu Thr Gly Leu Pro His Ala Pro Gly Leu Asp Ala Leu Leu Phe Gly Ile Phe Leu Val Val Tyr Val Leu Thr Val Leu Gly Asn Leu Leu Ile Leu Leu Val Ile Arg Val Asp Ser His Leu His Thr Pro Met Tyr Tyr Phe Leu Thr Asn Leu Ser Phe Ile Asp Met Trp Phe Ser Thr Val Thr Val Pro Lys Met Leu Met Thr Leu Val Ser Pro Ser Gly Arg Ala Ile Ser Phe His Ser Cys Val Ala Gln Leu Tyr Phe Phe His Phe Leu Gly Ser Thr Glu Cys Phe Leu Tyr Thr Val Met Ser Tyr Asp Arg Tyr Leu Ala Ile Ser Tyr Pro Leu Arg Tyr Thr Ser Met Met Ser Gly Ser Arg Cys Ala Leu Leu Ala Thr Gly Thr Trp Leu Ser Gly Ser Leu His Ser Ala Val Gln Thr Ile Leu Thr Phe His Leu Pro Tyr Cys Gly Pro Asn Gln Ile Gln His Tyr Phe Cys Asp Ala Pro Pro Ile Leu Lys Leu Ala Cys Ala Asp Thr Ser Ala Asn Val Met Val Ile Phe Val Asp Ile Gly Ile Val Ala Ser Gly Cys Phe Val Leu Ile Val Leu Ser Tyr Val Ser Ile Val Cys Ser Ile Leu Arg Ile Arg Thr Ser Asp Gly Arg Arg Arg Ala Phe Gln Thr Cys Ala Ser His Cys Ile Val Val Leu Cys Phe Phe Val Pro Cys Val Val Ile Tyr Leu Arg Pro Gly Ser Met Asp Ala Met Asp Gly Val

40/82 Val Ala Ile Phe Tyr Thr Val Leu Thr Pro Leu Leu Asn Pro Val Val Tyr Thr Leu Arg Asn Lys Glu Val Lys Lys Ala Val Leu Lys Leu Arg Asp Lys Val Ala His Pro Gln Arg Lys <210> 37 <211> 337 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474916CD1 <400> 37 Met Glu Gly Lys Asn Gln Thr Asn Ile Ser Glu Phe Leu Leu Leu Gly Phe Ser Ser Trp Gln Gln Gln Gln Val Leu Leu Phe Ala Leu Phe Leu Cys Leu Tyr Leu Thr Gly Leu Phe Gly Asn Leu Leu Ile Leu Leu Ala Ile Gly Ser Asp His Cys Leu His Thr Pro Met Tyr Phe Phe Leu Ala Asn Leu Ser Leu Val Asp Leu Cys Leu Pro Ser Ala Thr Val Pro Lys Met Leu Leu Asn Ile Gln Thr Gln Thr Gln Thr Ile Ser Tyr Pro Gly Cys Leu Ala Gln Met Tyr Phe Cys Met Met Phe Ala Asn Met Asp Asn Phe Leu Leu Thr Val Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys His Pro Leu His Tyr Ser Thr Ile Met Ala Leu Arg Leu Cys Ala Ser Leu Val Ala Ala Pro Trp Val Ile Ala Ile Leu Asn Pro Leu Leu His Thr Leu Met Met Ala His Leu His Phe Cys Ser Asp Asn Val Ile His His Phe Phe Cys Asp Ile Asn Ser Leu Leu Pro Leu Ser Cys Ser Asp Thr Ser Leu Asn Gln Leu Ser Val Leu Ala Thr Val Gly Leu Ile Phe Val Val Pro Ser Val Cys Ile Leu Val Ser Tyr Ile Leu Ile Val Ser Ala Val Met Lys Val Pro Ser Ala Gln Gly Lys Leu Lys Ala Phe Ser Thr Cys Gly Ser His Leu Ala Leu Val Ile Leu Phe Tyr Gly Ala Asn Thr Gly Val Tyr Met Ser Pro Leu Ser Asn His Ser Thr Glu Lys Asp Ser Ala Ala Ser Val Ile Phe Met Val Val Ala Pro Val Leu Asn Pro Phe Ile Tyr Ser Leu Arg Asn Asn Glu Leu Lys Gly Thr

41/82 Leu Lys Lys Thr Leu Ser Arg Pro Gly Ala Val Ala His Ala Cys Asn Pro Ser Thr Leu Gly Gly Arg Gly Gly Trp Ile Met Arg Ser Gly Asp Arg Asp His Pro Gly <210> 38 <211> 325 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472365CD1 <400> 38 Met Phe Leu Pro Asn Asp Thr Gln Phe His Pro Ser Ser Phe Leu Leu Leu Gly Ile Pro Gly Leu Glu Thr Leu His Ile Trp Ile Gly Phe Pro Phe Cys Ala Val Tyr Met Ile Ala Leu Ile Gly Asn Phe Thr Ile Leu Leu Val Ile Lys Thr Asp Ser Ser Leu His Gln Pro Met Phe Tyr Phe Leu Ala Met Leu Ala Thr Thr Asp Val Gly Leu Ser Thr Ala Thr Ile Pro Lys Met Leu Gly Ile Phe Trp Ile Asn Leu Arg Gly Ile Ile Phe Glu Ala Cys Leu Thr Gln Met Phe Phe Ile His Asn Phe Thr Leu Met Glu Ser Ala Val Leu Val Ala Met Ala Tyr Asp Ser Tyr Val Ala Ile Cys Asn Pro Leu Gln Tyr Ser Ala Ile Leu Thr Asn Lys Val Val Ser Val Ile Gly Leu Gly Val Phe Val Arg Ala Leu Ile Phe Val Ile Pro Ser Ile Leu Leu Ile Leu Arg Leu Pro Phe Cys Gly Asn His Val Ile Pro His Thr Tyr Cys Glu His Met Gly Leu Ala His Leu Ser Cys Ala Ser Ile Lys Ile Asn Ile Ile Tyr Gly Leu Cys Ala Ile Cys Asn Leu Val Phe Asp Ile Thr Val Ile Ala Leu Ser Tyr Val His Ile Leu Cys Ala Val Phe Arg Leu Pro Thr His Glu Pro Arg Leu Lys Ser Leu Ser Thr Cys Gly Ser His Val Cys Val Ile Leu Ala Phe Tyr Thr Pro Ala Leu Phe Ser Phe Met Thr His Cys Phe Gly Arg Asn Val Pro Arg Tyr Ile His Ile Leu Leu Ala Asn Leu Tyr Val Val Val Pro

42/82 Pro Met Leu Asn Pro Val Ile Tyr Gly Val Arg Thr Lys Gln Ile Tyr Lys Cys Val Lys Lys Ile Leu Leu Gln Glu Gln Gly Met Glu Lys Glu Glu Tyr Leu Ile His Thr Arg Phe <210> 39 <211> 327 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475230CD1 <400> 39 Met Leu His Thr Asn Asn Thr Gln Phe His Pro Ser Thr Phe Leu Val Val Gly Val Pro Gly Leu Glu Asp Val His Val Trp Ile Gly Phe Pro Phe Phe Ala Val Tyr Leu Thr Ala Leu Leu Gly Asn Ile Ile Ile Leu Phe Val Ile Gln Thr Glu Gln Ser Leu His Gln Pro Met Phe Tyr Phe Leu Ala Met Leu Ala Gly Thr Asp Leu Gly Leu Ser Thr Ala Thr Ile Pro Lys Met Leu Gly Ile Phe Trp Phe Asn Leu Gly Glu Ile Ala Phe Gly Ala Cys Ile Thr Gln Met Tyr Thr Ile His Ile Cys Thr Gly Leu Glu Ser Val Val Leu Thr Val Thr Gly Ile Asp Arg Tyr Ile Ala Ile Cys Asn Pro Leu Arg Tyr Ser Met Ile Leu Thr Asn Lys Val Ile Ala Ile Leu Gly Ile Val Ile Ile Val Arg Thr Leu Val Phe Val Thr Pro Phe Thr Phe Leu Thr Leu Arg Leu Pro Phe Cys Gly Val Arg Ile Ile Pro His Thr Tyr Cys Glu His Met Gly Leu Ala Lys Leu Ala Cys Ala Ser Ile Asn Val Ile Tyr Gly Leu Ile Ala Phe Ser Val Gly Tyr Ile Asp Ile Ser Val Ile Gly Phe Ser Tyr Val Gln Ile Leu Arg Ala Val Phe His Leu Pro Ala Trp Asp Ala Arg Leu Lys Ala Leu Ser Thr Cys Gly Ser His Val Cys Val Met Leu Ala Phe Tyr Leu Pro Ala Leu Phe Ser Phe Met Thr His Arg Phe Gly His Asn Ile Pro His Tyr Ile His Ile Leu Leu Ala Asn Leu Tyr Val Val Phe Pro Pro Ala Leu Asn Ser Val Ile Tyr Gly Val Lys Thr Lys Gln Ile Arg Glu

43/82 Gln Val Leu Arg Ile Leu Asn Pro Lys Ser Phe Trp His Phe Asp Pro Lys Arg Ile Phe His Asn Asn Ser Val Arg Gln <210> 40 <211> 313 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475229CD1 <400> 40 Met Pro Ile Ala Asn Asp Thr Gln Phe His Thr Ser Ser Phe Leu Leu Leu Gly Ile Pro Gly Leu Glu Asp Val His Ile Trp Ile Gly Phe Pro Phe Phe Ser Val Tyr Leu Ile Ala Leu Leu Gly Asn Ala Ala Ile Phe Phe Val Ile Gln Thr Glu Gln Ser Leu His Glu Pro Met Tyr Tyr Cys Leu Ala Met Leu Asp Ser Ile Asp Leu Ser Leu Ser Thr Ala Thr Ile Pro Lys Met Leu Gly Ile Phe Trp Phe Asn Ile Lys Glu Ile Ser Phe Gly Gly Tyr Leu Ser Gln Met Phe Phe Ile His Phe Phe Thr Val Met Glu Ser Ile Val Leu Val Ala Met Ala Phe Asp Arg Tyr Ile Ala Ile Cys Lys Pro Leu Trp Tyr Thr Met Ile Leu Thr Ser Lys Ile Ile Ser Leu Ile Ala Gly Ile Ala Val Leu Arg Ser Leu Tyr Met Val Ile Pro Leu Val Phe Leu Leu Leu Arg Leu Pro Phe Cys Gly His Arg Ile Ile Pro His Thr Tyr Cys Glu His Met Gly Ile Ala Arg Leu Ala Cys Ala Ser Ile Lys Val Asn Ile Met Phe Gly Leu Gly Ser Ile Ser Leu Leu Leu Leu Asp Val Leu Leu Ile Ile Leu Ser His Ile Arg Ile Leu Tyr Ala Val Phe Cys Leu Pro Ser Trp Glu Ala Arg Leu Lys Ala Leu Asn Thr Cys Gly Ser His Ile Gly Val Ile Leu Ala Phe Ser Thr Pro Ala Phe Phe Ser Phe Phe Thr His Cys Phe Gly His Asp Ile Pro Gln Tyr Ile His Ile Phe Leu Ala Asn Leu Tyr Val Val Val Pro Pro Thr Leu Asn Pro Val Ile Tyr Gly Val Arg Thr Lys His Ile

44/82 Arg Glu Thr Val Leu Arg Ile Phe Phe Lys Thr Asp His <210> 41 <211> 311 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477367CD1 <400> 41 Met Ala His Thr Asn Glu Ser Met Val Ser Glu Phe Val Leu Leu Gly Leu Ser Asn Ser Trp Gly Leu Gln Leu Phe Phe Phe Ala Ile Phe Ser Ile Val Tyr Val Thr Ser Val Leu Gly Asn Val Leu Ile Ile Val Ile Ile Ser Phe Asp Ser His Leu Asn Ser Pro Met Tyr Phe Leu Leu Ser Asn Leu Ser Phe Ile Asp Ile Cys Gln Ser Asn Phe Ala Thr Pro Lys Met Leu Val Asp Phe Phe Ile Glu Arg Lys Thr Ile Ser Phe Glu Gly Cys Met Ala Gln Ile Phe Val Leu His Ser Phe Val Gly Ser Glu Met Met Leu Leu Val Ala Met Ala Tyr Asp Arg Phe Ile Ala Ile Cys Lys Pro Leu His Tyr Ser Thr Ile Met Asn Arg Arg Leu Cys Val Ile Phe Val Ser Ile Ser Trp Ala Val Gly Val Leu His Ser Val Ser His Leu Ala Phe Thr Val Asp Leu Pro Phe Cys Gly Pro Asn Glu Val Asp Ser Phe Phe Cys Asp Leu Pro Leu Val Ile Glu Leu Ala Cys Met Asp Thr Tyr Glu Met Glu Ile Met Thr Leu Thr Asn Ser Gly Leu Ile Ser Leu Ser Cys Phe Leu Ala Leu Ile Ile Ser Tyr Thr Ile Ile Leu Ile Gly Val Arg Cys Arg Ser Ser Ser Gly Ser Ser Lys Ala Leu Ser Thr Leu Thr Ala His Ile Thr Val Val Ile Leu Phe Phe Gly Pro Cys Ile Tyr Phe Tyr Ile Trp Pro Phe Ser Arg Leu Pro Val Asp Lys Phe Leu Ser Val Phe Tyr Thr Val Cys Thr Pro Leu Leu Asn Pro Ile Ile Tyr Ser Leu Arg Asn Glu Asp Val Lys Ala Ala Met Trp Lys Leu Arg Asn His His Val Asn Ser Trp Lys Asn

45/82 <210> 42 <211> 304 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477936CD1 <400> 42 Met Glu Arg Ala Asn His Ser Val Val Ser Glu Phe Ile Leu Leu Gly Leu Ser Lys Ser Gln Asn Leu Gln Ile Leu Phe Phe Leu Gly Phe Ser Val Val Phe Val Gly Ile Val Leu Gly Asn Leu Leu Ile Leu Val Thr Val Thr Phe Asp Ser Leu Leu His Thr Pro Met Tyr Phe Leu Leu Ser Asn Leu Ser Cys Ile Asp Met Ile Leu Ala Ser Phe Ala Thr Pro Lys Met Ile Val Asp Phe Leu Arg Glu Arg Lys Thr Ile Ser Trp Trp Gly Cys Tyr Ser Gln Met Phe Phe Met His Leu Leu Gly Gly Ser Glu Met Met Leu Leu Val Ala Met Ala Ile Asp Arg Tyr Val Ala Ile Cys Lys Pro Leu His Tyr Met Thr Ile Met Ser Pro Arg Val Leu Thr Gly Leu Leu Leu Ser Ser Tyr Ala Val Gly Phe Val His Ser Ser Ser Gln Met Ala Phe Met Leu Thr Leu Pro Phe Cys Gly Pro Asn Val Ile Asp Ser Phe Phe Cys Asp Leu Pro Leu Val Ile Lys Leu Ala Cys Lys Asp Thr Tyr Ile Leu Gln Leu Leu Val Ile Ala Asp Ser Gly Leu Leu Ser Leu Val Cys Phe Leu Leu Leu Leu Val Ser Tyr Gly Val Ile Ile Phe Ser Val Arg Tyr Arg Ala Ala Ser Arg Ser Ser Lys Ala Phe Ser Thr Leu Ser Ala His Ile Thr Val Val Thr Leu Phe Phe Ala Pro Cys Val Phe Ile Tyr Val Trp Pro Phe Ser Arg Tyr Ser Val Asp Lys Ile 260 . 265 270 Leu Ser Val Phe Tyr Thr Ile Phe Thr Pro Leu Leu Asn Pro Ile Ile Tyr Thr Leu Arg Asn Gln Glu Val Lys Ala Ala Ile Lys Lys Arg Leu Cys Ile <210> 43 <211> 311 <212> PRT

46/82 <213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475214CD1 <400> 43 Met Glu Lys Ile Asn Asn Val Thr Glu Phe Ile Phe Trp Gly Leu Ser Gln Ser Pro Glu Ile Glu Lys Val Cys Phe Val Val Phe Ser Phe Phe Tyr Ile Ile Ile Leu Leu Gly Asn Leu Leu Ile Met Leu Thr Val Cys Leu Ser Asn Leu Phe Lys Ser Pro Met Tyr Phe Phe Leu Ser Phe Leu Ser Phe Val Asp Ile Cys Tyr Ser Ser Val Thr Ala Pro Lys Met Ile Val Asp Leu Leu Ala Lys Asp Lys Thr Ile Ser Tyr Val Gly Cys Met Leu Gln Leu Leu Gly Val His Phe Phe Gly Cys Thr Glu Ile Phe Ile Leu Thr Val Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys Lys Pro Leu His Tyr Met Thr Ile Met Asn Arg Glu Thr Cys Asn Lys Met Leu Leu Gly Thr Trp Val Gly Gly Phe Leu His Ser Ile Ile Gln Val Ala Leu Val Val Gln Leu Pro Phe Cys Gly Pro Asn Glu Ile Asp His Tyr Phe Cys Asp Val His Pro Val Leu Lys Leu Ala Cys Thr Glu Thr Tyr Ile Val Gly Val Val Val Thr Ala Asn Ser Gly Thr Ile Ala Leu Gly Ser Phe Val Ile Leu Leu Ile Ser Tyr Ser Ile Ile Leu Val Ser Leu Arg Lys Gln Ser Ala Glu Gly Arg Arg Lys Ala Leu Ser Thr Cys Gly Ser His Ile Ala Met Val Val Ile Phe Phe Gly Pro Cys Thr Phe Met Tyr Met Arg Pro Asp Thr Thr Phe Ser Glu Asp Lys Met Val Ala Val Phe Tyr Thr Ile Ile Thr Pro Met Leu Asn Pro Leu Ile Tyr Thr Leu Arg Asn Ala Glu Val Lys Asn Ala Met Lys Lys Leu Trp Gly Arg Asn Val Phe Leu Glu Ala Lys Gly Lys <210> 44 <211> 311 <212> PRT
<213> Homo Sapiens <220>

47/82 <221> misc_feature <223> Incyte ID No: 55036157CD1 <400> 44 Met Ala Ala Glu Asn His Ser Phe Val Thr Lys Phe Ile Leu Val Gly Leu Thr Glu Lys Ser Glu Leu Gln Leu Pro Leu Phe Leu Val Phe Leu Gly Ile Tyr Val Val Thr Val Leu Gly Asn Leu Gly Met Ile Thr Leu Ile Gly Leu Ser Ser His Leu His Thr Pro Met Tyr Cys 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 Gly Tyr Val Ala Ile Cys Ser Pro Leu Leu Tyr Ser Ile Ile Ile Ser Asn Lys Ala Cys Phe Ser Leu Ile Leu Val Val Tyr Val Ile Gly Leu Ile Cys Ala Ser Ala His Ile Gly Cys Met Phe Arg Val Gln Phe Cys Lys Phe Asp Val Ile Asn His Tyr Phe Cys Asp Leu Ile Ser Ile Leu Lys Leu Ser Cys Ser Ser Thr Tyr Ile Asn Glu Leu Leu Ile Leu Ile Phe Ser Gly Ile Asn Ile Leu Val Pro Ser Leu Thr Ile Leu Ser Ser Tyr Ile Phe Ile Ile Ala Ser Ile Leu Arg Ile Arg Tyr Thr Glu Gly Arg Ser Lys Ala Phe Ser Thr Cys Ser Ser His Ile Ser Ala Val Ser 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 Leu Ile Tyr Ser Leu Arg Asn Lys Asp Val His Val Ala Leu Lys Lys Thr Leu Gly Lys Arg Thr Phe Leu <210> 45 <211> 329 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature °
<223> Incyte ID No: 7475226CD1

48/82 <400> 45 Met Thr Leu Val Ser Phe Phe Ser Phe Leu Ser Lys Pro Leu Ile Met Leu Leu Ser Asn Ser Ser Trp Arg Leu Ser Gln Pro Ser Phe Leu Leu Val Gly Ile Pro Gly Leu Glu Glu Ser Gln His Trp Ile Ala Leu Pro Leu Gly Ile Leu Tyr Leu Leu Ala Leu Val Gly Asn Val Thr Ile Leu Phe Ile Ile Trp Met Asp Pro Ser Leu His Gln Ser Met Tyr Leu Phe Leu Ser Met Leu Ala Ala Ile Asp Leu Val Leu Ala Ser Ser Thr Ala Pro Lys Ala Leu Ala Val Leu Leu Val His Ala His Glu Ile Gly Tyr Ile Val Cys Leu Ile Gln Met Phe Phe Ile His Ala Phe Ser Ser Met Glu Ser Gly Val Leu Val Ala Met Ala Leu Asp Arg Tyr Val Ala Ile Cys His Pro Leu His His Ser Thr Ile Leu His Pro Gly Val Ile Gly Arg Ile Gly Met Val Val Leu Val Arg Gly Leu Leu Leu Leu Ile Pro Phe Pro Ile Leu Leu Gly Thr Leu Ile Phe Cys Gln Ala Thr Ile Ile Gly His Ala Tyr Cys Glu His Met Ala Val Val Lys Leu Ala Cys Ser Glu Thr Thr Val Asn Arg Ala Tyr Gly Leu Thr Met Ala Leu Leu Val Ile Gly Leu Asp Val Leu Ala Ile Gly Val Ser Tyr Ala His Ile Leu Gln Ala Val Leu Lys Val Pro Gly Ser Glu Ala Arg Leu Lys Ala Phe Ser Thr Cys Gly Ser His Ile Cys Val Ile Leu Val Phe Tyr Val Pro Gly Ile Phe Ser Phe Leu Thr His Arg Phe Gly His His Val Pro His His Val His Val Leu Leu Ala Thr Arg Tyr Leu Leu Met Pro Pro Ala Leu Asn Pro Leu Val Tyr Gly Val Lys Thr Gln Gln Ile Arg Gln Arg Val Leu Arg Val Phe Thr Gln Lys Asp <210> 46 <211> 312 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477353CD1 <400> 46

49/82 Met GIu Asn Tyr Asn Gln Thr Ser Thr Asp Phe Ile Leu Leu Gly Leu Phe Pro Pro Ser Lys Ile Gly Leu Phe Leu Phe Ile Leu Phe Val Leu Ile Phe Leu Met Ala Leu Ile Gly Asn Leu Ser Met Ile Leu Leu Ile Phe Leu Asp Thr His Leu His Thr Pro Met Tyr Phe

50 55 60 Leu Leu Ser Gln Leu Ser Leu Ile Asp Leu Asn Tyr Ile Ser Thr Ile Val Pro Lys Met Ala Ser Asp Phe Leu Tyr Gly Asn Lys Ser Ile Ser Phe Ile Gly Cys Gly Ile Gln Ser Phe Phe Phe Met Thr Phe Ala Gly Ala Glu Ala Leu Leu Leu Thr Ser Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys Phe Pro Leu His Tyr Pro Ile Arg Met Ser Lys Arg Met Tyr Val Leu Met Ile Thr Gly Ser Trp Met Ile Gly Ser Ile Asn Ser Cys Ala His Thr Val Tyr Ala Phe Arg 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 Tyr Thr Val Phe Leu Ser Ser Thr Ile Phe Leu Val Phe Pro Phe Thr Gly Ile Ala Cys Ser Tyr Gly Trp 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 Thr Phe Tyr Tyr Ala Pro Phe Ala Tyr Thr Tyr Leu Cys Pro Arg Ser Leu Arg Ser Leu Thr Glu Asp Lys Val 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 Arg Val Ile Gln Asn Ile Phe Ser Val Lys Met <210> 47 <211> 347 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 55036208CD1 <400> 47 Met Ala Trp Glu Asn Gln Thr Phe Asn Ser Asp Phe Ile Leu Leu Gly Ile Phe Asn His Ser Pro Pro His Thr Phe Leu Phe Phe Leu Val Leu Gly Ile Phe Leu 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 Ser Leu Met Asp Leu Met Leu Ile Cys Thr Thr Val Pro Lys Met Ala Phe Asn Tyr Leu Ser Gly Ser Lys Ser Ile Ser Met Ala Gly Cys Val Thr Gln Ile Phe Phe Tyr Ile Ser Leu Ser Gly Ser Glu Cys Phe Leu Leu Ala Val Met Ala Tyr Asp Arg Tyr Ile Ala Ile Cys His Pro Leu Arg Tyr Thr Asn Leu Met Asn Pro Lys Ile Cys Gly Leu Met Ala Thr Phe Ser Trp Ile Leu Gly Ser Thr Asp Gly Ile Ile Asp Ala Val Ala Thr Phe Ser Phe Ser Phe Cys Gly Ser Arg Glu Ile Ala His Phe Phe Cys Glu Phe Pro Ser Leu Leu Ile Leu Ser Cys Asn Asp Thr Ser Ile Phe Glu Glu Val Ile Phe Ile Cys Cys Ile Val Met Leu Val Phe Pro Val Ala Ile Ile Ile Ala Ser Tyr Ala Gly Val Ile Leu Ala Val Ile His Met Gly Ser Gly Glu Gly Arg Arg Lys Thr Phe Thr Thr Cys Ser Ser His Leu Met Val Val Gly Met Tyr Tyr Gly Ala Ala Leu Phe Met Tyr Ile Arg Pro Thr Ser Asp His Ser Pro Thr Gln Asp Lys Met 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 Phe Met Lys Ile Leu Gly Lys Gly Lys Ser Glu Ser Glu Leu Pro His Lys Leu Tyr Val Leu Leu Phe Ala Lys Phe Phe Phe Leu Ile Ser Ile Phe Phe Tyr Asp Val Lys Ile Leu Ala Leu Ile Met Tyr Ile Ala <210> 48 <211> 318 <212> PRT
<213> Homo Sapiens <220>
<22.1> misc_feature <223> Incyte ID No: 55019501CD1 <400> 48

51/82 Met Arg Gly Asp Asn His Ser Cys Phe Trp Asp Thr Pro Lys Asp Phe Ile Leu Leu Gly Ile Ser Asp Arg Pro Trp Leu Glu Leu Pro Val Phe Ala Val Leu Leu Val Phe Tyr Ile Leu Ala Met Leu Gly Asn Ile Ser Ile Ile Leu Val Ser Gln Leu Asp Pro Gln Leu His Ser Pro Met Tyr Ile Phe Leu Ser His Leu Ser Phe Leu Asp Leu Cys Tyr Thr Thr Thr Thr Val Pro Gln Met Leu Phe Asn Met Gly Ser Ser Gln Lys Thr Ile Ser Tyr Gly Gly Cys Thr Val Gln Tyr Ala Ile Phe His Trp Leu Gly Cys Thr Glu Cys Val Val Leu Ala Ala Met Ala Leu Asp Arg Tyr Val Ala Ile Cys Glu Pro Leu Arg Tyr Ala Ile Ile Met His Arg Pro Leu Cys Gln Gln Leu Val Ala Met Ala Trp Leu Ser Gly Phe Gly Asn Ser Leu Val Gln Val Ile Leu Thr Val Gln Leu Pro Phe Cys Gly Arg Gln Val Leu Asn Asn Phe Phe Cys Glu Val Pro Ala Met Ile Lys Leu Ser Cys Ala Asp Thr Thr Ala Asn Asp Ala Thr Leu Ala Val Leu Val Ala Phe Phe Val Leu Val Pro Leu Ala Leu Ile Leu Leu Ser Tyr Gly Phe Ile Ala Arg Ala Val Met Arg Ile Gln Ser Ser Arg Gly Arg His Lys Ala Phe Gly Thr Cys Ser Ser His Leu Leu Val Val Ser Leu Phe Tyr Leu Pro Ala Ile Tyr Met Tyr Leu Gln Pro Pro Ser Ser Tyr Ser Gln Glu Gln Gly Lys Phe Ile Ser Leu Phe Tyr Ser Ile Ile Thr Pro Thr Leu Asn Pro Phe Ile Tyr Thr Leu Arg Asn Lys Asp Val Lys Gly Ala Leu Arg Arg Leu Leu Ala Arg Thr Gly Arg Leu Cys Gly Arg <210> 49 <211> 2181 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7485090CB1 <400> 49 atgcagaaaa ccaagcaaga tgaggactat gaaagagcca ttggatttag tgtcaaaatg 60

52/82 gatgacagtg attctgattt tgcactgact caaggtagca tgatcactcc ttcatgccaa 120 aaaggatatt ttccctgtgg gaatcttacc aagtgcttac cccgagcttt tcactgtgat 180 ggcaaggatg actgtgggaa cggggcggac gaagagaact gtggtgacac tagtggatgg 240 gcgaccatat ttggcacagt gcatggaaat gctaacagcg tggccttaac acaggagtgc 300 tttctaaaac agtatccaca atgctgtgac tgcaaagaaa ctgaattgga atgtgtaaat 360 ggtgacttaa agtctgtgcc gatgatttct aacaatgtga cattactgtc tcttaagaaa 420 aacaaaatcc acagtcttcc agataaagtt ttcatcaaat acacaaaact taaaaagata 480 tttcttcagc ataattgcat tagacacata tccaggaaag cattttttgg attatgtaat 540 ctgcaaatat tatatctcaa ccacaactgc atcacaaccc tcagacctgg aatattcaaa 600 gacttacatc agctaacttg gctaattcta gatgacaatc caataaccag aatttcacag 660 cgcttgttta cgggattaaa ttccttgttt ttcctgtcta tggttaataa ctacttagaa 720 gctcttccca agcagatgtg tgcccaaatg cctcaactca actgggtgga tttggaaggc 780 aatagaataa agtatctcac aaattctacg tttctgtcgt gcgattcgct cacagtgctg 840 gatctgtcta gcaatacgat aacggaacta tcacctcacc tttttaaaga cttgaagctt 900 ctacaaaagc tgaacctgtc atccaatcct cttatgtatc ttcacaagaa ccagtttgaa 960 agtcttaaac aacttcagtc tctagacctg gaaaggatag agattccaaa tataaacaca 1020 cgaatgtttc aacccatgaa gaatctttct cacataccct gttatttcaa aaactttcga 1080 tactgctcct atgctcccca tgtccgaata tgtatgccct tgacggacgg catttcttca 1140 tttgaggacc tcttggctaa caatatcctc agaatatttg tctgggttat agctttcatt 1200 acctgctttg gaaatctttt tgtcattggc atgagatctt tcattaaagc tgaaaataca 1260 actcacgcta tgtccatcaa aatcctttgt tgtgctgatt gcctgatggg tgtttacttg 1320 ttctttgttg gcattttcga tataaaatac cgagggcagt atcagaagta tgccttgctg 1380 tggatggaga gcgtgcagtg ccgcctcatg gggttcctgg ccatgctgtc caccgaagtc 1440 tctgttctgc tactgaccta cttgactttg gagaagttcc tggtcattgt cttccccttc 1500 agtaacattc gacctggaaa acggcagacc tcagtcatcc tcatttgcat ctggatggcg 1560 ggatttttaa tagctgtaat tccattttgg aataaggatt attttggaaa cttttatggg 1620 aaaaatggag tatgtttccc actttattat gaccaaacag aagatattgg aagcaaaggg 1680 tattctcttg gaattttcct aggtgtgaac ttgctggctt ttctcatcat tgtgttttcc 1740 tatattacta tgttctgttc cattcaaaaa accgccttgc agaccacaga agtaaggaat 1800 tgttttggaa gagaggtggc tgttgcaaat cgtttctttt ttatagtgtt ctctgatgcc 1860 atctgctgga ttcctgtatt tgtagttaaa atcctttccc tcttccgggt ggaaatacca 1920 gacacaatga cttcctggat agtgattttt ttccttccag ttaacagtgc tttgaatcca 1980 atcctctata ctctcacaac caactttttt aaggacaagt tgaaacagct gctgcacaaa 2040 catcagagga aatcaatttt caaaattaaa aaaaaaagtt tatctacatc cattgtgtgg 2100 atagaggact cctcttccct gaaacttggg gttttgaaca aaataacact tggagacagt 2160 ataatgaaac cagtttccta g 2181 <210> 50 <211> 3215 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474890CB1 <400> 50 atggtctgtt cggctgcccc actgctgctc ctggccacaa ctcttcccct gctggggtca 60 ccagttgccc aagcatccca acctggacag agtcaggctg gaggggaatc tggatctggg 120 cagctcctgg accaagagaa tggagcaggg gagtgtaatg tcaaccacaa ggggaatttc 180 tattgtgctt gcctctctgg ctaccagtgg aacaccagca tctgcctcca ttaccctcct 240 tgtcaaagcc tccacaacca ccagccttgt ggctgccttg tcttcagcca tcccgaaccc 300 gggtactgcc agttgctgcc acctgtcccc gggatcctca acctgaactc ccagctgcag 360 atgcctggtg acacgctgag cctgactctc catctgagcc aggaggccac caacctgagc 420 tggttcctga ggcacccagg gagccccagt cccatcctcc tgcagccagg gacacaggtg 480 tctgtgactt ccagccacgg ccaggctgcc ctcagcgtct ccaacatgtc ccatcactgg 540

53/82 gcaggtgagt acatgagctg cttcgaggcc cagggcttca agtggaacct gtatgaggtg 600 gtgagggtgc ccttgaaggc gacagatgtg gctcgacttc cataccagct gtccatctcc 660 tgtgccacct cccctggctt ccagctgagc tgctgcatcc ccagcacaaa cctggcctac 720 accgcggcct ggagccctgg agagggcagc aaagcttcct ccttcaacga gtcaggctct 780 cagtgctttg tgctggctgt tcagcgctgc ccgatggctg acaccacgta cgcttgtgac 840 ctgcagagcc tgggcctggc tccactcagg gtccccatct ccatcaccat catccaggat 900 ggagacatca cctgccctga ggacgcctcg gtgctcacct ggaatgtcac caaggctggc 960 cacgtggcac aggccccatg tcctgagagc aagaggggca tagtgaggag gctctgtggg 1020 gctgacggag tctgggggcc ggtccacagc agctgcacag atgcgaggct cctggccttg 1080 ttcactagaa ccaagctgct gcaggcaggc cagggcagtc ctgctgagga ggtgccacag 1140 atcctggcac agctgccagg gcaggcggca gaggcaagtt caccctccga cttactgacc 1200 ctgctgagca ccatgaaata cgtggccaag gtggtggcag aggccagaat acagcttgac 1260 cgcagagccc tgaagaatct cctgattgcc acagacaagg tcctagatat ggacaccagg 1320 tctctgtgga ccctggccca agcccggaag ccctgggcag gctcgactct cctgctggct 1380 gtggagaccc tggcatgcag cctgtgccca caggaccacc ccttcgcctt cagcttaccc 1440 aatgtgctgc tgcagagcca gctgtttgga cccacgtttc ctgctgacta cagcatctcc 1500 ttccctactc ggcccccact gcaggctcag attcccaggc actcactggc cccattggtc 1560 cgtaatggaa ctgaaataag tattactagc ctggtgctgc gaaaactgga ccaccttctg 1620 ccctcaaact atggacaagg gctgggggat tccctctatg ccactcctgg cctggtcctt 1680 gtcatttcca tcatggcagg tgaccgggcc ttcagccagg gagaggtcat catggacttt 1740 gggaacacag atggttcccc tcactgtgtc ttctgggatc acagtctctt ccagggcagg 1800 gggggttggt ccaaagaagg gtgccaggca caggtggcca gtgccagccc cactgctcag 1860 tgcctctgcc agcacctcac tgccttctcc gtcctcatgt ccccacacac tgttccggaa 1920 gaacccgctc tggcgctgct gactcaagtg ggcttgggag cttccatact ggcgctgctt 1980 gtgtgcctgg gtgtgtactg gctggtgtgg agagtcgtgg tgcggaacaa gatctcctat 2040 ttccgccacg ccgccctgct caacatggtg ttctgcttgc tggctgcaga cacttgcttc 2100 ctgggcgccc cattcctctc tccagggccc cgaagcccgc tctgccttgc tgccgccttc 2160 ctctgtcatt tcctctacct ggccaccttt ttctggatgc tggcgcaggc cctggtgttg 2220 gcccaccagc tgctctttgt ctttcaccag ctggcaaagc accgagttct ccccctcatg 2280 gtgctcctgg gctacctgtg cccactgggg ttggcaggtg tcaccctggg gctctaccta 2340 cctcaagggc aatacctgag ggagggggaa tgctggttgg atgggaaggg aggggcgtta 2400 tacaccttcg tggggccagt gctggccatc ataggcgtga atgggctggt actagccatg 2460 gccatgctga agttgctgag accttcgctg tcagagggac ccccagcaga gaagcgccaa 2520 gctctgctgg gggtgatcaa agccctgctc attcttacac ccatctttgg cctcacctgg 2580 ggggctgggc ctggccactc tgttagagga agtctccacg gtccctcatt acatcttcac 2640 cattctcaac accctccagg gcgtcttcat cctattgttt ggttgcctca tggacaggaa 2700 gatacaagaa gctttgcgca aacgcttctg ccgcgcccaa gcccccagct ccaccatctc 2760 cctggccaca aatgaaggct gcatcttgga acacagcaaa ggaggaagcg acactgccag 2820 gaagacagat gcttcagagt gaaccacaca cggacccatg ttcctgcaag ggagttgagg 2880 ctgtgtgctt gaacccacca gatgagcctg gcccatgctc tgaactcttc ccgcctcccg 2940 gagctcagcc cttgagaaag gcaggcttat atttccctta gtgacactca tttatcttac 3000 agctcacccc ttctcatttc taaagtatcc agcaagaata gcaggaaaaa ttagctaaag 3060 gcacctaatg aataagcctg cctttgctcc agaaataatc gacagatatc aaagtgcgga 3120 ataattacaa gtaaactttc tcaaccagtt tttaactaca acatacatgt cgtgaatgaa 3180 tatatttgat aaaaatggtt ttaattgacc caaaa 3215 <210> 51 <211> 1671 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474936CB1 <400> 51

54/82 gctcttcagt gaggtgggct cagggagggc tctgtgcctc cgttcagcag agctgcagct 60 gctgcccagc tctcaggagg caagctggac tccctcactc ggctgcagga gcaaggacag 120 tgaggctcaa ccccgcctga gccatgccag ccaacttcac agagggcagc ttcgattcca 180 gtgggaccgg gcagacgctg gattcttccc cagtggcttg cactgaaaca gtgactttta 240 ctgaagtggt ggaaggaaag gaatggggtt ccttctacta ctcctttaag actgagcaat 300 tgataactct gtgggtcctc tttgttttta ccattgttgg aaactccgtt gtgctttttt 360 ccacatggag gagaaagaag aagtcaagaa tgaccttctt tgtgactcag ctggccatca 420 cagattcttt cacaggactg gtcaacatct tgacagatat tatttggcga ttcactggag 480 acttcacggc acctgacctg gtttgccgag tggtccgcta tttgcaggtt gtgctgctgt 540 acgcctctac ctacgtcctg gtgtccctca gcatagacag ataccatgcc atcgtctacc 600 ccatgaagtt ccttcaagga gaaaagcaag ccagggtcct cattgtgatc gcctggagcc 660 tgtcttttct gttctccatt cccaccctga tcatatttgg gaagaggaca ctgtccaacg 720 gtgaagtgca gtgctgggcc ctgtggcctg acgactccta ctggacccca tacatgacca 780 tcgtggcctt cctggtgtac ttcatccctc tgacaatcat cagcatcatg tatggcattg 840 tgatccgaac tatttggatt aaaagcaaaa cctacgaaac agtgatttcc aactgctcag 900 atgggaaact gtgcagcagc tataaccgag gactcatctc aaaggcaaaa atcaaggcta 960 tcaagtatag catcatcatc attcttgcct tcatctgctg ttggagtcca tacttcctgt 1020 ttgacatttt ggacaatttc aacctccttc cagacaccca ggagcgtttc tatgcctctg 1080 tgatcattca gaacctgcca gcattgaata gtgccatcaa ccccctcatc tactgtgtct 1140 tcagcagctc catctctttc ccctgcaggg agcgaagatc acaggattcc agaatgacgt 1200 tccgggagag aaccgagagg catgagatgc agattctgtc caagccagaa ttcatctaga 1260 ccctagggca gtgccagtgc taggctgagc accatcagct ctcccaggtc cttgtcacct 1320 gcttgggcac gtgcatggaa cccgagccaa cttcacccca ccctcgtcat tacctgggag 1380 atgcacaaga caaatgttct aatgactgca tgcactgctt aagtattggc caacacgaac 1440 tccccagtta ttcatgccag ccaggaagga aacgccttcc ttccccacca ttcccagccc 1500 tccttcccac tggccagcac ctgaacccag tgaacacagg catcagtggt ccagggtcct 1560 ggcttggagc cagtgagtag acaggcaagc agaggggaca aaggtagctg ggttatacat 1620 gaatattctc attacaatag aagaaaataa aagacttaat taagccaaaa a 1671 <210> 52 <211> 1336 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90012430CB1 <400> 52 cctgctgttg aaacagaatc ctatttggaa ggcagacatg tggcccatct ctgtagccat 60 cactgagaaa tctggatttt caagcaatga ttttcaacaa ttataaaatg gaagttgtag 120 actggataag agatgctcag ctaagggagt tcctggatgg tctttagatt gatacaccaa 180 tcctctgaaa ttgcatgcaa aaatgtgact tcccaagtat gcctggccac aatacctcca 240 ggaattcctc ttgcgatcct atagtgacac cccacttaat cagcctctac ttcatagtgc 300 ttattggcgg gctggtgggt gtcatttcca ttcttttcct cctggtgaaa atgaacaccc 360 ggtcagtgac caccatggcg gtcattaact tggtggtggt ccacagcgtt tttctgctga 420 cagtgccatt tcgcttgacc tacctcatca agaagacttg gatgtttggg ctgcccttct 480 gcaaatttgt gagtgccatg ctgcacatcc acatgtacct cacgttccta ttctatgtgg 540 tgatcctggt caccagatac ctcatcttct tcaagtgcaa agacaaagtg gaattctaca 600 gaaaactgca tgctgtggct gccagtgctg gcatgtggac gctggtgatt gtcattgtgg 660 tacccctggt tgtctcccgg tatggaatcc atgaggaata caatgaggag cactgtttta 720 aatttcacaa agagcttgct tacacatatg tgaaaatcat caactatatg atagtcattt 780 ttgtcatagc cgttgctgtg attctgttgg tcttccaggt cttcatcatt atgttgatgg 840 tgcagaagct acgccactct ttactatccc accaggagtt ctgggctcag ctgaaaaacc 900 tattttttat aggggtcatc cttgtttgtt tccttcccta ccagttcttt aggatctatt 960 acttgaatgt tgtgacgcat tccaatgcct gtaacagcaa ggttgcattt tataacgaaa 1020

55/82 tcttcttgag tgtaacagca attagctgct atgatttgct tctctttgtc tttgggggaa 1080 gccattggtt taagcaaaag ataattggct tatggaattg tgttttgtgc cgttagccac 1140 aaactacagt attcatattt gcttccttta tattgggaat aaaaatgggt ataggggagg 1200 taagaatggt atttcattac ttgatcaaaa ccatgccttg atgtacccaa aacaaaagga 1260 ctataaaatg caagagccct cattgtagtc cttatgggat ccctcccatc tctgagtgat 1320 ggcgtacaag acccgt 1336 <210> 53 <211> 1340 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90012586CB1 <400> 53 cctgctgttg aaacagaatc ctatttggaa ggcagacatg tggcccatct ctgtagccat 60 cactgagaaa tctggatttt caagggcctt tctctctgtt gcccaggctg gagtttagcg 120 actcaatcat ggctcactga ctgcagcatc gacctccggg gctcaagtga tcctttcatc 180 tcagcctcct cagtagctga gactacaggt gacttcccaa gtatgcctgg ccacaatacc 240 tccaggaatt cctcttgcga tcctatagtg acaccccact taatcagcct ctacttcata 300 gtgcttattg gcgggctggt gggtgtcatt tccattcttt tcctcctggt gaaaatgaac 360 acccggtcag tgaccaccat ggcggtcatt aacttggtgg tggtccacag cgtttttctg 420 ctgacagtgc catttcgctt gacctacctc atcaagaaga cttggatgtt tgggctgccc 480 ttctgcaaat ttgtgagtgc catgctgcac atccacatgt acctcacgtt cctattctat 540 gtggtgatcc tggtcaccag atacctcatc ttcttcaagt gcaaagacaa agtggaattc 600 tacagaaaac tgcatgctgt ggctgccagt gctggcatgt ggacgctggt gattgtcatt 660 gtggtacccc tggttgtctc ccggtatgga atccatgagg aatacaatga ggagcactgt 720 tttaaatttc acaaagagct tgcttacaca tatgtgaaaa tcatcaacta tatgatagtc 780 atttttgtca tagccgttgc tgtgattctg ttggtcttcc aggtcttcat cattatgttg 840 atggtgcaga agctacgcca ctctttacta tcccaccagg agttctgggc tcagctgaaa 900 aacctatttt ttataggggt catccttgtt tgtttccttc cctaccagtt ctttaggatc 960 tattacttga atgttgtgac gcattccaat gcctgtaaca gcaaggttgc attttataac 1020 gaaatcttct tgagtgtaac agcaattagc tgctatgatt tgcttctctt tgtctttggg 1080 ggaagccatt ggtttaagca aaagataatt ggcttatgga attgtgtttt gtgccgttag 1140 ccacaaacta cagtattcat atttgcttcc tttatattgg gaataaaaat gggtataggg 1200 gaggtaagaa tggtatttca ttacttgatc aaaaccatgc cttgatgtac ccaaaacaaa 1260 aggactataa aatgcaagag ccctcattgt agtccttatg ggatccctcc catctctgag 1320 tgatggcgta caagacccgt 1340 <210> 54 <211> 1460 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 90012670CB1 <400> 54 cctgctgttg aaacagaatc ctatttggaa ggcagacatg tggcccatct ctgtagccat 60 cactgagaaa tctggatttt caagcaatga ttttcaacaa ttataaaatg gaagttgtag 120 attggataag agatgctcag ctaagggagt tcctggatgg tctttagatt gatacaccaa 180 tcctctgaaa ttgcatgcaa aaatggcctt tctctctgtt gcccaggctg gagtttagcg 240 actcaatcat ggctcactga ctgcagcatc gacctccggg gctcaagtga tcctttcatc 300

56/82 tcagcctcct cagtagctga gactacaggt gacttcccaa gtatgcctgg ccacaatacc 360 tccaggaatt cctcttgcga tcctatagtg acaccccact taatcagcct ctacttcata 420 gtgcttattg gcgggctggt gggtgtcatt tccattcttt tcctcctggt gaaaatgaac 480 acccggtcag tgaccaccat ggcggtcatt aacttggtgg tggtccacag cgtttttctg 540 ctgacagtgc catttcgctt gacctacctc atcaagaaga cttggatgtt tgggctgccc 600 ttctgcaaat ttgtgagtgc catgctgcac atccacatgt acctcacgtt cctattctat 660 gtggtgatCC tggtCaccag atacctcatc ttcttcaagt gcaaagacaa agtggaattc 720 tacagaaaac tgcatgctgt ggctgccagt gctggcatgt ggacgctggt gattgtcatt 780 gtggtacccc tggttgtctc ccggtatgga atccatgagg aatacaatga ggagcactgt 840 tttaaatttc acaaagagct tgcttacaca tatgtgaaaa tcatcaacta tatgatagtc 900 atttttgtca tagccgttgc tgtgattctg ttggtcttcc aggtcttcat cattatgttg 960 atggtgcaga agctacgcca ctctttacta tcccaccagg agttctgggc tcagctgaaa 1020 aacctatttt ttataggggt catccttgtt tgtttccttc cctaccagtt ctttaggatc 1080 tattacttga atgttgtgac gcattccaat gcctgtaaca gcaaggttgc attttataac 1140 gaaatcttct tgagtgtaac agcaattagc tgctatgatt tgcttctctt tgtctttggg 1200 ggaagccatt ggtttaagca aaagataatt ggcttatgga attgtgtttt gtgccgttag 1260 ccacaaacta cagtattcat atttgcttcc tttatattgg gaataaaaat gggtataggg 1320 gaggtaagaa tggtatttca ttacttgatc aaaaccatgc cttgatgtac ccaaaacaaa 1380 aggactataa aatgcaagag ccctcattgt agtccttatg ggatccctcc catctctgag 1440 tgatggcgta caagacccgt 1460 <210> 55 <211> 4052 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2880041CB1 <400> 55 gaaagattgg acctccgaaa caatcttatt agtagtatag atccaggtgc cttctgggga 60 ctgtcatctc taaaaagatt ggatctgaca aacaatcgaa taggatgtct gaatgcagac 120 atatttcgag gactcaccaa tctggttcgg ctaaaccttt cggggaattt gttttcttca 180 ttatctcaag gaacttttga ttatcttgcg tcattacggt ctttggaatt ccagactgag 240 tatcttttgt gtgactgtaa catactgtgg atgcatcgct gggtaaagga gaagaacatc 300 acggtacggg ataccaggtg tgtttatcct aagtcactgc aggcccaacc agtcacaggc 360 gtgaagcagg agctgttgac atgcgaccct ccgcttgaat tgccgtcttt ctacatgact 420 ccatctcatc gccaagttgt gtttgaagga gacagccttc ctttccagtg catggcttca 480 tatattgatc aggacatgca agtgttgtgg tatcaggatg ggagaatagt tgaaaccgat 540 gaatcgcaag gtatttttgt tgaaaagaac atgattcaca actgctcctt gattgcaagt 600 gccctaacca tttctaatat tcaggctgga tctactggaa attggggctg tcatgtccag 660 accaaacgtg ggaataatac gaggactgtg gatattgtgg tattagagag ttctgcacag 720 tactgtccgc cagagagggt ggtaaacaac aaaggtgact tcagatggcc cagaacattg 780 gcaggcatta ctgcatatct gcagtgtacg cggaacaccc atggcagtgg gatatatccc 840 ggaaacccac aggatgagag aaaagcttgg cgcagatgtg atagaggtgg cttttgggca 900 gatgatgatt attctcgctg tcagtatgca aatgatgtca ctagagttct ttatatgttt 960 aatcagatgc ccctcaatct taccaatgcc gtggcaacag ctcgacagtt actggcttac 1020 actgtggaag cagccaactt ttctgacaaa atggatgtta tatttgtggc agaaatgatt 1080 gaaaaatttg gaagatttac caaggaggaa aaatcaaaag agctaggtga cgtgatggtt 1140 gacattgcaa gtaacatcat gttggctgat gaacgtgtcc tgtggctggc gcagagggaa 1200 gctaaagcct gcagtaggat tgtgcagtgt cttcagcgca ttgctaccta ccggctagcc 1260 ggtggagctc acgtttattc aacatattca cccaatattg ctctggaagc ttatgtcatc 1320 aagtctactg gcttcacggg gatgacctgt accgtgttcc agaaagtggc agcctctgat 1380 cgtacaggac tttcggatta tgggaggcgg gatccagagg gaaacctgga taagcagctg 1440 agctttaagt gcaatgtttc aaatacattt tcgagtctgg caCtaaagaa tactattgtg 1500

57/82 gaggcttcta ttcagcttcc tccttccctt ttctcaccaa agcaaaaaag agaactcaga 1560 ccaactgatg actctcttta caagcttcaa ctcattgcat tccgcaatgg aaagcttttt 1620 ccagccactg gaaattcaac aaatttggct gatgatggaa aacgacgtac tgtggttacc 1680 cctgtgattc tcaccaaaat agatggtgtg aatgtagata cccaccacat ccctgttaat 1740 gtgacactgc gtcgaattgc acatggagca gatgctgttg cagcccggtg ggatttcgat 1800 ttgctgaacg gacaaggagg ctggaagtca gatgggtgcc atatactcta ttcagatgaa 1860 aatatcacta cgattcagtg ctactccctt agtaactatg cagttttaat ggatttgacg 1920 ggatctgaac tatacaccca ggcggccagc ctcctgcatc ctgtggttta tactaccgct 1980 atcattctcc tcttatgtct cttagccgtc attgtcagtt acatatacca tcacagtttg 2040 attagaatca gcctcaagag ctggcacatg cttgtgaact tgtgctttca tattttccta 2100 acctgtgtgg tctttgtggg aggaataacc cagactagga atgccagcat ctgccaagca 2160 gttgggataa ttcttcacta ttccaccctt gccacagtac tatgggtagg agtgacagct 2220 cgaaatatct acaaacaagt cactaaaaaa gctaaaagat gccaggatcc tgatgaacca 2280 ccacctccac caagaccaat gctcagattt tacctgattg gtggtggtat ccccatcatt 2340 gtttgcggca taactgcagc agcgaacatt aagaattacg gcagtcggcc aaacgcaccc 2400 tattgctgga tggcatggga accctccttg ggagccttct atgggccagc cagcttcatc 2460 acttttgtaa actgcatgta ctttctgagc atatttattc agttgaaaag acaccctgag 2520 cgcaaatatg agcttaagga gcccacggag gagcaacaga gattggcagc caatgaaaat 2580 ggcgaaataa atcatcagga ttcaatgtct ttgtctctga tttctacatc agccttggaa 2640 aatgagcaca cttttcattc tcagctcttg ggggccagcc ttactttgct cttatatgtt 2700 gcactgtgga tgtttggggc tttggctgtt tctttgtatt accctttgga cttggttttt 2760 agcttcgttt ttggagccac aagtttaagc ttcagtgcgt tcttcatggt ccaccattgt 2820 gttaataggg aggatgttag acttgcgtgg atcatgactt gctgcccagg acggagctcg 2880 tattcagtgc aagtcaacgt ccagcccccc aactctaatg ggacgaatgg agaggcaccc 2940 aaatgcccca atagcagtgc ggagtcttca tgcacaaaca aaagtgcttc aagcttcaaa 3000 aattcctccc agggctgcaa attaacaaac ttgcaggcgg ctgcagctca gtgccatgcc 3060 aattctttac ctttgaactc cacccctcag cttgataata gtctgacaga acattcaatg 3120 gacaatgata ttaaaatgca cgtggcgcct ttagaagttc agtttcgaac aaatgtgcac 3180 tcaagccgcc accataaaaa cagaagtaaa ggacaccggg caagccgact cacagtcctg 3240 agagaatatg cctacgatgt cccaacgagc gtggaaggaa gcgtgcagaa cggcttacct 3300 aaaagccggc tgggcaataa cgaaggacac tcgaggagcc gaagagctta tttagcctac 3360 agagagagac agtacaaccc accccagcaa gacagcagcg atgcttgtag cacacttccc 3420 aaaagtagca gaaattttga aaagccagtt tcaaccacta gtaaaaaaga tgcgttaagg 3480 aagccagctg tggttgaact tgaaaatcag caaaaatctt atggcctcaa cttggccatt 3540 cagaatggac caattaaaag caatgggcag gagggaccct tgctcggtac cgatagcact 3600 ggcaatgtta ggactggatt atggaaacac gaaactactg tgtaacattg ctgggcttcc 3660 taggcagaaa ttcatataaa ctgtgatact cacattcctt gaagctatga gcatttaaaa 3720 actgtttaca gccaccatag ggattcaaaa gaatttggaa taaactttga agttttggat 3780 tttacttatt tttatcccca aattgttgct attttttagg atctgaaaca aaatctttct 3840 aaaacattgt tttagttgtc aaagcaccaa caggacattt tgggatgtga aatgtaattt 3900 cttggaatct gtaatttgta cttaatattt caggctgtat ttatatataa taaataggtg 3960 ttgttattgt caaacaaaaa aagggcggcc ccgatagtga gcccgcgacc ggaatatccg 4020 ccggccgcag cgggcgggat cgatagcttc ac 4052 <210> 56 <211> 1142 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90012123C81 <400> 56 agcactacag cctaagcttc cagagtggac acggagaagg gcatcagaaa accattctgg 60 tgaggtccac acccataagt caccatgtac aaggactgca tcgagtccac tggagactat 120

58/82 tttcttctct gtgacgccga ggggccatgg ggcatcattc tggagtccct ggccatactt 180 ggcatcgtgg tcacaattct gctactctta gcatttctct tcctcatgcg aaagatccaa 240 gactgcagcc agtggaatgt cctccccacc cagctcctct tcctcctgag tgtcctgggg 300 ctcttcggac tcgcttttgc cttcatcatc gagctcaatc aacaaactgc ccccgtacgc 360 tactttctct ttggggttct ctttgctctc tgtttctcat gcctcttagc tcatgcctcc 420 aatctagtga agctggttcg gggttgtgtc tccttctcct ggacgacaat tctgtgcatt 480 gctattggtt gcagtctgtt gcaaatcatt attgccactg agtatgtgac tctcatcatg 540 accagaggta tgatgtttgt gaatatgaca ccctgccagc tcaatgtgga ctttgttgta 600 ctcctggtct atgtcctctt cctgatggcc ctcacattct tcgtctccaa agccaccttc 660 tgtggcccgt gtgagaactg gaagcagcat ggaaggctca tctttatcac tgtgctcttc 720 tccatcatca tctgggtggt gtggatctcc atgctcctga gaggcaaccc gcagttccag 780 cgacagcccc agtgggatga cccggtcgtc tgcattgctc tggtcaccaa cgcatgggtt 840 ttcctgctgc tgtacatcgt ccctgagctc tgcattccct acagatcgtg tagacaggag 900 tgccctttac aaggcaatgc ctgccccgtc acagcctacc aacacagctt ccaagtggag 960 aaccaggagc tctccagagc ccgagacagt gatggagctg aggaggatgt agcattaact 1020 tcatatggta ctcccattca gccgcagact gttgatccca cacaagagtg tttcatccca 1080 caggctaaac taagccccca gcaagatgca ggaggagtat aaaagagcga attccagcac 1140 ac 1142 <210> 57 <211> 1054 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90012163CB1 <400> 57 agcactacag cctaagcttc cagagtggac acggagaagg gcatcagaaa accattctgg 60 tgaggtccac acccataagt caccatgtac aaggactgca tcgagtccac tggagactat 120 tttcttctct gtgacgccga ggggccatgg ggcatcattc tggagtccct ggccatactt 180 ggcatcgtgg tcacaattct gctactctta gcatttctct tcctcatgcg aaagatccaa 240 gactgcagcc agtggaatgt cctccccacc cagctcctct tcctcctgag tgtcctgggg 300 ctcttcggac tcgcttttgc cttcatcatc gagctcaatc aacaaactgc ccccgtacgc 360 tactttctct ttggggttct ctttgctctc tgtttctcat gcctcttagc tcatgcctcc 420 aatctagtga agctggttcg gggttgtgtc tccttctcct ggacgacaat tctgtgcatt 480 gctattggtt gcagtctgtt gcaaatcatt attgccactg agtatgtgac tctcatcatg 540 accagaggta tgatgtttgt gaatatgaca ccctgccagc tcaatgtgga ctttgttgta 600 ctcctggtct atgtcctctt cctgatggcc ctcacattct tcgtctccaa agccaccttc 660 tgtggcccgt gtgagaactg gaagcagcat ggaaggctca tctttatcac tgtgctcttc 720 tccatcatca tctgggtggt gtggatctcc atgctcctga gaggcaaccc gcagttccag 780 cgacagcccc agtgggatga cccggtcgtc tgcattgctc tggtcaccaa cgcatgggtt 840 ttcctgctgc tgtacatcgt ccctgagctc tgcattctct acagatcgtg tagacaggag 900 tgccctttac aaggcaatgc ctgccccgtc acagcctacc aacacagctt ccaagtggag 960 aaccaggagc tctccagaga ctgttgatcc cacacaagag tgtttcatcc cacaggctaa 1020 actaagcccc cagcaagatg caggaggagt ataa 1054 <210> 58 <211> 1251 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472462CB1

59/82 <400> 58 cctataccat tacctctagc acactgataa gagtctttct gtttcttcag gaacttgaca 60 agagaggacc ttaaagaaag tgagatcctt catacttagg aattcagtga cacttgctgg 120 gagaatgcct tctatcaatg acacccactt ctatcccccc ttcttcctcc tgctaggaat 180 accaggactg gacactttac atatctggat ttctttccca ttctgtattg tgtacctgat 240 tgccattgtg gggaatatga ccattctctt tgtgatcaaa actgaacata gtctacacca 300 gcccatgttc tacttcctgg ccatgttgtc tatgattgat ctgggtctgt ccacatccac 360 tatccccaaa atgctaggaa tcttctggtt caacctccaa gagatcagct ttgggggatg 420 ccttcttcag atgttcttta ttcacatgtt tacaggcatg gagactgttc tgttggtggt 480 catggcttat gaccgctttg ttgccatctg caaccctctc cagtacacca tgatcctcac 540 caataaaacc atcagtatcc tagcttctgt ggttgttgga agaaatttag ttcttgtaac 600 cccatttgtg tttctcattc tgcgtctgcc attctgtggg cataacatcg tacctcacac 660 atactgtgag cacaggggtc tggccgggtt ggcctgtgca cccattaaga tcaacataat 720 ctatgggctc atggtgattt cttatattat tgtggatgtg atcttaattg cctcttccta 780 tgtgcttatc cttagagctg tttttcgcct tccctctcaa gatgtccgac taaaggcctt 840 caatacctgt ggttctcatg tctgtgttat gctgtgcttt tacacaccag catttttttc 900 ttttatgaca catcgttttg gccaaaacat tccccactat atccatattc ttttggctaa 960 cctgtatgtg gttgtcccac ctgcccttaa ccctgtcatt tatggagtca ggaccaagca 1020 gatccgagag caaattgtga aaatatttgt acagaaagaa taattctgta ttaaagtttg 1080 gataaatata tctatataca acccaaatta tcatcatctg agctcccttt ttaatcttct 1140 gtaacagttg gtcatgcttg tcagattttt tccttttgac tggaagtgct ttagtgttga 1200 cagataatgc aaacttaaaa cctttgctgc ctgtttcccc tacttctctc c 1251 <210> 59 <211> 1187 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474873CB1 <400> 59 aaagacatag cttctaagca tttaacatac aatacttttt tgggtaggca ggtgacattg 60 tcactcattt aaccctatgt gatgtgttat ctttctcagc tatgcctcag ccttggggaa 120 cacactttac atatggggat ggtgagacat accaatgaga gcaacctagc aggtttcatc 180 cttttagggt tttctgatta tcctcagtta cagaaggttc tatttgtgct catattgatt 240 ctgtatttac taactatttt ggggaatacc accatcattc tggtttctcg tctggaaccc 300 aagcttcata tgccgatgta tttcttcctt tctcatctct ccttcctgta ccgctgcttc 360 accagcagtg ttattcccca gctcctggta aacctgtggg aacccatgaa aactatcgcc 420 tatggtggct gtttggttca cctttacaac tcccatgccc tgggatccac tgagtgcgtc 480 ctcccggctg tgatgtcctg tgaccgctat gtggctgtct gccgtcctct ccattacact 540 gtcttaatgc atatccatct ctgcatggcc ttggcatcta tggcatggct cagtggaata 600 gccaccaccc tggtacagtc caccctcacc ctgcagctgc ccttctgtgg gcatcgccaa 660 gtggatcatt tcatctgcga ggtccctgtg ctcatcaagc tggcttgtgt gggcaccacg 720 tttaacgagg ctgagctttt tgtggctagt atccttttcc ttatagtgcc tgtctcattc 780 atcctggtct cctctggcta cattgcccac gcagtgttga ggattaagtc agctaccagg 840 agacagaaag cattcgggac ctgcttctcc cacctgacag tggtcaccat cttttatgga 900 accatcatct tcatgtatct gcagccagcc aagagtagat ccagggacca gggcaagttt 960 gtttctctct tctacactgt ggtaacccgc atgcttaacc ctcttattta taccttgagg 1020 atcaaggagg tgaaaggggc attaaagaaa gttctagcaa aggctctggg agtaaatatt 1080 ttatgattat taaaaaaaaa tttaagtgac actgtgatga attttttttt tggtaaaaag 1140 tagcatcttt taataagagg attttttatg ggctcttctc acatttt 1187 <210> 60 <211> 1201

60/82 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475172CB1 <400> 60 gggaggatgc atcagcagag agcccaggat gtttcactag caccaaaggc tcaagactag 60 ccgtccaaga ttagcctttt aatggggttc ttgtctccca tgcatccctg caggcctccc 120 acccagagga gaatggctgc aggaaatcac tctacagtga cagagttcat tctcaagggt 180 ttaacgaaga gagcagacct ccagctcccc CtCtttCtCC tcttcctcgg gatctacttg 240 gtcaccatcg tggggaacct gggcatgatc actctaattt gtctgaactc tcagctgcac 300 acccccatgt actactttct cagcaatctg tcactcatgg atctctgcta ctcctccgtc 360 attaccccta agatgctggt gaactttgtg tcagagaaaa acatcatctc ctacgcaggg 420 tgcatgtcac agctctactt cttccttgtt tttgtcattg ctgagtgtta catgctgaca 480 gtgatggcct acgaccgcta tgttgccatc tgccaccctt tgctttacaa catcattatg 540 tctcatcaca cctgcctgct gctggtggct gtggtctacg ccatcggact cattggctcc 600 acaatagaaa ctggcctcat gttaaaactg ccctattgtg agcacctcat cagtcactac 660 ttctgtgaca tcctccctct catgaagctg tcctgctcta gcacctatga tgttgagatg 720 acagtcttct tttcggctgg attcaacatc atagtcacga gcttaacagt tcttgtttct 780 tacaccttca ttctctccag catcctcggc atcagcacca cagaggggag atccaaagcc 840 ttcagcacct gcagctccca ccttgcagcc gtgggaatgt tctatggatc aactgcattc 900 atgtacttaa aaccctccac aatcagttcc ttgacccagg agaatgtggc ctctgtgttc 960 tacaccacgg taatccccat gttgaatccc ctaatctaca gcctgaggaa caaggaagta 1020 aaggctgccg tgcagaaaac gctgaggggt aaactgtttt gatgcaaatg ttattgttcc 1080 ttttcaattt agtggtaatt gttataaata ccagagtgac agcttctgaa tgctggccag 1140 ctgtggatgg aaaataatca cttctccaca tggctgggtg aatgggcatt tttccttctt 1200 t 1201 <210> 61 <211> 2436 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475259CB1 <400> 61 gtaccgatga tgaataatga tgcagcttta ctactgagaa gggccccgtg cgtacagcaa 60 caaagtagtc actctgtctg gcttttatta gccacttagc acctattttc ttgtattttg 120 ttatagccct gtgtttcctt ctctgtcctt ccatattctc tataatgact actcagcacg 180 attattcaat tgcttagaaa agaaaaacac aggaaggaca gagaagtgga tattttagtc 240 agaagaaaac tccaatatct tacacttttc cccttggaag ctctgccttt ctgtggcgca 300 ctgttgcttg tttccttcca aagcctgcct ctcctatcta ccttcctcac agtttaaggt 360 gcaaggcaga ggcatccttt aaaaattaat tttcctagtc tgacaggaga atttcttgaa 420 cccgggaggc ggaggttgtg gtgaggcaag attgagtcat tgcactccag cctgggcaat 480 aagagcaagg ctccatctca aaaaataata ataataatta attttcctag tctgaaatgc 540 atttatttgt atctgctttt gacctattga agaaaccatg tcagctttct cacctcacac 600 ccgggacaga cagacgttaa aaaatgacca aacctacaga aaatatttcc agataatgaa 660 atttgagtat tgctttgctt tttgcacatc agttgaagat gtttactaga aaaaaaaaag 720 gtcattcagg ggtccaacag caagtatttc agatgatttt ggcatggagg taaagcttaa 780 gagatatttc taactggttt cttcaggatt ccagaatcag cttgagtaac tcattacaga 840 aaggaatgaa gcaatattca gtgggtaatc aacattccaa ttataggagt ctcttgtttc 900 cttttctgtg ttcacagatg acacagttga cggccagtgg gaatcagaca atggtgactg 960

61/82 agttcctctt ctctatgttc ccgcatgcgc acagaggtgg cctcttattc tttattccct 1020 tgcttctcat ctacggattt atcctaactg gaaacctaat aatgttcatt gtcatccagg 1080 tgggcatggc cctgcacacc cctttgtatt tctttatcag tgtcctctcc ttcctggaga 1140 tctgctatac cacaaccacc atccccaaga tgctgtcctg cctaatcagt gagcagaaga 1200 gcatttccgt ggctggctgc ctcctgcaga tgtacttttt ccactcactt ggtatcacag 1260 aaagctgtgt cctgacagca atggccattg acaggtacat agctatctgc aatccactcc 1320 gttacccaac catcatgatt cccaaacttt gtatccagct gacagttgga tcctgctttt 1380 gtggcttcct ccttgtgctt cctgagattg catggatttc caccttgcct ttctgtggct 1440 ccaaccagat ccaccagata ttctgtgatt tcacacctgt gctgagcttg gcctgcacag 1500 atacattcct agtggtcatt gtggatgcca tccatgcagc ggaaattgta gcctccttcc 1560 tggtcattgc tctatcctac atccggatta ttatagtgat tctgggaatg cactcagctg 1620 aaggtcatca caaggccttt tccacctgtg ctgctcacct tgctgtgttc ttgctatttt 1680 ttggcagtgt ggctgtcatg tatttgagat tctcagccac ctactcagtg ttttgggaca 1740 cagcaattgc tgtcactttt gttatccttg ctcccttttt caaccccatc atctatagcc 1800 tgaaaaacaa ggacatgaaa gaggctattg gaaggctttt ccactatcag aagagggctg 1860 gttgggctgg gaaatagata cagatcctgg agactctaaa aagcctcttg gaagagcaaa 1920 atttcactgt tatttatctt ttccatgtct atcctctttc tgtattgctg caactacttg 1980 ttctattatt tttaaaaaga atgataaact gctgtataca ggctgtggat ggtaagtgga 2040 tggccagttt acatcaatgc ctctaccatg cttgttatag ctgaagcagt atagatcaac 2100 tctcttgctt ttaacacagg ttggcttcct cctcgcactc cagcagctag gcccctgcta 2160 gtctcttcag tgctctgaca tgctagtggg gccgggaagc agaatagaag aggcatgaca 2220 gacaagaaag ttctgagaat tgctactgct aacttcccca attccctttc tggaaatgca 2280 tcccctgtca tttggcattc catataaata aatcgtttcc tgatactaga gagaggtctt 2340 ctctactttt agctcattta attcccaaac tcatttcagc aaacgtgaac tgattttgta 2400 catgatgtgt tacagaattt tagaccccaa gaggtt 2436 <210> 62 <211> 1050 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475267CB1 <400> 62 tatctatctc ttgtaggtaa ttcagaccct aatacctgga aggtattact ctctgaatgg 60 atcaccacat gcctcccaac aatgtgactg aattcattct cttggggctc acacagaatc 120 cacacttgca gaaaatactc tttattgtat ttttatttat ttttctattt accatgctgg 180 ccaatctgtt cattgtcatc accatctcct gtagccccac actttcatca cccatgtact 240 tctttctcac ttacttatcc tttatagatg cctcctacac ctctgtcaca acccccaaaa 300 tgatcaccga cctgctctac cagaggagaa ctatttcctt ggctggctgc ctgactcagc 360 tctttgtgga gcacttgctg ggaggctcag agatcatcct ccttattgtc atggcctatg 420 accgctacgt ggccatctgc aagcccctgc actacacaac cattatgcaa caagggatct 480 gccaccttct ggtggtgata gcctggattg gaggcatcct gcatgccact gtgcagattc 540 ttttcatgac cgacttgccc ttctgtggtc ccaatgtcat tgaccacttt atgtgtgatc 600 tcttcccatt gttgaaactt gcctgcagag acacctacag acttgggatg ctggtggcag 660 ccaacagtgg agccatgtgc ttgctcatct tttccctgct cgtcatctcc tacatagtca 720 tcctgagctc cctgaaatcc tatagctctg aaggacagca caaagccctc tccacctgtg 780 gctcccactt tactgtcgtt gtactctttt ttgtgccttg catattcacc tacatgcatc 840 ctgtggtcac ctactctgtg gacaagttgg tgactgtgtt ctttgcaatc ctcactccca 900 tgttaaatcc tataatttac actgtgagaa acacagaggt aaaaaatgcc gtgaggagtt 960 tgttgaggaa aagagtaaca gtttatgcat aatggcaaga aagtgatgat atatataaac 1020 atggaggatt atatctgtgg taaattgtgc 1050 <210> 63

62/82 <211> 1451 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475271CB1 <400> 63 tcaagaatta agtccctaag tacacacact cctcatgtta tctcctaaca acacagggat 60 tctttccatt ttcagttgtt tattctgtgc aattactgcc attcaatcac ccaagcagga 120 tgaatcacag cgttgtaact gagttcatta ttctgggcct caccaaaaag cctgaactcc 180 agggaattat cttcctcttt tttctcattg tctatcttgt ggcttttctc ggcaacatgc 240 tcatcatcat tgccaaaatc tataacaaca ccttgcatac gcccatgtat gttttccttc 300 tgacactggc tgttgtggac atcatctgca caacaagcat cataccgaag atgctgggga 360 ccatgctaac atcagaaaat accatttcat atgcaggctg catgtcccag ctcttcttgt 420 tcacatggtc tctgggagct gagatggttc tcttcaccac catggcctat gaccgctatg 480 tggccatttg tttccctctt cattacagta ctattatgaa ccaccatatg tgtgtagcct 540 tgctcagcat ggtcatggct attgcagtca ccaattcctg ggtgcacaca gctcttatca 600 tgaggttgac tttctgtggg ccaaacacca ttgaccactt cttctgtgag atacccccat 660 tgctggcttt gtcctgtagc cctgtaagaa tcaatgaggt gatggtgtat gttgctgata 720 ttaccctggc cataggggac tttattctta cctgcatctc ctatggtttt atcattgttg 780 ctattctccg tatccgcaca gtagaaggca agaggaaggc cttctcaaca tgctcatctc 840 atctcacagt ggtgaccctt tactattctc ctgtaatcta cacctatatc cgccctgctt 900 ccagctatac atttgaaaga gacaaggtgg tagctgcact ctatactctt gtgactccca 960 cattaaaccc gatggtgtac agcttccaga atagggagat gcaggcagga attaggaagg 1020 tgtttgcatt tctgaaacac tagtagtttc aacatgcaac atcacttctg tactccagaa 1080 ccatcttcta gagcatctca gattttactg gtttttcata cttacctcca ctccaatttt 1140 cccttccctc ttattcctgc cttcttccta gcagtctcat tgtctccaaa attctgtact 1200 ctttatgtga agaatattca taaagcaata tgcacaatac cctcacataa atatatgtca 1260 taatatatat tccaacattt tccaaaaata tgtacataac ttcgaatact tatatatgca 1320 tatacacaaa tatttaccta tatgtgcatg tgcacatcat acatgcaaat atcacaaaac 1380 attttgtgta ttttgtgcca tttatttgtt ggtatgtgaa tgtgagctgg agagaagtag 1440 tgtgtgtgat a 1451 <210> 64 <211> 1288 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7475305CB1 <400> 64 agtgtggctt caggaccagg gtgttggcta tcatgaaatg aggaagcata aacagtagaa 60 gtgatttctt aggttgttga gatagataga ataatataaa tgtggcatac cttgtgttta 120 gttcaagaac tataatctag atgtaacacc tgaaaataaa ctcttttatt gatattctac 180 aggcagaaga aatgaagata gcaaacaaca cagtagtgac agaatttatc ctccttggtc 240 tgactcagtc tcaagatatt cagctcttgg tctttgtgct gatcttaatt ttctacctta 300 tcatcctccc tggaaatttt ctcattattt tcaccataag gtcagaccct gggctcacag 360 cccccctcta tttatttctg ggcaacttgg ccttcctgga tgcatcctac tccttcattg 420 tggctcccag gatgttggtg gacttcctct ctgagaaaaa ggtaatctcc tacagaggct 480 gcatcactca gctctttttc ttgcacttcc ttggaggagg ggagggatta ctccttgttg 540 tgatggcctt tgaccgctac atcgccatct gccggcctct gcactgttca actgtcatga 600 accctagagc ctgctatgca atgatgttgg ctctgtggct tgggggtttt gtccactcca 660

63/82 gctcccactt tactgtcgtt gtactctttt ttgtgccttg ttatccaggt ggtcctcatc ctccgcttgc ctttttgtgg cccaaaccag Ctggacaact 720 tcttctgtga tgtccgacag gtcatcaagc tggcttgcac cgacatgttt gtggtggagc 780 ttctaatggt cttcaacagt ggcctgatga cactcctgtg ctttctgggg cttctggctt 840 cctatgcagt catcctctgc catgttcgta gggcagcttc tgaagggaag aacaaggcca 900 tgtccacgtg caccactcgt gtcattatta tacttcttat gtttggacct gctatcttca 960 tctacatgtg ccctttcagg gccttaccag ctgacaagat ggtttctctc tttcacacag 1020 tgatctttcc attgatgaat cctatgattt atacccttcg caaccaggaa gtgaaaactt 1080 ccatgaagag gttattgagt cgacatgtag tctgtcaagt ggattttata ataagaaact 1140 gagaaggagg aattctggct ggaattcata tcattcattt aacaagtcct gtttttcact 1200 gagtacctcc catttgccag gtaccattgt aggcaatgga ggagagttat gcataatgag 1260 agaataaact tattatattt aaagaata 1288 <210> 65 <211> 1271 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No. 7476160CB1 <400> 65 gtagacagca catctagtac tacaagtctt ttgatttggc atcaaatcca acataccttt 60 tattatgctt ttcacttttt gctcttctgt tagaagttgt ttcactaaaa atatattata 120 gctccatcat gtattcatct gactaaattc cactgtgcat cttctttctt attgcagctc 180 aaatgatgag acttatgaaa gaggttcgag gcagaaatca aacagaagta acagaatttc 240 tcctcttagg actttccgac aatccagatc tacaaggagt cctctttgca ttgtttctgt 300 tgatctatat ggcaaacatg gtgggcaatt tggggatgat tgtattgatt aagattgatc 360 tctgtctcca cacccccatg tatttctttc tcagtagcct ctcttttgta gatgcctctt 420 actcttcttc cgtcactccc aagatgctgg tgaacctcat ggctgagaat aaggccattt 480 cttttcatgg atgtgctgcc cagttctact tctttggctc cttcctgggg actgagtgct 540 tcctgttggc catgatggca tatgaccgct atgcagccat ttggaacccc ctgctctacc 600 cagttctcgt gtctgggaga atttgctttt tgctaatagc tacctccttc ttagcaggtt 660 gtggaaatgc agccatacat acagggatga cttttaggtt gtccttttgt ggttctaata 720 ggatcaacca tttctactgt gacaccccgc cactgctcaa actctcttgc tctgataccc 780 acttcaatgg cattgtgatc atggcattct caagttttat tgtcatcagc tgtgttatga 840 ttgtcctcat ttcctacctg tgtatcttca ttgccgtctt gaagatgcct tcgttagagg 900 gcaggcacaa agccttctcc acctgtgcct cttacctcat ggctgtcacc atattctttg 960 gaacaatcct cttcatgtac ttgcgcccta catctagcta ctcaatggag caagacaagg 1020 ttgtctctgt cttttataca gtaataatcc ctgtgctaaa tcccctcatc tatagtttaa 1080 aaaataagga tgtaaaaaag gccctaaaga agatcttatg gaaacacatc ttgtagagcc 1140 atgttaccca tcatttgtta cgtagaagaa aatacatttt catgttaact gtattctctg 1200 attgtttaag ctgtttctct gtgttaaagt agataattta aaatgaagta tactttttaa 1260 tatcctagta t 1271 <210> 66 <211> 954 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7476781CB1 <400> 66 atgggggaca accaatcacg ggtcacagaa ttcatcctgg ttggattcca gctcagtgtg 60

64/82 gagatggaag tgctcctctt ctggatcttc tccctgttat atctcttcag cctgctgggg 120 aatggggtca tctttgggct catctgcctg gactctaagc ttcacacccc catgtacttc 180 ttcctctcac acctggccgt cattgacatg tcctatgctt ccaacaatgt tcccaagatg 240 ctggcaaacc tagtgaacca gaaaagaact atctcgttca tctcttgcat aatgcagact 300 tttttgtatt tggcttttgc tgttacagtg tgcctgattt tggtggtgat gtcctatgac 360 agatttgtgg ccatctgcca tcccctgcat tacactgtca tcatgagctg gagagtgtgc 420 actgtcctgg ctgtggcttc ctgggtgttc agcttcctcc tggctctggt ccatttagtt 480 ctcattctga ggctgccctt ctgtgggccc caggaggtga accacttctt cggtgaaatc 540 ctgtctgtcc tcaagttggc ctgtgctgac acctggctca accaggtggt catctttgca 600 gcctgcatgt tcatcctggt agggccgctc tgcctggtgc tggtctccta cttgcacatc 660 ctggcggcca tcttgaggat ccagtctggg gagggccgca gaaaggcctt ctctacctgc 720 tcctcccacc tctgcgtggt ggggcttttc tttggcagcg ccattgtcat gtacatggcc 780 cccaagtcaa gccattctca agaacggagg aagatccttt ccctgtttta cagccttttc 840 aacccgatcc tgaaccccct catctacagc cttaggaatg cagaggtgaa aggggctcta 900 aagagagtcc tttggaaaca gagatcaatt gaagaatcat ttgagatatc ctga 954 <210> 67 <211> 1451 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7487603CB1 -<400> 67 agttcatata aataaaacat gccagatatg tgaaggaggg ggttatgttt tatatatgtg 60 tgacgttaat ccttctatgc atacgtacag gtgaacataa cataaaaaaa tgttcccggc 120 aaattggaca tctgtaaaag tatttttctt cctgggattt tttcactacc ccaaagttca 180 ggtcatcata tttgcggtgt gcttgctgat gtacctgatc accttgctgg gcaacatttt 240 tctgatctcc atcaccattc tagattccca cctgcacacc cctatgtacc tcttcctcag 300 caatctctcc tttctggaca tctggtactc ctcttctgcc ctctctccaa tgctggcaaa 360 ctttgtttca gggagaaaca ctatttcatt ctcagggtgc gccactcaga tgtacctctc 420 ccttgccatg ggctccactg agtgtgtgct cctgcccatg atggcatatg accggtatgt 480 ggccatctgc aaccccctga gataccctgt catcatgaat aggagaacct gtgtgcagat 540 tgcagctggc tcctggatga caggctgtct cactgccatg gtggaaatga tgtctgtgct 600 gccactgtct ctctgtggta atagcatcat caatcatttc acttgtgaaa ttctggccat 660 cttgaaattg gtttgtgtgg acacctccct ggtgcagtta atcatgctgg tgatcagtgt 720 acttcttctc cccatgccaa tgctactcat ttgtatctct tatgcattta tcctcgccag 780 tatcctgaga atcagctcag tggaaggtcg aagtaaagcc ttttcaacgt gcacagccca 840 cctgatggtg gtagttttgt tctatgggac ggctctctcc atgcacctga agccctccgc 900 tgtagattca caggaaatag acaaatttat ggctttggtg tatgccggac aaacccccat 960 gttgaatcct atcatctata gtctacggaa caaagaggtg aaagtggcct tgaaaaaatt 1020 gctgattaga aatcatttta atactgcctt catttccatc ctcaaataac aatcacactc 1080 atatagataa tcaacattac ccagaaaact gcataatagt ttacttaaac caaccctgga 1140 aactacttat tttcaataga agtttactat tatatcctct attctgattt gtcttataag 1200 taaaactttt catattaaca aatcatttat gaagaataaa ttaagtttcc aagaaagcaa 1260 ttagcattta ttgaatatta gtataacatt aaaattagat aattgcctat tatttcatat 1320 ttactttcta tagcatctca gtgtccagct gtgacataag cataataaca ataaatatgc 1380 caaaactgta aaattttgag actagtcaat tttgaaataa tttactccaa ataattcaca 1440 atttcccctc a 1451 <210> 68 <211> 1511 <212> DNA
<213> Homo sapiens

65/82 <220>
<221> misc_feature <223> Incyte ID No: 58015601CB1 <400> 68 ggatataaaa ccatcttgta ttgcagcatt ttgtcagtgt catctctaat ttcactatgt 60 taataactaa ggattccacc ctataaaaga agcagagtac ctgaattctc ctaaatgaca 120 cgtgtttcca tgcatgtatg tgtatacagt aatacaagat atattatatt acaccttatg 180 ttaatttttt tttatataag aagtattata gctatacttt ttttccgatt actctattgg 240 tagaagagga tttttttaat ttcatgagca taattgagtt ggttccagta acatatttga 300 aaacaaattc aacaaagaat ccattcaaaa taatacattt cttaatgctc cctctgaaac 360 actcagcaaa tattgtgcat ctttgaccca cagctctgac cttcctgtcc tagatgaggg 420 tttgtctttc tctgccacaa gagcatggaa ggcaaccaga catggatcac agacatcacc 480 ctgctgggat tccaggctgg tccagcactg gcgattctcc tctgtggact cttctctgtc 540 ttctatacac tcaccctgct ggggaatggg gtcatctttg ggattatctg cctggactct 600 aagcttcaca cacccatgta cttcttcctc tcacacctgg ccatcattga catgtcctat 660 gcttccaaca atgttcccaa gatgttggca aacctaatga accagaaaag aaccatctcc 720 tttgttccat gcataatgca gacttttttg tatttggctt ttgctgttac agagtgcctg 780 attttggtgg tgatgtccta tgataggtat gtggccatct gccacccttt ccagtacact 840 gtcatcatga gctggagagt gtgcacgatc ctggttctca cgtcctggtc atgtgggttt 900 gccctgtccc tggtacatga aattctcctt ctaaggttgc ccttctgtgg gccccgggat 960 gtgaaccacc tcttctgtga aattctgtct gtcctcaagc tggcctgtgc tgacacctgg 1020 gttaaccaag tggtcatatt tgctacctgt gtgtttgtct tagtcgggcc tctttccttg 1080 attctggtct cctacatgca catcctcggg gccatcctga agatccagac aaaggagggc 1140 cgcataaagg ccttctccac ctgctcctcc cacctgtgtg tggttggact attctttggc 1200 atagccatgg tggtttacat ggtcccagac tctaatcaac gagaggagca ggagaaaatg 1260 ctgtccctgt ttcacagtgt cttgaaccca atgctgaacc ccctgatcta cagcctgagg 1320 aatgctcagt tgaagggcgc cctccacaga gcactccaga ggaagaggtc catgagaacg 1380 gtgtatgggc tttgccttta aaacatgtgg tttgctgaag caagaatttt gaatatattt 1440 tgcagaagaa gtttaatata aaaatggtga gtgattgaat tcaagctttg aaaatagggc 1500 aatattcaat g 1511 <210> 69 <211> 1056 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 6541249CB1 <400> 69 atgtctgggg acaacagctc cagcctgacc ccaggattct ttatcttgaa tggcgttcct 60 gggctggaag ccacacacat ctggatctcc ctgccattct gctttatgta catcattgct 120 gtcgtgggga actgtgggct catctgcctc atcagccatg aggaggccct gcaccggccc 180 atgtactact tcctggccct gctctccttc actgatgtca ccttgtgcac caccatggta 240 cctaatatgc tgtgcatatt ctggttcaac ctcaaggaga ttgactttaa cgcctgcctg 300 gcccagatgt tttttgtcca tatgctgaca gggatggagt ctggggtgct catgctcatg 360 gccctggacc gctatgtggc catctgctac cccttacgct atgccaccat ccttaccaac 420 cctgtcatcg ccaaggctgg tcttgccacc ttcttgagga atgtgatgct catcatccca 480 ttcactctcc tcaccaagcg cctgccctat tgccggggga acttcatccc ccacacctac 540 tgtgaccata tgtctgtggc caaggtatcc tgtggcaatt tcaaggtcaa tgctatttat 600 ggtctgatgg ttgctctcct gattggtgtg tttgatatct gctgtatctc tgtatcttac 660 actatgattt tgcaggctgt tatgagcctg tcatcagcag atgctcgtca caaagccttc 720 agcacctgca catctcacat gtgttccatt gtgatcacct atgttgctgc ttttttcact 780 tttttcactc atcgttttgt aggacacaat atcccaaacc acatacacat catcgtggcc 840

66/82 aacctttatc tgctactgcc tcctaccatg aacccaattg tttatggagt caagaccaag 900 cagattcagg aaggtgtaat taaattttta cttggagaca afiaagaatgt ccaagggttc 960 tgtttttccc aagtcatcag tttagggtct ccatttaaaa tggatctaaa tgggaacaat 1020 agactccagg ttcttcgaaa ggagcgggaa gaataa 1056 <210> 70 <211> 1351 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472078CB1 <400> 70 tgtccaatgt ttgatacaaa aaaaaatgtt tgatatgaaa aaaatcaaac attggacaat 60 atgtacatga gtttaaagtc aggaaaaaaa atgaaacctt caaggtagtt cttcttactt 120 ctcaaagatg tttagctgta catcttattt atttttttct ctaccctctc atgtgctgga 180 ctatgccctc tccatttaca ggtagctcta ctagaaatat ggagagcgga aaccaatcaa 240 cagtgactga atttatcttc actggattcc ctcagcttca ggatggtagt ctcctgtact 300 tctttccttt acttttcatc tatactttta ttatcattga taacttatta atcttctctg 360 ctgtaaggct ggacacccat ctccacaacc ccatgtataa ttttatcagt atattttcct 420 ttctggagat ctggtacacc acagccacca ttcccaagat gctctccaac ctcatcagtg 480 aaaagaaggc catctcaatg actggctgca tcttgcagat gtatttcttc cactcacttg 540 aaaactcaga ggggatcttg ctgaccacca tggccattga cagatacgtt gccatctgca 600 accctcttcg ctatcaaatg atcatgaccc cccggctctg tgctcaactc tctgcaggtt 660 cctgcctctt cggtttcctt atcctgcttc ccgagattgt gatgatttcc acactgcctt 720 tctgtgggcc caaccaaatc catcagatct tctgtgactt ggtccctgtg ctaagcctgg 780 cctgtacaga cacgtccatg attctgattg aggatgtgat tcatgctgtg accatcatca 840 ttaccttcct aatcattgcc ctgtcctatg taagaattgt cactgtgata ttgaggattt 900 cctcttctga agggaggcaa aaggcttttt ctacctgtgc aggccacctc atggtcttcc 960 tgatattctt tggcagtgta tcactcatgt acttgcgttt cagcgacact tatccaccag 1020 ttttggacac agccattgca ctgatgttta ctgtacttgc tccattcttc aatcccatca 1080 tttatagcct gagaaacaag gacatgaaca atgcgattaa aaaactgttc tgtcttcaaa 1140 aagtgttgaa caagcctgga ggttaataca gagccacagg ttcccttccg ttgcttttgt 1200 tttgttttgt tttttgagat ggagtttcac tcttgttgcc tgggctggag tgcaatggca 1260 tgatcttggc tcactgcaac ctccacctcc tggttttgaa caattctcct gcctcagcct 1320 ccctagtagc cgcgattaca ggcatgcacc a 1351 <210> 71 <211> 1202 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472087CB1 <400> 71 tatagtatac gaatgaaccc cttatcctca caaaactgag ttttaaccaa aagcatgact 60 gcttttcatt tatatggttt cagatccggc taacgagctc atatctccct cattatgtct 120 gttctcaata actccgaagt caagcttttc cttctgattg ggatcccagg actggaacat 180 gcccacattt ggttctccat ccccatttgc ctcatgtacc tgcttgccat catgggcaac 240 tgcaccattc tctttattat aaagacagag ccctcgcttc atgagcccat gtattatttc 300 cttgccatgt tggctgtctc tgacatgggc ctgtccctct cctcccttcc taccatgttg 360 agggtcttct tgttcaatgc catgggaatt tcacctaatg cctgctttgC tcaagaattc 420

67/82 ttcattcatg gattcactgt catggaatcc tcagtacttc taattatgtc tttggaccgc 480 tttcttgcca ttcacaatcc cttaagatac agttctatcc tcactagcaa cagggttgct 540 aaaatgggac ttattttagc cattaggagc attctcttag tgattccatt tcccttcacc 600 ttaaggagat taaaatattg tcaaaagaat cttctttctc actcatactg tcttcatcag 660 gataccatga agctggcctg ctctgacaac aagaccaatg tcatctatgg cttcttcatt 720 gctctctgta ctatgctgga cttggcactg attgttttgt cttatgtgct gatcttgaag 780 actatactca gcattgcatc tttggcagag aggcttaagg ccctaaatac ctgtgtctcc 840 cacatctgtg ctgtgctcac cttctatgtg cccatcatca ccctggctgc catgcatcac 900 tttgccaagc acaaaagccc tcttgttgtg atccttattg cagatatgtt cttgttggtg 960 ccgcccctta tgaaccccat tgtgtactgt gtaaagactc gacaaatctg ggagaagatc 1020 ttggggaagt tgcttaatgt atgtgggaga taagaacttg aacaattagg taataaatta 1080 tcaaccagta ggcatttact gtcatttgct atgtgcttaa tgccatagaa gtcactaatg 1140 aaggactgga tgatggaagt gaaaagctat gtagtgcaga atttataata aagttgagaa 1200 t 1201 <210> 72 <211> 1251 <212> DNA
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 7472089CB1 <400> 72 tttgaaaatt agaaataata actcagtgaa tcttgaagtg ccagagagat gttacaagtg 60 ataaactatg tattatgtgt tatgttaaat gactgaaaca tccctgtctt ctcagtgctt 120 ccctatgtcg gtcctcaata ataccattgc tgagcctctg atcttcctcc tgatgggcat 180 tccaggcctg aaagccaccc agtactggat ctccatccct ttttgtctcc tatatgttgt 240 tgccgtctct ggaaatagca tgatcctgtt tgtggtcctc tgtgaacgga gcctccataa 300 gcctatgtac tatttcctct ctatgctttc agccacagac ctgagcttgt ccctgtgtac 360 actttctact acccttggtg tcttctggtt tgaagcccga gaaatcaacc taaatgcctg 420 cattgcccag atgttctttc tacacggatt tactttcatg gagtctgggg ttctactggc 480 catggccttt gatcgttttg tggccatctg ttacccactg agatacacta ccatccttac 540 caatgcccga attgccaaga ttgggatgag catgttgata agaaatgttg ccgtcatgtt 600 gccagtcatg ctctttgtca agaggttgtc cttctgcagt tctatggtcc tttcacattc 660 ttactgctac catgttgatc tcatccaact ctcctgcaca gacaatagga tcaacagcat 720 ccttggtctg tttgcgcttt tgtccactac agggtttgac tgcccttgca tcctgctctc 780 ctatatcctg atcattcgat ctgtcctcag cattgcttcc tcagaagaga ggcggaaagc 840 cttcaacacc tgcacatccc acatcagtgc tgtttccatc ttctacctcc ctctcatcag 900 tttgtctctt gtccatcgct atggccattc agcacctcca tttgtccaca tcatcatggc 960 caatgtcttt ctgctaatcc ctcctgtgct caaccctatt atttacagtg taaagattaa 1020 gcagattcaa aaggccatta tcaaggtctt aattcagaag cactccaaat ctaatcatca 1080 gctatttctg attagagata aagccattta tgaataagtg ctttgctaca atggagtaat 1140 tgtcccaaag tgcccacaca tgcctccaac agtccaaact aagcaattta taggttatgt 1200 gacatttatt tagtcaattt ctttgtcaat aaattcatgt cattgccaac t 1251 <210> 73 <211> 1221 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474902CB1

68/82 <400> 73 tctttaattt ccaccaggtg caatcaccag tactgcctca atttacttca ggattttgga 60 gggcacccac cttccccctt gtctcctcac acaatgaccc tgggatccct gggaaacagc 120 agcagcagcg tttctgctac cttcctgctg agtggcatcc ctgggctgga gcgcatgcac 180 atctggatct ccatcccact gtgcttcatg tatctggttt ccatcccggg caactgcaca 240 attcttttta tcattaaaac agagcgctca cttcatgaac ctatgtatct cttcctgtcc 300 atgctggctc tgattgacct gggtctctcc ctttgcactc tccctacagt cctgggcatc 360 ttttgggttg gagcacgaga aattagccat gatgcctgct ttgctcagct ctttttcatt 420 cactgcttct ccttcctcga gtcctctgtg ctactgtcta tggcctttga ccgctttgtg 480 gctatctgcc accccttgca ctatgtttcc attctcacca acacagtcat tggcaggatt 540 ggcctggtct ctctgggtcg tagtgtagca ctcatttttc cattaccttt tatgctcaaa 600 agattcccct attgtggctc cccagttctc tcacattctt attgtctcca ccaagaagtg 660 atgaaattgg cctgtgccga catgaaggcc aacagcatct acggcatgtt tgtcatcgtc 720 tctacagtgg gtatagactc actgctcatc ctcttctctt atgctctgat cctgcgcacc 780 gtgctgtcca tcgcctccag ggctgagaga ttcaaggccc ttaacacctg tgtttcccac 840 atctgtgctg tgctgctctt ctacactccc atgattggcc tctctgtcat ccatcgcttt 900 ggaaagcagg caccccacct ggtccaggtg gtcatgggtt tcatgtatct tctctttcct 960 cctgtgatga atcccattgt ctacagtgtg aagaccaaac agatccggga tcgagtgacg 1020 catgcctttt gttactaact gtgtctagtg ttagagccac tgtctcctga aacgtgccct 1080 tgtttgccta tcctttataa tttctaacat gcataaaata aaggagacat ttacttactc 1140 aacaaacatg tacagggatg agttacagtc cttacagaac cctcactctg ttgtggccag 1200 ggcagggggg ggtaaaatgt a 1221 <210> 74 <211> 1276 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475057CB1 <400> 74 cctcactacc ccttcctccc actacaatac atatagactt ttaggagcaa aggattctcc 60 tatcttagct agaattgcga ccttaattat ccatccaaac ccccctaacc aaaagttata 120 aacctgtgat cctcccagcc ctgcactact cctacacgta tgccatggat gggtttgtgc 180 cactgtgaga cccccaaaat ctctcccctt atcttccctg tcaggagtca tgcccccatg 240 agccctcaag ttgtgcccac cacagaatgg cagaaactct acaactcaat tccaccttcc 300 tacacccaaa cttcttcata ctgactggct ttccagggct aggaagtgcc cagacttggc 360 tgacactggt ctttgggccc atttatctgc tggccctgct gggcaatgga gcactgccgg 420 cagtggtgtg gatagactcc acactgcacc agcccatgtt tctactgttg gccatcctgg 480 cagccacaga cctgggctta gccacatcta tagccccagg gttgctggct gtgctgtggc 540 ttgggccccg atctgtgcca tatgctgtgt gcctggtcca gatgttcttt gtacatgcac 600 tgactgccat ggaatcaggt gtgcttttgg ccatggcctg tgatcgtgct gcggcaatag 660 ggcgtccact gcactaccct gtcctggtca ccaaagcctg tgtgggttat gcagccttgg 720 ccctggcact gaaagctgtg gctattgttg tacctttccc actgctggtg gcaaagtttg 780 agcacttcca agccaagacc ataggccata cctattgtgc acacatggca gtggtagaac 840 tggtggtggg taacacacag gccaccaact tatatggtct ggcactttca ctggccatct 900 caggtatgga tattctgggt atcactggct cctatggact cattgcccat gctgtgctgc 960 agctacctac ccgggaggcc catgccaagg cctttggtac atgtagttct cacatctgtg 1020 tcattctggc cttctacata cctggtctct tctcctacct cacacaccgc tttggtcatc 1080 acactgtccc aaagcctgtg cacatccttc tctccaacat ctacttgctg ctgccacctg 1140 ccctcaaccc cctcatctat ggggcccgca ccaagcagat cagagaccga ctcctggaaa 1200 ccttcacatt cagaaaaagc ccgttgtaat gtccagtggt aacaatggag cctaagagtg 1260 gaggtgaggg gacaat 1276

69/82 <210> 75 <211> 1509 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475261CB1 <400> 75 gcctggggca gtcagggctc atcccctgga ggacaccgga caccctgtgg atgcccttca 60 tggttattgg gtcccttgga gaagtccagg tggtgaaaaa taggacacag ctttgagaga 120 tgagggagac tggtggtgac aggctcagga gggcattcca gacccagtgt tacaggctga 180 gacctagaat ctatatcatc agaggttagt gcttaacatg tatctgggtg gggagctgct 240 tttgctcccc actggtgctc tggggagcca cgtggcctct ctttacacca aaactCCCta 300 tccaggtcca gctccactct ccctctgccc cagcttccct gcagcccttt cctcttgctc 360 tttgatgttt tgtaggcctg cagctcccaa gcacagaggc atgagtgggg agaatgtcac 420 cagggtcggc accttcatcc tggtgggctt ccccacggcc ccagggctgc agtacctgct 480 cttcctcctc ttcctgctca cctacctctt tgtcctggtg gagaacctgg ccatcatcct 540 caccgtctgg agcagcacct ccctccacag gcccatgtac tactttctga gctccatgtc 600 tttcctagag atctggtacg tgtctgacat cacccccaag atgctggagg gcttcctcct 660 ccagcagaaa cgcatctctt tcgtcgggtg catgacgcag ctctacttct tcagctccct 720 ggtgtgcacc gagtgtgtgc ttctggcctc catggcctac gaccgctacg tggccatctg 780 ccacccgctg cgctaccacg tccttgtgac cccggggctg tgcctccagc tggtgggctt 840 ctcctttgtg agtggcttca ccatctccat gatcaaggtc tgttttatct ccagcgtcac 900 gttctgtggc tccaacgtct tgaaccactt cttctgtgac atttccccca tcctcaagct 960 ggcctgcacg gacttctcca ctgcagagct ggtggatttc attctggcct tcatcatcct 1020 ggtgtttcca ctcctggcca ccatgctgtc atatgcgcac atcaccctgg ctgtcctgcg 1080 catcccctcg gccaccggct gctggagagc cttcttcacc tgcgcctctc acctcaccgt 1140 ggtcaccgtc ttctatacag ccttgctttt catgtatgtc cggccccagg ccattgattc 1200 ccggagctcc aacaagctca tctctgtttt gtacacagtt atcaccccca tcttgaaccc 1260 cttgatatac tgcctgagga ataaggaatt taagaatgcc ttgaaaaaag ccttcggctt 1320 gacgagctgc gccgtagagg ggaggctttc tagtcttctg gaacttcatc tccaaataca 1380 cagccagcct ctctgaggag gccatttgac tgtttgcatt attgtagtac cgcatttatt 1440 taaaaattac ttccaaatct atttttctcc ttcaggaaaa aaactgaggt tgaggttaat 1500 agcataggt 1509 <210> 76 <211> 1301 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475262CB1 <400> 76 actatgaata tatacttggt gagtgactgt ttcattactt agaattttta agatccccaa 60 gaccaattgt agatatttct cttatatttt ccattgctgc ttaagtaatg cttaccaact 120 aaccaaccaa ccaaggagaa aagatcctct ctatgacaga gtttcatctg caaagccaaa 180 tgccctcaat aagactcatc ttcagaaggc tgtccttagg cagaattaaa cccagtcaga 240 gccccaggtg ttcaacctca tttatggtgg tgccttcttt ctccatcgca gagcactgga 300 gaaggatgaa aggggcaaac ctgagccaag ggatggagtt tgagctcttg ggcctcacca 360 ctgaccccca gctccagagg ctgctcttcg tggtgttcct gggcatgtac acagccactc 420 tgctggggaa cctggtcatg ttcctcctga tccatgtgag tgccaccctg cacacaccca 480 tgtactccct cctgaagagc ctctccttct tggatttctg ctactcctcc acggttgtgc 540

70/82 cccagaccct ggtgaacttc ttggccaaga ggaaagtgat ctcttatttt ggctgcatga 600 ctcagatgtt cttctatgcg ggttttgcca ccagtgagtg ctatctcatc gctgccatgg 660 cctatgaccg ctatgccgct atttgtaacc ccctgctcta ctcaaccatc atgtctcctg 720 aggtctgtgc ctcgctgatt gtgggctcct acagtgcagg attcctcaat tctcttatcc 780 acactggctg tatctttagt ctgaaattct gcggtgctca tgtcgtcact cacttcttct 840 gtgatgggcc acccatcctg tccttgtctt gtgtagacac ctcactgtgt gagatcctgc 900 tcttcatttt tgctggtttc aaccttttga gctgcaccct caccatcttg atctcctact 960 tcttaattct caacaccatc ctgaaaatga gctcggccca gggcaggttt aaggcatttt 1020 ccacctgtgc atcccacctc actgccatct gcctcttctt tggcacaaca ctttttatgt 1080 acctgcgccc caggtccagc tactccttga cccaggaccg cacagttgct gtcatctaca 1140 cagtggtgat cccagtgctg aaccccctca tgtactcttt gagaaacaag gatgtgaaga 1200 aagctttaat aaaggtttgg ggtaggaaaa caatggaatg atttctcaat gcattaccac 1260 atatctttag aaagtcaagg gaacttttac cttaggtggt g 1301 <210> 77 <211> 1051 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475266CB1 <400> 77 gaagagcagt gagggtccat gttaaggtaa ttcatacttt ctattttcac agaaatgcct 60 aaagaagaat gaccatggaa aattattcta tggcagctca gtttgtctta gatggtttaa 120 cacagcaagc agagctccag ctgcccctct tcctcctgtt cctgggaatc tatgtggtca 180 cagtagtggg caacctgggc atgattctcc tgattgcagt cagccctcta cttcacaccc 240 ccatgtacta tttcctcagc agcttgtcct tcgtcgattt ctgctattcc tctgtcatta 300 ctcccaaaat gctggtgaac ttcctaggaa agaagaatac aatcctttac tctgagtgca 360 tggtccagct cgttttcttt gtggtctttg tggtggctga gggttacctc ctgactgcca 420 tggcatatga tcgctatgtt gccatctgta gcccactgct ttataatgcg atcatgtcct 480 catgggtctg ctcactgcta gtgctggctg ccttcttctt gggctttctc tctgccttga 540 ctcatacaag tgccatgatg aaactgtcct tttgcaaatc ccacattatc aaccattact 600 tctgtgatgt tcttcccctc ctcaatctct cctgctccaa cacacacctc aatgagcttc 660 tactttttat cattgcgggg tttaacacct tggtgcccac cctagctgtt gctgtctcct 720 atgccttcat cctctacagc atccttcaca tccgctcctc agagggccgg tccaaagctt 780 ttggaacatg cagctctcat ctcatggctg tggtgatctt ctttgggtcc attaccttca 840 tgtatttcaa gcccccttca agtaactccc tggaccagga gaaggtgtcc tctgtgttct 900 acaccacggt gatccccatg ctgaaccctt taatatacag tctgaggaat aaggatgtga 960 agaaagcatt aaggaaggtc ttagtaggaa aatgagtcct gatttggggg atttatagat 1020 gggaaaaatg aatagttcca atgaacatat a 1051 <210> 78 <211> 1490 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475284CB1 <400> 78 gggtggtgag ggaagaaaaa ttacttattg ggtgcgatgt acaccatttg ggtgatgagt 60 atgctataaa cccaggcttc agtacttggc aatatatcca tgtaacaaaa tatatccatg 120 taacaaaaat gcacttgtgc ctcttaaatc tgtaaaaatg aaaagttaca aacaagaaaa 180

71/82 gaaaccactg cagaatccag tacagagcaa agcaggcaga tcaagatgat tctaccagga 240 agcattcctc tgtaactatc tagatctctt tcttcttttc catttcattc atctaaacac 300 aggtaattta catgcatatt aaatttaacc acttatttct tttcacagac acccaagagt 360 tgattcctcc ccaggaatga gaaatcacac aatggtgact gaattcatcc ttctgggaat 420 ccctgagaca gagggcctag agacagccct tttattcctg ttctcctcat tttatttatg 480 caccctcttg ggaaacgtgc ttatccttac agctatcatc tcctccactc gacttcacac 540 tcctatgtat tttttcttgg gaaacctctc catctttgac ctgggtttct cttcaacgac 600 tgttcccaag atgttgttct acctttcggg gaacagccat gctatctcat atgcaggctg 660 cgtgtcccag cttttcttct accatttcct aggctgtact gagtgtttcc tctacacagt 720 gatggcctgt gaccgctttg ttgccatatg ttttcctttg agatacacgg tcatcatgaa 780 ccacagggtg tgctttatgt tggccacggg gacctggatg attggctgtg tccatgccat 840 gatcctaact cccctcacct tccagttacc ttactgtggc cctaacaagg tgggctatta 900 cttctgtgat attcctgcag tgttacctct agcctgtaag gacacatcct tagcccagag 960 ggtaggtttt acaaatgttg gtcttttgtc tctcatttgc ttttttctca tccttgtttc 1020 ctatacttgc attgggattt ccatatcaaa aatccgctca gcagagggca ggcagcgggc 1080 cttctccacc tgcagcgctc acctcactgc aatcctttgt gcttatgggc cagtcatcgt 1140 tatctatcta caacccaatc ccagtgcctt gcttggttcc ataattcaga tattgaataa 1200 tctggtaacc ccaatgttga atccactaat ctatagcctt aggaataagg atgtaaaatc 1260 agatcagccc tgaggaatgt atttcccaag aaaagctttg ctctgggaaa taaatgagaa 1320 catttaaagc tttgctggat ttaagatttt gattacattt tgaagtttga ctctccactc 1380 cctgagaaaa tttcccatct gctgctgcca aggcaagttg aaacgaaatg tactccaaat 1440 caatctacta cttaacctcg ctcttttaaa atatctgtct gttggtgtat 1490 <210> 79 <211> 1288 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475309CB1 <400> 79 catatatcac aatgttctta aatcttgctt tagttaccct caacaactct gaacatttat 60 ttgtgtagaa tttggattaa atggggcaaa gagatattat ttctaatgtt tctttttctc 120 cctgagtgaa gatcctgaat ctgaagacac attcatcagt catgtcccag gtgactaaca 180 ccacacaaga aggcatctac ttcatcctca cggacatccc tggatttgag gcctcccaca 240 tctggatctc catccccgtc tgctgtctct acaccatctc catcatgggc aataccacca 300 tcctcactgt cattcgcaca gagccatctg tccaccagcg catgtatctg tttctctcca 360 tgctggccct gacggacctg ggtctcaccc tcaccaccct acccacagtc atgcagcttc 420 tctggttcaa cgttcgtaga atcagctctg aggcctgttt tgctcagttt ttcttccttc 480 atggattctc ctttatggag tcttctgtcc tcctggctat gtccgttgac tgctatgtgg 540 ccatctgctg tcccctccat tatgcctcca tcctcaccaa tgaagtcatt ggtagaactg 600 ggttagccat catttgctgc tgtgttctgg cggttcttcc ctcccttttc ttactcaagc 660 gactgccttt ctgccactcc caccttctct ctcgctccta ttgcctccac caggatatga 720 tccgcctggt ctgtgctgac atcaggctca acagctggta tggatttgct cttgccttgc 780 tcattattat cgtggatcct ctgctcattg tgatctccta tacacttatt ctgaaaaata 840 tcttgggcac agccacctgg gctgagcgac tccgtgccct caataactgc ctgtcccaca 900 ttctagctgt cctggtcctc tacattccca tggttggtgt atctatgact catcgctttg 960 ccaagcatgc ctctccactg gtccatgtta tcatggccaa tatctacctg ctggcacccc 1020 cggtgatgaa ccccatcatt tacagtgtaa agaacaagca gatccaatgg ggaatgttaa 1080 atttcctttc cctcaaaaat atgcattcaa gatgagggaa tgcatttctt aaattactga 1140 caagtatgag tcataggctt aaggggggaa tatattcaga attaggaaac tataaaataa 1200 aacttcatca taatattaag gcagtatgac aagtccctgg cttttagcat ggaatttttg 1260 gctgaggtga gctagcagca gtgattct 1288

72/82 <210> 80 <211> 1124 <212> DNA
<213> Homo Sapiens <220>
<.221> misc_feature <223> Incyte ID No: 7477359CB1 <400> 80 ctccatgatc ctctcagctc atgtgttctg ttattctaaa tttaattgtt ttggatgtac 60 ccattccatt cctgccttag gtgcggatcc ccctggaggg atgggattgg gcaatgagag 120 ttccctaatg gatttcatcc ttctaggctt ctcagaccac cctcgtctgg aggctgttct 180 ctttgtattt gtccttttct tctacctcct gacccttgtg ggaaacttca ccataatcat 240 catctcatat ctggatcccc ctcttcatac cccaatgtac ttttttctca gcaacctctc 300 tttactggac atctgcttca ctactagcct tgctcctcag accttagtta acttgcaaag 360 accaaagaag acgatcactt acggtggttg tgtggcgcaa ctctatattt ctctggcact 420 gggctccact gaatgtatcc tcttggctga catggccttg gatcggtaca ttgctgtctg 480 caaacccctc cactatgtag tcatcatgaa cccacggctt tgccaacagc tggcatctat 540 ctcctggctc agtggtttgg ctagttccct aatccatgca acttttacct tgcaattgcc 600 tctctgtggc aaccataggc tggaccattt tatttgcgaa gtaccagctc ttctcaagtt 660 ggcttgtgtg gacaccactg tcaatgaatt ggtgcttttt gttgttagtg ttctgtttgt 720 tgtcattcca ccagcactca tctccatctc ctatggcttc ataactcaag ctgtgctgag 780 gatcaaatca gtagaggcaa ggcacaaagc cttcagcacc tgctcctccc accttacagt 840 ggtgattata ttctatggca ccataatcta cgtgtacctg caacctagtg acagctatgc 900 ccaggaccaa gggaagttta tctccctctt ctacaccatg gtgaccccca ctttaaatcc 960 tatcatctat actttaagga acaaggatat gaaagaggct ctgaggaaac ttctctcggg 1020 aaaattgtga ttcctatgga catgatttgc aaggaattca ttataagcca ggtagcttat 1080 tcagcttcta tttagtcaaa ctcatagttc taaatttcac atgt 1124 <210> 81 <211> 1447 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 58004547CB1 <400> 81 gcctgcagct cccaagcaca gaggcatgag tggggagaat gtcaccaagg tcagcacctt 60 catcctggtg ggcctcccca cggccccagg gctgcagtac ctgctcttcc tcctcttcct 120 gctcacctac ctctttgtcc tggtggagaa cctggccatc atcctcatcg tctggagcag 180 cacctccctc cacaggccca tgtactactt tctgagctcc atgtctttcc tggagatctg 240 gtacgtgtct gacatcaccc ccaagatgct ggagggcttc ctcctccagc agaaacgcat 300 ctctttcgtc gggtgcatga cgcagctcta cttcttcagc tccctggtgt gcaccgagtg 360 tgtgcttctg gcctccatgg cctacgaccg ctacgtggcc atctgccacc cgctgcgcta 420 ccacgtcctt gtgaccccgg ggctgtgcct ccagctggtg ggcttctcct ttgtgagtgg 480 cttcaccatc tccatgatca aggtctgttt tatctccagc gtcacgttct gtggctccaa 540 cgtcttgaac cacttcttct gtgacatttc ccccatcctc aagctggcct gcacggactt 600 ctccactgca gagctggtgg atttcatcct ggccttcatc atcctggtgt ttccactcct 660 ggccaccata ctgtcatatt ggcacatcac cctggctgtc ctgcgcatcc cctcggccac 720 cggctgctgg agagccttct ctacctgcgc ctctcacctc accgtggtca ccgtcttcta 780 tacagccttg cttttcatgt atgtccggcc ccaagccatt gattcccaga gctccaacaa 840 gctcatctct gccgtgtaca ctgttgtcac gccaataatt aaccctttga tttactgcct 900 gaggaacaag gaatttaagg acgccttgaa aaaggccttg ggcttgggtc aaacttcaca 960

73/82 ctaagacaac taaatgtcct agagtaaaat ctgtagtgat gaaacaacat tgtatgtggc 1020 actttgtgct ctttaaatta atttttaagt taaatttaac aaacctacag aactaagtga 1080 aatttaacaa acctacacaa ctgtgcacaa gtaacaagcc ttcgtctgca tatgttttca 1140 caaaaggact gtgctcatgg aaccagctcc cagatcaaga actagaagct gccttcacgc 1200 cctctcccag tcattaaccc ttctccacaa aaagcactgt cctgacttcc aaaaccagat 1260 gctagcattg gtgtttctga ggggcttaca cataaagtat gtgccctgtt atgttcagct 1320 tctttcattc accgtgttgc ttgagtgaat ccttgatgtt tttgcatgta acaataattc 1380 atgcattctc attgctatcc aatattcata ataaataata catttatact gtataataaa 1440 aaaaaaa 1447 <210> 82 <211> 1026 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7476156CB1 <400> 82 tgattgctcg tctttagttt gaatattttc agtgacaggg aatttactac atccatcaaa 60 aaactaaatc caggcacaca tacacttgaa gcaatggata aagaaaacag ctcaatggtg 120 actgagttta tcttcatggg catcacccag gaccctcaga tggagatcat cttcttcgtg 180 gtcttcctca tagtttacct ggttaatgta gtggggaata ttggtatgat tatcctgatt 240 acaacagaca ctcagcttca cacacccatg tattttttcc tctgcaacct ctcctttgtt 300 gacctgggct actcctcagc cattgccccc aggatgctgg ctgacttcct aacaaatcac 360 aaagttatct ccttctccag ctgtgccacc cagtttgctt tttttgtagg ttttgtggat 420 gctgagtgct atgtcctggc agccatggcc tatggtcgtt ttgtggccat ttgtcgaccc 480 ctccactata gcaccttcat gtccaagcag gtctgcttgg ctctcatgct gggctcttac 540 ctggctggtc tagtgagttt agtagcccac actaccctca ccttcagcct gagttactgt 600 ggttccaata tcatcaatca tttcttctgc gaaatcccac cactcttggc cctctcttgc 660 tcagacacct acatcagtga gatcttgctc ttcagtctgt gtggcttcat tgaattcagc 720 accatcctca tcatcttcat ctcctatacc tttatccttg ttgcaatcat cagaatgcgt 780 tcagctgaag gccgccttaa ggctttctcc acctgcgggt ctcaccttac tggcatcacc 840 ctcttctatg gcacagtcat gtttatgtac ctgaggccaa catccagcta ctccctggac 900 caagacaagt gggcctctgt gttctacacg gttatcatcc ccatgttaaa tcccttgatc 960 tacagtttgc ggaacaagga tgtgaaagct gctttcaaaa agctaattgg aaaaaaatct 1020 caataa 1026 <210> 83 <211> 1481 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475114CB1 <400> 83 gactcttgct agtctgtata gactaatttt ttaatttttt tctcctttat tctttttttg 60 catattttct caccttgaca ggcactgaag agaatctagt atggccaatg tcaccttggt 120 gacaggattt cttcttatgg ggttttctaa tatccagaag ctgcggattt tatatggtgt 180 gctcttccta ctgatttacc tggcagccct aatgagtaac cttctcatca ttactctcat 240 taccctggac gtaaagctcc aaacacccat gtacttcttc ctgaagaact tatccttttt 300 ggatgtcttc ctggtgtctg ttccaatccc aaaattcatt gtcaacaacc taacccacaa 360 caattccatt tccattctag gatgtgcctt ccagctactt ttaatgactt ccttctcagc 420

74/82 aggagagata tttatcctca ctgccatgtc ctatgaccgc tatgtagcca tctgctgtcc 480 cctgaactac gaggtaatca tgaatactgg agtctgtgtg ttaatggcaa gtgtttcctg 540 ggccattgga gggctctttg gtactgcgta cacagctggc acattttcca tgcctttctg 600 tggctccagt gtgattccac agtttttctg tgatgttcct tcattactaa ggatttcctg 660 ttctgaaaca ctaatggtaa tttatgcagg tattggagtt ggtgcatgtt taagcatttc 720 ttgtttcatc tgtattgtga tctcttacat ttatatcttc tccactgtac tgaagatccc 780 taccactaaa ggtcagtcca aagctttttc cacatgcttc ccccatctca ctgttttcac 840 tgtttttatc ataactgctt attttgttta tcttaagcca ccttcaaatt caccatctgt 900 tattgacagg ctgctttctg tgatctacac tgtgatgcct ccagtattta accctgtaac 960 ctacagcctg cggaacaatg acatgaaatg tgctctgata aggttgctgc agaaaacata 1020 tggtcaggag gcttacttca tttaacactt tcaagttctg tcagtgatac agtgccttac 1080 agatcacaag aaactttcct tatttgtaac tttggaaaga cctgagaaaa gaaagcaata 1140 tactcaaatt attttttccc tgaagaaata aatactcaag agcctaactg actatttcta 1200 agtcacttaa ttgcattaca tcaggataat acatagtgtt atagtaatca tttggtattc 1260 ttctatgaca aagcatttct gcctttgtat tataactttc tgaaagaatt ggttctgttt 1320 aacatgatgc tttcatcttt ggtctcttaa taacccattt tctctatttt attcactgag 1380 aacaaattaa aaatgctgag gtgattcaat gagtgcagta taatgcattt gcattatact 1440 acccagataa aattcacatg actcccctga gaatctagct a 1481 <210> 84 <211> 1106 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No. 55003505C81 <400> 84 ccacatagag aatggattct catttctcaa ttaagtgcta aatgctgggt gctctttata 60 tccccagagg gagagagacc aagggtgaga agaaatgtcc aacgccagcc tcgtgacagc 120 attcatcctc acaggccttc cccatgcccc agggctggac gccctcctct ttggaatctt 180 cctggtggtt tacgtgctca ctgtgctggg gaacctcctc atcctgctgg tgatcagggt 240 ggattctcac ctccacaccc ccatgtacta cttcctcacc aacctgtcct tcattgacat 300 gtggttctcc actgtcacgg tgcccaaaat gctgatgacc ttggtgtccc caagcggcag 360 ggctatctcc ttccacagct gcgtggctca gctctatttt ttccacttcc tggggagcac 420 cgagtgtttc ctctacacag tcatgtccta tgatcgctac ttggccatca gttacccgct 480 caggtacacc agcatgatga gtgggagcag gtgtgccctc ctggccaccg gcacttggct 540 cagtggctct ctgcactctg ctgtccagac catattgact ttccatttgc cctactgtgg 600 acccaaccag atccagcact acttctgtga cgcaccgccc atcctgaaac tggcctgtgc 660 agacacctca gccaacgtga tggtcatctt tgtggacatt gggatagtgg cctcaggctg 720 ctttgtcctg atagtgctgt cctatgtgtc catcgtctgt tccatcctgc ggatccgcac 780 ctcagatggg aggcgcagag cctttcagac ctgtgcctcc cactgtattg tggtcctttg 840 cttctttgtt ccctgtgttg tcatttatct gaggccaggc tccatggatg ccatggatgg 900 agttgtggcc attttctaca ctgtgctgac gccccttctc aaccctgttg tgtacaccct 960 gagaaacaag gaggtgaaga aagctgtgtt gaaacttaga gacaaagtag cacatcctca 1020 gaggaaataa atactaggaa gtaaatacac tagtttgttt aaaaatagta atctaattta 1080 gttattcatg tgaaattgat tatatg 1106 <210> 85 <211> 1601 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature

75/82 <223> Incyte ID No: 7474916CB1 <400> 85 gtaggacata cctaaggttc agtgaggttt gagagagagc atcagagaga aggtaggatg 60 gcctaaagaa agaacattta gtaagtggta ctacactagg tgaatatata aaaaataaca 120 agggacattt tttttactgg caaaaatatt tcattctctg ggtcttcatg cagatatatt 180 caagcaatgg aagggaaaaa tcaaaccaat atctctgaat ttctcctcct gggcttctca 240 agttggcaac aacagcaggt gctactcttt gcacttttcc tgtgtctcta tttaacaggg 300 ctgtttggaa acttactcat cttgctggcc attggctcgg atcactgcct tcacacaccc 360 atgtatttct tccttgccaa tctgtccttg gtagacctct gccttccctc agccacagtc 420 cccaagatgc tactgaacat ccaaacccaa acccaaacca tctcctatcc cggctgcctg 480 gctcagatgt atttctgtat gatgtttgcc aatatggaca attttcttct cacagtgatg 540 gcatatgacc gttacgtggc catctgtcac cctttacatt actccaccat tatggccctg 600 cgcctctgtg cctctctggt agctgcacct tgggtcattg ccattttgaa ccctctcttg 660 cacactctta tgatggccca tctgcacttc tgctctgata atgttatcca ccatttcttc 720 tgtgatatca actctctcct ccctctgtcc tgttccgaca ccagtcttaa tcagttgagt 780 gttctggcta cggtggggct gatctttgtg gtaccttcag tgtgtatcct ggtatcctat 840 atcctcattg tttctgctgt gatgaaagtc ccttctgccc aaggaaaact caaggctttc 900 tctacctgtg gatctcacct tgccttggtc attcttttct atggagcaaa cacaggggtc 960 tatatgagcc ccttatccaa tcactctact gaaaaagact cagccgcatc agtcattttt 1020 atggttgtag cacctgtgtt gaatccattc atttacagtt taagaaacaa tgaactgaag 1080 gggactttaa aaaagaccct aagccggccg ggcgcggtgg ctcacgcctg taatcccagc 1140 actttgggag gccgaggcgg gtggatcatg aggtcaggag atcgagacca tcctggctaa 1200 caaggtgaaa ccccgtctct actaaaaata caaaaaatta gccgggcgcg gtggcgggcg 1260 cctgtagtcc cagctactcg ggaggctgag gcaggagaat ggcgtgaacc cgggaagcgg 1320 agcttgcagt gagccgagat tgcgccactg cagtccgcag tccggcctgg gcgacagagc 1380 gagactccgt ctcaaaaaaa aaaaaaaaaa aaaaaaaaac cctaagccaa agaaaaatct 1440 tctcccactg atttatacag gctgcaggga gttcaacagg agtcatattt taatatcatt 1500 ttcattctca tcataatggc aaagctgtta ttaaatatct aattagactt attactaacc 1560 taagtttact acaaactcta ggagtaacat aatcttatta t 1601 <210> 86 <211> 1327 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472365CB1 <400> 86 acttacctaa agtgctctgt atacagtatg tttcaaagtg atagaatttc ctgcaaaaaa 60 tcatgtgcac aaatgtatgt ttcttatatt aaatttttgt ctccgaactg cagaagcctg 120 tgtggttaca tgcagattgg gtgagcatac atttctgtag actgtggact tatgcattca 180 caagcaggat gttccttccc aatgacaccc agtttcaccc ctcctccttc ctgttgctgg 240 ggatcccagg actagaaaca cttcacatct ggatcggctt tcccttctgt gctgtgtaca 300 tgatcgcact catagggaac ttcactattc tacttgtgat caagactgac agcagcctac 360 accagcccat gttctacttc ctggccatgt tggccaccac tgatgtgggt ctctcaacag 420 ctaccatccc taagatgctt ggaatcttct ggatcaacct cagagggatc atctttgaag 480 cctgcctcac ccagatgttt tttatccaca acttcacact tatggagtca gcagtccttg 540 tggcaatggc ttatgacagc tatgtggcca tctgcaatcc actccaatat agcgccatcc 600 tcaccaacaa ggttgtttct gtgattggtc ttggtgtgtt tgtgagggct ttaattttcg 660 tcattccctc tatacttctt atattgcggt tgcccttctg tgggaatcat gtaattcccc 720 acacctactg tgagcacatg ggtcttgctc atctatcttg tgccagcatc aaaatcaata 780 ttatttatgg tttatgtgcc atttgtaatc tggtgtttga catcacagtc attgccctct 840 cttatgtgca tattctttgt gctgttttcc gtcttcctac tcatgagccc cgactcaagt 900

76/82 ccctcagcac atgtggttca catgtgtgtg taatccttgc cttctataca ccagccctct 960 tttcctttat gactcattgc tttggccgaa atgtgccccg ctatatccat atactcctag 1020 ccaatctcta tgttgtggtg ccaccaatgc tcaatcctgt catatatgga gtcagaacca 1080 agcagatcta taaatgtgta aagaaaatat tattgcagga acaaggaatg gaaaaggaag 1140 agtacctaat acatacgagg ttctgaatgc aattttatga aatttcagtg agagaaatgt 1200 cttgtcataa aaattatatt ctaatatgtg gctttattgg ctctcttctg tatttaaata 1260 cattgaattt ctccatctgc ttttcatacc acattttgag atctgttgct gcattttttt 1320 ttttttt 1327 <210> 87 <211> 1163 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475230CB1 <400> 87 ggtctcttat gctatattat ggagttaaag aatggatttt ctgtttcccc aagaaataga 60 caccatgctg tgagagaagt tatggtttct cactggaagc aagaaaactc atgcaagaaa 120 tgtgtctgta gggaatggca ccaatatgct tcataccaac aatacacagt ttcacccttc 180 caccttcctc gtagtggggg tcccagggct ggaagatgtg catgtatgga ttggcttccc 240 cttctttgcg gtgtatctaa cagcccttct agggaacatc attatcctgt ttgtgataca 300 gactgaacag agcctccacc aacccatgtt ttacttccta gccatgttgg ccggcactga 360 tctgggcttg tctacagcaa ccatccccaa gatgctggga attttctggt ttaatcttgg 420 agagattgca tttggtgcct gcatcacaca gatgtatacc attcatatat gcactggcct 480 ggagtctgtg gtactgacag tcacgggcat agatcgctat attgccatct gcaaccccct 540 gagatatagc atgatcctta ccaacaaggt aatagccatt ctgggcatag tcatcattgt 600 caggactttg gtatttgtga ctccattcac atttctcacc ctgagattgc ctttctgtgg 660 tgtccggatt atccctcata cctattgtga acacatgggc ttggcaaagt tagcttgtgc 720 cagtattaat gttatatatg gattgattgc cttctcagtg ggatacattg acatttctgt 780 gattggattt tcctatgtcc agatcctccg agctgtcttc catctcccag cctgggatgc 840 ccggcttaag gcactcagca catgtggctc tcacgtctgt gttatgttgg ctttctacct 900 gccagccctc ttttccttca tgacacaccg ctttggccac aacatccctc attacatcca 960 cattcttctg gccaatctgt atgtggtttt tccccctgct cttaactctg ttatctatgg 1020 ggtcaaaaca aaacagatac gagagcaggt acttaggata ctcaacccta aaagcttttg 1080 gcattttgac cccaagagga tcttccacaa caattcagtt agacaataat gagatcataa 1140 caaaataaac actggaaaca ttt 1163 <210> 88 <211> 1121 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475229CB1 <400> 88 ttaccaggaa tcaggataaa gtgagaagtg gagcaagaat cactaatgga aagtcaataa 60 ttgtcactga tacacacaac agctttttgt gacagaaaga atgcctatag ctaacgacac 120 ccagttccat acttcttcat tcctactgct gggtatccca gggctagaag atgtgcacat 180 ctggattgga ttcccttttt tctctgtgta tcttattgca ctcctgggaa atgctgctat 240 cttctttgtg atccaaactg agcagagtct ccatgagccc atgtactact gcctggccat 300 gttggattcc attgacctga gcttgtctac ggccaccatt cccaaaatgc tgggcatctt 360

77/82 ctggttcaat atcaaggaaa tatcttttgg aggctacctt tctcagatgt tcttcatcca 420 tttcttcact gtcatggaga gcatcgtatt ggtggccatg gcctttgacc gctacattgc 480 catttgcaaa cctctttggt acaccatgat cctcaccagc aaaatcatca gcctcattgc 540 aggcattgct gtcctgagga gcttgtacat ggtcattcca ctggtgtttc tcctcttaag 600 gttgcccttc tgtggacatc gtatcatccc tcatacttac tgtgagcaca tgggcattgc 660 ccgtctggcc tgtgccagca tcaaagtcaa cattatgttt ggtcttggca gtatttctct 720 cttgttattg gatgtgctcc ttattattct ctcccatatc aggatcctct atgctgtctt 780 ctgcctgccc tcctgggaag ctcgactcaa agctctcaac acctgtggct ctcacattgg 840 tgttatctta gccttttcta caccagcatt tttctctttc tttacacact gctttggcca 900 tgatattccc caatatatcc acattttctt ggctaatcta tatgtggttg ttcctcccac 960 cctcaatcct gtaatctatg gggtcagaac caaacatatt agggagacag tgctgaggat 1020 tttcttcaag acagatcact aaccagttgg agtttggagg gtctctctta gcattcatga 1080 tgaagcagcc actagggagg agagaagaga caccaaggaa t 1121 <210> 89 <211> 958 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477367CB1 <400> 89 taggtaactg aatattggat acatggctca cacaaatgaa tcgatggtgt ctgagtttgt 60 acttttggga ctctctaatt cctggggact tcaacttttc tttttcgcca tcttctctat 120 agtctatgtg acatcagtgc taggcaatgt cttaattatt gtcattattt cttttgactc 180 ccatttgaac tctcctatgt acttcttgct cagtaatctt tctttcattg atatctgtca 240 gtctaacttt gccaccccca agatgcttgt agactttttt attgagcgca agactatctc 300 ctttgagggt tgcatggccc agatattcgt tcttcacagt tttgttggga gtgagatgat 360 gttgcttgta gctatggcat atgacagatt tatagccata tgtaagcctc tgcactacag 420 tacaattatg aaccggaggc tctgtgtaat ttttgtgtct atttcctggg cggtgggcgt 480 tcttcattct gtgagccact tggcttttac agtggacctg ccattctgtg gtcccaatga 540 ggtggatagc ttcttttgtg accttccctt ggtgatagag ctggcttgca tggatacata 600 tgaaatggaa attatgaccc taacgaacag tggcctgata tcattgagct gtttcctggc 660 tttaattatt tcctacacca tcattttgat cggtgtccga tgcaggtcct ccagtgggtc 720 atctaaggct ctttctacat taactgccca catcacagtg gtcattcttt tcttcgggcc 780 ttgcatttat ttctatatat ggccttttag cagacttcct gtggacaaat ttctttctgt 840 gttctacact gtttgtactc ccttgttgaa ccccatcatc tactctttga ggaatgaaga 900 tgttaaagca gccatgtgga agctgagaaa ccatcatgtg aactcctgga aaaactag 958 <210> 90 <211> 1101 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477936CB1 <400> 90 caaatgtgga catcaacatg aacatttctt tccaatatgc atcatttccc taccccttat 60 tctcacttat tttgatcatt atgggataaa gttgacgata tggaaagagc aaaccattca 120 gtggtatcgg aatttatttt gttgggactt tccaaatctc aaaatcttca gattttattc 180 ttcttgggat tctctgtggt cttcgtgggg attgtgttag gaaacctgct catcttggtg 240 actgtgacct ttgattcgct ccttcacaca ccaatgtatt ttctgcttag caacctctcc 300

78/82 tgcattgata tgatcctggc ttcttttgct acccctaaga tgattgtaga tttcctccga 360 gaacgtaaga ccatctcatg gtggggatgt tattcccaga tgttctttat gcacctcctg 420 ggtgggagtg agatgatgtt gcttgtagcc atggcaatag acaggtatgt tgccatatgc 480 aaacccctcc attacatgac catcatgagc ccacgggtgc tcactgggct actgttatcc 540 tcctatgcag ttggatttgt gcactcatct agtcaaatgg ctttcatgtt gactttgccc 600 ttctgtggtc ccaatgttat agacagcttt ttctgtgacc ttccccttgt gattaaactt 660 gcctgcaagg acacctacat cctacagctc ctggtcattg ctgacagtgg gctcctgtca 720 ctggtctgct tcctcctctt gcttgtctcc tatggagtca taatattctc agttaggtac 780 cgtgctgcta gtcgatcctc taaggctttc tccactctct cagctcacat cacagttgtg 840 actctgttct ttgctccgtg tgtctttatc tacgtctggc ccttcagcag atactcggta 900 gataaaattc tttctgtgtt ttacacaatt ttcacacctc tcttaaatcc tattatttat 960 acattaagaa atcaagaggt aaaagcagcc attaaaaaaa gactctgcat ataaatttaa 1020 agcatacttt ttagatgaga cttttgaaga gacactctct tgtttgtttg aacatttaag 1080 aaactcgttt ttaatgtatc a 1101 <210> 91 <211> 1192 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475214CB1 <400> 91 agcatttttt cctcttttga tctactgtga ttttccgttg gttctgcgtt agaaataaaa 60 taaatacagt cctttttatg ttttcttttt ttcaggtaat ttaattgtct cctaagaact 120 tgacccattc catggaaaaa ataaacaacg taactgaatt cattttctgg ggtctttctc 180 agagcccaga gattgagaaa gtttgttttg tggtgttttc tttcttctac ataatcattc 240 ttctgggaaa tctcctcatc atgctgacag tttgcctgag caacctgttt aagtcaccca 300 tgtatttctt tctcagcttc ttgtcttttg tggacatttg ttactcttca gtcacagctc 360 ccaagatgat tgttgacctg ttagcaaagg acaaaaccat ctcctatgtg gggtgcatgt 420 tgcaactgct tggagtacat ttctttggtt gcactgagat cttcatcctt actgtaatgg 480 cctatgatcg ttatgtggct atctgtaaac ccctacatta tatgaccatc atgaaccggg 540 agacatgcaa taaaatgtta ttagggacgt gggtaggtgg gttcttacac tccattatcc 600 aagtggctct ggtagtccaa ctaccctttt gtggacccaa tgagatagat cactactttt 660 gtgatgttca ccctgtgttg aaacttgcct gcacagaaac atacattgtt ggtgttgttg 720 tgacagccaa cagtggtacc attgctctgg ggagttttgt tatcttgcta atctcctaca 780 gcatcatcct agtttccctg agaaagcagt cagcagaagg caggcgcaaa gccctctcca 840 cctgtggctc ccacattgcc atggtcgtta tctttttcgg cccctgtact tttatgtaca 900 tgcgccctga tacgaccttt tcagaggata agatggtggc tgtattttac accattatca 960 ctcccatgtt aaatcctctg atttatacac tgagaaatgc agaagtaaag aatgcaatga 1020 agaaactgtg gggcagaaat gttttcttgg aggctaaagg gaaatagttg gacttaataa 1080 tttaagctag atgaccttaa aatttctcag ccttggttaa ctcatctgtg caatcaagac 1140 aataacaacc tcatgggatt tggagtggaa aaatgagaca ataacacatt ca 1192 <210> 92 <211> 1341 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 55036157CB1 <400> 92

79/82 tttgcaagta ttagcttaaa aatactgact tgaagatcat tgaaatattc aaaatacaaa 60 atagacactc aaagttttta attattataa acataggtaa aatattatca tatagaatac 120 cagttactag aaaataaaga ttactacatg ctaaattggg ataaattatg attcagttaa 180 ttgaaccgga gttaaatgat catatataaa caagggatca ctttcctaca aaaggagaat 240 aacaatacaa ttcacctaaa taccatgttt tttctctccc ctgcagaaac tcatcaaaga 300 atggcagcag aaaaccattc ttttgtgact aagtttattc tggttgggct aacagagaag 360 tcagagctac agctgcccct cttcctcgtc ttcctgggaa tctatgtagt cacagtgctg 420 gggaacctgg gcatgatcac actgattggg ctcagttctc acctgcacac acctatgtac 480 tgtttcctca gcagtctgtc cttcattgac ttctgccatt ccactgtcat tacccctaag 540 atgctggtga actttgtgac agagaagaac atcatctcct accctgaatg catgactcag 600 ctctacttct tcctcgtttt tgctattgca gagtgtcaca tgttggctgc aatggcatat 660 gacggctacg tggccatctg tagccccttg ctgtacagca tcatcatatc caataaggct 720 tgcttttctc tgattttagt ggtgtatgta ataggcctga tttgtgcgtc agctcatata 780 ggctgtatgt ttagggttca attctgcaaa tttgatgtga tcaaccatta tttctgtgat 840 cttatttcta tcttgaagct ctcctgttct agtacttaca ttaatgagtt actgatttta 900 atctttagtg gaattaacat CCttgtCCCC agcctgacca tcctcagctc ttacatcttc 960 atcattgcca gcatcctccg cattcgctac actgagggca ggtccaaagc cttcagcact 1020 tgcagctccc acatctcggc tgtttctgtt ttctttgggt ctgcagcatt catgtacctg 1080 cagccatcat ctgtcagctc catggaccag gggaaagtgt cctctgtgtt ttatactatt 1140 gttgtgccca tgctgaaccc cctgatctac agcctgagga ataaagatgt ccacgttgcc 1200 ctgaagaaaa cgctagggaa aagaacattc ttatgaacag aagtacaatg aaaaagattg 1260 cattagatct aagtttttgg ctatgatatt gtatgaaatg atgtctttca ctttagtgca 1320 tctgtcaaca ttctttctaa t 1341 <210> 93 <211> 1114 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7475226CB1 <400> 93 taatgtagca ggaatgcaga aatcatgact ttggtttctt ttttctcttt cctctccaag 60 ccattgataa tgctccttag caattcaagc tggaggctat cccagccttc ttttctcctg 120 gtagggattc caggtttaga ggaaagccag cactggattg cactgcccct gggcatcctt 180 tacctccttg ctttagtggg caatgttacc attctcttca tcatctggat ggacccatcc 240 ttgcaccaat ctatgtacct cttcctgtcc atgctagctg ccatcgacct ggttctggcc 300 tcctccactg cacccaaagc ccttgcagtg ctcctggttc atgcccacga gattgggtac 360 atcgtctgcc tgatccagat gttcttcatc catgcattct cctccatgga gtcaggggta 420 cttgtggcca tggctctgga tcgctatgta gccatttgtc accccttgca ccattccaca 480 atcctgcatc caggggtcat agggcgcatc ggaatggtgg tgctggtgag gggattacta 540 ctccttatcc ccttccccat tttgttggga acacttatct tctgccaagc caccatcata 600 ggccatgcct attgtgaaca tatggctgtt gtgaaacttg cctgctcaga aaccacagtc 660 aatcgagctt atgggctgac tatggccttg cttgtgattg ggctggatgt tctggccatt 720 ggtgtttcct atgcccacat cctccaggca gtgctgaagg taccagggag tgaggcccga 780 cttaaggcgt ttagcacatg tggctctcat atttgtgtca tcctggtctt ctatgtccct 840 ggaattttct ccttcctcac tcaccgcttt ggtcatcatg taccccatca tgtccatgtt 900 cttctggcca cacggtatct cctcatgcca cctgcgctca atcctcttgt ctatggagtg 960 aagactcagc agatccgcca gcgagtgctc agagtgttta cacaaaagga ttgatctgaa 1020 catattctca ttgtttcctt cggaggcttc ttctgcggac cacagccagg agcctgtgac 1080 tggtgtagab tacatgaata cagaccactc tgca 1114 <210> 94 <211> 960

80/82 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477353CB1 <400> 94 gaaggatcgt atgaatgccc catggaaaat tacaatcaaa cgtcaactga tttcatctta 60 ttggggctgt tcccaccatc aaaaattggc cttttcctct tcattctctt tgttctcatt 120 ttcctaatgg ctctaattgg aaacctatcc atgattcttc tcatcttctt ggacacccat 180 ctccacacac ccatgtattt cctgcttagt cagctctccc tcattgacct aaattacatc 240 tctacgattg ttcctaagat ggcttctgat tttctgtatg gaaacaagtc tatctccttc 300 attgggtgtg ggattcagag tttcttcttc atgacttttg caggtgcaga agcgctgctc 360 ctgacatcaa tggcctatga tcgttatgtg gccatttgct ttcctctcca ctatcccatc 420 cgtatgagca aaagaatgta tgtgctgatg ataacaggat cttggatgat aggctccatc 480 aactcttgtg ctcacacagt atatgcattc cgtatcccat attgcaagtc cagagccatc 540 aatcattttt tctgtgatgt tccagctatg ttgacattag cctgtacaga cacctgggtc 600 tatgagtaca cagtgttttt gagcagcacc atctttcttg tgtttccctt cactggcatt 660 gcgtgttcct atggctgggt tctccttgct gtctaccgca tgcactctgc agaagggagg 720 aaaaaggcct attcgacctg cagcacccac ctcactgtag taactttcta ctatgcaccc 780 tttgcttata cctatctatg tccaagatcc ctgcgatctc tgacagagga caaggttctg 840 gctgttttct acaccatcct caccccaatg ctcaacccca tcatctacag cctgagaaac 900 aaggaggtga tgggggccct gacacgagtg attcagaata tcttctcggt gaaaatgtag 960 <210> 95 <211> 1269 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 55036208CB1 <400> 95 ctccataatt ttataagtga tgctgtattg aaagaacata tttcgttcag attctatact 60 tcttgacctt ttagtttcct acttctattc atgctgtatt gatcacccaa ctacagaatt 120 taccaaaatc acacgattta taagacactg ggtaaatgtt taccaaatta ataagatggt 180 tttgtggtac taggtaaaaa gcacattcat catggcatgg gagaatcaga ccttcaactc 240 cgacttcatc ctccttggaa tcttcaatca cagcccacca cacacgttcc tcttctttct 300 ggtcctgggc atctttttag tggccttcat gggaaactct gtcatggttc tcctcatcta 360 cctggacacc cagctccaca cccccatgta cttcctcctc agccaactgt ccctcatgga 420 cctcatgctc atctgcacca ccgtacccaa gatggccttc aactacttgt ctggcagcaa 480 gtccatttct atggctggtt gtgtcacaca aattttcttc tatatatcac tgtctggctc 540 tgaatgtttt cttttggctg ttatggctta tgaccgctat attgctattt gccaccctct 600 aagatatacc aatctcatga atcctaaaat ttgtggactt atggctacct tctcctggat 660 cctgggctct acagatggaa tcattgatgc tgtagccaca ttttccttct ccttttgtgg 720 gtctcgggaa atagcccact tcttctgtga attcccttcc ctactaatcc tctcatgcaa 780 tgacacatca atatttgaag aggttatttt catctgctgt atagtaatgc ttgttttccc 840 tgttgcaatc atcattgctt cctatgctgg agttattctg gctgtcattc acatgggatc 900 tggagagggt cgtcgcaaaa ctttcacgac ctgttcctct cacctcatgg tggtgggaat 960 gtactatgga gcagctttgt tcatgtacat acggcccaca tctgatcact ccccaacgca 1020 ggacaagatg gtgtctgtat tctacaccat cctcactccc atgctgaatc ccctcatcta 1080 cagcctccgc aacaaggagg tgactagagc attcatgaag atcttaggaa agggcaagtc 1140 tgagagtgag ttacctcata aactttatgt tttgctgttt gctaaattct tctttctaat 1200 atccatcttt ttctatgatg tcaaaatact agcattgatt atgtacattg cctaacatat 1260

81/82 ttatgggca <210> 96 <211> 2197 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 55019501CB1 <400> 96 tcatccccac tcccactgta acagatgcag gaattgaagt ttagcaagac tggcttggtg 60 aaggtcagag taggaggggg tgtgagatgc gcatactgct gtcttgacgc cccatccagg 120 gggctaaaga gggtcaggca ggaggaagcc tgagagaaag cagacaccgt gagaaaatga 180 ctcatttcat cccgtgccag tcactcaccc aaactctctt cactcatcat cgcagctccc 240 agaagatgcc ttagcatgag aggtgacaac cacagctgct tctgggacac cccaaaggac 300 tttatcctcc tgggcatttc cgacaggcca tggctggagc tcccagtctt tgcagtcctc 360 ctggtgttct acattctggc tatgctgggg aacatctcta tcatcctggt atcccagctg 420 gatcctcagc tccacagccc catgtacata ttcctcagcc acttgtcctt cctggatctc 480 tgctacacca ccaccactgt ccctcagatg ctgttcaaca tggggagctc ccagaagacc 540 atcagttacg gtggctgcac ggtgcaatac gccattttcc actggctggg ctgcaccgag 600 tgcgttgtct tggcggccat ggctctggac cgctacgtgg ccatctgtga gccactccgc 660 tatgctatca tcatgcaccg cccactctgc cagcagctcg tggctatggc ctggctcagc 720 ggcttcggca actcccttgt tcaggtcatc ctgacagtgc aattgccttt ctgtggccgg 780 caggtgctga acaacttctt ctgcgaggtg ccagccatga tcaagctgtc ctgtgctgat 840 actacggcga atgatgccac cctggctgtg ctggtggcct tctttgtgct ggtccctctg 900 gccctcatcc tcctctccta cggcttcatt gctcgggcag tgatgaggat ccagtcctcc 960 aggggacggc acaaggcctt cgggacttgc-tcttcccacc tgttggtggt ctccctcttc 1020 tacctgcccg ccatttacat gtacctgcag ccgccatcca gttactcaca ggagcagggc 1080 aagttcatct ccctcttcta ttctataatc acccccaccc ttaacccttt catctacacc 1140 ttgaggaata aggacgtgaa gggagctctc cgaagactcc tggcaaggac cggaaggctg 1200 tgtggaaggt gaaaaatgta atcccggaag acctacacct ctggcagaac tccgcagcaa 1260 ggactctggg gggtgggtag ggttgtagct caggggtggt acagcacttc cttggcatgt 1320 gggaaggact gagttcaacc cctagggctg taggaaattc ttggaacagt aggttctttg 1380 agagatttga aagaaaacaa aaaacatcca aaagcatttc accttccgtg tggtgatgag 1440 gctcaaagtc ccgcatctga gcgttaagtt ccattctcag agcacagttc taagaagcca 1500 tatatcttca agaagcgctt aaatgacttg atgctcacaa gtactcgtta cgctgtttaa 1560 cttctaattc cctggctctt cctgttttct tcctcttcct gtatttcctc tctcgtttcc 1620 catgtatctt tttaaacccc tttcttccat tacagtcact ccagatgtgt acaaaatgaa 1680 cttgacacag agtcttacct cgtttataat aaacgtctgt tcttcgtgga tcaaaatcaa 1740 tacattttgt atttattttc caaaatgtca taagaatgac atgactgcat ccttctattc 1800 ataaccataa acaattttct tttccctctg gtttaaactg tccttacaga aagttttccc 1860 accgttgagt gggaactttc tgctagaagg ctagatgact ccctgttagg gtacaggggt 1920 acaaacaagt acatctcccc tctcagcttc tagatggacg tattccagtg gagaatgcag 1980 taacaaatac aatacagtta aaatgataaa acctaacata agagctgaag atgtttaact 2040 cagtctagag ctcctgccta gcttggtcct ggcttcactg cccagaacca cacacaagaa 2100 atgttgacag tgattcattc aagctaacat ttgtggaagg tttctgtgat gctggtgata 2160 cactgagtgt ttgtaagtaa cgatctgtgt tatactg 2197

82/82

Claims (157)

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:1-48, 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:1-36 and SEQ ID NO:38-48, c) a naturally occurring polypeptide comprising an amino acid sequence at least 91%
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:37, d) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, and e) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-48.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-48.

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:49-96.

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:1-48.

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:49-96, 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:49-84 and SEQ ID NO:86-96, c) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 91% identical to the polynucleotide sequence of SEQ ID NO:85, d) a polynucleotide complementary to a polynucleotide of a), e) a polynucleotide complementary to a polynucleotide of b), and f) an RNA equivalent of a)-e).

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:1-48.

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 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 F(ab')2 fragment, or e) a humanized antibody.

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

33. A method of diagnosing a condition or disease associated with the expression of 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:1-48, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from said animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which binds specifically to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-48.

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:1-48, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-48.

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:1-48 in a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-48 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-48 from a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID

NO:1-48.

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:1-48, ii) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-48, 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:1-48.
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 polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:23.

85. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:24.

86. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:25.

87. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:26.

88. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:27.

89. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:28.

90. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:29.

91. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:30.

92. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:31.

93. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:32.

94. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:33.

95. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:34.

96. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:35.

97. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:36.

98. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:37.

99. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:38.

100. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:39.

101. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:40.

102. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:41.

103. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:42.

104. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:43.

105. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:44.

106. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:45.

107. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:46.

108. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:47.

109. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:48.

110. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:49.

111. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:50.

112. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:51.

113. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:52.

114. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:53.

115. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:54.

116. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:55.

117. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:56.

118. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:57.

119. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:58.

120. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:59.

121. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:60.

122. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:61.

123. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:62.

124. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:63.

1-25. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:64.

126. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:65.

127. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:66.

128. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:67.

129. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:68.

130. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:69.

131. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:70.

132. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:71.

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

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

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

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

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

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

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

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

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

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

143. A polynucleotide of claim 12, comprising the polynucleotide Sequence of SEQ ID

NO:82.

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

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

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

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

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

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

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

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

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

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

154. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:93.

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

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

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