CA2425963A1 - Human kinases - Google Patents

Abstract

The invention provides human human kinases (PKIN) and polynucleotides which identify and encode PKIN. The invention also provides expression vectors, ho st cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of PKIN.

Description

HUMAN KINASES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human kinases and to the use of these sequences in the diagnosis, treatment, and prevention of cancer, immune disorders, disorders affecting growth and development, cardiovascular diseases, and lipid disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of human kinases.
1o BACKGROUND OF THE INVENTION
Kinases comprise the largest known enzyme superfamily and vary widely in their target molecules. Kinases catalyze the transfer of high energy phosphate groups from a phosphate donor to a phosphate acceptor. Nucleotides usually serve as the phosphate donor in these reactions, with most kinases utilizing adenosine triphosphate (ATP). The phosphate acceptor can be any of a variety of molecules, including nucleosides, nucleotides, lipids, carbohydrates, and proteins. Proteins are phosphorylated on hydroxyamino acids. Addition of a phosphate group alters the local charge on the acceptor molecule, causing internal conformational changes and potentially influencing intermolecular contacts. Reversible protein phosphorylation is the primary method for regulating protein activity in eukaryotic cells. In general, proteins are activated by phosphorylation in response to extracellular signals such as hormones, neurotransmitters, and growth and differentiation factors. The activated proteins initiate the cell's intracellular response by way of intracellular signaling pathways and second messenger molecules such as cyclic nucleotides, calcium-calinodulin, inositol, and various mitogens, that regulate protein phosphorylation.
Kinases are involved in all aspects of a cell's function, from basic metabolic processes, such as glycolysis, to cell-cycle regulation, differentiation, and communication with the extracellular environment through signal transduction cascades. Inappropriate phosphorylation of proteins in cells has been linked to changes in cell cycle progression and cell differentiation.
Changes in the cell cycle have been linked to induction of apoptosis or cancer. Changes in cell differentiation have been linked to diseases and disorders of the reproductive system, immune system, and skeletal muscle.
There are two classes of protein kinases. One class, protein tyrosine kinases (PTKs), phosphorylates tyrosine residues, and the other class, protein serine/threonine kinases (STKs), phosphorylates serine and threonine residues. Some PTKs and STKs possess structural characteristics of both families and have dual specificity for both tyrosine and serine/threonine residues. Alinost all kinases contain a conserved 250-300 amino acid catalytic domain containing specific residues and sequence motifs characteristic of the kinase family. The protein kinase catalytic domain can be further divided into 11 subdomains. N-terminal subdomains I-IV
fold into a two-lobed structure which binds and orients the ATP donor molecule, and subdomain V
spans the two lobes. C-terminal subdomains VI-XI bind the protein substrate and transfer the gamma phosphate from ATP to the hydroxyl group of a tyrosine, serine, or threonine residue. Each of the 11 subdomains contains specific catalytic residues or amino acid motifs characteristic of that subdomain. For example, subdomain I contains an 8-amino acid glycine-rich ATP binding consensus motif, subdomain 1I
contains a critical lysine residue required for maximal catalytic activity, and subdomains VI through IX
comprise the highly conserved catalytic core. PTKs and STKs also contain distinct sequence motifs in subdomains VI and VIII which may confer hydroxyamino acid specificity.
In addition, kinases may also be classified by additional amino acid sequences, generally between 5 and 100 residues, which either flank or occur within the kinase domain. These additional amino acid sequences regulate kinase activity and determine substrate specificity. (Reviewed in Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Book, Vol I p.p. 17-20 Academic Press, San Diego, CA.). In particular, two protein kinase signature sequences have been identified in the kinase domain, the first containing an active site lysine residue involved in ATP binding, and the second containing an aspartate residue important for catalytic activity. If a protein analyzed includes the two protein kinase signatures, the probability of that protein being a protein kinase is close to 100%
(PROSITE: PDOC00100, November 1995).
Protein Tyrosine Kinases Protein tyrosine kinases (PTKs) may be classified as either transmembrane, receptor PTKs or nontransmembrane, nonreceptor PTK proteins. Transmembrane tyrosine kinases function as receptors for most growth factors. Growth factors bind to the receptor tyrosine ~kinase (RTK), which causes the receptor to phosphorylate itself (autophosphorylation) and specific intracellular second messenger proteins. Growth factors (GF) that associate with receptor PTKs include epidermal GF, platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascular endothelial GF, and macrophage colony stimulating factor.
Nontransmembrane, nonreceptor PTKs lack transmembrane regions and, instead, form signaling complexes with the cytosolic domains of plasma membrane receptors.
Receptors that function through non-receptor PTKs include those for cytokines and hormones (growth hormone and prolactin), and antigen-specific receptors on T and B lymphocytes.
Many PTKs were first identified as oncogene products in cancer cells in which PTK

activation was no longer subject to normal cellular controls. In fact, about one third of the known oncogenes encode PTKs. Furthermore, cellular transformation (oncogenesis) is often accompanied by increased tyrosine phosphorylation activity (Charbonneau, H. and Tonks, N.
K. (1992) Annu. Rev.
Cell Biol. 8:463-93). Regulation of PTK activity may therefore be an important strategy in controlling some types of cancer.
Protein Serine/Threonine Kinases Protein serine/threonine kinases (STKs) are nontransmembrane proteins. A
subclass of STKs are known as ERKs (extracellular signal regulated kinases) or MAPS
(mitogen-activated protein kinases) and are activated after cell stimulation by a variety of hormones and growth factors.
i0 Cell stimulation induces a signaling cascade leading to phosphorylation of MEK (MAP/BRK kinase) which, in turn, activates ERK via serine and threonine phosphorylation. A
varied number of proteins represent the downstream effectors for the active ERK and implicate it in the control of cell proliferation and differentiation, as well as regulation of the cytoskeleton.
Activation of ERK is normally transient, and cells possess dual specificity phosphatases that are responsible for its down-regulation. Also, numerous studies have shown that elevated ERK activity is associated with some cancers. Other STKs include the second messenger dependent protein kinases such as the cyclic-AMP dependent protein kinases (PKA), calcium-calmodulin (CaM) dependent protein kinases, and the mitogen-activated protein kinases (MAP); the cyclin-dependent protein kinases; checkpoint and cell cycle kinases; proliferation-related kinases; 5'-AMP-activated protein kinases; and kinases involved in apoptosis.
The second messenger dependent protein kinases primarily mediate the effects of second messengers such as cyclic AMP (cAMP), cyclic GMP, inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cyclic ADPribose, arachidonic acid, diacylglycerol and calcium-calmodulin. The PKAs are involved in mediating hormone-induced cellular responses and are activated by cAMP
produced within the cell in response to hormone stimulation. cAMP is an intracellular mediator of hormone action in all animal cells that have been studied. Hormone-induced cellular responses include thyroid hormone secretion, cortisol secretion, progesterone secretion, glycogen breakdown, bone resorption, and regulation of heart rate and force of heart muscle contraction. PKA is found in all animal cells and is thought to account for the effects of cAMP in most of these cells. Altered PKA
expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, K.J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York, NY, pp. 416-431, 1887).
The casein kinase I (CKI) gene family is another subfamily of serine/threonine protein kinases. This continuously expanding group of kinases have been implicated in the regulation of numerous cytoplasmic and nuclear processes, including cell metabolism, and DNA
replication and repair. CKI enzymes are present in the membranes, nucleus, cytoplasm and cytoskeleton of eukaryotic cells, and on the mitotic spindles of mammalian cells (Fish, K.J.
et al., (1995) J. Biol. Chem.
270:14875-14883.
The CKI family members all have a short amino-terminal domain of 9-76 amino acids, a highly conserved kinase domain of 284 amino acids, and a variable carboxyl-terminal domain that ranges from 24 to over 200 amino acids in length (Cegielska, A. et al., (1998) J.
Biol. Chem. 273:1357-1364.) The CKI family is comprised of highly related proteins, as seen by the identification of isoforms of casein kinase I from a variety of sources. There are at least five mammalian isoforms, a, ~3, y, 8, and s. Fish et al., identified CKI-epsilon from a human placenta cDNA library. It is a basic protein of 416 amino acids and is closest to CKI-delta. Through recombinant expression, it was determined to phosphorylate known CKI substrates and was inhibited by the CKI-specific inhibitor CKI-7. The human gene for CKI-epsilon was able to rescue yeast with a slow-growth phenotype caused by deletion of the yeast CKI locus, HRR250 (Fish et al, supra.) The mammalian circadian mutation tau was found to be a semidominant autosomal allele of CKI-epsilon that markedly shortens period length of circadian rhythms in Syrian hamsters. The tau locus is encoded by casein kinase I-epsilon, which is also a homolog of the Drosophila circadian gene double-time. Studies of both the wildtype and tau mutant CKI-epsilon enzyme indicated that the mutant enzyme has a noticeable reduction in the maximum velocity and autophosphorylation state.
Further, in vitro, CKI-epsilon is able to interact with mammalian PERIOD
proteins, while the mutant enzyme is deficient in its ability to phosphorylate PERIOD. Lowrey et al., have proposed that CKI-epsilon plays a major role in delaying the negative feedback signal within the transcription-translation-based autoregulatory loop that composes the core of the circadian mechanism.
Therefore the CKI-epsilon enzyme is an ideal target for pharmaceutical compounds influencing circadian rhythms, jet-lag and sleep, in addition to other physiologic and metabolic processes under circadian regulation (Lowrey, P.L. et al., (2000) Science 288:483-491.) Calcium-Calmodulin Dependent Protein Kinases Calcium-calmodulin dependent (CaM) kinases are involved in regulation of smooth muscle contraction, glycogen breakdown (phosphorylase kinase), and neurotransmission (CaM kinase I and CaM kinase II). CaM dependent protein kinases are activated by calmodulin, an intracellular calcium receptor, in response to the concentration of free calcium in the cell. Many CaM kinases are also activated by phosphorylation. Some CaM kinases are also activated by autophosphorylation or by other regulatory kinases. CaM kinase I phosphorylates a variety of substrates including the neurotransmitter-related proteins synapsin I and II, the gene transcription regulator, CREB, and the cystic fibrosis conductance regulator protein, CFTR (Haribabu, B. et al.
(1995) EMBO Journal 14:3679-3686). CaM kinase II also phosphorylates synapsin at different sites and controls the synthesis of catecholamines in the brain through phosphorylation and activation of tyrosine hydroxylase. CaM kinase II controls the synthesis of catecholamines and seratonin, through phosphorylation/activation of tyrosine hydroxylase and tryptophan hydroxylase, respectively (Fujisawa, H. (1990) BioEssays 12:27-29). The mRNA encoding a calinodulin-binding protein kinase-like protein was found to be enriched in mammalian forebrain. This protein is associated with vesicles in both axons and dendrites and accumulates largely postnatally. The amino acid sequence of this protein is similar to CaM-dependent STKs, and the protein binds calmodulin in the presence of calcium (Godbout, M. et al. (1994) J. Neurosci. 14:1-13).
Mito~en-Activated Protein Kinases The mitogen-activated protein kinases (MAP) which mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades are another STK family that regulates intracellular signaling pathways. Several subgroups have been identified, and each manifests different substrate specificities and responds to distinct extracellular stimuli (Egan, S.E. and Weinberg, R.A.
(1993) Nature 365:781-783). MAP kinase signaling pathways are present in mammalian cells as well as in yeast. The extracellular stimuli which activate MAP kinase pathways include epidermal growth factor (EGF), ultraviolet light, hyperosmolar medium, heat shock, endotoxic lipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).
Altered MAP kinase expression is implicated in a variety of disease conditions including cancer, inflammation, immune disorders, and disorders affecting growth and development.
Cyclin-Dependent Protein Kinases The cyclin-dependent protein kinases (CDKs) are STKs that control the progression of cells through the cell cycle. The entry and exit of a cell from mitosis are regulated by the synthesis and destruction of a family of activating proteins called cyclins. Cyclins are small regulatory proteins that bind to and activate CDKs, which then phosphorylate and activate selected proteins involved in the mitotic process. CDKs are unique in that they require multiple inputs to become activated. In addition to cyclin binding, CDK activation requires the phosphorylation of a specific threonine residue and the dephosphorylation of a specific tyrosine residue on the CDK.
Another family of STKs associated with the cell cycle are the NIMA (never in mitosis)-related kinases (Neks). Both CDKs and Neks are involved in duplication, maturation, and separation of the microtubule organizing center, the centrosome, in animal cells (Fry, A.M., et al. (1998) EMBO
J. 17:470-481). The NIM-related kinases also include NIK1 histidine kinases, which function in signal transmission (Yamada-Okabe, T. et al. (1999) J. Bacteriol. 181:7243-7247).
Checkpoint and Cell Cycle Kinases In the process of cell division, the order and timing of cell cycle transitions are under control of cell cycle checkpoints, which ensure that critical events such as DNA
replication and chromosome segregation are carried out with precision. If DNA is damaged, e.g. by radiation, a checkpoint pathway is activated that arrests the cell cycle to provide time for repair.
If the damage is extensive, apoptosis is induced. In the absence of such checkpoints, the damaged DNA is inherited by aberrant to cells which may cause proliferative disorders such as cancer. Protein kinases play an important role in this process. For example, a specific kinase, checkpoint kinase 1 (Chkl), has been identified in yeast and mammals, and is activated by DNA damage in yeast. Activation of Chkl leads to the arrest of the cell at the G2/M transition. (Sanchez, Y. et al. (1997) Science 277:1497-1501.) Specifically, Chkl phosphorylates the cell division cycle phosphatase CDC25, inhibiting its normal function which is 15 to dephosphorylate and activate the cyclin-dependent kinase Cdc2. Cdc2 activation controls the entry of cells into mitosis. (Peng, C-Y et al. (1997) Science 277:1501- 1505.) Thus, activation of Chkl prevents the damaged cell from entering mitosis. A similar deficiency in a checkpoint kinase, such as Chkl, may also contribute to cancer by failure to arrest cells with damaged DNA at other checkpoints such as G2/M.
20 Proliferation-Related Kinases Proliferation-related kinase is a serum/cytokine inducible STK that is involved in regulation of the cell cycle and cell proliferation in human megakarocytic cells (Li, B. et al. (1996) J. Biol. Chem.
271:19402-8). Proliferation-related kinase is related to the polo (derived from Drosophila polo gene) family of STKs implicated in cell division. Proliferation-related kinase is downregulated in lung tumor 25 tissue and may be a proto-oncogene whose deregulated expression in normal tissue leads to oncogenic transformation.
The RET (rearranged during transfection) proto-oncogene encodes a tyrosine kinase receptor involved in both multiple endocrine neoplasia type 2, an inherited cancer syndrome, and Hirschsprung disease, a developmental defect of enteric neurons. RET and its functional ligand, glial cell line-3o derived neurotrophic factor, play key roles in the development of the human enteric nervous system (Pachnis, V. et al. (1998) Am. J. Physiol. 275:6183-6186).
=AMP-activated protein kinase A ligand-activated STK protein kinase is 5 =AMP-activated protein kinase (AMPK) (Gao, G.

et al. (1996) J. Biol Chem. 271:8675-8681). Mammalian AMPK is a regulator of fatty acid and sterol synthesis through phosphorylation of the enzymes acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase and mediates responses of these pathways to cellular stresses such as heat shock and depletion of glucose and ATP. AMPK is a heterotrimeric complex comprised of a catalytic alpha subunit and two non-catalytic beta and gamma subunits that are believed to regulate the activity of the alpha subunit. Subunits of AMPK have a much wider distribution in non-lipogenic tissues such as brain, heart, spleen, and lung than expected.
This distribution suggests that its role may extend beyond regulation of lipid metabolism alone.
Kinases in Apoptosis l0 Apoptosis is a highly regulated signaling pathway leading to cell death that plays a crucial role in tissue development and homeostasis. Deregulation of this process is associated with the pathogenesis of a number of diseases including autoimmune disease, neurodegenerative disorders, and cancer. Various STKs play key roles in this process. ZIP kinase is an STK
containing a C-terminal leuciue zipper domain in addition to its N-terminal protein kinase domain.
This C-terminal domain appears to mediate homodimerization and activation of the kinase as well as interactions with transcription factors such as activating transcription factor, ATF4, a member of the cyclic-AMP
responsive element binding protein (ATF/CREB) family of transcriptional factors (Sanjo, H. et al.
(1998) J. Biol. Chem, 273:29066-29071). DRAKl and DRAK2 are STKs that share homology with the death-associated protein kinases (DAP kinases), known to function in interferon-y induced apoptosis (Sanjo et al. su ra). Like ZIP kinase, DAP kinases contain a C-terminal protein-protein interaction domain, in the form of ankyrin repeats, in addition to the N-terminal kinase domain. ZIP, DAP, and DRAK kinases induce morphological changes associated with apoptosis when transfected into NIH3T3 cells (Sanjo et al. su ra). However, deletion of either the N-terminal kinase catalytic domain or the C-terminal domain of these proteins abolishes apoptosis activity, indicating that in addition to the kinase activity, activity in the C-terminal domain is also necessary for apoptosis, possibly as an interacting domain with a regulator or a specific substrate.
RICK is another STK recently identified as mediating a specific apoptotic pathway involving the death receptor, CD95 (Inohara, N. et al. (1998) J. Biol. Chem. 273:12296-12300). CD95 is a member of the tumor necrosis factor receptor superfamily and plays a critical role in the regulation and homeostasis of the immune system (Nagata, S. (1997) Cell 88:355-365). The CD95 receptor signaling pathway involves recruitment of various intracellular molecules to a receptor complex following ligand binding. This process includes recruitment of the cysteine protease caspase-8 which, in turn, activates a caspase cascade leading to cell death. RICK is composed of an N-terminal kinase catalytic domain and a C-terminal "caspase-recruitment" domain that interacts with caspase-like domains, indicating that RICK plays a role in the recruitment of caspase-8.
This interpretation is supported by the fact that the expression of RICK in human 293T cells promotes activation of caspase-8 and potentiates the induction of apoptosis by various proteins involved in the CD95 apoptosis pathway (Inohara et al. su ra .
Mitochondrial Protein Kinases A novel class of eukaryotic kinases, related by sequence to prokaryotic histidine protein kinases, are the mitochondrial protein kinases (MPKs) which seem to have no sequence similarity with other eukaryotic protein kinases. These protein kinases are located exclusively in the mitochondrial l0 matrix space and may have evolved from genes originally present in respiration-dependent bacteria which were endocytosed by primitive eukaryotic cells. MPKs are responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase complexes (Harris, R.A. et al. (1995) Adv. Enzyme Regul. 34:147-162). Five MPKs have been identified. Four members correspond to pyruvate dehydrogenase kinase isozymes, regulating the activity of the pyruvate dehydrogenase complex, which is an important regulatory enzyme at the interface between glycolysis and the citric acid cycle. The fifth member corresponds to a branched-chain alpha-ketoacid dehydrogenase kinase, important in the regulation of the pathway for the disposal of branched-chain amino acids. (Harris, R.A. et al. (1997) Adv. Enzyme Regul.
37:271-293). Both starvation and the diabetic state are known to result in a great increase in the activity of the pyruvate dehydrogenase kinase in the liver, heart and muscle of the rat. This increase contributes in both disease states to the phosphorylation and inactivation of the pyruvate dehydrogenase complex and conservation of pyruvate and lactate for gluconeogenesis (Harris (1995) supra).
KINASES WITH NON-PROTEIN SUBSTRATES
Liyid and Inositol kinases Lipid kinases phosphorylate hydroxyl residues on lipid head groups. A family of kinases involved in phosphorylation of phosphatidylinositol (PI) has been described, each member phosphorylating a specific carbon on the inositol ring (Leevers, S.J. et al.
(1999) C~rr. Opin. Cell. Biol.
11:219-225). The phosphorylation of phosphatidylinositol is involved in activation of the protein kinase C signaling pathway. The inositol phospholipids (phosphoinositides) intracellular signaling pathway begins with binding of a signaling molecule to a G-protein linked receptor in the plasma membrane.
This leads to the phosphorylation of phosphatidylinositol (PI) residues on the inner side of the plasma .

membrane by inositol kinases, thus converting PI residues to the biphosphate state (PIPZ). PIPZ is then cleaved into inositol triphosphate (1P3) and diacylglycerol. These two products act as mediators for separate signaling pathways. Cellular responses that are mediated by these pathways are glycogen breakdown in the liver in response to vasopressin, smooth muscle contraction in response to acetylcholine, and thrombin-induced platelet aggregation.
PI 3-kinase (PI3K), which phosphorylates the D3 position of PI and its derivatives, has a central role in growth factor signal cascades involved in cell growth, differentiation, and metabolism.
PI3K is a heterodimer consisting of an adapter subunit and a catalytic subunit. The adapter subunit acts as a scaffolding protein, interacting with specific tyrosine-phosphorylated proteins, lipid moieties, to and other cytosolic factors. When the adapter subunit binds tyrosine phosphorylated targets, such as the insulin responsive substrate (IRS)-1, the catalytic subunit is activated and converts PI (4,5) bisphosphate (PIPZ) to PI (3,4,5) P3 (PIPS). PIPS then activates a number of other proteins, including PKA, protein kinase B (PKB), protein kinase C (PKC), glycogen synthase kinase (GSK)-3, and p70 ribosomal s6 kinase. PI3K also interacts directly with the cytoskeletal organizing proteins, Rac, rho, and cdc42 (Shepherd, P.R., et al. (1998) Biochem. J. 333:471-490). Animal models for diabetes, such as obese and fat mice, have altered PI3K adapter subunit levels. Specific mutations in the adapter subunit have also been found in an insulin-resistant Danish population, suggesting a role for PI3K in type-2 diabetes (Shepard, su ra).
PKC is also activated by diacylglycerol (DAG). Phorbol esters (PE) are analogs of DAG and tumor promoters that cause a variety of physiological changes when administered to cells and tissues.
PE and DAG bind to the N-terminal region of PKC. This region contains one or more copies of a cysteine-rich domain about 50 amino-acid residues long and essential for DAG/PE-binding.
Diacylglycerol kinase (DGK), the enzyme that converts DAG into phosphatidate, contains two copies of the DAG/PE-binding domain in its N-terminal section (Azzi, A. et al. ( 1992) Eur. J. Biochem.
208:547-557).
An example of lipid kinase phosphorylation activity is the phosphorylation of D-erythro-sphingosine to the sphingolipid metabolite, sphingosine-1-phosphate (SPP). SPP has emerged as a novel lipid second-messenger with both extracellular and intracellular actions (Kohama, T. et al. (1998) J. Biol. Chem. 273:23722-23728). Extracellularly, SPP is a ligand for the G-protein coupled receptor EDG-1 (endothelial-derived, G-protein coupled receptor).
Intracellularly, SPP
regulates cell growth, survival, motility, and cytoskeletal changes. SPP
levels are regulated by sphingosine kinases that specifically phosphorylate D-erythro-sphingosine to SPP. The importance of sphingosine kinase in cell signaling is indicated by the fact that various stimuli, including platelet-derived growth factor (PDGF), nerve growth factor, and activation of protein kinase C, increase cellular levels of SPP by activation of sphingosine kinase, and the fact that competitive inhibitors of the enzyme selectively inhibit cell proliferation induced by PDGF (Kohama et al. su ra).
Purine Nucleotide Kinases The purine nucleotide kinases, adenylate kinase (ATP:AMP phosphotransferase, or AdK) and guanylate kinase ( ATP:GMP phosphotransferase, or GuK) play a key role in nucleotide metabolism and are crucial to the synthesis and regulation of cellular levels of ATP and GTP, respectively. These two molecules are precursors in DNA and RNA synthesis in growing cells and provide the primary source of biochemical energy in cells (ATP), and signal transduction pathways (GTP). Inhibition of various steps in the synthesis of these two molecules has been the basis of many antiproliferative drugs for cancer and antiviral therapy (Pillwein, K. et al. (1990) Cancer Res.
50:1576-1579).
AdK is found in almost all cell types and is especially abundant in cells having high rates of ATP synthesis and utilization such as skeletal muscle. In these cells AdK is physically associated with mitochondria and myofibrils, the subcellular structures that are involved in energy production and utilization, respectively. Recent studies have demonstrated a major function for AdK in transferring high energy phosphoryls from metabolic processes generating ATP to cellular components consuming ATP ( Zeleznikar, R.J. et al. (1995) J. Biol. Chem. 270:7311-7319). Thus AdK
may have a pivotal role in maintaining energy production in cells, particularly those having a high rate of growth or metabolism such as cancer cells, and may provide a target for suppression of its activity to treat certain cancers. Alternatively, reduced AdK activity may be a source of various metabolic, muscle-energy disorders that can result in cardiac or respiratory failure and may be treatable by increasing AdK activity.
GuK, in addition to providing a key step in the synthesis of GTP for RNA and DNA synthesis, also fulfills an essential function in signal transduction pathways of cells through the regulation of GDP
and GTP. Specifically, GTP binding to membrane associated G proteins mediates the activation of cell receptors, subsequent intracellular activation of adenyl cyclase, and production of the second messenger, cyclic AMP. GDP binding to G proteins inhibits these processes. GDP
and GTP levels also control the activity of certain oncogenic proteins such as p21'~ known to be involved in control of cell proliferation and oncogenesis (Bos, J.L. (1989) Cancer Res. 49:4682-4689). High ratios of GTP:GDP caused by suppression of GuK cause activation of p21'~ and promote oncogenesis.
Increasing GuK activity to increase levels of GDP and reduce the GTP:GDP ratio may provide a therapeutic strategy to reverse oncogenesis.
GuK is an important enzyme in the phosphorylation and activation of certain antiviral drugs l0 useful in the treatment of herpes virus infections. These drugs include the guanine homologs acyclovir and buciclovir (Miller, W.H. and Miller R.L. (1980) J. Biol. Chem. 255:7204-7207; Stenberg, K. et al.
(1986) J. Biol. Chem. 261:2134-2139). Increasing GuK activity in infected cells may provide a therapeutic strategy for augmenting the effectiveness of these drugs and possibly for reducing the necessary dosages of the drugs.
Pyrimidine Kinases The pyrirnidine kinases are deoxycytidine kinase and thymidine kinase 1 and 2.
Deoxycytidine kinase is located in the nucleus, and thymidine kinase 1 and 2 are found in the cytosol (Johansson, M.
et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:11941-11945). Phosphorylation of to deoxyribonucleosides by pyrimidine kinases provides an alternative pathway for de novo synthesis of DNA precursors. The role of pyrimidine kinases, like purine kinases, in phosphorylation is critical to the activation of several chemotherapeutically important nucleoside analogues (Arner E.S. and Eriksson, S. (1995) Pharmacol. Ther. 67:155-186).
The discovery of new human kinases, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cancer, immune disorders, disorders affecting growth and development, cardiovascular diseases, and lipid disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of human kinases.
2o SUMMARY OF THE INVENTION
The invention features purified polypeptides, human kinases, referred to collectively as "PKIN" and individually as "PKIN-1," "PKIN-2," "PKIN-3," "PKIN-4," "PKIN-5,"
"PKIN-6,'>
«P~-7~» «P~-8~» «P~-9~» «P~-10~» «P~-11~» «P~-12,» «P~-13~» «P~-14~»
«P~-15 » «P~_16 » «P~-17 » «P~-18 » «P~-19 » «P~-20 » «P~-21 " and "PKIN-> > > > > > >
22." In one aspect, the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:I-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ )D NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of 3o SEQ 117 NO:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-22.
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 1D N0:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ 1D N0:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 1D N0:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ
1D NO:1-22. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID N0:23-44.
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 1D NO:1-22, b) a polypeptide comprising a naturally occurnng amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID N0:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 1D N0:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ D7 NO:1-22. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID N0:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D NO:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
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 1D NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ 1D NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
ID NO:1-22.
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 N0:23-44, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ )17 N0:23-44, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). 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 N0:23-44, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence 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 )D
N0:23-44, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of SEQ )D N0:23-44, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
13' 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:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ )17 NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D N0:1-22, and d) an irntnunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ D7 NO:1-22. The invention additionally to provides a method of treating a disease or condition associated with decreased expression of functional PKIN, 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 >D NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-22, and d) an immunogenic fragment of a polypeptide having an. amino acid sequence selected from the group consisting of SEQ )D NO:1-22. 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 PKIN, 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 )D NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ 117 NO:1-22, c) a biologically active fragment of 3o a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D N0:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-22. 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 PK1N, 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 1D NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ll7 N0:1-22, c) a biologically active fragment of a polypeptide l0 having an amino acid sequence selected from the group consisting of SEQ )D
NO:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 117 NO:1-22. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
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 ID NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ )D NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 1D
NO:l-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D N0:1-22. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ )D N0:23-44, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
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
>D N0:23-44, 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 N0:23-44, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44, iii} a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probability score for the match between each polypeptide and its GenBank homolog is 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 S 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.
2o 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
"PKIN" refers to the amino acid sequences of substantially purified PKIN
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.
3o The term "agonist" refers to a molecule which intensifies or mimics the biological activity of PKIN. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of PKIN either by directly interacting with PKIN or by acting on components of the biological pathway in which PKIN
participates.

An "allelic variant" is an alternative form of the gene encoding PKIN. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding PKIN include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as PKIN or a polypeptide with at least one functional characteristic of PKIN. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding PKIN, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding PKIN. 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 PKIN. 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 PKIN is retained. For example, negatively charged amino acids may include aspartic acid and glutarnic acid, and positively charged amino acids may include lysine and 2o 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 phenylalauine 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 PKIN. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of PKIN either by directly interacting with PKIN or by acting on components of the biological pathway in which PKIN
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding an epitopic determinant.
Antibodies that bind PKIN polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (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'-NH2), 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 "spiegeliner" refers to an aptamer which includes L-DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a 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 l0 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 PKIN, 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 PKIN or fragments of PKIN 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., NaCl), 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, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or polypeptide.
Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A
derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
A "fragment" is a unique portion of PKIN or the polynucleotide encoding PKIN
which is identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For 2o 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 m N0:23-44 comprises a region of unique polynucleotide sequence that specifically identifies SEQ >l7 N0:23-44, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:23-44 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ
B7 N0:23-44 from related polynucleotide sequences. The precise length of a fragment of SEQ ID
N0:23-44 and the region of SEQ ID N0:23-44 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A fragment of SEQ )I7 NO:1-22 is encoded by a fragment of SEQ m N0:23-44. A
fragment of SEQ ID NO:1-22 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-22. For example, a fragment of SEQ ID NO:1-22 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-22.
The precise length of a fragment of SEQ >I7 NO:1-22 and the region of SEQ 117 N0:1-22 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 2o 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.nlin.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:' S0 l0 Expect: l0 Word Size: I1 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 S0, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of 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: I1 and Extension Gap: 1 penalties Gap x drop-off. 50 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 )D 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 pg/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 (T"~ 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 T", and conditions for nucleic acid hybridization are well known and can be found in Sarnbrook, 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 maybe 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 p,g/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"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 PKIN
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 PKIN 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 PKIN. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of PKIN.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an PKIN may involve lipidation, glycosylation, 3o 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 PKIN.
"Probe" refers to nucleic acid sequences encoding PKIN, 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 to be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU
primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/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, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and S' 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 PKIN, nucleic acids encoding PKIN, 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% 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" 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 3o 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.
l0 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 human kinases (PKIN), the polynucleotides encoding PKIN, and the use of these compositions for the diagnosis, treatment, or prevention of cancer, immune disorders, disorders affecting growth and development, cardiovascular diseases, and lipid disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ 117 NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide 117) 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 117) 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 m) 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 score for the match between each polypeptide and its GenBank homolog. Column 5 shows the annotation of the GenBank homolog 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 MOT1FS
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 human kinases.
For example, SEQ ID N0:1 is 91 % identical to human casein kinase I-alpha (GenBank ID
g852055) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.9e-167, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:1 also contains a eukaryotic protein kinase domain as determined by searching for statistically significant matches in the hidden Markov model (IEVVIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIIVVIPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ
l0 ID N0:1 is a protein kinase.
For example, SEQ B7 NO:10 is 91% identical to Mus musculus FYVE finger-containing phosphoinositide kinase (GenBank ID g4200446) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:10 also contains a phosphatidyl inositol 4-phosphate S-kinase domain as determined by searching for statistically significant matches in the hidden Markov model (HIVIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from PRODOM analysis provides further corroborative evidence that SEQ ID N0:10 is a phosphoinositide kinase.
For example, SEQ ID N0:12 is 71 % identical to human serine/threonine protein kinase (GenBank D7 g7160989) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.7e-148, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ D7 N0:12 also contains a eukaryotic protein kinase domain as determined by searching for statistically significant matches in the hidden Markov model (HIVIM)-based PFAM database of conserved protein family domains.
(See Table 3.) Data from BLIIVVIPS and MOTIFS analyses provide further corroborative evidence that SEQ ID
N0:12 is protein kinase.
For example, SEQ ID N0:13 is 86% identical to murine pantothenate kinase 1 beta (GenBank 117 g6690020) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.) The BLAST probability score is 1.6e-129, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. Pantothenate kinase (PanK) is proposed to be the master regulator of CoA biosynthesis in mammalian cells, by controlling flux through the CoA biosynthetic pathway. Changes in the level of tissue PanK activitiy is reflected by the concurrent changes in the levels of CoA as seen in various metabolic states. Alterations in CoA levels and PanK activity are seen during starvation/feeding, pathological states such as diabetes and by treatment with hypolipidemic drugs (Rock, C.O. et al., (2000) J. Biol. Chem. 275:1377-1383.) For example, SEQ 1D N0:16 is 68% identical to Mus musculus Nck-interacting kinase-like embryo specific kinase (GenBank ID 86472874) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ 1D N0:16 also contains a eukaryotic protein kinase domain as determined by searching for statistically significant matches in the hidden Markov model (HIVIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIIVVIPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ D7 N0:16 is a protein kinase.
For example, SEQ ID N0:19 is 99% identical to human RET tyrosine kinase receptor (GenBank 1D 85419753) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:19 also contains a eukaryotic protein kinase domain as determined by searching for statistically significant matches in the hidden Markov model (HIVIM)-based PFAM database of conserved protein family domains.
(See Table 3.) Data from BLIIVVIPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ 1D N0:19 is a tyrosine kinase.
For example, SEQ 1D N0:22 is 33% identical to Gallus gallus smooth muscle myosin light 2o chain kinase precursor (GenBank 1D 8212661) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.2 e-60, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:22 also contains two eukaryotic protein kinase domains as determined by searching for statistically significant matches in the hidden Markov model (HIVIM)-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 N0:22 is a protein kinase.
SEQ >D N0:2-9, SEQ 1D N0:11, SEQ 1D N0:14-15, SEQ 1D N0:17-18, and SEQ 1D
N0:20-21 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO:1-22 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. Columns 1 and 2 list the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide of the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column 4 lists fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ID N0:23-44 or that distinguish between SEQ D7 N0:23-44 and related polynucleotide sequences. Column 5 shows identification numbers corresponding to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or sequence assemblages comprised of both cDNA and genomic DNA. These sequences were used to assemble the full length polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences in column 5 relative to their respective full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for example, to Incyte cDNAs along with their corresponding cDNA libraries. For example, 18381287 is the identification number of an Incyte cDNA sequence, and CARDNOTO1 is the cDNA
library from which it is derived. Incyte cDNAs for which cDNA libraries are not indicated were derived from pooled cDNA libraries (e.g., 71583296V1). Alternatively, the identification numbers in column 5 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotide sequences. In addition, the identification numbers in column 5 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST"). Alternatively, the identification numbers in column 5 may be derived from the 2o 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 identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching"
algorithm. For example, FL_~c:~~~XXX Nl Nz_YYYYY N3 NQ represents a "stitched"
sequence in which ~'~~~XXX 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 identification numbers in column 5 may refer to assemblages of exons brought together by an "exon-stretching" algorithm. For example, FLXX~:XXX~IAAAA~BBBBB_1 N is the identification number of a "stretched" sequence, with I~:~~~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, to 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 column 5 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 PKIN variants. A preferred PKIN variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the PKIN amino acid sequence, and which contains at least one functional or structural characteristic of PKIN.
The invention also encompasses polynucleotides which encode PKIN. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:23-44, which encodes PKIN. The polynucleotide sequences of SEQ ID N0:23-44, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding PKIN. In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding PKIN. A particular aspect of the invention encompasses a variant of a polynucleotide sequence corrlprising a sequence selected from the group consisting of SEQ )D N0:23-44 which has at least about 70%, or alternatively at least about 85%, or even at least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ
l0 ID N0:23-44. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of PKIN.
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 PKIN, 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 PKIN, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode PKIN and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring PKIN under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding PKIN 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 PKIN 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 PKIN
and PKIN 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 PKIN 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
N0:23-44 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-. 511.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or to combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carned out using either the ABI 373 or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and BiotechnoloQV, Wiley VCH, New York NY, pp.
856-853.) The nucleic acid sequences encoding PKIN 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 inhuman 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. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode PKIN may be cloned in recombinant DNA molecules that direct expression of PKIN, 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 PKIN.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter PKIN-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.

The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.

5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of PKIN, 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 PKIN 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, PKIN itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide.
2o 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 PKIN, 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, su ra, pp. 28-53.) In order to express a biologically active PKIN, the nucleotide sequences encoding PKIN 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 PKIN. Such elements may vary in their strength and specificity.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding PKIN. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding PKIN 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 l0 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 PKIN and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biology, 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 PKIN. 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, su ra; 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 Technology (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. I_m_m__unol. 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 PKIN. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding PKIN 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 PKIN into the vector's multiple cloning l0 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 PKIN are needed, e.g. for the production of 15 antibodies, vectors which direct high level expression of PKIN 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 PKIN. 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 20 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 PKIN. Transcription of sequences 25 encoding PKIN may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.

6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These 30 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 PKIN
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 PKIN in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc.
S Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are to constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355.) For long term production of recombinant proteins in mammalian systems, stable expression of PKIN in cell lines is preferred. For example, sequences encoding PK1N can be transformed into cell 15 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 20 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 25 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) 30 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), B glucuronidase and its substrate B-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 PKIN is inserted within a marker gene sequence, transformed cells containing sequences encoding PKIN can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding PKIN under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates l0 expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding PKIN
and that express PKIN 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 PKIN 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 PKIN 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 I-m_m__unoloay, 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 PKIN
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, the sequences encoding PKIN, 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 PKIN 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 PKIN may be designed to contain signal sequences which direct to secretion of PKIN 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 PKIN 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 PKIN protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of PKIN 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), calinodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calinodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the PKIN encoding sequence and the heterologous protein sequence, so that PKIN

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 PKIN 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.
PKIN of the present invention or fragments thereof may be used to screen for compounds l0 that specifically bind to PKIN. At least one and up to a plurality of test compounds may be screened for specific binding to PKIN. 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 PKIN, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which PKIN
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 PKIN, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E.
coli. Cells expressing PKIN or cell membrane fractions which contain PKIN are then contacted with a test compound and binding, stimulation, or inhibition of activity of either PKIN 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 PKIN, either in solution or affixed to a solid support, and detecting the binding of PKIN 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 cornpound(s) may be free in solution or affixed to a solid support.
PKIN of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of PKIN. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for PKIN

activity, wherein PKIN is combined with at least one test compound, and the activity of PKIN in the presence of a test compound is compared with the activity of PKIN in the absence of the test compound. A change in the activity of PKIN in the presence of the test compound is indicative of a compound that modulates the activity of PKIN. Alternatively, a test compound is combined with an in vitro or cell-free system comprising PKIN under conditions suitable for PKIN
activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of PKIN 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 PKIN or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP
system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D.
(1996) Clip. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding PKIN 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 PKIN 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 PKIN 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 PKIN, e.g., by secreting PKIN 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 PKIN and human kinases. In addition, the expression of PKIN is closely associated with brain, breast tumor, cardiovascular, digestive, fallopian tube tumor, fetal stomach, nervous, ovarian tumor, pancreatic tumor, peritoneal tumor, pituitary gland, placental, prostate tumor, neural, spinal cord, and testicular tissues, and with umbilical cord blood dendritic cells.
Therefore, PKIN appears to play a role in cancer, immune disorders, disorders affecting growth and development, cardiovascular diseases, and lipid disorders. In the treatment of disorders associated with increased PKIN expression or activity, it is desirable to decrease the expression or activity of PKIN.
In the treatment of disorders associated with decreased PKIN expression or activity, it is desirable to increase the expression or activity of PKIN.
Therefore, in one embodiment, PKIN 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 PKIN.
Examples of such disorders include, but are not limited to, a cancer, such as 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, leukemias such as multiple myeloma, and lymphomas such as Hodgkin's disease; an immune 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, 3o myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helininthic infections, and trauma; a growth and developmental 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, renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a cardiovascular disease, such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and 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, congenital lung anomalies, atelectasis, pulmonary congestion and edema, pulinonary embolism, pulmonary hemorrhage, pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive pulmonary disease, restrictive pulmonary disease, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in collagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors, inflammatory and noninflammatory pleural effusions, pneumothorax, pleural tumors, drug-induced lung disease, radiation-induced lung disease, and complications of lung transplantation; and a lipid disorder such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine paltnitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GMZ gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes mellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin:cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, and obesity.
In another embodiment, a vector capable of expressing PK1N 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 PKIN including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified PI~NN in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PKIN including, but not limited to, those provided above.
In still another~embodiment, an agonist which modulates the activity of PKIN
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PKIN including, but not limited to, those listed above.
In a further embodiment, an antagonist of PKIN may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of PKIN.
Examples of such disorders include, but are not limited to, those cancer, immune disorders, disorders affecting growth and development, cardiovascular diseases, and lipid disorders described above.
In one aspect, an antibody which specifically binds PKIN 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 PKIN.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding PKIN may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of PKIN 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 PKIN may be produced using methods which are generally known in the art.
In particular, purified PKIN may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind PKIN.
Antibodies to PKIN may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with PKIN 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 Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to PKIN
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 PKIN
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 PKIN 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 3o 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., Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (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 PKIN-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 PKIN may also be generated.
Forexample, such fragments include, but are not limited to, F(ab~2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab~2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between PKIN and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering PKIN 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 PKIN. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of PKIN-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The Ke determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple PKIN epitopes, represents the average affinity, or avidity, of the antibodies for PKIN. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular PKIN epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in which the PKIN-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of PKIN, 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 PKIN-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and Coligan et al. su ra.) In another embodiment of the invention, the polynucleotides encoding PKIN, 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 PKIN. Such technology is well known in the art, and antisense oligonucleotides or larger fragments 2o can be designed from various locations along the coding or control regions of sequences encoding PKIN. (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 Clip. 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 Morris, M.C. et al. (1997) Nucleic Acids Res.
25(14):2730-2736.) In another embodiment of the invention, polynucleotides encoding PKIN 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 (SC>D)-Xl disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, l0 R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi). In the case where a genetic deficiency in PKIN expression or regulation causes disease, the expression of PKIN 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 PKIN are treated by constructing mammalian expression vectors encoding PKIN
and introducing these vectors by mechanical means into PKIN-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 PKIN 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).
PKIN
may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci.
USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) C~rr. 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 Blau, H.M. supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding PKIN from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KTT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to PKIN expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding PKIN 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.5. 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 PKIN to cells which have one or more genetic abnormalities with respect to the expression of PKIN. 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.
to 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 PKIN to target cells which have one or more genetic abnormalities with respect to the expression of PKIN. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing PKIN 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 herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well 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 PKIN to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) C~.irr. 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 PKIN into the alphavirus genome in place of the capsid-coding region results in the production of a large number of PKIN-coding RNAs and the synthesis of high levels of PKIN 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 PKIN 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 Immunolo 'sac 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 PKIN.
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 PKIN. 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' andlor 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-, thin-, 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 2o compound which is effective in altering expression of a polynucleotide encoding PKIN. 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 PI~NN
expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding PKIN may be therapeutically useful, and in the treatment of disorders associated with decreased PK1N expression or activity, a compound which specifically promotes expression of the polynucleotide encoding PKIN 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 PKIN 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 PKIN 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 to polynucleotide encoding PKIN. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharom~pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem.
Biophys. Res. Commun.
268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified 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.
Biotechnol. 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remin tg on's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of PKIN, antibodies to PKIN, and mimetics, agonists, antagonists, or inhibitors of PK1N.
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, intxaperitoneal, 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 PKIN or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, PKIN or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example PKIN
or fragments thereof, antibodies of PKIN, and agonists, antagonists or inhibitors of PKIN, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the EDso (the dose therapeutically effective in 50% of the population) or LDso (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LDso/EDSO ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the EDso with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~cg to 100,000 ~.cg, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind PKIN may be used for the diagnosis of disorders characterized by expression of PKIN, or in assays to monitor patients being treated with PKIN or agonists, antagonists, or inhibitors of PKIN. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for PKIN include methods which utilize the antibody and a label to detect PKIN 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 PKIN, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of PKIN expression. Normal or standard values for PKIN expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to PKIN under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of PKIN
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 PKIN 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 PKIN
may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of PKIN, and to monitor regulation of PKIN levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding PKIN or closely related molecules may be used to identify nucleic acid sequences which encode PKIN. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding PKIN, allelic variants, or related sequences.
2o Probes may also be used for the detection of related sequences, and may have at least 50%
sequence identity to any of the PKIN encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ 1D
N0:23-44 or from genomic sequences including promoters, enhancers, and introns of the PK1N
gene.
Means for producing specific hybridization probes for DNAs encoding PKIN
include the cloning of polynucleotide sequences encoding PKIN or PKIN 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 PKIN may be used for the diagnosis of disorders associated with expression of PKIN. Examples of such disorders include, but are not limited to, a cancer, such as 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, leukemias such as multiple myeloma and lymphomas such as Hodgkin's disease; an immune disorder, such as acquired imrnunodeficiency syndrome (A)DS), 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 growth and developmental 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, renal tubular acidosis, anemia, C~shing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a cardiovascular disease, such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mural annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of cardiac transplantation, congenital lung anomalies, atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary hemorrhage, pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive pulinonary disease, restrictive pulmonary disease, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in collagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors, inflammatory and noninflammatory pleural effusions, pneumothorax, pleural tumors, drug-induced lung disease, radiation-induced lung disease, and complications of lung transplantation; and a lipid disorder such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palinitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid 2o storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GMZ gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes mellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin:cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, and obesity. The polynucleotide sequences encoding PKIN 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 PKIN expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding PKIN may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding PKIN 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 PKIN 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 PKIN, 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 PKIN, 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 PKIN
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 PKIN, or a fragment of a polynucleotide complementary to the polynucleotide encoding PKIN, 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 PKIN 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 PKIN are used to amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived, for example, from diseased or normal to 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).
Methods which may also be used to quantify the expression of PKIN 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 rnicroarray 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, PKIN, fragments of PKIN, or antibodies specific for PKIN 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.
to A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of 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 3o 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 PKIN
to quantify the levels of PKIN expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson,.
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample.
A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. 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 PKIN
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (PACs), bacterial artificial chromosomes (BACs), bacterial Pl constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.

7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop 2o 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, Larder, 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-Uliich, et al. (1995) in Meyers, su ra, 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 PKIN 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, PKIN, 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 PKIN 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., Geysers, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with PKIN, or fragments thereof, and washed. Bound PKIN is then detected by methods well known in the art.
Purified PK1N 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 PKIN specifically compete with a test compound for binding PKIN. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with PKIN.
In additional embodiments, the nucleotide sequences which encode PKIN 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.
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 preferred specific 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, in particular U.S. Ser. No. 60/242,410, U.S. Ser. No. 60/244,068, U.S. Ser. No.
60/245,708, U.S. Ser.
No. 60/247,672, U.S. Ser. No. 60/249,565, U.S. Ser. No. 60/252,730, and U.S.
Ser. No. 60/250,807, are hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. 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 CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, su ra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XI,1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an l0 AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, Q1AWELL 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).
2o 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 Phartnacia 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, and hidden Markov model (HIvIM)-based protein family databases such as PFAM. (HMM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin.
Struct. Biol. 6:361-365.) l0 The queries were performed using programs based on BLAST, FASTA, BLIIVVIPS, and HIVIMER.
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, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (HIVIM)-based protein family databases such as PFAM. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ )D
N0:23-44. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative human kinases were initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg).
Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of l0 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 human kinases, the encoded polypeptides were analyzed by querying against PFAM models for human kinases. Potential human kinases were also identified by homology to Incyte cDNA sequences that had been annotated as human kinases. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA
or public cDNA
coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence. Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data ~~Stitched" Sequences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example )II were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals thus identified were then "stitched" together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.
"Stretched" Sequences Partial DNA sequences were extended to full length with an algorithm based on BLAST
analysis. First, partial cDNAs assembled as described in Example III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA
sequences were therefore "stretched" or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.
VI. Chromosomal Mapping of PKIN Encoding Polynucleotides The sequences which were used to assemble SEQ ID N0:23-44 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ )D N0:23-44 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as 1?hrap (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI "GeneMap'99" World Wide Web site (http://www.ncbi.nlin.nih.gov/genemap~, can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
In this manner, SEQ ID N0:29 was mapped to chromosome 1 within the interval from 199.20 to 203.00 centiMorgans, to chromosome 13 within the interval from 105.20 centiMorgans to the q terminus, and to chromosome 6 within the interval from 59.60 to 72.20 centiMorgans. More than one map location is reported for SEQ ID N0:29, indicating that sequences having different map locations were assembled into a single cluster. This situation occurs, for example, when sequences having strong similarity, but not complete identity, are assembled into a single cluster.
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 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two sequences and the 5 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 PKIN 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 PKIN. cDNA sequences and cDNA
library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of PKIN Encoding Polynucleotides Full length polynucleotide sequences were also produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH~)2S04, 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 p1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 p1 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 ~cl 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. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:23-44 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~.cCi of [Y 32P] 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 carned 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.
X. 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), su ra).
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; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.) Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
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/pl oligo-(dT) primer (2lmer), 1X first strand buffer, 0.03 units/~1 RNase inhibitor, 500 p,M dATP, 500 p,M dGTP, 500 pM dTTP, 40 ~.M
dCTP, 40 pM 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 O.SM sodium hydroxide and incubated for 20 minutes at 85°C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
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 p.1 SX SSC/0.2% SDS.
Microarray Preparation Sequences of the present invention are used to generate array elements. Each array element to is amplified from bacterial cells containing vectors with cloned cDNA
inserts. PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 pg.
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 p1 of the array element DNA, at an average concentration of 100 ng/pl, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 p1 of sample mixture consisting of 0.2 pg each of Cy3 and Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 p,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 for 10 min at 45° C in a first wash buffer (1X SSC, 0.1 SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried.
Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm x 1.8 cm array used in the present example is scanned with a 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 rim 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).
XI. Complementary Polynucleotides Sequences complementary to the PKIN-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring PKIN. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with to smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of PK1N. 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 PKIN-encoding transcript.
XII. Expression of PKIN
Expression and purification of PKIN is achieved using bacterial or virus-based expression systems. For expression of PKIN 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 PKIN upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of PKIN in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA
encoding PKIN 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 S~odoptera fru~iperda (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, PKIN 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 Lponicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Phartnacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from PKIN 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 PKIN obtained by these methods can be used directly in the assays shown in to Examples XVI, XVII, and XVI>I, where applicable.
XIII. ~nctional Assays PKIN function is assessed by expressing the sequences encoding PKIN 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 ~g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 Ecg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide;
changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of PKIN on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding PKIN 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 PKIN and other genes of interest can be analyzed by northern analysis or microarray techniques.
XIV. Production of PHIN Specific Antibodies PKIN substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the PKIN 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. 11.) Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (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-PKIN activity by, for example, binding the peptide or PKIN to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XV. Purification of Naturally Occurring PKIN Using Specific Antibodies Naturally occurring or recombinant PKIN is substantially purified by immunoaffinity chromatography using antibodies specific for PKIN. An immunoaffinity column is constructed by covalently coupling anti-PKIN 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 PKIN are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PKIN (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/PKIN 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 PKIN is collected.
XVI. Identification of Molecules Which Interact with PKIN
PKIN, or biologically active fragments thereof, are labeled with 1~I Bolton-Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled PKIN, washed, and any wells with labeled PKIN complex are assayed. Data obtained using different concentrations of PKIN are used to calculate values for the number, affinity, and association of PKIN with the candidate molecules.
Alternatively, molecules interacting with PKIN 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).
PKIN 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).
XVII. Demonstration of PKIN Activity Generally, protein kinase activity is measured by quantifying the phosphorylation of a protein substrate by PKIN in the presence of gamma-labeled 32P-ATP. PKIN is incubated with the protein substrate, 3zP-ATP, and an appropriate kinase buffer. The 32P incorporated into the substrate is separated from free'zP-ATP by electrophoresis and the incorporated 32P is counted using a radioisotope counter. The amount of incorporated 32P is proportional to the activity of PKIN. A
determination of the specific amino acid residue phosphorylated is made by phosphoamino acid analysis of the hydrolyzed protein.
In one alternative, protein kinase activity is measured by quantifying the transfer of gamma phosphate from adenosine triphosphate (ATP) to a serine, threonine or tyrosine residue in a protein substrate. The reaction occurs between a protein kinase sample with a biotinylated peptide substrate and gamma 32P-ATP. Following the reaction, free avidin in solution is added for binding to the biotinylated 32P-peptide product. The binding sample then undergoes a centrifugal ultrafiltration process with a membrane which will retain the product-avidin complex and allow passage of free gamma 3zP-ATP. The reservoir of the centrifuged unit containing the'zP-peptide product as retentate is then counted in a scintillation counter. This procedure allows assay of any type of protein kinase sample, depending on the peptide substrate and kinase reaction buffer selected. This assay is provided in kit form (ASUA, Affinity Ultrafiltration Separation Assay, Transbio Corporation, Baltimore MD, U.S. Patent No. 5,869,275). Suggested substrates and their respective enzymes are as follows:
Histone Hl (Sigma) and p34°a~2kinase, Annexin I, Angiotensin (Sigma) and EGF receptor kinase, Annexin II and src kinase, ERK1 & ERK2 substrates and MEK, and myelin basic protein and ERK
(Pearson, J.D. et al. (1991) Methods in Enzymology 200:62-81).
In another alternative, protein kinase activity of PKIN is demonstrated in vitro in an assay containing PKIN, SOwI of kinase buffer, leg substrate, such as myelin basic protein (MBP) or synthetic peptide substrates, 1 mM DTT, 10 ~g ATP, and O.S~Ci ['y-33P]ATP. The reaction is to incubated at 30°C for 30 minutes and stopped by pipetting onto P81 paper. The unincorporated ['y-33P~ATP is removed by washing and the incorporated radioactivity is measured using a radioactivity scintillation counter. Alternatively, the reaction is stopped by heating to 100 °C in the presence of SDS
loading buffer and visualized on a 12% SDS polyacrylamide gel by autoradiography. Incorporated radioactivity is corrected for reactions carried out in the absence of PKIN or in the presence of the inactive kinase, K38A.
In yet another alternative, adenylate kinase or guanylate kinase activity may be measured by the incorporation of 32P from gamma-labeled 32P -ATP into ADP or GDP using a gamma radioisotope counter. The enzyme, in a kinase buffer, is incubated together with the appropriate nucleotide mono-phosphate substrate (AMP or GMP) and 3zP-labeled ATP as the phosphate donor. The 2o reaction is incubated at 37°C and terminated by addition of trichloroacetic acid. The acid extract is neutralized and subjected to gel electrophoresis to separate the mono-, di-, and triphosphonucleotide fractions. The diphosphonucleotide fraction is cut out and counted. The radioactivity recovered is proportional to the enzyme activity.
In yet another alternative, other assays for PKIN include scintillation proximity assays (SPA), scintillation plate technology and filter binding assays. Useful substrates include recombinant proteins tagged with glutathione transferase, or synthetic peptide substrates tagged with biotin. Inhibitors of PKIN activity, such as small organic molecules, proteins or peptides, may be identified by such assays.
Kinase activity of PKIN may be determined by its ability to convert polyphosphate substrate (Polyp) to ATP in the presence of ADP. PKIN and Poly P are incubated at 37°C for 40 minutes and 3o then at 90°C for 2 minutes in a buffer containing 50 mM Tris-HCl, pH
7.4, 40 mM ammonium sulfate, 4 mM MgClz, and 5 ~M ADP. The reaction mixture is diluted 1:100 in 100 mM Tris-HCl (pH 8.0), 4 mM EDTA, which is then diluted 1:1 in luciferase reaction mixture (ATP
Bioluminescence Assay Kit CLS II; Boehringer Mannheim). The ATP generated is then quantitated using a luminometer (Kornberg, A. et al. (1999) Annu. Rev. Biochem. 68:89-125; Ault-Riche, D. et al. (1998) J. Bacteriol.
180:1841-1847).
Kinase activity of PKIN, as measured by phosphorylation of substrate, may be determined using an immune complex kinase assay well known in the art. COS7 cells are transfected with an expression plasmid constructed from a FLAG tag expression vector (pMEl8S-FLAG) containing PKIN DNA. A control transfection using vector alone without the PKIN DNA
insert is done in parallel. After 48 hours, the cells are lysed in buffer A (20 mM HEPES-NaOH, pH 7.5, 3 mM
MgCl2, 100 mM NaCl2, 1 mM dithiothreitol, 1 mM phenylinethanesulfonyl fluoride, 1 ~g/ml leupeptin, l mM EGTA, 1 mM Na3Vo4, 10 mM NaF, 20 mM ~i-glycerophosphate, and 0.5% Triton X-100) and to centrifuged at 14,000 rpm. Supernatants are incubated with anti-FLAG
antibody (M2 monoclonal antibody; Eastman Kodak Co.) in a SO% slurry of protein A-Sepharose (Amersham Pharmacia Biotech) for 1.5 hours at 4°C. Immune complexes are precipitated and washed twice in buffer A and twice in buffer B (20 mM HEPES-NaOH, pH 7.5, 1 mM dithiothreitol, 10 wM
Na3Vo4, 2 mM (3-glycerophosphate, 0.1 mM phenylmethanesulfonyl fluoride, 0.1 ~g/ml leupeptin, 0.1 mM EGTA.) Precipitates are incubated in buffer B containing 0.17 mg/ml myelin basic protein (MBP) (Sigma), 20 p,M ATP, and 5 ~Ci of ['y-'ZP]ATP (NEN Life Science Products) at 30°C
for 20 minutes. The reaction is stopped by the addition of 4X Laemmli sample buffer (SO mM Tris-HCI, pH 6.8, 2% SDS, 30 mM dithiothreitol, and 10% glycerol) and heated at 95°C for 5 minutes. Proteins are separated by SDS-polyacrylamide gel electrophoresis and radioactivity incorporated into MBP
is detected by 2o autoradiography (Nakano, K. et al. (2000) J. Biol. Chem. 275:20533-20539.) In yet another alternative, an assay for PanK activity of PKIN includes the enzyme preparation method as described in Vallari, D.S. et al., (1987) J. Biol. Chem.
262:2468-247.
Pantothenate kinase-specific activities in cell lysates are calculated as a function of protein concentration with the assay being linear with respect to both time and protein input. Protein concentrations are measured using the Bradford assay using bovine 7-globulin as a standard.
Standard assays contain D-[1-14C]pantothenate (45.5 p.M; specific activity SS
mCi/mmol), ATP (2.5 mM, pH 7.0), MgCl2 (2.5 mM), Tris-HCl (0.1 M, pH 7.5), and l5pg of protein from a soluble cell extract in a total volume of 40 p1. The mixture is incubated for 10 min. at 37 °C, and the reaction is stopped by depositing a 30-~,1 aliquot onto a Whatman DE81 ion-exchange filter disc which is then washed in three changes of 1 % acetic acid in 95 % ethanol (25 ml/disc) to remove unreacted pantothenate. 4'-Phosphopantothenate is quantitated by counting the dried disc in 3 ml of scintillation solution (Rock, su ra).
XVIII. Enhancement/Inhibition of Protein Kinase Activity ' Agonists or antagonists of PKIN activation or inhibition may be tested using assays described in section XVII. Agonists cause an increase in PKIN activity and antagonists cause a decrease in PKIN activity.
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 to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

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b N~ ~~a~~ N~ p ~~ o~A~ ~ ~ C~ v m ~_ N .J
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<110> INCYTE GENOMICS, INC.
GURURAJAN, Rajagopal BAUGHN, Mariah R.
WALIA, Narinder K.
ELLIOTT, Vicki S.
XU, Yuming ARVIZU, Chandra YAO, Monique G.
RAMKUMAR, Jayalaxmi DING, Li TANG, Y. Tom HAFALIA, April J.A.
NGUYEN, Danniel B.
GANDHI, Ameena R.
LU, Yan YUE, Henry BURFORD, Neil BANDMAN, Olga TRIBOULEY, Catherine LAL, Preeti G.
RECIPON, Shirley A.
LU, Dyung Aina M.
BOROWSKY, Mark L.
THORNTON, Michael SWARNAKER Anita THANGAVELU, Kavitha KHAN, Farrah A.
ISON, Craig H.
<120> HUMAN KINASES
<130> PI-0262 PCT
<140> To Be Assigned <141> Herewith <150> 60/242,410; 60/244,068; 60/245,708; 60/247,672; 60/249,565;
60/252,730; 60/250,807 <151> 2000-10-20; 2000-10-27; 2000-11-03; 2000-11-09; 2000-11-16 2000-11-22; 2000-12-O1 <160> 44 <170> PERL Program <210> 1 <211> 337 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7482896CD1 <400> 1 Met Thr Asn Asn Ser Gly Ser Lys Ala Glu Leu Val Val Gly Gly Val Tyr Leu Gly Ile Thr Thr Thr Asn Gly Glu Asp Val Ala Val Lys Leu Glu Ser Gln Lys Val Lys His Pro Gln Leu Leu Tyr Glu Ser Lys Leu Tyr Thr Ile Leu Gln Gly Gly Val Gly Ile Pro His Met His Trp Tyr Gly Gln Glu.Lys Asp Asn Asn Val Leu Val Met Asp Leu Leu Gly Pro Ser Leu Glu Asp Leu Phe Asn Phe Cys Ser Arg Arg Phe Thr Met Lys Thr Val Leu Met Leu Ala Asp Gln Met Ile Ser Arg Ile Glu Tyr Val His Thr Lys Asn Phe Leu His Arg Asp Ile Lys Pro Asp Asn Phe Leu Met Gly Thr Gly Arg His Cys Asn Lys Leu Phe Leu Ile Asp Phe Gly Leu Ala Lys Lys Tyr Arg Asp Asn Arg Thr Arg Gln His Ile Pro Tyr Arg Glu Asp Lys His Leu Ile Gly Thr Val Arg Tyr Ala Ser Ile Asn Ala His Leu Gly Ile Glu Gln Ser Arg Arg Asp Asp Met Glu Ser Leu Gly Tyr Val Phe Met Tyr Phe Asn Arg Thr Ser Leu Pro Trp Gln Gly Leu Arg Ala Met Thr Lys Lys Gln Lys Tyr Glu Lys Ile Ser Glu Lys Lys Met Ser Thr Pro Val Glu Val Leu Cys Lys Gly Phe Pro Ala Glu Phe Ala Met Tyr Leu Asn Tyr Cys Arg Gly Leu Arg Phe Glu Glu Val Pro Asp Tyr Met Tyr Leu Arg Gln Leu Phe Arg Ile Leu Phe Arg Thr Leu Asn His Gln Tyr Asp Tyr Thr Phe Asp Trp Thr Met 290 . 295 300 Leu Lys Gln Lys Ala Ala Gln Gln Ala Ala Ser Ser Ser Gly Gln Gly Gln Gln Ala Gln Thr Gln Thr Gly Lys Gln Thr Glu.Lys Asn Lys Asn Asn Val Lys Asp Asn <210> 2 <211> 475 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7483046CD1 <400> 2 Met Glu Arg Arg Leu Arg Ala Leu Glu Gln Leu Ala Arg Gly Glu Ala Gly Gly Cys Pro Gly Leu Asp Gly Leu Leu Asp Leu Leu Leu Ala Leu His His Glu Leu Ser Ser Gly Pro Leu Arg Arg Glu Arg Ser Val Ala Gln Phe Leu Ser Trp Ala Ser Pro Phe Val Ser Lys Val Lys Glu Leu Arg Leu Gln Arg Asp Asp Phe Glu Ile Leu Lys Val Ile Gly Arg Gly Ala Phe Gly Glu Val Thr Val Val Arg Gln Arg Asp Thr Gly Gln Ile Phe Ala Met Lys Met Leu His Lys Trp Glu Met Leu Lys Arg Ala Glu Thr Ala Cys Phe Arg Glu Glu Arg Asp Val Leu Val Lys Gly Asp Ser Arg Trp Val Thr Thr Leu His Tyr Ala Phe Gln Asp Glu Glu Tyr Leu Tyr Leu Val Met Asp Tyr Tyr Ala Gly Gly Asp Leu Leu Thr Leu Leu Ser Arg Phe Glu Asp Arg Leu Pro Pro Glu Leu Ala Gln Phe Tyr Leu Ala Glu Met Val Leu Ala Ile His Ser Leu His Gln Leu Gly Tyr Val His Arg Asp Val Lys Pro Asp Asn Val Leu Leu Asp Val Asn Gly His Ile Arg Leu Ala Asp Phe Gly Ser Cys Leu Arg Leu Asn Thr Asn Gly Met Val Asp Ser Ser Val Ala Val Gly Thr Pro Asp Tyr Ile Ser Pro Glu Ile Leu Gln Ala Met Glu Glu Gly Lys Gly His Tyr Gly Pro Gln Cys Asp Trp Trp Ser Leu Gly Val Cys Ala Tyr Glu Leu Leu Phe Gly Glu Thr Pro Phe Tyr Ala Glu Ser Leu Val Glu Thr Tyr Gly Lys Ile Met Asn His Glu Asp His Leu Gln Phe Pro Pro Asp Val Pro Asp Val Pro Ala Ser Ala Gln Asp Leu Ile Arg Gln Leu Leu Cys Arg Gln Glu Glu Arg Leu Gly Arg Gly Gly Leu Asp Asp Phe Arg Asn His Pro Phe Phe Glu Gly Val Asp Trp Glu Arg Leu Ala Ser Ser Thr Ala Pro Tyr Ile Pro Glu Leu Arg Gly Pro Met Asp Thr Ser Asn Phe Asp Val Asp Asp Asp Thr Leu Asn His Pro Gly Thr Leu Pro Pro Pro Ser His Gly Ala Phe Ser Gly His His Leu Pro Phe Val Gly Phe Thr Tyr Thr Ser Gly Ser His Ser Pro Glu Ser Ser Ser Glu Ala Trp Ala Ala Leu Glu Arg Lys Leu Gln Cys Leu Glu Gln Glu Lys Val Glu Leu Ser Arg Lys His Gln Glu Ala Leu His Ala Pro Thr Asp His Arg Glu Leu Glu Gln Leu Arg Lys Glu Val Gln Thr Leu Arg Asp Arg Leu Pro Gly Ile Pro Ser Ala His Pro His Pro Leu Leu Glu Phe Leu <210> 3 <211> 675 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 71636374CD1 <400> 3 Met Thr Thr Ser His Met Asn Gly His Val Thr Glu Glu Ser Asp Ser Glu Val Lys Asn Val Asp Leu Ala Ser Pro Glu Glu His Gln Lys His Arg Glu Met Ala Val Asp Cys Pro Gly Asp Leu Gly Thr Arg Met Met Pro Ile Arg Arg Ser Ala Gln Leu Glu Arg Ile Arg Gln Gln Gln Glu Asp Met Arg Arg Arg Arg Glu Glu Glu Gly Lys Lys Gln Glu Leu Asp Leu Asn Ser Ser Met Arg Leu Lys Lys Leu Ala Gln Ile Pro Pro Lys Thr Gly Ile Asp Asn Pro Met Phe Asp Thr Glu Glu Gly Ile Val Leu Glu Ser Pro His Tyr Ala Val Lys Ile Leu Glu Ile Glu Asp Leu Phe Ser Ser Leu Lys His Ile Gln His Thr Leu Val Asp Ser Gln Ser Gln Glu Asp Ile Ser Leu Leu Leu Gln Leu Val Gln Asn Lys Asp Phe Gln Asn Ala Phe Lys Ile His Asn Ala Ile Thr Val His Met Asn Lys Ala Ser Pro Pro Phe Pro Leu Ile Ser Asn Ala Gln Asp Leu Ala Gln Glu Val Gln Thr Val Leu Lys Pro Val His His Lys Glu Gly Gln Glu Leu Thr Ala Leu Leu Asn Thr Pro His Ile Gln Ala Leu Leu Leu Ala His Asp Lys Val Ala Glu Gln Glu Met Gln Leu Glu Pro Ile Thr Asp Glu Arg Val Tyr Glu Ser Ile Gly Gln Tyr Gly Gly Glu Thr Val Lys Ile Val Arg Ile Glu Lys Ala Arg Asp Ile Pro Leu Gly Ala Thr Val Arg Asn Glu Met Asp Ser Val Ile Ile Ser Arg Ile Val Lys Gly Gly Ala Ala Glu Lys Ser Gly Leu Leu His Glu Gly Asp Glu Val Leu Glu Ile Asn Gly Ile Glu Ile Arg Gly Lys Asp Val Asn Glu Val Phe Asp Leu Leu Ser Asp Met His Gly Thr Leu Thr Phe Val Leu Ile Pro Ser Gln Gln Ile Lys Pro Pro Pro Ala Lys Glu Thr Val Ile His Val Lys Ala His Phe Asp Tyr Asp Pro Ser Asp Asp Pro Tyr Val Pro Cys Arg Glu Leu Gly Leu Ser Phe Gln Lys Gly Asp Ile Leu His Val Ile Ser Gln Glu Asp Pro Asn Trp Trp Gln Ala Tyr Arg Glu Gly Asp Glu Asp Asn Gln Pro Leu Ala Gly Leu Val Pro Gly Lys Ser Phe Gln Gln Gln Arg Glu Ala Met Lys Gln Thr Ile Glu Glu Asp Lys Glu Pro,Glu Lys Ser Gly Lys Leu Trp Cys Ala Lys Lys Asn Lys Lys Lys Arg Lys Lys Val Leu Tyr Asn Ala Asn Lys Asn Asp Asp Tyr Asp Asn Glu Glu Ile Leu Thr Tyr Glu Glu Met Ser Leu Tyr His Gln Pro Ala Asn Arg Lys Arg Pro Ile Ile Leu Ile Gly Pro Gln Asn Cys Gly Gln Asn Glu Leu Arg Gln Arg Leu Met Asn Lys Glu Lys Asp Arg Phe Ala Ser Ala Val Pro His Thr Thr Arg Ser Arg Arg Asp Gln Glu Val Ala Gly Arg Asp Tyr His Phe Val Ser Arg Gln Ala Phe Glu Ala Asp Ile Ala Ala Gly Lys Phe Ile Glu His Gly Glu Phe Glu Lys Asn Leu Tyr Gly Thr Ser Ile Asp Ser Val Arg Gln Val Ile Asn Ser Gly Lys Ile Cys Leu Leu Ser Leu Arg Thr Gln Ser Leu Lys Thr Leu Arg Asn Ser Asp Leu Lys Pro Tyr Ile Ile Phe Ile Ala Pro Pro Ser Gln Glu Arg Leu Arg Ala Leu Leu Ala Lys Glu Gly Lys Asn Pro Lys Pro Glu Glu Leu Arg Glu Ile Ile Glu Lys Thr Arg Glu Met Glu Gln Asn Asn Gly His Tyr Phe Asp Thr Ala Ile Val Asn Ser Asp Leu Asp Lys Ala Tyr Gln Glu Leu Leu Arg Leu Ile Asn Lys Leu Asp Thr Glu Pro Gln Trp Val Pro Ser Thr Trp Leu Arg <210> 4 <211> 835 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7480597CD1 <400> 4 Met Ala Glu Gly Lys Glu Gly Gln Val Pro Ser Tyr Met Asp Gly Ser Arg Gln Arg Glu Asn Glu Glu Asp Ala Lys Ala Glu Thr Pro Asp Val Thr Ile Arg Ser Tyr Glu Ile Tyr Ser Leu Pro Trp Asn Arg Gln Gln Gly Leu Cys Asp His Ser Leu Lys Tyr Leu Ser Ser Arg Ile Thr Glu Arg Lys Leu Gln Gly Ser Trp Leu Pro Ala Ser Arg Gly A~sn Leu Glu Lys Pro Phe Leu Gly Pro Arg Gly Pro Val Val Pro Leu Phe Cys Pro Arg Asn Gly Leu His Ser Ala His Pro Glu Asn Ser Pro Leu Lys Pro Arg Val Val Thr Val Val Lys Leu Gly Gly Gln Arg Pro Arg Lys Ile Thr Leu Leu Leu Asn Arg Arg Ser Val Gln Thr Phe Glu Gln Leu Leu Ala Asp Ile Ser Glu Ala Leu Gly Ser Pro Arg Trp Lys Asn Asp Arg Val Arg Lys Leu Phe Asn Leu Lys Gly Arg Glu Ile Arg Ser Val Ser Asp Phe Phe Arg Glu Gly Asp Ala Phe Ile Ala Met Gly Lys Glu Pro Leu Thr Leu Lys Ser Ile Gln Val Ala Val Glu Glu Leu Tyr Pro Asn Lys Ala Arg Ala Leu Thr Leu Ala Gln His Ser Arg Ala Pro Ser Pro Arg Leu Arg Ser Arg Leu Phe Ser Lys Ala Leu Lys Gly Asp His Arg Cys Gly Glu Thr Glu Thr Pro Lys Ser Cys Ser Glu Val Ala Gly Cys Lys Ala Ala Met Arg His Gln Gly Lys Ile Pro Glu Glu Leu Ser Leu Asp Asp Arg Ala Arg Thr Gln Lys Lys Trp Gly Arg Gly Lys Trp Glu Pro Glu Pro Ser Ser Lys Pro Pro Arg Glu Ala Thr Leu Glu Glu Arg His Ala Arg Gly Glu Lys His Leu Gly Val Glu Ile Glu Lys Thr Ser Gly Glu Ile Ile Arg Cys Glu Lys Cys Lys Arg Glu Arg Glu Leu Gln Gln Ser Leu Glu Arg Glu Arg Leu Ser Leu Gly Thr Ser Glu Leu Asp Met Gly Lys Gly Pro Met Tyr Asp Val Glu Lys Leu Val Arg Thr Arg Ser Cys Arg Arg Ser Pro Glu Ala Asn Pro Ala Ser Gly Glu Glu Gly Trp Lys Gly Asp Ser His Arg Ser Ser Pro Arg Asn Pro Thr Gln Glu Leu Arg Arg Pro Ser Lys Ser Met Asp Lys Lys Glu Asp Arg Gly Pro Glu Asp Gln Glu Ser His Ala Gln Gly Ala Ala Lys Ala Lys Lys Asp Leu Val Glu Val Leu Pro Val Thr Glu Glu Gly Leu Arg Glu Val Lys Lys Asp Thr Arg Pro Met Ser Arg Ser Lys His Gly Gly Trp Leu Leu Arg Glu His Gln Ala Gly Phe Glu Lys Leu Arg Arg Thr Arg Gly Glu Glu Lys Glu Ala Glu Lys Glu Lys Lys Pro Cys Met Ser Gly Gly Arg Arg Met Thr Leu Arg Asp Asp Gln Pro Ala Lys Leu Glu Lys Glu Pro Lys Thr Arg Pro Glu Glu Asn Lys Pro Glu Arg Pro Ser Gly Arg Lys Pro Arg Pro Met Gly Ile Ile Ala Ala Asn Val Glu Lys His Tyr Glu Thr Gly Arg Val Ile Gly Asp Gly Asn Phe Ala Val Val Lys Glu Cys Arg His Arg Glu Thr Arg Gln Ala Tyr Ala Met Lys Ile Ile Asp Lys Ser Arg Leu Lys Gly Lys Glu Asp Met Val Asp Ser Glu Ile Leu Ile Ile Gln Ser Leu Ser His Pro Asn Ile Val Lys Leu His Glu Val Tyr Glu Thr Asp Met Glu Ile Tyr Leu Ile Leu Glu Tyr Val Gln Gly Gly Asp Leu Phe Asp Ala Ile Ile Glu Ser Val Lys Phe Pro Glu Pro Asp Ala Ala Leu Met Ile Met Asp Leu Cys Lys Ala Leu Val His Met His Asp Lys Ser Ile Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Val Gln Arg Asn Glu Asp Lys Ser Thr Thr Leu Lys Leu Ala Asp Phe Gly Leu Ala Lys His Val Val Arg Pro Ile Phe Thr Val Cys Gly Thr Pro Thr Tyr Val Ala Pro Glu Ile Leu Ser Glu Lys Gly Tyr Gly Leu Glu Val Asp Met Trp Ala Ala Gly Val Ile Leu Tyr Ile Leu Leu Cys Gly Phe Pro Pro Phe Arg Ser Pro Glu Arg Asp Gln Asp Glu Leu Phe Asn Ile Ile Gln Leu Gly His Phe Glu Phe Leu Pro Pro Tyr Trp Asp Asn Ile Ser Asp Ala Ala Lys Asp Leu Val Ser Arg Leu Leu Val Val Asp Pro Lys Lys Arg Tyr Thr Ala His Gln Val Leu Gln His Pro Trp Ile Glu Thr Ala Gly Lys Thr Asn Thr Val Lys Arg Gln Lys Gln Val Ser Pro Ser Ser Glu Gly His Phe Arg Ser Gln His Lys Arg Val Val Glu Gln Val Ser <210> 5 <211> 373 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3227248CD1 <400> 5 Met Lys Leu Ile Asn Gly Lys Lys Gln Thr Phe Pro Trp Phe Gly Met Asp Ile Gly Gly Thr Leu Val Lys Leu Val Tyr Phe Glu Pro Lys Asp Ile Thr Ala Glu Glu Glu Gln Glu Glu Val Glu Asn Leu Lys Ser Ile Arg Lys Tyr Leu Thr Ser Asn Thr Ala Tyr Gly Lys Thr Gly Ile Arg Asp Val His Leu Glu Leu Lys Asn Leu Thr Met Cys Gly Arg Lys Gly Asn Leu His Phe Ile Arg Phe Pro Ser Cys Ala Met His Arg Phe Ile Gln Met Gly Ser Glu Lys Asn Phe Ser Ser Leu His Thr Thr Leu Cys Ala Thr Gly Gly Gly Ala Phe Lys Phe Glu Glu Asp Phe Arg Met Ile Ala Asp Leu Gln Leu His Lys Leu Asp Glu Leu Asp Cys Leu Ile Gln Gly Leu Leu Tyr Val Asp Ser Val Gly Phe Asn Gly Lys Pro Glu Cys Tyr Tyr Phe Glu Asn Pro Thr Asn Pro Glu Leu Cys Gln Lys Lys Pro Tyr Cys Leu Asp Asn Pro Tyr Pro Met Leu Leu Val Asn Met Gly Ser Gly Val Ser Ile Leu Ala Val Tyr Ser Lys Asp Asn Tyr Lys Arg Val Thr Gly Thr Ser Leu Gly Gly Gly Thr Phe Leu Gly Leu Cys Cys Leu Leu Thr Gly Cys Glu Thr Phe Glu Glu Ala Leu Glu Met Ala Ala Lys Gly Asp Ser Thr Asn Val Asp Lys Leu Val Lys Asp Ile Tyr Gly Gly Asp Tyr Glu Arg Phe Gly Leu Gln Gly Ser Ala Val Ala Ser Ser Phe Gly Asn Met Met Ser Lys Glu Lys Arg Asp Ser Ile Ser Lys Glu Asp Leu Ala Arg Ala Thr Leu Val Thr Ile Thr Asn Asn Ile Gly Ser Ile Ala Arg Met Cys Ala Leu Asn Glu Asn Ile Asp Arg Val Val Phe Val Gly Asn Phe Leu Arg Ile Asn Met Val Ser Met Lys Leu Leu Ala Tyr Ala Met Asp Phe Trp Ser Lys Gly Gln Leu Lys Ala Leu Phe Leu Glu His Glu Gly Tyr Phe Gly Ala Val Gly Ala Leu Leu Glu Leu Phe Lys Met Thr Asp Asp Lys <210> 6 <211> 735 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 4207273CD1 <400> 6 Met Pro Gln Ile Ala Lys Lys Gln Ser Thr His Arg Thr Gln Lys Pro Lys Lys Gln Ser Phe Pro Cys Ile Cys Lys Asn Pro Gly Thr Gln Lys Ser Cys Val Pro Leu Ser Val Gln Pro Thr Glu Pro Arg Leu Asn Tyr Leu Asp Leu Lys Tyr Ser Asp Met Phe Lys Glu Ile Asn Ser Thr Ala Asn Gly Pro Gly Ile Tyr Glu Met Phe Gly Thr Pro Val Tyr Cys His Val Arg Glu Thr Glu Arg Asp Glu Asn Thr Tyr Tyr Arg Glu Ile Cys Ser Ala Pro Ser Gly Arg Arg Ile Thr Asn Lys Cys Arg Ser Ser His Ser Glu Arg Lys Ser Asn Ile Arg Thr Arg Leu Ser Gln Lys Lys Thr His Met Lys Cys Pro Lys Thr Ser Phe Gly Ile Lys Gln Glu His Lys Val Leu Ile Ser Lys Glu Lys Ser Ser Lys Ala Val His Ser Asn Leu His Asp Ile Glu Asn Gly Asp Gly Ile Ser Glu Pro Asp Trp Gln Ile Lys Ser Ser Gly Asn Glu Phe Leu Ser Ser Lys Asp Glu Ile His Pro Met Asn Leu Ala Gln Thr Pro Glu Gln Ser Met Lys Gln Asn Glu Phe Pro Pro Val Ser Asp Leu Ser Ile Val Glu Glu Val Ser Met Glu Glu Ser Thr Gly Asp Arg Asp Ile Ser Asn Asn Gln Ile Leu Thr Thr Ser Leu Arg Asp Leu Gln Glu Leu Glu Glu Leu His His Gln Ile Pro Phe Ile Pro Ser Glu Asp Ser Trp Ala Val Pro Ser Glu Lys Asn Ser Asn Lys Tyr Val Gln Gln Glu Lys Gln Asn Thr Ala Ser Leu Ser Lys Val Asn Ala Ser Arg Ile Leu Thr Asn Asp Leu Glu Phe Asp Ser Val Ser Asp His Ser Lys Thr Leu Thr Asn Phe Ser Phe Gln Ala Lys Gln Glu Ser Ala Ser Ser Gln Thr Tyr Gln Tyr Trp Val His Tyr Leu Asp His Asp Ser Leu Ala Asn Lys Ser Ile Thr Tyr Gln Met Phe Gly Lys Thr Leu Ser Gly Thr Asn Ser Ile Ser Gln Glu Ile Met Asp Ser Val Asn Asn Glu Glu Leu Thr Asp Glu Leu Leu Gly Cys Leu Ala Ala Glu Leu Leu Ala Leu Asp Glu Lys Asp Asn Asn Ser Cys Gln Lys Met Ala Asn Glu Thr Asp Pro Glu Asn Leu Asn Leu Val Leu Arg Trp Arg Gly Ser Thr Pro Lys Glu Met Gly Arg Glu Thr Thr Lys Val Lys Ile Gln Arg His Ser Ser Gly Leu Arg Ile Tyr Asp Arg Glu Glu Lys Phe Leu Ile Ser Asn Glu Lys Lys Ile Phe Ser Glu Asn Ser Leu Lys Ser Glu Glu Pro Ile Leu Trp Thr Lys Gly Glu Ile Leu Gly Lys Gly Ala Tyr Gly Thr Val Tyr Cys Gly Leu Thr Ser Gln Gly Gln Leu Ile Ala Val Lys Gln Val Ala Leu Asp Thr Ser Asn Lys Leu Ala Ala Glu Lys Glu Tyr Arg Lys Leu Gln Glu Glu Val Asp Leu Leu Lys Ala Leu Lys His Val Asn Ile Val Ala Tyr Leu Gly Thr Cys Leu Gln Glu Asn Thr Val Ser Ile Phe Met Glu Phe Val Pro Gly Gly Ser Ile Ser Ser Ile Ile Asn Arg Phe Gly Pro Leu Pro Glu Met Val Phe Cys Lys Tyr Thr Lys Gln Ile Leu Gln Gly Val Ala Tyr Leu His Glu Asn Cys Val Val His Arg Asp Ile Lys Gly Asn Asn Val Met Leu Met Pro Thr Gly Ile Ile Lys Leu Ile Asp Phe Gly Cys Ala Arg Arg Leu Ala Trp Ala Gly Leu Asn Gly Thr His Ser Asp Met Leu Lys Ser Met His Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Ile Asn Glu Ser Gly Tyr Gly Arg Lys Ser Asp Ile Trp Ser Ile Gly Cys Thr Val Phe Glu Met Ala Thr Gly Lys Pro Pro Leu Ala Ser Met Asp Arg Met Ala Ala Met Phe Tyr Ile Gly Ala His Arg Gly Leu Met Pro Pro Leu Pro Asp His Phe Ser Glu Asn Ala Ala Asp Phe Val Arg Met Cys Leu Thr Arg Asp Gln His Glu Arg Pro Ser Ala Leu Gln Leu Leu Lys His Ser Phe Leu Glu Arg Ser His <210> 7 <211> 506 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7483334CD1 <400> 7 Met Asp Asp Tyr Met Val Leu Arg Met Ile Gly Glu Gly Ser Phe Gly Arg Ala Leu Leu Val Gln Leu Glu Ser Ser Asn Gln Met Phe Ala Met Lys Glu Ile Arg Leu Pro Lys Ser Phe Ser Asn Thr Gln Asn Ser Arg Lys Glu Ala Val Leu Leu Ala Lys Met Lys His Pro Asn Ile Val Ala Phe Lys Glu Ser Phe Glu Ala Glu Gly His Leu Tyr Ile Val Met Glu Tyr Cys Asp Gly Gly Asp Leu Met Gln Lys Ile Lys Gln Gln Lys Gly Lys Leu Phe Pro Glu Asp Met Ile Leu Asn Trp Phe Thr Gln Met Cys Leu Gly Val Asn His Ile His Lys Lys Arg Val Leu His Arg Asp Ile Lys Ser Lys Asn Ile Phe Leu Thr Gln Asn Gly Lys Val Lys Leu Gly Asp Phe Gly Ser Ala Arg Leu Leu Ser Asn Pro Met Ala Phe Ala Cys Thr Tyr Val Gly Thr Pro Tyr Tyr Val Pro Pro Glu Ile Trp Glu Asn Leu Pro Tyr Asn Asn Lys Ser Asp Ile Trp Ser Leu Gly Cys Ile Leu Tyr Glu Leu Cys Thr Leu Lys His Pro Phe Gln Ala Asn Ser Trp Lys Asn Leu Ile Leu Lys Val Cys Gln Gly Cys Ile Ser Pro Leu Pro Ser His Tyr Ser Tyr Glu Leu Gln Phe Leu Val Lys Gln Met Phe Lys Arg Asn Pro Ser His Arg Pro Ser Ala Thr Thr Leu Leu Ser Arg Gly Ile Val Ala Arg Leu Val Gln Lys Cys Leu Pro Pro Glu Ile Ile Met Glu Tyr Gly Glu Glu Val Leu Glu Glu Ile Lys Asn Ser Lys His Asn Thr Pro Arg Lys Lys Thr Asn Pro Ser Arg Ile Arg Ile Ala Leu Gly Asn Glu Ala Ser Thr Val Gln Glu Glu Glu Gln Asp Arg Lys Gly Ser His Thr Asp Leu Glu Ser Ile Asn Glu Asn Leu Val Glu Ser Ala Leu Arg Arg Val Asn Arg Glu Glu Lys Gly Asn Lys Ser Val His Leu Arg Lys Ala Ser Ser Pro Asn Leu His Arg Arg Gln Trp Glu Lys Asn Val Pro Asn Thr Ala Leu Thr Ala Leu Glu Asn Ala Ser Ile Leu Thr Ser Ser Leu Thr Ala Glu Asp Asp Arg Gly Gly Ser Val Ile Lys Tyr Ser Lys Asn Thr Thr Arg Lys Gln Trp Leu Lys Glu Thr Pro Asp Thr Leu Leu Asn Ile Leu Lys Asn Ala Asp Leu Ser Leu Ala Phe Gln Thr Tyr Thr Ile Tyr Arg Pro Gly Ser Glu Gly Phe Leu Lys Gly. Pro Leu Ser Glu Glu Thr Glu Ala Ser Asp Ser Val Asp Gly Gly His Asp Ser Val Ile Leu Asp Pro Glu Arg Leu Glu Pro Gly Leu Asp Glu Glu Asp Thr Asp Phe Glu Glu Glu Asp Asp Asn Pro Asp Trp Val Ser Glu Leu Lys Lys Arg Ala Gly Trp Gln Gly Leu Cys Asp Arg <210> 8 <211> 2014 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7483337CD1 <400> 8 Met Glu Thr Leu Asn Gly Ala Gly Asp Thr Gly Gly Lys Pro Ser Thr Arg Gly Gly Asp Pro Ala Ala Arg Ser Arg Arg Thr Glu Gly Ile Arg Ala Ala Tyr Arg Arg Gly Asp Arg Gly Gly Ala Arg Asp Leu Leu Glu Glu Ala Cys Asp Gln Cys Ala Ser Gln Leu Glu Lys Gly Gln Leu Leu Ser Ile Pro Ala Ala Tyr Gly Asp Leu Glu Met Val Arg Tyr Leu Leu Ser Lys Arg Leu Val Glu Leu Pro Thr Glu Pro Thr Asp Asp Asn Pro Ala Val Val Ala Ala Tyr Phe Gly His Thr Ala Val Val Gln Asn Thr Leu Pro Thr Glu Pro Thr Asp Asp Asn Pro Ala Val Val Ala Ala Tyr Phe Gly His Thr Ala Val Val Gln Glu Leu Leu Glu Ser Leu Pro Gly Pro Cys Ser Pro Gln Arg Leu Leu Asn Trp Met Leu Ala Leu Ala Cys Gln Arg Gly His Leu Gly Val Val Lys Leu Leu Val Leu Thr His Gly Ala Asp Pro Glu Ser Tyr Ala Val Arg Lys Asn Glu Phe Pro Val Ile Val Arg Leu Pro Leu Tyr Ala Ala Ile Lys Ser Gly Asn Glu Asp Ile Ala Ile Phe Leu Leu Arg His Gly Ala Tyr Phe Cys Ser Tyr Ile Leu Leu Asp Ser Pro Asp Pro Ser Lys His Leu Leu Arg Lys Tyr Phe Ile Glu Ala Ser Pro Leu Pro Ser Ser Tyr Pro Gly Lys Thr Ala Leu Arg Val Lys Trp Ser His Leu Arg Leu Pro Trp Val Asp Leu Asp Trp Leu Ile Asp Ile Ser Cys Gln Ile Thr Glu Leu Asp Leu Ser Ala Asn Cys Leu Ala Thr Leu Pro Ser Val Ile Pro Trp Gly Leu Ile Asn Leu Arg Lys Leu Asn Leu Ser Asp Asn His Leu Gly Glu Leu Pro Gly Val Gln Ser Ser Asp Glu Ile Ile Cys Ser Arg Leu Leu Glu Ile Asp Ile Ser Ser Asn Lys Leu Ser His Leu Pro Pro Gly Phe Leu His Leu Ser Lys Leu Gln Lys Leu Thr Ala Ser Lys Asn Cys Leu Glu Lys Leu Phe Glu Glu Glu Asn Ala Thr Asn Trp Ile Gly Leu Arg Lys Leu Gln Glu Leu Asp Ile Ser Asp Asn Lys Leu Thr Glu Leu Pro Ala Leu Phe Leu His Ser Phe Lys Ser Leu Asn Ser Leu Asn Val Ser Arg Asn Asn Leu Lys Val Phe Pro Asp Pro Trp Ala Cys Pro Leu Lys Cys Cys Lys Ala Ser Arg Asn Ala Leu Glu Cys Leu Pro Asp Lys Met Ala Val Phe Trp Lys Asn His Leu Lys Asp Val Asp Phe Ser Glu Asn Ala Leu Lys Glu Val Pro Leu Gly Leu Phe Gln Leu Asp Ala Leu Met Phe Leu Arg Leu Gln Gly Asn Gln Leu Ala Ala Leu Pro Pro Gln Glu Lys Trp Thr Cys Arg Gln Leu Lys Thr Leu Asp Leu Ser Arg Asn Gln Leu Gly Lys Asn Glu Asp Gly Leu Lys Thr Lys Arg Ile Ala Phe Phe Thr Thr Arg Gly Arg Gln Arg Ser Gly Thr Glu Ala Glu Thr Thr Met Glu Phe Ser Ala Ser Leu Val Thr Ile Val Phe Leu Ser Asn Asn Cys Asn Leu Cys Ala Tyr Thr Cys Ala Ala Ser Val Leu Glu Phe Pro Ala Phe Leu Ser Glu Ser Leu Glu Val Leu Cys Leu Asn Asp Asn His Leu Asp Thr Val Pro Pro Ser Val Cys Leu Leu Lys Ser Leu Ser Glu Leu Tyr Leu Gly Asn Asn Pro Gly Leu Arg Glu Leu Pro Pro Glu Leu Gly Gln Leu Gly Asn Leu Trp Gln Leu Asp Thr Glu Asp Leu Thr Ile Ser Asn Val Pro Ala Glu Ile Gln Lys Glu Gly Pro Lys Ala Met Leu Ser Tyr Leu Arg Ala Gln Leu Arg Lys Ala Glu Lys Cys Lys Leu Met Lys Met Ile Ile Val Gly Pro Pro Arg Gln Gly Lys Ser Thr Leu Leu Glu Ile Leu Gln Thr Gly Arg Ala Pro Gln Val Val His Gly Glu Ala Thr Ile Arg Thr Thr Lys Trp Glu Leu Gln Arg Pro Ala Gly Ser Arg Ala Lys Val Lys Asp Gly Leu Arg Ala Glu Ser Leu Trp Val Glu Ser Val Glu Phe Asn Val Trp Asp Ile Gly Gly Pro Ala Ser Met Ala Thr Val Asn Gln Cys Phe Phe Thr Asp Lys Ala Leu Tyr Val Val.Va1 Trp Asn Leu Ala Leu Gly Glu Glu Ala Val Ala Asn Leu Gln Phe Trp Leu Leu Asn Ile Glu Ala Lys Ala Pro Asn Ala Val Val Leu Val Val Gly Thr His Leu Asp Leu Ile Glu Ala Lys Phe Arg Val Glu Arg Ile Ala Thr Leu Arg Ala Tyr Val Leu Ala Leu Cys Arg Ser Pro Ser Gly Ser Arg Ala Thr Gly Phe Pro Asp Ile Thr Phe Lys His Leu His Glu Ile Ser Cys Lys Ser Leu Glu Gly Gln Glu Gly Leu Arg Gln Leu Ile Phe His Val Thr Cys Ser Met Lys Asp Val Gly Ser Thr Ile Gly Cys Gln Arg Leu Ala Gly Arg Leu Ile Pro Arg Ser Tyr Leu Ser Leu Gln Glu Ala Val Leu Ala Glu Gln Gln Arg Arg Ser Arg Asp Asp Asp Val Gln Tyr Leu Thr Asp Arg Gln Leu Glu Gln Leu Val Glu Gln Thr Pro Asp Asn Asp Ile Lys Asp Tyr Glu Asp Leu Gln Ser Ala Ile Ser Phe Leu Ile Glu Thr Gly Thr Leu Leu His Phe Pro Asp Thr Ser His Gly Leu Arg Asn Leu Tyr Phe Leu Asp Pro Ile Trp Leu Ser Glu Cys Leu Gln Arg Ile Phe Asn Ile Lys Gly Ser Arg Ser Val Ala Lys Asn Gly Val Ile Arg Ala Glu Asp Leu Arg Met Leu Leu Val Gly Thr Gly Phe Thr Gln Gln Thr Glu Glu Gln Tyr Phe Gln Phe Leu Ala Lys Phe Glu Ile Ala Leu Pro Val Ala Asn Asp Ser Tyr Leu Leu Pro His Leu Leu Pro Ser Lys Pro Gly Leu Asp Thr His Gly Met Arg His Pro Thr Ala Asn Thr Ile Gln Arg Val Phe Lys Met Ser Phe Val Pro Val Gly Phe Trp Gln Arg Phe Ile Ala Arg Met Leu Ile Ser Leu Ala Glu Met Asp Leu Gln Leu Phe Glu Asn Lys Lys Asn Thr Lys Ser Arg Asn Arg Lys Val Thr Ile Tyr Ser Phe Thr Gly Asn Gln Arg Asn Arg 1100 110.5 1110 Cys Ser Thr Phe Arg Val Lys Arg Asn Gln Thr Ile Tyr Trp Gln Glu Gly Leu Leu Val Thr Phe Asp Gly Gly Tyr Leu Ser Val Glu Ser Ser Asp Val Asn Trp Lys Lys Lys Lys Ser Gly Gly Met Lys Ile Val Cys Gln Ser Glu Val Arg Asp Phe Ser Ala Met Ala Phe Ile Thr Asp His Val Asn Ser Leu Ile Asp Gln Trp Phe Pro Ala Leu Thr Ala Thr Glu Ser Asp Gly Thr Pro Leu Met Glu Gln Tyr Val Pro Cys Pro Val Cys Glu Thr Ala Trp Ala Gln His Thr Asp Pro Ser Glu Lys Ser Glu Asp Val Gln Tyr Phe Asp Met Glu Asp Cys Val Leu Thr Ala Ile Glu Arg Asp Phe Ile Ser Cys Pro Arg His Pro Asp Leu Pro Val Pro Leu Gln Glu Leu Val Pro Glu Leu Phe Met Thr Asp Phe Pro Ala Arg Leu Phe Leu Glu Asn Ser Lys Leu Glu His Ser Glu Asp Glu Gly Ser Val Leu Gly Gln Gly Gly Ser Gly Thr Val Ile Tyr Arg Ala Arg Tyr Gln Gly Gln Pro Val Ala Val Lys Arg Phe His Ile Lys Lys Phe Lys Asn Phe Ala Asn Val Pro Ala Asp Thr Met Leu Arg His Leu Arg Ala Thr Asp Ala Met Lys Asn Phe Ser Glu Phe Arg Gln Glu Ala Ser Met Leu His Ala Leu Gln His Pro Cys Ile Val Ala Leu Ile Gly Ile Ser Ile His Pro Leu Cys Phe Ala Leu Glu Leu Ala Pro Leu Ser Ser Leu Asn Thr Val Leu Ser Glu Asn Ala Arg Asp Ser Ser Phe Ile Pro Leu Gly His Met Leu Thr Gln Lys Ile Ala Tyr Gln Ile Ala Ser Gly Leu Ala Tyr Leu His Lys Lys Asn Ile Ile Phe Cys Asp Leu Lys Ser Asp Asn Ile Leu Val Trp Ser Leu Asp Val Lys Glu His Ile Asn Ile Lys Leu Ser Asp Tyr Gly Ile Ser Arg Gln Ser Phe His Glu Gly Ala Leu Gly Val Glu Gly Thr Pro Gly Tyr Gln Ala Pro Glu Ile Arg Pro Arg Ile Val Tyr Asp Glu Lys Val Asp Met Phe Ser Tyr Gly Met Val Leu Tyr Glu Leu Leu Ser Gly Gln Arg Pro Ala Leu Gly His His Gln Leu Gln Ile Ala Lys Lys Leu Ser Lys Gly Ile Arg Pro Val Leu Gly Gln Pro Glu Glu Val Gln Phe Arg Arg Leu Gln Ala Leu Met Met Glu Cys Trp Asp Thr Lys Pro Glu Lys Arg Pro Leu Ala Leu Ser Val Val Ser Gln Met Lys Asp Pro Thr Phe Ala Thr Phe Met Tyr Glu Leu Cys Cys Gly Lys Gln Thr Ala Phe Phe Ser Ser Gln Gly Gln Glu Tyr Thr Val Val Phe Trp Asp Gly Lys Glu Glu Ser Arg Asn Tyr Thr Val Val Asn Thr Glu Lys Gly Leu Met Glu Val Gln Arg Met Cys Cys Pro Gly Met Lys Val Ser Cys Gln Leu Gln Val Gln Arg Ser Leu Trp Thr Ala Thr Glu Asn Ser Tyr Leu Val Leu Ala Gly Leu Ala Asp Gly Leu Val Ala Val Phe Pro Val Val Arg Gly Thr Pro Lys Asp Ser Cys Ser Tyr Leu Cys Ser His Thr Ala Asn Arg Ser Lys Phe Ser Ile Ala Asp Glu Asp Ala Arg Gln Asn Pro Tyr Pro Val Lys Ala Met Glu Val Val Asn Ser Gly Ser Glu Val Trp Tyr Ser Asn Gly Pro Gly Leu Leu Val Ile Asp Cys Ala Ser Leu Glu Ile Cys Arg Arg Leu Glu Pro Tyr Met Ala Pro Ser Met Val Thr Ser Val Val Cys Ser Ser Glu Gly Arg Gly Glu Glu Val Val Trp Cys Leu Asp Asp Lys Ala Asn Ser Leu Val Met Tyr His Ser Thr Thr Tyr Gln Leu Cys Ala Arg Tyr Phe Cys Gly Val Pro Ser Pro Leu Arg Asp Met Phe Pro Val Arg Pro Leu Asp Thr Glu Pro Pro Ala Ala Ser His Thr Ala Asn Pro Lys Val Pro Glu Gly Asp Ser Ile Ala Asp Val Ser Ile Met Tyr Ser Glu Glu Leu Gly Thr Gln Ile Leu Ile His Gln Glu Ser Leu Thr Asp Tyr Cys Ser Met Ser Ser Tyr Ser Ser Ser Pro Pro Arg Gln Ala Ala Arg Ser Pro Ser Ser Leu Pro Ser Ser Pro Ala Ser Ser Ser Ser Val Pro Phe Ser Thr Asp Cys Glu Asp Ser Asp Met Leu His Thr Pro Gly Ala Ala Ser Asp Arg Ser Glu His Asp Leu Thr Pro Met Asp Gly Glu Thr Phe Ser Gln His Leu Gln Ala Val Lys Ile Leu Ala Val Arg Asp Leu Ile Trp Val Pro Arg Arg Gly Gly Asp Val Ile Val Ile Gly Leu Glu Lys Asp Ser Gly Ala Gln Arg Gly Arg Val Ile Ala Val Leu Lys Ala Arg Glu Leu Thr Pro His Gly Val Leu Val Asp Ala Ala Val Val Ala Lys Asp Thr Val Val Cys Thr Phe Glu Asn Glu Asn Thr Glu Trp Cys Leu Ala Val Trp Arg Gly Trp Gly Ala Arg Glu Phe Asp Ile Phe Tyr Gln Ser Tyr Glu Glu Leu Gly Arg Leu Glu Ala Cys Thr Arg Lys Arg Arg <210> 9 <211> 348 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 6035509CD1 <400> 9 -Met Met Leu Gly Leu Glu Ser Leu Pro Asp Pro Thr Asp Thr Trp Glu Ile Ile Glu Thr Ile Gly Lys Gly Thr Tyr Gly Lys Val Tyr Lys Val Thr Asn Lys Arg Asp Gly Ser Leu Ala Ala Val Lys Ile Leu Asp Pro Val Ser Asp Met Asp Glu Glu Ile Glu Ala Glu Tyr Asn Ile Leu Gln Phe Leu Pro Asn His Pro Asn Val Val Lys Phe Tyr Gly Met Phe Tyr Lys Ala Asp His Cys Val Gly Gly Gln Leu Trp Leu Val Leu Glu Leu Cys Asn Gly Gly Ser Val Thr Glu Leu Val Lys Gly Leu Leu Arg Cys Gly Gln Arg Leu Asp Glu Ala Met Ile Ser Tyr Ile Leu Tyr Gly Ala Leu Leu Gly Leu Gln His Leu His Asn Asn Arg Ile Ile His Arg Asp Val Lys Gly Asn Asn Ile Leu Leu Thr Thr Glu Gly Gly Val Lys Leu Val Asp Phe Gly Val Ser Ala Gln Leu Thr Ser Thr Arg Leu Arg Arg Asn Thr Ser Val Gly Thr Pro Phe Trp Met Ala Pro Glu Val Ile Ala Cys Glu Gln Gln Tyr Asp Ser Ser Tyr Asp Ala Arg Cys Asp Val Trp Ser Leu Gly Ile Thr Ala Ile Glu Leu Gly Asp Gly Asp Pro Pro Leu Phe Asp Met His Pro Val Lys Thr Leu Phe Lys Ile Pro Arg Asn Pro Pro Pro Thr Leu Leu His Pro Glu Lys Trp Cys Glu Glu Phe Asn His Phe Ile Ser Gln Cys Leu Ile Lys Asp Phe Glu Arg Arg Pro Ser Val Thr His Leu Leu Asp His Pro Phe Ile Lys Gly Val His Gly Lys Val Leu Phe Leu Gln Lys Gln Leu Ala Lys Val Leu Gln Asp Gln Lys His Gln Asn Pro Val Ala Lys Thr Arg His Glu Arg Met His Thr Arg Arg Pro Tyr His Val Glu Asp Ala Glu Lys Tyr Cys Leu Glu Asp Asp Leu Val Asn Leu Glu Val Leu Asp Glu Val Leu Asn Ile <210> 10 <211> 2042 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7373485CD1 <400> 10 Met Ala Thr Asp Asp Lys Thr Ser Pro Thr Leu Asp Ser Ala Asn Asp Leu Pro Arg Ser Pro Thr Ser Pro Ser His Leu Thr His Phe Lys Pro Leu Thr Pro Asp Gln Asp Glu Pro Pro Phe Lys Ser Ala Tyr Ser Ser Phe Val Asn Leu Phe Arg Phe Asn Lys Glu Arg Ala ~ 50 55 60 Glu Gly Gly Gln Gly Glu Gln Gln Pro Leu Ser Gly Ser Trp Thr Ser Pro Gln Leu Pro Ser Arg Thr Gln Ser Val Arg Ser Pro Thr Pro Tyr Lys Lys Gln Leu Asn Glu Glu Leu Gln Arg Arg Ser Ser Ala Leu Asp Thr Arg Arg Lys Ala Glu Pro Thr Phe Gly Gly His Asp Pro Arg Thr Ala Val Gln Leu Arg Ser Leu Ser Thr Val Leu Lys Arg Leu Lys Glu Ile Met Glu Gly Lys Ser Gln Asp Ser Asp Leu Lys Gln Tyr Trp Met Pro Asp Ser Gln Cys Lys Glu Cys Tyr Asp Cys Ser Glu Lys Phe Thr Thr Phe Arg Arg Arg His His Cys Arg Leu Cys Gly Gln Ile Phe Cys Ser Arg Cys Cys Asn Gln Glu Ile Pro Gly Lys Phe Met Gly Tyr Thr Gly Asp Leu Arg Ala Cys Thr Tyr Cys Arg Lys Ile Ala Leu Ser Tyr Ala His Ser Thr Asp Ser Asn Ser Ile Gly Glu Asp Leu Asn Ala Leu Ser Asp Ser Ala Cys Ser Val Ser Val Leu Asp Pro Ser Glu Pro Arg Thr Pro Val Gly Ser Arg Lys Ala Ser Arg Asn Ile Phe Leu Glu Asp Asp Leu Ala Trp Gln Ser Leu Ile His Pro Asp Ser Ser Asn Thr Pro Leu Ser Thr Arg Leu Val Ser Val Gln Glu Asp Ala Gly Lys Ser Pro Ala Arg Asn Arg Ser Ala Ser Ile Thr Asn Leu Ser Leu Asp Arg Ser Gly Ser Pro Met Val Pro Ser Tyr Glu Thr Ser Val Ser Pro Gln Ala Asn Arg Thr Tyr Val Arg Thr Glu Thr Thr Glu Asp Glu Arg Lys Ile Leu Leu Asp Ser Val Gln Leu Lys Asp Leu Trp Lys Lys Ile Cys His His Ser Ser Gly Met Glu Phe Gln Asp His Arg Tyr Trp Leu Arg Thr His Pro Asn Cys Ile Val Gly Lys Glu Leu Val Asn Trp Leu Ile Arg Asn Gly His Ile Ala Thr Arg Ala Gln Ala Ile Ala Ile Gly Gln Ala Met Val Asp Gly Arg Trp Leu Asp Cys Val Ser His His Asp Gln Leu Phe Arg Asp Glu Tyr Ala Leu Tyr Arg Pro Leu Gln Ser Thr Glu Phe Ser Glu Thr Pro Ser Pro Asp Ser Asp Ser Val Asn Ser Val Glu Gly His Ser Glu Pro Ser Trp Phe Lys Asp Ile Lys Phe Asp Asp Ser Asp Thr Glu Gln Ile Ala Glu Glu Gly Asp Asp Asn Leu Ala Lys Tyr Leu Ile Ser Asp Thr Gly Gly Gln Gln Leu Ser Ile Ser Asp Ala Phe Ile Lys Glu Ser Leu Phe Asn Arg Arg Val Glu Glu Lys Ser Lys Glu Leu Pro Phe Thr Pro Leu Gly Trp His His Asn Asn Leu Glu Leu Leu Arg Glu Glu Asn Gly Glu Lys Gln Ala Met Glu Arg Leu Leu Ser Ala Asn His Asn His Met Met Ala Leu Leu Gln Gln Leu Leu His Ser Asp Ser Leu Ser Ser Ser Trp Arg Asp Ile Ile Val Ser Leu Val Cys Gln Val Val Gln Thr Val Arg Pro Asp Val Lys Asn Gln Asp Asp Asp Met Asp Ile Arg Gln Phe Val His Ile Lys Lys Ile Pro Gly Gly Lys Lys Phe Asp Ser Val Val Val Asn Gly Phe Val Cys Thr Lys Asn Ile Ala His Lys Lys Met Asn Ser Cys Ile Lys Asn Pro Lys Ile Leu Leu Leu Lys Cys Ser Ile Glu Tyr Leu Tyr Arg 650 ' 655 660 Glu Glu Thr Lys Phe Thr Cys Ile Asp Pro Ile Val Leu Gln Glu Arg Glu Phe Leu Lys Asn Tyr Val Gln Arg Ile Val Asp Val Arg Pro Thr Leu Val Leu Val Glu Lys Thr Val Ser Arg Ile Ala Gln Asp Met Leu Leu Glu His Gly Ile Thr Leu Val Ile Asn Val Lys Ser Gln Val Leu Glu Arg Ile Ser Arg Met Thr Gln Gly Asp Leu Val Met Ser Met Asp Gln Leu Leu Thr Lys Pro Arg Leu Gly Thr Cys His Lys Phe Tyr Met Gln Ile Phe Gln Leu Pro Asn Glu Gln Thr Lys Thr Leu Met Phe Phe Glu Gly Cys Pro Gln His Leu Gly Cys Thr Ile Lys Leu Arg Gly Gly Ser Asp Tyr Glu Leu Ala Arg Val Lys Glu Ile Leu Ile Phe Met Ile Cys Val Ala Tyr His Ser Gln Leu Glu Ile Ser Phe Leu Met Asp Glu Phe Ala Met Pro Pro Thr Leu Met Gln Asn Pro Ser Phe His Ser Leu Ile Glu Gly Arg Gly His Glu Gly Ala Val Gln Glu Gln Tyr Gly Gly Gly Ser Ile Pro Trp Asp Pro Asp Ile Pro Pro Glu Ser Leu Pro Cys Asp Asp Ser Ser Leu Leu Glu Ser Arg Ile Val Phe Glu Lys Gly Glu Gln Glu Asn Lys Asn Leu Pro Gln Ala Val Ala Ser Val Lys His Gln Glu His Ser Thr Thr Ala Cys Pro Ala Gly Leu Pro Cys Ala Phe Phe Ala Pro Val Pro Glu Ser Leu Leu Pro Leu Pro Val Asp Asp Gln Gln Asp Ala Leu Gly Ser Glu Leu Pro Glu Ser Leu Gln Gln Thr Val Val Leu Gln Asp Pro Lys Ser Gln Ile Arg Ala Phe Arg Asp Pro Leu Gln Asp Asp Thr Gly Leu Tyr Val Thr Glu Glu Val Thr Ser Ser Glu Asp Lys Arg Lys Thr Tyr Ser Leu Ala Phe Lys Gln Glu Leu Lys Asp Val Lle Leu Cys Ile Ser Pro Val Ile Thr Phe Arg Glu Pro Phe Leu Leu Thr Glu Lys Gly Met Arg Cys Ser Thr Arg Asp Tyr Phe Ala Glu Gln Val Tyr Trp Ser Pro Leu Leu Asn Lys Glu Phe Lys Glu Met Glu Asn Arg Arg Lys Lys Gln Leu Leu Arg Asp Leu Ser Gly Leu Gln Gly Met Asn Gly Ser Ile Gln Ala Lys Ser Ile Gln Val Leu Pro Ser His Glu Leu Val Ser Thr Arg Ile Ala Glu His Leu Gly Asp Ser Gln Ser Leu Gly Arg Met Leu Ala Asp Tyr Arg Ala Arg Gly Gly Arg Ile Gln Pro Lys Asn Ser Asp Pro Phe Ala His Ser Lys Asp Ala Ser Ser Thr Ser Ser Gly Lys Ser Gly Ser Lys Asn Glu Gly Asp Glu Glu Arg Gly Leu Ile Leu Ser Asp Ala Val Trp Ser Thr Lys Val Asp Cys Leu Asn Pro Ile Asn His Gln Arg Leu Cys Val Leu Phe Ser Ser Ser Ser Ala Gln Ser Ser Asn Ala Pro Ser Ala Cys Val Ser Pro Trp Ile Val Thr Met Glu Phe Tyr Gly Lys Asn Asp Leu Thr Leu Gly Ile Phe Leu Glu Arg Tyr Cys Phe Arg Pro Ser Tyr Gln Cys Pro Ser Met Phe Cys Asp Thr Pro Met Val His His Ile Arg Arg Phe Val His Gly Gln Gly Cys Val Gln Ile Ile Leu Lys Glu Leu Asp Ser Pro Val Pro Gly Tyr Gln His Thr Ile Leu Thr Tyr Ser Trp Cys Arg Ile Cys Lys Gln Val Thr Pro Val Val Ala Leu Ser Asn Glu Ser Trp Ser Met Ser Phe Ala Lys Tyr Leu Glu Leu Arg Phe Tyr Gly His Gln Tyr Thr Arg Arg Ala Asn Ala Glu Pro Cys Gly His Ser Ile His His Asp Tyr.His Gln Tyr Phe Ser Tyr Asn Gln Met Val Ala Ser Phe Ser Tyr Ser Pro Ile Arg Leu Leu Glu Val Cys Val Pro Leu Pro Lys Ile Phe Ile Lys Arg Gln Ala Pro Leu Lys Val Ser Leu Leu Gln Asp Leu Lys Asp Phe Phe Gln Lys Val Ser Gln Val Tyr Val Ala Ile Asp Glu Arg Leu Ala Ser Leu Lys Thr Asp Thr Phe Ser Lys Thr Arg Glu Glu Lys Met Glu Asp Ile Phe Ala Gln Lys Glu Met Glu Glu Gly Glu Phe Lys Asn Trp Ile Glu Lys Met Gln Ala Arg Leu Met Ser Ser Ser Val Asp Thr Pro Gln Gln Leu Gln Ser Val Phe Glu Ser Leu Ile Ala Lys Lys Gln Ser Leu Cys Glu Val Leu Gln Ala Trp Asn Asn Arg Leu Gln Asp Leu Phe Gln Gln Glu Lys Gly Arg Lys Arg Pro Ser Val Pro Pro Ser Pro Gly Arg Leu Arg Gln Gly Glu Glu Ser Lys Ile Ser Ala Met Asp Ala Ser Pro Arg Asn Ile Ser Pro Gly Leu Gln Asn Gly Glu Lys Glu Asp Arg Phe Leu Thr Thr Leu Ser Ser Gln Ser Ser Thr Ser Ser Thr His Leu Gln Leu Pro Thr Pro Pro Glu Val Met Ser Glu Gln Ser Val Gly Gly Pro Pro Glu Leu Asp Thr Ala Ser Ser Ser Glu Asp Val Phe Asp Gly His Leu Leu Gly Ser Thr Asp Ser Gln Val Lys Glu Lys Ser Thr Met Lys Ala Ile Phe Ala Asn Leu Leu Pro Gly Asn Ser Tyr Asn Pro Ile Pro Phe Pro Phe Asp Pro Asp Lys His Tyr Leu Met Tyr Glu His Glu Arg Val Pro Ile Ala Val Cys Glu Lys Glu Pro Ser Ser Ile Ile Ala Phe Ala Leu Ser Cys Lys Glu Tyr Arg Asn Ala Leu Glu Glu Leu Ser Lys Ala Thr Gln Trp Asn Ser Ala Glu Glu Gly Leu Pro Thr Asn Ser Thr Ser Asp Ser Arg Pro Lys Ser Ser Ser Pro Ile Arg Leu Pro Glu Met Ser Gly Gly Gln Thr Asn Arg Thr Thr Glu Thr Glu Pro Gln Pro Thr Lys Lys Ala Ser Gly Met Leu Ser Phe Phe Arg Gly Thr Ala Gly Lys Ser Pro Asp Leu Ser Ser Gln Lys Arg Glu Thr Leu Arg Gly Ala Asp Ser Ala Tyr Tyr Gln Val Gly Gln Thr Gly Lys Glu Gly Thr Glu Asn Gln Gly Val Glu Pro Gln Asp Glu Val Asp Gly Gly Asp Thr Gln Lys Lys Gln Leu Ile Asn Pro His Val Glu Leu Gln Phe Ser Asp Ala Asn Ala Lys Phe Tyr Cys Arg Leu Tyr Tyr Ala Gly Glu Phe His Lys Met Arg Glu Val Ile Leu Asp Ser Ser Glu Glu Asp Phe Ile Arg Ser Leu Ser His Ser Ser Pro Trp Gln Ala Arg Gly Gly Lys Ser Gly Ala Ala Phe Tyr Ala Thr Glu Asp Asp Arg Phe Ile Leu Lys Gln Met Pro Arg Leu Glu Val Gln Ser Phe Leu Asp Phe Ala Pro His Tyr Phe Asn Tyr Ile Thr Asn Ala Val Gln Gln Lys Arg Pro Thr Ala Leu Ala Lys Ile Leu Gly Val Tyr Arg Ile Gly Tyr Lys Asn Ser Gln Asn Asn Thr Glu Lys Lys Leu Asp Leu Leu Val Met Glu Asn Leu Phe Tyr Gly Arg Lys Met Ala Gln Val Phe Asp Leu Lys Gly Ser Leu Arg Asn Arg Asn Val Lys Thr Asp Thr Gly Lys Glu Ser Cys Asp Val Val Leu Leu Asp Glu Asn Leu Leu Lys Met Val Arg Asp Asn Pro Leu Tyr Ile Arg Ser His Ser Lys Ala Val Leu Arg Thr Ser Ile His Ser Asp Ser His Phe Leu Ser Ser His Leu Ile Ile Asp Tyr Ser Leu Leu Val Gly Arg Asp Asp Thr Ser Asn Glu Leu Val Val Gly Ile Ile Asp Tyr Ile Arg Thr Phe Thr Trp Asp Lys Lys Leu Glu Met Val Val Lys Ser Thr Gly Ile Leu Gly Gly Gln Gly Lys Met Pro Thr Val Val Ser Pro Glu Leu Tyr Arg Thr Arg Phe Cys Glu Ala Met Asp Lys Tyr Phe Leu Met Val Pro Asp His Trp Thr Gly Leu Gly Leu Asn Cys <210> 11 <211> 551 <212> PRT
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 5734965CD1 <400> 11 Met Ser Gly Gly Glu Gln Lys Pro Glu Arg Tyr Tyr Val Gly Val Asp Val Gly Thr Gly Ser Val Arg Ala Ala Leu Val Asp Gln Ser Gly Val Leu Leu Ala Phe Ala Asp Gln Pro Ile Lys Asn Trp Glu Pro Gln Phe Asn His His Glu Gln Ser Ser Glu Asp Ile Trp Ala Ala Cys Cys Val Val Thr Lys Lys Val Val Gln Gly Ile Asp Leu Asn Gln Ile Arg Gly Leu Gly Phe Asp Ala Thr Cys Ser Leu Val Val Leu Asp Lys Gln Phe His Pro Leu Pro Val Asn Gln Glu Gly Asp Ser His Arg Asn Val Ile Met Trp Leu Asp His Arg Ala Val Ser Gln Val Asn Arg Ile Asn Glu Thr Lys His Ser Val Leu Gln Tyr Val Gly Gly Val Met Ser Val Glu Met Gln Ala Pro Lys Leu Leu Trp Leu Lys Glu Asn Leu Arg Glu Ile Cys Trp Asp Lys Ala Gly His Phe Phe Asp Leu Pro Asp Phe Leu Ser Trp Lys Ala Thr Gly Val Thr Ala Arg Ser Leu Cys Ser Leu Val Cys Lys Trp Thr Tyr Ser Ala Glu Lys Gly Trp Asp Asp Ser Phe Trp Lys Met Ile Gly Leu Glu Asp Phe Val Ala Asp Asn Tyr Ser Lys Ile Gly Asn Gln Val Leu Pro Pro Gly Ala Ser Leu Gly Asn Gly Leu Thr Pro Glu.Ala Ala Arg Asp Leu Gly Leu Leu Pro Gly Ile Ala Val Ala Ala Ser Leu Ile Asp Ala His Ala Gly Gly Leu Gly Val Ile Gly Ala Asp Val Arg Gly His Gly Leu Ile Cys Glu Gly Gln Pro Val Thr Ser Arg Leu Ala Val Ile Cys Gly Thr Ser Ser Cys His Met Gly Ile Ser Lys Asp Pro Ile Phe Val Pro Gly Val Trp Gly Pro Tyr Phe Ser Ala Met Val Pro Gly Phe Trp Leu Asn Glu Gly Gly Gln Ser Val Thr Gly Lys Leu Ile Asp His Met Val Gln Gly His Ala Ala Phe Pro Glu Leu Gln Val Lys Ala Thr Ala Arg Cys Gln Ser Ile Tyr Ala Tyr Leu Asn Ser His Leu Asp Leu Ile Lys Lys Ala Gln Pro Val Gly Phe Leu Thr Val Asp Leu His Val Trp Pro Asp Phe His Gly Asn Arg Ser Pro Leu Ala Asp Leu Thr Leu Lys Gly Met Val Thr Gly Leu Lys Leu Ser Gln Asp Leu Asp Asp Leu Ala Ile Leu Tyr Leu Ala Thr Val Gln Ala Ile Ala Leu Gly Thr Arg Phe Ile Ile Glu Ala Met Glu Ala Ala Gly His Ser Ile Ser Thr Leu Phe Leu Cys Gly Gly Leu Ser Lys Asn Pro Leu Phe Val Gln Met His Ala Asp Ile Thr Gly Met Pro Val Val Leu Ser Gln Glu Val Glu Ser Val Leu Val Gly Ala Ala Val Leu Gly Ala Cys Ala Ser Gly Asp Phe Ala Ser Val Gln Glu Ala Met Ala Lys Met Ser Lys Val Gly Lys Val Val Phe Pro Arg Leu Gln Asp Lys Lys Tyr Tyr Asp Lys Lys Tyr Gln Val Phe Leu Lys Leu Val Glu His Gln Lys Glu Tyr Leu Ala Ile Met Asn Asp Asp <210> 12 <211> 485 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473788CD1 <400> 12 Met Arg Ser Gly Ala Glu Arg Arg Gly Ser Ser Ala Ala Ala Ser Pro Gly Ser Pro Pro Pro Gly Arg Ala Arg Pro Ala Gly Ser Asp Ala Pro Ser Ala Leu Pro Pro Pro Ala Ala Gly Gln Pro Arg Ala Arg Asp Ser Gly Asp Val Arg Ser Gln Pro Arg Pro Leu Phe Gln Trp Ser Lys Trp Lys Lys Arg Met Gly Ser Ser Met Ser Ala Ala Thr Ala Arg Arg Pro Val Phe Asp Asp Lys Glu Asp Val Asn Phe Asp His Phe Gln Ile Leu Arg Ala Ile Gly Lys Gly Ser Phe Gly Lys Val Cys Ile Val Gln Lys Arg Asp Thr Glu Lys Met Tyr Ala Met Lys Tyr Met Asn Lys Gln Gln Cys Ile Glu Arg Asp Glu Val Arg Asn Val Phe Arg Glu Leu Glu Ile Leu Gln Glu Ile Glu His Val Phe Leu Val Asn Leu Trp Tyr Ser Phe Gln Asp Glu Glu Asp Met Phe Met Val Val Asp Leu Leu Leu Gly Gly Asp Leu Arg Tyr His Leu Gln Gln Asn Val Gln Phe Ser Glu Asp Thr Val Arg Leu Tyr Ile Cys Glu Met Ala Leu Ala Leu Asp Tyr Leu Arg Gly Gln His Ile Ile His Arg Asp Val Lys Pro Asp Asn Ile Leu Leu Asp Glu Arg Gly His Ala His Leu Thr Asp Phe Asn Ile Ala Thr Ile Ile Lys Asp Gly Glu Arg Ala Thr Ala Leu Ala Gly Thr Lys Pro Tyr Met Ala Pro Glu Ile Phe His Ser Phe Val Asn Gly Gly Thr Gly Tyr Ser Phe Glu Val Asp Trp Trp Ser Val Gly Val Met Ala Tyr Glu Leu Leu Arg Gly Trp Arg Pro Tyr Asp Ile His Ser Ser Asn Ala Val Glu Ser Leu Val Gln Leu Phe Ser Thr Val Ser Val Gln Tyr Val Pro Thr Trp Ser Lys Glu Met Val Ala Leu Leu Arg Lys Leu Leu Thr Val Asn Pro Glu His Arg Leu Ser Ser Leu Gln Asp Val Gln Ala Ala Pro Ala Leu Ala Gly Val Leu Trp Asp His Leu Ser Glu Lys Arg Val Glu Pro Gly Phe Val Pro Asn Lys Gly Arg Leu His Cys Asp Pro Thr Phe Glu Leu Glu Glu Met Ile Leu Glu Ser Arg Pro Leu His Lys Lys Lys Lys Arg Leu Ala Lys Asn Lys Ser Arg Asp Asn Ser Arg Asp Ser Ser Gln Ser Glu Asn Asp Tyr Leu Gln Asp Cys Leu Asp Ala Ile Gln Gln Asp Phe Val Ile Phe Asn Arg Glu Lys Leu Lys Arg Ser Gln Asp Leu Pro Arg Glu Pro Leu Pro Ala Leu Ser Pro Gly Met Leu Arg Ser Leu Trp Arg Thr Arg Arg Thr Leu Arg Leu Pro Met Cys Gly Pro Ile Cys Pro Ser Ala Gly Ser Gly <210> 13 <211> 282 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3107989CD1 <400> 13 Met Pro Ala Phe Ile Gln Met Gly Arg Asp Lys Asn Phe Ser Ser Leu His Thr Val Phe Cys Ala Thr Gly Gly Gly Ala Tyr Lys Phe Glu Gln Asp Phe Leu Thr Ile Gly Asp Leu Gln Leu Cys Lys Leu Asp Glu Leu Asp Cys Leu Ile Lys Gly Ile Leu Tyr Ile Asp Ser Val Gly Phe Asn Gly Arg Ser Gln Cys Tyr Tyr Phe Glu Asn Pro Ala Asp Ser Glu Lys Cys Gln Lys Leu Pro Phe Asp Leu Lys Asn Pro Tyr Pro Leu Leu Leu Val Asn Ile Gly Ser Gly Val Ser Ile Leu Ala Val Tyr Ser Lys Asp Asn Tyr Lys Arg Val Thr Gly Thr Ser Leu Gly Gly Gly Thr Phe Phe Gly Leu Cys Cys Leu Leu Thr Gly Cys Thr Thr Phe Glu Glu Ala Leu Glu Met Ala Ser Arg Gly Asp Ser Thr Lys Val Asp Lys Leu Val Arg Asp Ile Tyr Gly Gly Asp Tyr Glu Arg Phe Gly Leu Pro Gly Trp Ala Val Ala Ser Ser Phe Gly Asn Met Met Ser Lys Glu Lys Arg Asp Ser Ile Ser Lys Glu Asp Leu Ala Arg Ala Thr Leu Val Thr Ile Thr Asn Asn Ile Gly Ser Ile Ala Arg Met Cys Ala Leu Asn Glu Asn Ile Asp Arg Val Val Phe Val Gly Asn Phe Leu Arg Ile Asn Met Val Ser Met Lys Leu Leu Ala Tyr Ala Met Asp Phe Trp Ser Lys Gly Gln Leu Lys Ala Leu Phe Leu Glu His Glu Gly Tyr Phe Gly Ala Val Gly Ala Leu Leu Glu Leu Phe Lys Met Thr Asp Asp Lys <210> 14 <211> 151 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7482887CD1 <400> 14 Met Ala Asn Thr Glu Ser Ile Ile Ile Asn Pro Ser Ala Val Gln His Ser Leu Val Gly Glu Ile Ile Lys Tyr Ser Glu Gln Lys Gly Phe Tyr Leu Val Thr Met Lys Phe Leu Arg Ala Ser Glu Lys Pro Leu Lys Pro His Tyr Thr Asn Leu Lys Asp His Pro Phe Phe Pro Asp Leu Val Lys Tyr Met Asn Ser Gly Gln Val Val Ala Met Val Leu Glu Gly Leu Asn Val Ala Lys Thr Gly Leu Arg Met Leu Gly Glu Thr Asn Ser Leu Gly Ser Met Leu Glu Thr Ile Ile Arg Arg Asp Phe Cys Ala Lys Ile Gly Gly Asn Val Ile Gly Gly Ser Asp Ser Leu Gln Ser Ala Glu Lys Glu Ile Ser Leu Trp Phe Lys Pro Lys Glu Pro Val Asp Tyr Arg Ser Cys Ala Tyr Asp Trp Val Tyr Ala <210> 15 <211> 410 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2963414CD1 <400> 15 Met Val Val Gln Asn Ser Ala Asp Ala Gly Asp Met Arg Ala Gly Val Gln Leu Glu Pro Phe Leu His Gln Val Gly Gly His Met Ser Val Met Lys Tyr Asp Glu His Thr Val Cys Lys Pro Leu Val Ser Arg Glu Gln Arg Phe Tyr Glu Ser Leu Pro Leu Ala Met Lys Arg Phe Thr Pro Gln Tyr Lys Gly Thr Val Thr Val His Leu Trp Lys Asp Ser Thr Gly His Leu Ser Leu Val Ala Asn Pro Val Lys Glu Ser Gln Glu Pro Phe Lys Val Ser Thr Glu Ser Ala Ala Val Ala Ile Trp Gln Thr Leu Gln Gln Thr Thr Gly Ser Asn Gly Ser Asp Cys Thr Leu Ala Gln Trp Pro His Ala Gln Leu Ala Arg Ser Pro Lys Glu Ser Pro Ala Lys Ala Leu Leu Arg Ser Glu Pro His Leu Asn Thr Pro Ala Phe Ser Leu Val Glu Asp Thr Asn Gly Asn Gln Val Glu Arg Lys Ser Phe Asn Pro Trp Gly Leu Gln Cys His Gln Ala His Leu Thr Arg Leu Cys Ser Glu Tyr Pro Glu Asn Lys Arg His Arg Phe Leu Leu Leu Glu Asn Val Val Ser Gln Tyr Thr His Pro Cys Val Leu Asp Leu Lys Met Gly Thr Arg Gln His Gly Asp Asp Ala Ser Glu Glu Lys Lys Ala Arg His Met Arg Lys Cys Ala Gln Ser Thr Ser Ala Cys Leu Gly Val Arg Ile Cys Gly Met Gln Val Tyr Gln Thr Asp Lys Lys Tyr Phe Leu Cys Lys Asp Lys Tyr Tyr Gly Arg Lys Leu Ser Val Glu Gly Phe Arg Gln Ala Leu Tyr Gln Phe Leu His Asn Gly Ser His Leu Arg Arg Glu Leu Leu Glu Pro Ile Leu His Gln Leu Arg Ala Leu Leu Ser Ile Ile Arg Ser Gln Ser Ser Tyr Arg Phe Tyr Ser Ser Ser Leu Leu Val Ile Tyr Asp Gly Gln Glu Pro Pro Glu Arg Ala Pro Gly Ser Pro His Pro His Glu Ala Pro Gln Ala Ala His Gly Ser Ser Pro Gly Gly Leu Thr Lys Val Asp Ile Arg Met Ile Asp Phe Ala His Thr Thr Tyr Lys Gly Tyr Trp Asn Glu His Thr Thr Tyr Asp Gly Pro Asp Pro Gly Tyr Ile Phe Gly Leu Glu Asn Leu Ile Arg Ile Leu Gln Asp Ile Gln Glu Gly Glu <210> 16 <211> 1581 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477139CD1 <400> 16 Met Ala Gly Pro Gly Gly Trp Arg Asp Arg Glu Val Thr Asp Leu Gly His Leu Pro Asp Pro Thr Gly Ile Phe Ser Leu Asp Lys Thr Ile Gly Leu Gly Thr Tyr Gly Arg Ile Tyr Leu Gly Leu His Glu Lys Thr Gly Ala Phe Thr Ala Val Lys Val Met Asn Ala Arg Lys Thr Pro Leu Pro Glu Ile Gly Arg Arg Val Arg Val Asn Lys Tyr Gln Lys Ser Val Gly Trp Arg Tyr Ser Asp Glu Glu Glu Asp Leu Arg Thr Glu Leu Asn Leu Leu Arg Lys Tyr Ser Phe His Lys Asn Ile Val Ser Phe Tyr Gly Ala Phe Phe Lys Leu Ser Pro Pro Gly Gln Arg His Gln Leu Trp Met Val Met Glu Leu Cys Ala Ala Gly Ser Val Thr Asp Val Val Arg Met Thr Ser Asn Gln Ser Leu Lys Glu Asp Trp Ile Ala Tyr Ile Cys Arg Glu Ile Leu Gln Gly Leu Ala His Leu His Ala His Arg Val Ile His Arg Asp Ile Lys Gly Gln Asn Val Leu Leu Thr His Asn Ala Glu Val Lys Leu Val Asp Phe Gly Val Ser Ala Gln Val Ser Arg Thr Asn Gly Arg Arg Asn Ser Phe Ile Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Ile Asp Cys Asp Glu Asp Pro Arg Arg Ser Tyr Asp Tyr Arg Ser Asp Val Trp Ser Val Gly Ile Thr Ala Ile Glu Met Ala Glu Gly Ala Pro Pro Leu Cys Asn Leu Gln Pro Leu Glu Ala Leu Phe Val Ile Leu Arg Glu Ser Ala Pro Thr Val Lys Ser Ser Gly Trp Ser Arg Lys Phe His Asn Phe Met Glu Lys Cys Thr Ile Lys Asn Phe Leu Phe Arg Pro Thr Ser Ala Asn Met Leu Gln His Pro Phe Val Arg Asp Ile Lys Asn Glu Arg His Val Val Glu Ser Leu Thr Arg His Leu Thr Gly Ile Ile Lys Lys Arg Gln Lys Lys Gly Ile Pro Leu Ile Phe Glu Arg Glu Glu Ala Ile Lys Glu Gln Tyr Thr Val Arg Arg Phe Arg Gly Pro Ser Cys Thr His Glu Leu Leu Arg Leu Pro Thr Ser Ser Arg Cys Arg Pro Leu Arg Val Leu His Gly Glu Pro Ser Gln Pro Arg Trp Leu Pro Asp Arg Glu Glu Pro Gln Val Gln Ala Leu Gln Gln Leu Gln Gly Ala Ala Arg Val Phe Met Pro Leu Gln Ala Leu Asp Ser Ala Pro Lys Pro Leu Lys Gly Gln Ala Gln Ala Pro Gln Arg Leu Gln Gly Ala Ala Arg Val Phe Met Pro Leu Gln Ala Gln Val Lys Ala Lys Ala Ser Lys Pro Leu Gln Met Gln Ile Lys Ala Pro Pro Arg Leu Arg Arg Ala Ala Arg Val Leu Met Pro Leu Gln Ala Gln Val Arg Ala Pro Arg Leu Leu Gln Val Gln Ser Gln Val Ser Lys Lys Gln Gln Ala Gln Thr Gln Thr Ser Glu Pro Gln Asp Leu Asp Gln Val Pro Glu Glu Phe Gln Gly Gln Asp Gln Val Pro Glu Gln Gln Arg Gln Gly Gln Ala Pro Glu Gln Gln Gln Arg His Asn Gln Val Pro Glu Gln Glu Leu Glu Gln Asn Gln Ala Pro Glu Gln Pro Glu Val Gln Glu Gln Ala Ala Glu Pro Ala Gln Ala Glu Thr Glu Ala Glu Glu Pro Glu Ser Leu Arg.Val Asn Ala Gln Val Phe Leu Pro Leu Leu Ser Gln Asp His His Val Leu Leu Pro Leu His Leu Asp Thr Gln Val Leu Ile Pro Val Glu Gly Gln Thr Glu Gly Ser Pro Gln Ala Gln Ala Trp Thr Leu Glu Pro Pro Gln Ala Ile Gly~Ser Val Gln Ala Leu Ile Glu Gly Leu Ser Arg Asp Leu Leu Arg Ala Pro Asn Ser Asn Asn Ser Lys Pro Leu Gly Pro Leu Gln Thr Leu Met Glu Asn Leu Ser Ser Asn Arg Phe Tyr Ser Gln Pro Glu Gln Ala Arg Glu Lys Lys Ser Lys Val Ser Thr Leu Arg Gln Ala Leu Ala Lys Arg Leu Ser Pro Lys Arg Phe Arg Ala Lys Ser Ser Trp Arg Pro Glu Lys Leu Glu Leu Ser Asp Leu Glu Ala Arg Arg Gln Arg Arg Gln Arg Arg Trp Glu Asp Ile Phe Asn Gln His Glu Glu Glu Leu Arg Gln Val Asp Lys Thr Ser Trp Arg Gln Trp Gly Pro Ser Asp Gln Leu Ile Asp Asn Ser Phe Thr Gly Met Gln Asp Leu Lys Lys Tyr Leu Lys Gly Lys Thr Thr Phe His Asn Val Gln Val Val Ile Tyr Arg Ala Val Lys Gly Asn Asp Asp Val Ala Thr Arg Ser Thr Val Pro Gln Arg Ser Leu Leu Glu Gln Ala Gln Lys Pro Ile Asp Ile Arg Gln Arg Ser Ser Gln Asn Arg Gln Asn Trp Leu Ala Ala Ser Gly Asp Ser Lys His Lys Ile Leu Ala Gly Lys Thr Gln Ser Tyr Cys Leu Thr Ile Tyr Ile Ser Glu Val Lys Lys Glu Glu Phe Gln Glu Gly Met Asn Gln Lys Cys ,Gln Gly Ala Gln Val Gly Leu Gly Pro Glu Gly His Cys Ile Trp Gln Leu Gly Glu Ser Ser Ser Glu Glu Glu Ser Pro Val Thr Gly Arg Arg Ser Gln Ser Ser Pro Pro Tyr Ser Thr Ile Asp Gln Lys Leu Leu Val Asp Ile His Val Pro Asp Gly Phe Lys Val Gly Lys Ile Ser Pro Pro Val Tyr Leu Thr Asn Glu Trp Val Gly Tyr Asn Ala Leu Ser Glu Ile Phe Arg Asn Asp Trp Leu Thr Pro Ala Pro Val Ile Gln Pro Pro Glu Glu Asp Gly Asp Tyr Val Glu Leu Tyr Asp Ala Ser Ala Asp Thr Asp Gly Asp Asp Asp Asp Glu Ser Asn Asp Thr Phe Glu Asp Thr Tyr Asp His Ala Asn Gly Asn Asp Asp Leu Asp Asn Gln Val Asp Gln Ala Asn Asp Val Cys Lys Asp His Asp Asp Asp Asn Asn Lys Phe Val Asp Asp Val Asn Asn Asn Tyr Tyr Glu Ala Pro Ser Cys Pro Ser Leu Leu Ser Gly Gln Ala Met Ala Glu Met Glu Ala Ala Ser Lys Met Val Met Met Glu Val Val 1055 1060 .1065 Glu Lys Arg Lys Pro Thr Glu Ala Met Glu Ala Ile Gln Pro Ile Glu Ala Met Glu Glu Val Gln Pro Val Arg Asp Asn Ala Ala Ile Gly Asp Gln Glu Glu His Ala Ala Asn Ile Gly Ser Glu Arg Arg Gly Ser Glu Gly Asp Gly Gly Lys Gly Val Val Arg Thr Ser Glu Glu Ser Gly Ala Leu Gly Leu Asn Gly Glu Glu Asn Cys Ser Glu Thr Asp Gly Pro Gly Leu Lys Arg Pro Ala Ser Gln Asp Phe Glu Tyr Leu Gln Glu Glu Pro Gly Gly Gly Asn Glu Ala Ser Asn Ala Ile Asp Ser Gly Ala Ala Pro Ser Ala Pro Asp His Glu Ser Asp Asn Lys Asp Ile Ser Glu Ser Ser Thr Gln Ser Asp Phe Ser Ala Asn His Ser Ser Pro Ser Lys Gly Ser Gly Met Ser Ala Asp Ala Asn Phe Ala Ser Ala Ile Leu Tyr Ala Gly Phe Val Glu Val Pro Glu Glu Ser Pro Lys Gln Pro Ser Glu Val Asn Val Asn Pro Leu Tyr Val Ser Pro Ala Cys Lys Lys Pro Leu Ile His Met Tyr Glu Lys Glu Phe Thr Ser Glu Ile Cys Cys Gly Ser Leu Trp Gly Val Asn Leu Leu Leu Gly Thr Arg Ser Asn Leu Tyr Leu Met Asp Arg Ser Gly Lys Ala Asp Ile Thr Lys Leu Ile Arg Arg Arg Pro Phe Arg Gln Ile Gln Val Leu Glu Pro Leu Asn Leu Leu Ile Thr Ile Ser Gly His Lys Asn Arg Leu Arg Val Tyr His Leu Thr Trp Leu Arg Asn Lys Ile Leu Asn Asn Asp Pro Glu Ser Lys Arg Arg Gln Glu Glu Met Leu Lys Thr Glu Glu Ala Cys Lys Ala Ile Asp Lys Leu Thr Gly Cys Glu His Phe Ser Val Leu Gln His Glu Glu Thr Thr Tyr Ile Ala Ile Ala Leu Lys Ser Ser Ile His Leu Tyr Ala Trp Ala Pro Lys Ser Phe Asp Glu Ser Thr Ala Ile Lys Val Phe Pro Thr Leu Asp His Lys Pro Val Thr Val Asp Leu Ala Ile Gly Ser Glu Lys Arg Leu Lys Ile Phe Phe Ser Ser Ala Asp Gly Tyr His Leu Ile Asp Ala Glu Ser Glu Val Met Ser Asp Val Thr Leu Pro Lys Asn Asn Ile Ile Ile Leu Pro Asp Cys Leu Gly Ile Gly Met Met Leu Thr Phe Asn Ala Glu Ala Leu Ser Val Glu Ala Asn Glu Gln Leu Phe Lys Lys Ile Leu Glu Met Trp Lys Asp Ile Pro Ser Ser Ile Ala Phe Glu Cys Thr Gln Arg Thr Thr Gly Trp Gly Gln Lys Ala Ile Glu Val Arg Ser Leu Gln Ser Arg Val Leu Glu Ser Glu Leu Lys Arg Arg Ser Ile Lys Lys Leu Arg Phe Leu Cys Thr Arg Gly Asp Lys Leu Phe Phe Thr Ser Thr Leu Arg Asn His His Ser Arg Val Tyr Phe Met Thr Leu Gly Lys Leu Glu Glu Leu Gln Ser Asn Tyr Asp Val <210> 17 <211> 1084 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 55009053CD1 <400> 17 Met Glu Thr Gln Ala Val Ala Thr Ser Pro Asp Gly Arg Tyr Leu Lys Phe Asp Ile Glu Ile Gly Arg Gly Ser Phe Lys Thr Val Tyr Arg Gly Leu Asp Thr Asp Thr Thr Val Glu Val Ala Trp Cys Glu Leu Gln Thr Arg Lys Leu Ser Arg Ala Glu Arg Gln Arg Phe Ser Glu Glu Val Glu Met Leu Lys Gly Leu Gln His Pro Asn Ile Val Arg Phe Tyr Asp Ser Trp Lys Ser Val Leu Arg Gly Gln Val Cys Ile Val Leu Val Thr Glu Leu Met Thr Ser Gly Thr Leu Lys Thr Tyr Leu Arg Arg Phe Arg Glu Met Lys Pro Arg Val Leu Gln Arg Trp Ser Arg Gln Ile Leu Arg Gly Leu His Phe Leu His Ser Arg Val Pro Pro Ile Leu His Arg Asp Leu Lys Cys Asp Asn Val Phe Ile Thr Gly Pro Ser Gly Ser Val Lys Ile Gly Asp Leu Gly Leu Ala Thr Leu Lys Arg Ala Ser Phe Ala Lys Ser Val Ile Gly Thr Pro Glu Phe Met Ala Pro Glu Met Tyr Glu Glu Lys Tyr Asp Glu Ala Val Asp Val Tyr Ala Phe Gly Met Cys Met Leu Glu Met Ala Thr Ser Glu Tyr Pro Tyr Ser Glu Cys Gln Asn Ala Ala Gln Ile Tyr Arg Lys Val Thr Ser Gly Arg Lys Pro Asn Ser Phe His Lys Val Lys Ile Pro Glu Val Lys Glu Ile Ile Glu Gly Cys Ile Arg Thr Asp Lys Asn Glu Arg Phe Thr Ile Gln Asp Leu Leu Ala His Ala Phe Phe Arg Glu Glu Arg Gly Val His Val Glu Leu Ala Glu Glu Asp Asp Gly Glu Lys Pro Gly Leu Lys Leu Trp Leu Arg Met Glu Asp Ala Arg Arg Gly Gly Arg Pro Arg Asp Asn Gln Ala Ile Glu Phe Leu Phe Gln Leu Gly Arg Asp Ala Ala Glu Glu Val Ala Gln Glu Met Val Ala Leu Gly Leu Val Cys Glu Ala Asp Tyr Gln Pro Val Ala Arg Ala Val Arg Glu Arg Val Ala Ala Ile Gln Arg Lys Arg Glu Lys Leu Arg Lys Ala Arg Glu Leu Glu Ala Leu Pro Pro Glu Pro Gly Pro Pro Pro Ala Thr Val Pro Met Ala Pro Gly Pro Pro Ser Val Phe Pro Pro Glu Pro Glu Glu Pro Glu Ala Asp Gln His Gln Pro Phe Leu Phe Arg His Ala Ser Tyr Ser Ser Thr Thr Ser Asp Cys Glu Thr Asp Gly Tyr Leu Ser Ser Ser Gly Phe Leu Asp Ala Ser Asp Pro Ala Leu Gln Pro Pro Gly Gly Val Pro Ser Ser Leu Ala Glu Ser His Leu Cys Leu Pro Ser Ala Phe Ala Leu Ser Ile Pro Arg Ser Gly Pro Gly Ser Asp Phe Ser Pro Gly Asp Ser Tyr Ala Ser Asp Ala Ala Ser Gly Leu Ser Asp Val Gly Glu Gly Met Gly Gln Met Arg Arg Pro Pro Gly Arg Asn Leu Arg Arg Arg Pro Arg Ser Arg Leu Arg Val Thr Ser Val Ser Asp Gln Asn Asp Arg Val Val Glu Cys Gln Leu Gln Thr His Asn Ser Lys Met Val Thr Phe Arg Phe Asp Leu Asp Gly Asp Ser Pro Glu Glu Ile Ala Ala Ala Met Val Tyr Asn Glu Phe Ile Leu Pro Ser Glu Arg Asp Gly Phe Leu Arg Arg Ile Arg Glu Ile Ile Gln Arg Val Glu Thr Leu Leu Lys Arg Asp Thr Gly Pro Met Glu Ala Ala Glu Asp Thr Leu Ser Pro Gln Glu Glu Pro Ala Pro Leu Pro Ala Leu Pro Val Pro Leu Pro Asp Pro Ser Asn Glu Glu Leu Gln Ser Ser Thr Ser Leu Glu His Arg Ser Trp Thr Ala Phe Ser Thr Ser Ser Ser 5er Pro Gly Thr Pro Leu Ser Pro Gly Asn Pro Phe Ser Pro Gly Thr Pro Ile Ser Pro Gly Pro Ile Phe Pro Ile Thr Ser Pro Pro Cys His Pro Ser Pro Ser Pro Phe Ser Pro Ile Ser Ser Gln Val Ser Ser Asn Pro Ser Pro His Pro Thr Ser Ser Pro Leu Pro Phe Ser Ser Ser Thr Pro Glu Phe Pro Val Pro Leu Ser Gln Cys Pro Trp Ser Ser Leu Pro Thr Thr Ser Pro Pro Thr Phe Ser Pro Thr Cys Ser Gln Val Thr Leu Ser Ser Pro Phe Phe Pro Pro Cys Pro Ser Thr Ser Ser Phe Pro Ser Thr Thr Ala Ala Pro Leu Leu Ser Leu Ala Ser Ala Phe Ser Leu Ala Val Met Thr Val Ala Gln Ser Leu Leu Ser Pro Ser Pro Gly Leu Leu Ser Gln Ser Pro Pro Ala Pro Pro Ser Pro Leu Pro Ser Leu Pro Leu Pro Pro Pro Val Ala Pro Gly Gly Gln Glu Ser Pro Ser Pro His Thr Ala Glu Val Glu Ser Glu Ala Ser Pro Pro Pro Ala Arg Pro Leu Pro Gly Glu Ala Arg Leu Ala Pro Ile Ser Glu Glu Gly Lys Pro Gln Leu Val Gly Arg Phe Gln Val Thr Ser Ser Lys Glu Pro Ala Glu Pro Leu Pro Leu Gln Pro Thr Ser Pro Thr Leu Ser Gly Ser Pro Lys Pro Ser Thr Pro Gln Leu Thr Ser Glu Ser Ser Asp Thr Glu Asp Ser Ala Gly Gly Gly Pro Glu Thr Arg Glu Ala Leu Ala Glu Ser Asp Arg Ala Ala Glu Gly Leu Gly Ala Gly Val Glu Glu Glu Gly Asp Asp Gly Lys Glu Pro Gln Val Gly Gly Ser Pro Gln Pro Leu Ser His Pro Ser Pro Val Trp Met Asn Tyr Ser Tyr Ser Ser Leu Cys Leu Ser Ser Glu Glu Ser Glu Ser Ser Gly Glu Asp Glu Glu Phe Trp Ala Glu Leu Gln Ser Leu Arg Gln Lys His Leu Ser Glu Val Glu Thr Leu Gln Thr Leu Gln Lys Lys Glu Ile Glu Asp Leu Tyr Ser Arg Leu Gly Lys Gln Pro Pro Pro Gly Ile Val Ala Pro Ala Ala Met Leu Ser Ser Arg Gln Arg Arg Leu Ser Lys Gly Ser Phe Pro Thr Ser Arg Arg Asn Ser Leu Gln Arg Ser Glu Pro Pro Gly Pro Gly Ile Met Arg Arg Asn Ser Leu Ser Gly Ser Ser Thr Gly Ser Gln Glu Gln Arg Ala Ser Lys Gly Val Thr Phe Ala Gly Asp Val Gly Arg Met <210> 18 <211> 600 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474648CD1 <400> 18 Met Gly Glu Ser Gly Asn His His Phe Gln Gln Thr Asn Thr Gly Thr Glu Asn Gln Thr Ala His Val Leu Thr His Lys Trp Glu Leu Asp Asn Glu Asn Ile Trp Ala Gln Gly Gly Glu His His Lys Leu Gly Pro Val Met Gly Trp Lys Ala Arg Ser Gly Lys Thr Leu Gly Glu Ile Pro Asn Val Gly Thr Leu Thr Leu Leu Thr Gly Tyr Gly Gly Cys Gln Leu Pro Cys Cys Lys Asp Thr Gln Ala Ala Tyr Gly Glu Thr His Val Val Arg Ser Gly Gly Leu Leu Pro Thr Ala Ser Trp Glu Leu Arg Pro Ala Asp Ser His Thr Val Thr Ser Asp Asp Pro Gly Val Ser Val Val Ser Gly Tyr Pro Gly Gly Cys Leu Pro Asp His Asp Pro Pro Val Gly Phe Leu Ser Glu Gly Pro Ala Pro Arg Ser Cys Ser Leu Ile Lys Gly Gly Gly Thr Gly Leu Ala Ala Ser Arg Val Pro Arg Ser Arg Glu Arg Arg Ala Cys Cys Gly Tyr Gly Val Arg Arg Gln Gln Glu Gly Gly Pro Gly Ala Thr Ser Ala Gly Leu Gly Gln Ala Arg Arg Ser Lys Pro Ser Arg Arg Arg Arg Arg Gly Ala Trp Ala Arg Gly Gly Gly Pro Gly Gly Ala Glu Asp Thr Gly Gly Ser Leu Pro Ser Gln Val Arg Pro Pro Gly Pro Cys Gln Cys Pro Val Gln Phe Leu Phe Asp Ile Ser Glu Gln Gly Val Gln Arg Met Gly Lys Lys Arg Ala Gly Ala Ala Ala Asn Lys Gly Arg Asn Ser Tyr Leu Arg Arg Tyr Asp Ile Lys Ala Leu Ile Gly Thr Gly Ser Phe Ser Arg Val Val Arg Val Glu Gln Lys Thr Thr Lys Lys Pro Phe Ala Ile Lys Val Met Glu Thr Arg Glu Arg Glu Gly Arg Glu Ala Cys Val Ser Glu Leu Ser Val Leu Arg Arg Val Ser His Arg Tyr Ile Val Gln Leu Met Glu Ile Phe Glu Thr Glu Asp Gln Val Tyr Met Val Met Glu Leu Ala Thr Gly Gly Glu Leu Phe Asp Arg Leu Ile Ala Gln Gly Ser Phe Thr Glu Arg Asp Ala Val Arg Ile Leu Gln Met Val Ala Asp Gly Ile Arg Tyr Leu His Ala Leu Gln Ile Thr His Arg Asn Leu Lys Pro Glu Asn Leu Leu Tyr Tyr His Pro Gly Glu Glu Ser Lys Ile Leu Ile Thr Asp Phe Gly Leu Ala Tyr Ser Gly Lys Lys Ser Gly Asp Trp Thr Met Lys Thr Leu Cys Gly Thr Pro Glu Tyr Ile Ala Pro Glu Val Leu Leu Arg Lys Pro Tyr Thr Ser Ala Val Asp Met Trp Ala Leu Gly Val Ile Thr Tyr Ala Leu Leu Ser Gly Phe Leu Pro Phe Asp Asp Glu Ser Gln Thr Arg Leu Tyr Arg Lys Ile Leu Lys Gly Lys Tyr Asn Tyr Thr Gly Glu Pro Trp Pro Ser Ile Ser His Leu Ala Lys Asp Phe Ile Asp Lys Leu Leu Ile Leu Glu Ala Gly His Arg Met Ser Ala Gly Gln Ala Leu Asp His Pro Trp Val Ile Thr Met Ala Ala Gly Ser Ser Met Lys Asn Leu Gln Arg Ala Ile Ser Arg Asn Leu Met Gln Arg Ala Ser Pro His Ser Gln Ser Pro Gly Ser Ala Gln Ser Ser Lys Ser His Tyr Ser His Lys Ser Arg His Met Trp Ser Lys Arg Asn Leu Arg Ile Val Glu Ser Pro Leu Ser Ala Leu Leu <210> 19 <211> 1114 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7483053CD1 <400> 19 Met Ala Lys Ala Thr Ser Gly Ala Ala Gly Leu Arg Leu Leu Leu Leu Leu Leu Leu Pro Leu Leu Gly Lys Val Ala Leu Gly Leu Tyr 20 25 ~ 30 Phe Ser Arg Asp Ala Tyr Trp Glu Lys Leu Tyr Val Asp Gln Ala Ala Gly Thr Pro Leu Leu Tyr Val His Ala Leu Arg Asp Ala Pro Glu Glu Val Pro Ser Phe Arg Leu Gly Gln His Leu Tyr Gly Thr Tyr Arg Thr Arg Leu His Glu Asn Asn Trp Ile Cys Ile Gln Glu Asp Thr Gly Leu Leu Tyr Leu Asn Arg Ser Leu Asp His Ser Ser Trp Glu Lys Leu Ser Val Arg Asn Arg Gly Phe Pro Leu Leu Thr Val Tyr Leu Lys Val Phe Leu Ser Pro Thr Ser Leu Arg Glu Gly Glu Cys Gln Trp Pro Gly Cys Ala Arg Val Tyr Phe Ser Phe Phe Asn Thr Ser Phe Pro Ala Cys Ser Ser Leu Lys Pro Arg Glu Leu Cys Phe Pro Glu Thr Arg Pro Ser Phe Arg Ile Arg Glu Asn Arg Pro Pro Gly Thr Phe His Gln Phe Arg Leu Leu Pro Val Gln Phe Leu Cys Pro Asn Ile Ser Val Ala Tyr Arg Leu Leu Glu Gly Glu Gly Leu Pro Phe Arg Cys Ala Pro Asp Ser Leu Glu Val Ser Thr Arg Trp Ala Leu Asp Arg Glu Gln Arg Glu Lys Tyr Glu Leu Val Ala Val Cys Thr Val His Ala Gly Ala Arg Glu Glu Val Val Met Val Pro Phe Pro Val Thr Val Tyr Asp Glu Asp Asp Ser Ala Pro Thr Phe Pro Ala Gly Val Asp Thr Ala Ser Ala Val Val Glu Phe Lys Arg Lys Glu Asp Thr Val Val Ala Thr Leu Arg Val Phe Asp Ala Asp Val Val Pro Ala Ser Gly Glu Leu Val Arg Arg Tyr Thr Ser Thr Leu Leu Pro Gly Asp Thr Trp Ala Gln Gln Thr Phe Arg Val Glu His Trp Pro Asn Glu Thr Ser Val Gln Ala Asn Gly Ser Phe Val Arg Ala Thr Val His Asp Tyr Arg Leu Val Leu Asn Arg Asn Leu Ser Ile Ser Glu Asn Arg Thr Met Gln Leu Ala Val Leu Val Asn Asp Ser Asp Phe Gln Gly Pro Gly Ala Gly Val Leu Leu Leu His Phe Asn Val Ser Val Leu Pro Val Ser Leu His Leu Pro Ser Thr Tyr Ser Leu Ser Val Ser Arg Arg Ala Arg Arg Phe Ala Gln Ile Gly Lys Val Cys Val Glu Asn Cys Gln Ala Phe Ser Gly Ile Asn Val Gln Tyr Lys Leu His Ser Ser Gly Ala Asn Cys Ser Thr Leu Gly Val Val Thr Ser Ala Glu Asp Thr Ser Gly Ile Leu Phe Val Asn Asp Thr Lys Ala Leu Arg Arg Pro Lys Cys Ala Glu Leu His Tyr Met Val Val Ala Thr Asp Gln Gln Thr Ser Arg Gln Ala Gln Ala Gln Leu Leu Val Thr Val Glu Gly Ser Tyr Val Ala Glu Glu Ala Gly Cys Pro Leu Ser Cys Ala Val Ser Lys Arg Arg Leu Glu Cys Glu Glu Cys Gly Gly Leu Gly Ser Pro Thr Gly Arg Cys Glu Trp Arg Gln Gly Asp Gly Lys Gly Ile Thr Arg Asn Phe Ser Thr Cys Ser Pro Ser Thr Lys Thr Cys Pro Asp Gly His Cys Asp Val Val Glu Thr Gln Asp Ile Asn Ile Cys Pro Gln Asp Cys Leu Arg Gly Ser Ile Val Gly Gly His Glu Pro Gly Glu Pro Arg Gly Ile Lys Ala Gly Tyr Gly Thr Cps Asn Cys Phe Pro Glu Glu Glu Lys Cys Phe Cys Glu Pro Glu Asp Ile Gln Asp Pro Leu Cys Asp Glu Leu Cys Arg Thr Val Ile Ala Ala Ala Val Leu Phe Ser Phe Ile Val Ser Val Leu Leu Ser Ala Phe Cys Ile His Cys Tyr His Lys Phe Ala His Lys Pro Pro Ile Ser Ser Ala Glu Met Thr Phe Arg Arg Pro Ala Gln Ala Phe Pro Val Ser Tyr Ser Ser Ser Ser Ala Arg Arg Pro Ser Leu Asp Ser Met Glu Asn Gln Val Ser Val Asp Ala Phe Lys Ile Leu Glu Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu Val Leu Gly Lys Thr Leu Gly Glu Gly Glu Phe Gly Lys Val Val Lys Ala Thr Ala Phe His Leu Lys Gly Arg Ala Gly Tyr Thr Thr Val Ala Val Lys Met Leu Lys Glu Asn Ala Ser Pro Ser Glu Leu Arg Asp Leu Leu Ser Glu Phe Asn Val Leu Lys Gln Val Asn His Pro His Val Ile Lys Leu Tyr Gly Ala Cys Ser Gln Asp Gly Pro Leu Leu Leu Ile Val Glu Tyr Ala Lys Tyr Gly Ser Leu Arg Gly Phe Leu Arg Glu Ser Arg Lys Val Gly Pro Gly Tyr Leu Gly Ser Gly Gly Ser Arg Asn Ser Ser Ser Leu Asp His Pro Asp Glu Arg Ala Leu Thr Met Gly Asp Leu Ile Ser Phe Ala Trp Gln Ile Ser Gln Gly Met Gln Tyr Leu Ala Glu Met Lys Leu Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Ala Glu Gly Arg Lys Met Lys Ile Ser Asp Phe Gly Leu Ser Arg Asp Val Tyr Glu Glu Asp Ser Tyr Val Lys Arg Ser Gln Gly Arg Ile Pro Val Lys Trp Met Ala Ile Glu Ser Leu Phe Asp His Ile Tyr Thr Thr Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Val Thr Leu Gly Gly Asn Pro Tyr Pro Gly Ile Pro Pro Glu Arg Leu Phe Asn Leu Leu Lys Thr Gly His Arg Met Glu Arg Pro Asp Asn Cys Ser Glu Glu Met Tyr Arg Leu Met Leu Gln Cys Trp Lys Gln Glu Pro Asp Lys Arg Pro Val Phe Ala Asp Ile Ser Lys Asp Leu Glu Lys Met Met Val Lys Arg Arg Asp Tyr Leu Asp Leu Ala Ala Ser Thr Pro Ser Asp Ser Leu Ile Tyr Asp Asp Gly Leu Ser Glu Glu Glu Thr Pro Leu Val Asp Cys Asn Asn Ala Pro Leu Pro Arg Ala Leu Pro Ser Thr Trp Ile Glu Asn Lys Leu Tyr Gly Met Ser Asp Pro Asn Trp Pro Gly Glu Ser Pro Val Pro Leu Thr Arg Ala Asp Gly Thr Asn Thr Gly Phe Pro Arg Tyr Pro Asn Asp Ser Val Tyr Ala Asn Trp Met Leu Ser Pro Ser Ala Ala Lys Leu Met Asp Thr Phe Asp Ser <210> 20 <211> 567 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7483117CD1 <400> 20 Met Asp Asp Lys Asp Ile Asp Lys Glu Leu Arg Gln Lys Leu Asn Phe Ser Tyr Cys Glu Glu Thr Glu Ile Glu Gly Gln Lys Lys Val Glu Glu Ser Arg Glu Ala Ser Ser Gln Thr Pro Glu Lys Gly Glu Val Gln Asp Ser Glu Ala Lys Gly Thr Pro Pro Trp Thr Pro Leu Ser Asn Val His Glu Leu Asp Thr Ser Ser Glu Lys Asp Lys Glu Ser Pro Asp Gln Ile Leu Arg Thr Pro Val Ser His Pro Leu Lys Cys Pro Glu Thr Pro Ala Gln Pro Asp Ser Arg Ser Lys Leu Leu Pro Ser Asp Ser Pro Ser Thr Pro Lys Thr Met Leu Ser Arg Leu Val Ile Ser Pro Thr Gly Lys Leu Pro Ser Arg Gly Pro Lys His Leu Lys Leu Thr Pro Ala Pro Leu Lys Asp Glu Met Thr Ser Leu Ala Leu Val Asn Ile Asn Pro Phe Thr Pro Glu Ser Tyr Lys Lys Leu Phe Leu Gln Ser Gly Gly Lys Arg Lys Ile Arg Gly Asp Leu Glu Glu Ala Gly Pro Glu Glu Gly Lys Gly Gly Leu Pro Ala Lys Arg Cys Val Leu Arg Glu Thr Asn Met Ala Ser Arg Tyr Glu Lys Glu Phe Leu Glu Val Glu Lys Ile Gly Val Gly Glu Phe Gly Thr Val Tyr Lys Cys Ile Lys Arg Leu Asp Gly Cys Val Tyr Ala Ile Lys Arg Ser Met Lys Thr Phe Thr Glu Leu Ser Asn Glu Asn Ser Ala Leu His Glu Val Tyr Ala His Ala Val Leu Gly His His Pro His Val Val Arg Tyr Tyr Ser Ser Trp Ala Glu Asp Asp His Met Ile Ile Gln Asn Glu Tyr Cys Asn Gly Gly Ser Leu Gln Ala Ala Ile Ser Glu Asn Thr Lys Ser Gly Asn His Phe Glu Glu Pro Lys Leu Lys Asp Ile Leu Leu Gln Ile Ser Leu Gly Leu Asn Tyr Ile His Asn Ser Ser Met Val His Leu Asp Ile Lys Pro Ser Asn Ile Phe Ile Cys His Lys Met Gln Ser Glu Ser Ser Gly Val Ile Glu Glu Val Glu Asn Glu Ala Asp Trp Phe Leu Ser Ala Asn Val Met Tyr Lys Ile Gly Asp Leu Gly His Ala Thr Ser Ile Asn Lys Pro Lys Val Glu Glu Gly Asp Ser Arg Phe Leu Ala Asn Glu Ile Leu Gln Glu Asp Tyr Arg His Leu Pro Lys Ala Asp Ile Phe Ala Leu Gly Leu Thr Ile Ala Val Ala Ala Gly Ala Glu Ser Leu Pro Thr Asn Gly Ala Ala Trp His His Ile Arg Lys Gly Asn Phe Pro Asp Val Pro Gln Glu Leu Ser Glu Ser Phe Ser Ser Leu Leu Lys Asn Met Ile Gln Pro Asp Ala Glu Gln Arg Pro Ser Ala Ala Ala Leu Ala Arg Asn Thr Val Leu Arg Pro Ser Leu Gly Lys Thr Glu Glu Leu Gln Gln Gln Leu Asn Leu Glu Lys Phe Lys Thr Ala Thr Leu Glu Arg Glu Leu Arg Glu Ala Gln Gln Ala Gln Ser Pro Gln Gly Tyr Thr His His Gly Asp Thr Gly Val Ser Gly Thr His Thr Gly Ser Arg Ser Thr Lys Arg Leu Val Gly Gly Lys Ser Ala Arg Ser Ser Ser Phe Thr Ser Gly Glu Arg Glu Pro Leu His <210> 21 <211> 2054 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7484498CD1 <400> 21 Met Leu Lys Phe Lys Tyr Gly Ala Arg Asn Pro Leu Asp Ala Gly Ala Ala Glu Pro Ile Ala Ser Arg Ala Ser Arg Leu Asn Leu Phe Phe Gln Gly Lys Pro Pro Phe Met Thr Gln Gln Gln Met Ser Pro Leu Ser Arg Glu Gly Ile Leu Asp Ala Leu Phe Val Leu Phe Glu Glu Cys Ser Gln Pro Ala Leu Met Lys Ile Lys His Val Ser Asn Phe Val Arg Lys Tyr Ser Asp Thr Ile Ala Glu Leu Gln Glu Leu Gln Pro Ser Ala Lys Asp Phe Glu Val Arg Ser Leu Val Gly Cys Gly His Phe Ala Glu Val Gln Val Val Arg Glu Lys Ala Thr Gly Asp Ile Tyr Ala Met Lys Val Met Lys Lys Lys Ala Leu Leu Ala Gln Glu Gln Val Ser Phe Phe Glu Glu Glu Arg Asn Ile Leu Ser Arg Ser Thr Ser Pro Trp Ile Pro Gln Leu Gln Tyr Ala Phe Gln Asp Lys Asn His Leu Tyr Leu Val Met Glu Tyr Gln Pro Gly Gly Asp Leu Leu Ser Leu Leu Asn Arg Tyr Glu Asp Gln Leu Asp Glu Asn Leu Ile Gln Phe Tyr Leu Ala Glu Leu Ile Leu Ala Val His Ser Val His Leu Met Gly Tyr Val His Arg Asp Ile Lys Pro Glu Asn Ile Leu Val Asp Arg Thr Gly His Ile Lys Leu Val Asp Phe Gly Ser Ala Ala Lys Met Asn Ser Asn Lys Met Val Asn Ala Lys Leu Pro Ile Gly Thr Pro Asp Tyr Met Ala Pro Glu Val Leu Thr Val Met Asn Gly Asp Gly Lys Gly Thr Tyr Gly Leu Asp Cys Asp Trp Trp Ser Val Gly Val Ile Ala Tyr Glu Met Ile Tyr Gly Arg Ser Pro Phe Ala Glu Gly Thr Ser Ala Arg Thr Phe Asn Asn Ile Met Asn Phe Gln Arg Phe Leu Lys Phe Pro Asp Asp Pro Lys Val Ser Ser Asp Phe Leu Asp Leu Ile Gln Ser Leu Leu Cys Gly Gln Lys Glu Arg Leu Lys Phe Glu Gly Leu Cys Cys His Pro Phe Phe Ser Lys Ile Asp Trp Asn Asn Ile Arg Asn Ser Pro Pro Pro Phe Val Pro Thr Leu Lys Ser Asp Asp Asp Thr Ser Asn Phe Asp Glu Pro Glu Lys Asn Ser Trp Val Ser Ser Ser Pro Cys Gln Leu Ser Pro Ser Gly Phe Ser Gly Glu Glu Leu Pro Phe Val Gly Phe Ser Tyr Ser Lys Ala Leu Gly Ile Leu Gly Arg Ser Glu Ser Val Val Ser Gly Leu Asp Ser Pro Ala Lys Thr Ser Ser Met Glu Lys Lys Leu Leu Ile Lys Ser Lys Glu Leu Gln Asp Ser Gln Asp Lys Cys His Lys Met Glu Gln Glu Met Thr Arg Leu His Arg Arg Val Ser Glu Val Glu'Ala Val Leu Ser Gln Lys Glu Val Glu Leu Lys Ala Ser Glu Thr Gln Arg Ser Leu Leu Glu Gln Asp Leu Ala Thr Tyr Ile Thr Glu Cys Ser Ser Leu Lys Arg Ser Leu Glu Gln Ala Arg Met Glu Val Ser Gln Glu Asp Asp Lys Ala Leu Gln Leu Leu His Asp Ile Arg Glu Gln Ser Arg Lys Leu Gln Glu Ile Lys Glu Gln Glu Tyr Gln Ala Gln Val Glu Glu Met Arg Leu Met Met Asn Gln Leu Glu Glu Asp Leu Val Ser Ala Arg Arg Arg Ser Asp Leu Tyr Glu Ser Glu Leu Arg Glu Ser Arg Leu Ala Ala Glu Glu Phe Lys Arg Lys Ala Thr Glu Cys Gln His Lys Leu Leu Lys Ala Lys Asp Gln Gly Lys Pro Glu Val Gly Glu Tyr Ala Lys Leu Glu Lys Ile Asn Ala Glu Gln Gln Leu Lys Ile Gln Glu Leu Gln Glu Lys Leu Glu Lys Ala Val Lys Ala Ser Thr Glu Ala Thr Glu Leu Leu Gln Asn Ile Arg Gln Ala Lys Glu Arg Ala Glu Arg Glu Leu Glu Lys Leu Gln Asn Arg Glu Asp Ser Ser Glu Gly Ile Arg Lys Lys Leu Val Glu Ala Glu Glu Arg Arg His Ser Leu Glu Asn Lys Val Lys Arg Leu Glu Thr Met Glu Arg Arg Glu Asn Arg Leu Lys Asp Asp Ile Gln Thr Lys Ser Gln Gln Ile Gln Gln Met Ala Asp Lys Ile Leu Glu Leu Glu Glu Lys His Arg Glu Ala Gln Val Ser Ala Gln His Leu Glu Val His Leu Lys Gln Lys Glu Gln His Tyr Glu Glu Lys Ile Lys Val Leu Asp Asn Gln Ile Lys Lys Asp Leu Ala Asp Lys Glu Thr Leu Glu Asn Met Met Gln Arg His Glu Glu Glu Ala His Glu Lys Gly Lys Ile Leu Ser Glu Gln Lys Ala Met Ile Asn Ala Met Asp Ser Lys Ile Arg Ser Leu Glu Gln Arg Ile Val Glu Leu Ser Glu Ala Asn Lys Leu Ala Ala Asn Ser Ser Leu Phe Thr Gln Arg Asn Met Lys Ala Gln Glu Glu Met Ile Ser Glu Leu Arg Gln Gln Lys Phe Tyr Leu Glu Thr Gln Ala Gly Lys Leu Glu Ala Gln Asn Arg Lys Leu Glu Glu Gln Leu Glu Lys Ile Ser His Gln Asp His Ser Asp Lys Asn Arg Leu Leu Glu Leu Glu Thr Arg Leu Arg Glu Val Ser Leu Glu His Glu Glu Gln Lys Leu Glu Leu Lys Arg Gln Leu Thr Glu Leu Gln Leu Ser Leu Gln Glu Arg Glu Ser Gln Leu Thr Ala Leu Gln Ala Ala Arg Ala Ala Leu Glu Ser Gln Leu Arg Gln Ala Lys Thr Glu Leu Glu Glu Thr Thr Ala Glu Ala Glu Glu Glu Ile Gln Ala Leu Thr Ala His Arg Asp Glu Ile Gln Arg Lys Phe Asp Ala Leu Arg Asn Ser Cys Thr Val Ile Thr Asp Leu Glu Glu Gln Leu Asn Gln Leu Thr Glu Asp Asn Ala Glu Leu Asn Asn Gln Asn Phe Tyr Leu Ser Lys Gln Leu Asp Glu Ala Ser Gly Ala Asn Asp Glu Ile Val Gln Leu Arg Ser Glu Val Asp His Leu Arg Arg Glu Ile Thr Glu Arg Glu Met Gln Leu Thr Ser Gln Lys Gln Thr Met Glu Ala Leu Lys Thr Thr Cys Thr Met Leu Glu Glu Gln Val Met Asp Leu Glu Ala Leu Asn Asp Glu Leu Leu Glu Lys Glu Arg Gln Trp Glu Ala Trp Arg Ser Val Leu Gly Asp Glu Lys Ser Gln Phe Glu Cys Arg Val Arg Glu Leu Gln Arg Met Leu Asp Thr Glu Lys Gln Ser Arg Ala Arg Ala Asp Gln Arg Ile Thr Glu Ser Arg Gln Val Val Glu Leu Ala Val Lys Glu His Lys Ala Glu Ile Leu Ala Leu Gln Gln Ala Leu Lys Glu Gln Lys Leu Lys Ala Glu Ser Leu Ser Asp Lys Leu Asn Asp Leu Glu Lys Lys His Ala Met Leu Glu Met Asn Ala Arg Ser Leu Gln Gln Lys Leu Glu Thr Glu Arg Glu Leu Lys Gln Arg Leu Leu Glu Glu Gln Ala Lys Leu Gln Gln Gln Met Asp Leu Gln Lys Asn His Ile Phe Arg Leu Thr Gln Gly Leu Gln Glu Ala Leu Asp Arg Ala Asp Leu Leu Lys Thr Glu Arg Ser Asp Leu Glu Tyr Gln Leu Glu Asn Ile Gln Val Leu Tyr Ser His Glu Lys Val Lys Met Glu Gly Thr Ile Ser Gln Gln Thr Lys Leu Ile Asp Phe Leu Gln Ala Lys Met Asp Gln Pro Ala Lys Lys Lys Lys Val Pro Leu Gln Tyr Asn Glu Leu Lys Leu Ala Leu Glu Lys Glu Lys Ala Arg Cys Ala Glu Leu Glu Glu Ala Leu Gln Lys Thr Arg Ile Glu Leu Arg Ser Ala Arg Glu Glu Ala Ala His Arg Lys Ala Thr Asp His Pro His Pro Ser Thr Pro Ala Thr Ala Arg Gln Gln Ile Ala Met Ser Ala Ile Val Arg Ser Pro Glu His Gln Pro Ser Ala Met Ser Leu Leu Ala Pro Pro Ser Ser Arg Arg Lys Glu Ser Ser Thr Pro Glu Glu Phe Ser Arg Arg Leu Lys Glu Arg Met His His Asn Ile Pro His Arg Phe Asn Val Gly Leu Asn Met Arg Ala Thr Lys Cys Ala Val Cys Leu Asp Thr Val His Phe Gly Arg Gln Ala Ser Lys Cys Leu Glu Cys Gln Val Met Cys His Pro Lys Cys Ser Thr Cys Leu Pro Ala Thr Cys Gly Leu Pro Ala Glu Tyr Ala Thr His Phe Thr Glu Ala Phe Cys Arg Asp Lys Met Asn Ser Pro Gly Leu Gln Thr Lys Glu Pro Ser Ser Ser Leu His Leu Glu Gly Trp Met Lys Val Pro Arg Asn Asn Lys Arg Gly Gln Gln Gly Trp Asp Arg Lys Tyr Ile Val Leu Glu Gly Ser Lys Val Leu Ile Tyr Asp Asn Glu Ala Arg Glu Ala Gly Gln Arg Pro Val Glu Glu Phe Glu Leu Cys Leu Pro Asp Gly Asp Val Ser Ile His Gly Ala Val Gly Ala Ser Glu Leu Ala Asn Thr Ala Lys Ala Asp Val Pro Tyr Ile Leu Lys Met Glu Ser His Pro His Thr Thr Cys Trp Pro Gly Arg Thr Leu Tyr Leu Leu Ala Pro Ser Phe Pro Asp Lys Gln Arg Trp Val Thr Ala Leu Glu Ser Val Val Ala Gly Gly Arg Val Ser Arg Glu Lys Ala Glu Ala Asp Ala Lys Leu Leu Gly Asn Ser Leu Leu Lys Leu Glu Gly Asp Asp Arg Leu Asp Met Asn Cys Thr Leu Pro Phe Ser Asp Gln Val Val Leu Val Gly Thr Glu Glu Gly Leu Tyr Ala Leu Asn Val Leu Lys Asn Ser Leu Thr His Val Pro Gly Ile Gly Ala Val Phe Gln Ile Tyr Ile Ile Lys Asp Leu Glu Lys Leu Leu Met Ile Ala Gly Glu Glu Arg Ala Leu Cys Leu Val Asp Val Lys Lys Val Lys Gln Ser Leu Ala Gln Ser His Leu Pro Ala Gln Pro Asp Ile Ser Pro Asn Ile Phe Glu 170p 1705 1710 Ala Val Lys Gly Cys His Leu Phe Gly Ala Gly Lys Ile Glu Asn Gly Leu Cys Ile Cys Ala Ala Met Pro Ser Lys Val Val Ile Leu Arg Tyr Asn Glu Asn Leu Ser Lys Tyr Cys Ile Arg Lys Glu Ile Glu Thr Ser Glu Pro Cys Ser Cys Ile His Phe Thr Asn Tyr Ser Ile Leu Ile Gly Thr Asn Lys Phe Tyr Glu Ile Asp Met Lys Gln Tyr Thr Leu Glu Glu Phe Leu Asp Lys Asn Asp His Ser Leu Ala Pro Ala Val Phe Ala Ala Ser Ser Asn Ser Phe Pro Val Ser Ile Val Gln Val Asn Ser Ala Gly Gln Arg Glu Glu Tyr Leu Leu Cys Phe His Glu Phe Gly Val Phe Val Asp Ser Tyr Gly Arg Arg Ser Arg Thr Asp Asp Leu Lys Trp Ser Arg Leu Pro Leu Ala Phe Ala Tyr Arg Glu Pro Tyr Leu Phe Val Thr His Phe Asn Ser Leu Glu Val Ile Glu Ile Gln Ala Arg Ser Ser Ala Gly Thr Pro Ala Arg Ala Tyr Leu Asp Ile Pro Asn Pro Arg Tyr Leu Gly Pro Ala Ile Ser Ser Gly Ala Ile Tyr Leu Ala Ser Ser Tyr Gln Asp Lys Leu Arg Val Ile Cys Cys Lys Gly Asn Leu Val Lys Glu Ser Gly Thr Glu His His Arg Gly Pro Ser Thr Ser Arg Ser Ser Pro Asn Lys Arg Gly Pro Pro Thr Tyr Asn Glu His Ile Thr Lys Arg Val Ala 1'955 1960 1965 Ser Ser Pro Ala Pro Pro Glu Gly Pro Ser His Pro Arg Glu Pro Ser Thr Pro His Arg Tyr Arg Glu Gly Arg Thr Glu Leu Arg Arg Asp Lys Ser Pro Gly Arg Pro Leu Glu Arg Glu Lys Ser Pro Gly Arg Met Leu Ser Thr Arg Arg Glu Arg Ser Pro Gly Arg Leu Phe Glu Asp Ser Ser Arg Gly Arg Leu Pro Ala Gly Ala Val Arg Thr Pro Leu Ser Gln Val Asn Lys Val Trp Asp Gln Ser Ser Val <210> 22 <211> 1665 <212> PRT
<213> Homo sapiens <220>
<221> mist feature <223> Incyte ID No: 7638121CD1 <400> 22 Met Gly Cys Cys Arg Leu Gly Cys Gly Gly Cys Ser Val Ala His Ser Val Ser Gln Gly Leu Thr Asn His Pro Ser Met Val Gly Cys Gly Trp His Pro Gly Leu Cys Gly Trp Gly Gly Gly Leu His Ser Ser Leu Pro Ala Leu Pro Gly Pro Pro Ser Met Gln Val Thr Ile Glu Asp Val Gln Ala Gln Thr Gly Gly Thr Ala Gln Phe Glu Ala Ile Ile Glu Gly Asp Pro Gln Pro Ser Val Thr Trp Tyr Lys Asp 80 . 85 90 Ser Val Gln Leu Val Asp Ser Thr Arg Leu Ser Gln Gln Gln Glu Gly Thr Thr Tyr Ser Leu Val Leu Arg His Met Ala Ser Lys Asp Ala Gly Val Tyr Thr Cys Leu Ala Gln Asn Thr Gly Gly Gln Val Leu Cys Lys Ala Glu Leu Leu Val Leu Gly Gly Asp Asn Glu Pro Asp Ser Glu Lys Gln Ser His Arg Arg Lys Leu His Ser Phe Tyr Glu Val Lys Glu Glu Ile Gly Arg Gly Val Phe Gly Phe Val Lys Arg Val Gln His Lys Gly Asn Lys Ile Leu Cys Ala Ala Lys Phe Ile Pro Leu Arg Ser Arg Thr Arg Ala Gln Ala Tyr Arg Glu Arg Asp Ile Leu Ala Ala Leu Ser His Pro Leu Val Thr Gly Leu Leu Asp Gln Phe Glu Thr Arg Lys Thr Leu Ile Leu Ile Leu Glu Leu Cys Ser Ser Glu Glu Leu Leu Asp Arg Leu Tyr Arg Lys Gly Val Val Thr Glu Ala Glu Val Lys Val Tyr Ile Gln Gln Leu Val Glu Gly Leu His Tyr Leu His Ser His Gly Val Leu His Leu Asp Ile Lys Pro Ser Asn Ile Leu Met Val His Pro Ala Arg Glu Asp Ile Lys Ile Cys Asp Phe Gly Phe Ala Gln Asn Ile Thr Pro Ala Glu Leu Gln Phe Ser Gln Tyr Gly Ser Pro Glu Phe Val Ser Pro Glu Ile Ile Gln Gln Asn Pro Val Ser Glu Ala Ser Asp Ile Trp Ala Met Gly Val Ile Ser Tyr Leu Ser Leu Thr Cys Ser Ser Pro Phe Ala Gly Glu Ser Asp Arg Ala Thr Leu Leu Asn Val Leu Glu Gly Arg Val Ser Trp Ser Ser Pro Met Ala Ala His Leu Ser Glu Asp Ala Lys Asp Phe Ile Lys Ala Thr Leu Gln Arg Ala Pro Gln Ala Arg Pro Ser Ala Ala Gln Cys Leu Ser His Pro Trp Phe Leu Lys Ser Met Pro Ala Glu Glu Ala His Phe Ile Asn Thr Lys Gln Leu Lys Phe Leu Leu Ala Arg Ser Arg Trp Gln Arg Ser Leu Met Ser Tyr Lys Ser Ile Leu Val Met Arg Ser Ile Pro Glu Leu Leu Arg Gly Pro Pro Asp Ser Pro Ser Leu Gly Val Ala Arg His Leu Cys Arg Asp Thr Gly Gly Ser Ser Ser Ser Ser Ser Ser Ser Asp Asn Glu Leu Ala Pro Phe Ala Arg Ala Lys Ser Leu Pro Pro Ser Pro Val Thr His Ser Pro Leu Leu His Pro Arg Gly Phe Leu Arg Pro Ser Ala Ser Leu Pro Glu Glu Ala Glu Ala Ser Glu Arg Ser Thr Glu Ala Pro Ala Pro Pro Ala Ser Pro Glu Gly Ala Gly Pro Pro Ala Ala Gln Gly Cys Val Pro Arg His Ser Val Ile Arg Ser Leu Phe Tyr His Gln Ala Gly Glu Ser Pro Glu His Gly Ala Leu Ala Pro Gly Ser Arg Arg His Pro Ala Arg Arg Arg His Leu Leu Lys Gly Gly Tyr Ile Ala Gly Ala Leu Pro Gly Leu Arg Glu Pro Leu Met Glu His Arg Val Leu Glu Glu Glu Ala Ala Arg Glu Glu Gln Ala Thr Leu Leu Ala Lys Ala Pro Ser Phe Glu Thr Ala Leu Arg Leu Pro Ala Ser Gly Thr His Leu Ala Pro Gly His Ser His Ser Leu Glu His Asp Ser Pro Ser Thr Pro Arg Pro Ser Ser Glu Ala Cys Gly Glu Ala Gln Arg Leu Pro Ser Ala Pro Ser Gly Gly Ala Pro Ile Arg Asp Met Gly His Pro Gln Gly Ser Lys Gln Leu Pro Ser Thr Gly Gly His Pro Gly Thr Ala Gln Pro Glu Arg Pro Ser Pro Asp Ser Pro Trp Gly Gln Pro Ala Pro Phe Cys His Pro Lys Gln Gly Ser Ala Pro Gln Glu Gly Cys Ser Pro His Pro Ala Val Ala Pro Cys Pro Pro Gly Ser Phe Pro Pro Gly Ser Cys Lys Glu Ala Pro Leu Val Pro Ser Ser Pro Phe Leu Gly Gln Pro Gln Ala Pro Leu Ala Pro Ala Lys Ala Ser Pro Pro Leu Asp Ser Lys Met Gly Pro Gly Asp Ile Ser Leu Pro Gly Arg Pro Lys Pro Gly Pro Cys Ser Ser Pro Gly Ser Ala Ser Gln Ala Ser Ser Ser Gln Val Ser Ser Leu Arg Val Gly Ser Ser Gln Val Gly Thr Glu Pro Gly Pro Ser Leu Asp Ala Glu Gly Trp Thr Gln Glu Ala Glu Asp Leu Ser Asp Ser Thr Pro Thr Leu Gln Arg Pro Gln Glu Gln Val Thr Met Arg Lys Phe Ser Leu Gly Gly Arg Gly Gly Tyr Ala Gly Val Ala Gly Tyr Gly Thr Phe Ala Phe Gly Gly Asp Ala Gly Gly Met Leu Gly Gln Gly Pro Met Trp Ala Arg Ile Ala Trp Ala Val Ser Gln Ser Glu Glu Glu Glu Gln Glu Glu Ala Arg Ala Glu Ser Gln Ser Glu Glu Gln Gln Glu Ala Arg Ala Glu Ser Pro Leu Pro Gln Val Ser Ala Arg Pro Val Pro Glu Val Gly Arg Ala Pro Thr Arg Ser Ser Pro Glu Pro Thr Pro Trp Glu Asp Ile Gly Gln Val Ser Leu Val Gln Ile Arg Asp Leu Ser Gly Asp Ala Glu Ala Ala Asp Thr Ile Ser Leu Asp Ile Ser Glu Val Asp Pro Ala Tyr Leu Asn Leu Ser Asp Leu Tyr Asp Ile Lys Tyr Leu Pro Phe Glu Phe Met Ile Phe Arg Lys Val Pro Lys Ser Ala Gln Pro Glu Pro Pro Ser Pro Met Ala Glu Glu Glu Leu Ala Glu Phe Pro Glu Pro Thr Trp Pro Trp Pro Gly Glu Leu Gly Pro His Ala Gly Leu Glu Ile Thr Glu Glu Ser Glu Asp Val Asp Ala Leu Leu Ala Glu Ala Ala Val Gly Arg Lys Arg Lys Trp Ser Ser Pro Ser Arg Ser Leu Phe His Phe Pro Gly Arg His Leu Pro Leu Asp Glu Pro Ala Glu Leu Gly Leu Arg Glu Arg Val Lys Ala Ser Val Glu His Ile Ser Arg Ile Leu Lys Gly Arg Pro Glu Gly Leu Glu Lys Glu Gly Pro Pro Arg Lys Lys Pro Gly Leu Ala Ser Phe Arg Leu Ser Gly Leu Lys Ser Trp Asp Arg Ala Pro Thr Phe Leu Arg Glu Leu Ser Asp Glu Thr Val Val Leu Gly Gln Ser Val Thr Leu Ala Cys Gln Val Ser Ala Gln Pro Ala Ala Gln Ala Thr Trp Ser Lys Asp Gly Ala Pro Leu Glu Ser Ser Ser Arg Val Leu Ile Ser Ala Thr Leu Lys Asn Phe Gln Leu Leu Thr Ile Leu Val Val Val Ala Glu Asp Leu Gly Val Tyr Thr Cys Ser Val Ser Asn Ala Leu Gly Thr Val Thr Thr Thr Gly Val Leu Arg Lys Ala Glu Arg Pro Ser Ser Ser Pro Cys Pro Asp Ile Gly Glu Val Tyr Ala Asp Gly Val Leu Leu Val Trp Lys Pro Val Glu Ser Tyr Gly Pro Val Thr Tyr Ile Val Gln Cps Ser Leu Glu Gly Gly Ser Trp Thr Thr Leu Ala Ser Asp Ile Phe Asp Cys Cys Tyr Leu Thr Ser Lys Leu Ser Arg Gly Gly Thr Tyr Thr Phe Arg Thr Ala Cys Val Ser Lys Ala Gly Met Gly Pro Tyr Ser Ser Pro Ser Glu Gln Val Leu Leu Gly Gly Pro Ser His Leu Ala Ser Glu Glu Glu Ser Gln Gly Arg Ser Ala Gln Pro Leu Pro Ser Thr Lys Thr Phe Ala Phe Gln Thr Gln Ile Gln Arg Gly Arg Phe Ser Val Val Arg Gln Cys Trp Glu Lys Ala Ser Gly Arg Ala Leu Ala Ala Lys Ile Ile Pro Tyr His Pro Lys Asp Lys Thr Ala Val Leu Arg Glu Tyr Glu Ala Leu Lys Gly Leu Arg His Pro His Leu Ala Gln Leu His Ala Ala Tyr Leu Ser Pro Arg His Leu Val Leu Ile Leu Glu Leu Cys Ser Gly Pro Glu Leu Leu Pro Cys Leu Ala Glu Arg Ala Ser Tyr Ser Glu Ser Glu Val Lys Asp Tyr Leu Trp Gln Met Leu Ser Ala Thr Gln Tyr Leu His Asn Gln His Ile Leu His Leu Asp Leu Arg Ser Glu Asn Met Ile Ile Thr Glu Tyr Asn Leu Leu Lys Val Val Asp Leu Gly Asn Ala Gln Ser Leu Ser Gln Glu Lys Val Leu Pro Ser Asp Lys Phe Lys Asp Tyr Leu Glu Thr Met Ala Pro Glu Leu Leu Glu Gly Gln Gly Ala Val Pro Gln Thr Asp Ile Trp Ala Ile Gly Val Thr Ala Phe Ile Met Leu Ser Ala Glu Tyr Pro Val Ser Ser Glu Gly Ala Arg Asp Leu Gln Arg Gly Leu Arg Lys Gly Leu Val Arg Leu Ser Arg Cys Tyr Ala Gly Leu Ser Gly Gly Ala Val Ala Phe Leu Arg Ser Thr Leu Cys Ala Gln Pro Trp Gly Arg Pro Cys Ala Ser Ser Cys Leu Gln Cys Pro Trp Leu Thr Glu Glu Gly Pro Ala Cys Ser Arg Pro Ala Pro Val Thr Phe Pro Thr Ala Arg Leu Arg Val Phe Val Arg Asn Arg Glu Lys Arg Arg Ala Leu Leu Tyr Lys Arg His Asn Leu Ala Gln Val Arg <210> 23 <211> 1014 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7482896CB1 <400> 23 atgacaaaca acagcggctc caaagccgaa ctcgttgtgg gagggaaata caaactggtg 60 cggaagatcg ggtctggctc ctttggagac gtttatctgg gcatcaccac caccaacggc 120 gaggacgtag cagtgaagct ggaatctcag aaggtcaagc acccccagtt gctgtatgag 180 agcaaactct acacgattct tcaaggtggg gttggcatcc cccacatgca ctggtatggt 240 caggaaaaag acaacaatgt gctagtcatg gaccttctgg gacccagcct cgaagacctc 300 tttaatttct gttcaagaag gttcaccatg aaaactgtac ttatgttagc cgaccagatg 360 atcagcagaa ttgaatacgt gcatacaaag aattttctac accgagacat taaaccagat 420 aacttcctga tgggtactgg gcgtcactgt aataagttgt tccttattga ttttggtttg 480 gccaaaaagt acagagacaa caggaccagg caacacatac cgtacagaga agataaacac 540 ctcattggca ctgtccgata tgccagcatc aatgcacatc ttggtattga gcagagccgc 600 cgagatgaca tggaatcctt aggctacgtt ttcatgtatt ttaatagaac cagcctgccg 660 tggcaaggac taagggctat gacaaaaaaa caaaaatatg aaaagattag tgagaagaag 720 atgtccaccc ctgttgaagt tttatgtaag gggtttcctg cagaattcgc catgtacttg 780 aactactgtc gtgggctgcg ctttgaggaa gtcccagatt acatgtatct gaggcagcta 840 ttccgcattc ttttcaggac cctgaaccac caatatgact acacatttga ttggacgatg 900 ttaaagcaga aagcagcaca gcaggcagcc tcttccagtg ggcagggtca gcaggcccaa 960 acccagacag gcaagcaaac tgaaaaaaac aagaataatg tgaaagataa ctaa 1014 <210> 24 <211> 1530 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7483046CB1 <400> 24 cggcctgaca ggcgggcatg cgggcggcca gactgtagcc gagcagcgag gctccggccg 60 cagccatgga gcggcggctg cgcgcgctgg agcagctggc gcggggcgag gccggcggct 120 gcccggggct cgacggcctc ctagatctgc tgctggcgct gcaccacgag ctcagcagcg 180 gccccctacg gcgggagcgc agcgtggcgc agttcctgag ctgggccagc cccttcgtat 240 caaaggtgaa agaactgcgt ctgcagagag atgactttga gatcttgaag gtgatcggcc 300 gaggagcctt tggggaggtc accgtggtga ggcagaggga cactgggcag atttttgcca 360 tgaaaatgct gcacaagtgg gagatgctga agagggctga gacagcctgt ttccgggagg 420 agcgggatgt gctcgtgaaa ggggacagcc gttgggtgac cactctgcac tatgccttcc 480 aagacgagga gtacctgtac cttgtgatgg actactatgc tggtggggac ctcctgacgc 540 tgctgagccg cttcgaggac cgtctcccgc ccgagctggc ccagttctac ctggctgaga 600 tggtgctggc catccactcg ctgcaccagc tgggttatgt ccacagggat gtcaagccag 660 acaacgtcct gctggatgtg aacgggcaca ttcgcctggc tgacttcggc tcctgcctgc 720 gtctcaacac caacggcatg gtggattcat cagtggcagt agggacgccg gactatatct 780 cccctgagat cctgcaggcc atggaggagg gcaagggcca ctacggccca cagtgtgact 840 ggtggtcgct tggagtctgc gcctatgagc tgctctttgg ggagacgccc ttctatgctg 900 agtccttggt ggaaacctac ggcaagatca tgaaccacga ggaccacctg cagttccccc 960 cggacgtgcc tgacgtgcca gccagcgccc aagacctgat ccgccagctg ctgtgtcgcc 1020 aggaagagcg gctaggccgt ggtgggctgg atgacttccg gaaccatcct ttcttcgaag 1080 gcgtggactg ggagcggctg gcgagcagca cggcccccta tattcctgag ctgcgggggc 1140 ccatggacac ctccaacttt gatgtggatg acgacaccct caaccatcca gggaccctgc 1200 caccgccctc ccacggggcc ttctccggcc atcacctgcc attcgtgggc ttcacctaca 1260 cctcaggcag tcacagtcct gagagcagct ctgaggcttg ggctgccctg gagcggaagc 1320 tccagtgtct ggagcaggag aaggtggagc tgagcaggaa gcaccaagag gccctgcacg 1380 cccccacaga ccatcgggag ctggagcagc tacggaagga agtgcagact ctgcgggaca 1440 ggctgccagg tatcccttcc gcccaccccc accctctcct tgagtttctg tgaattaaaa 1500 tatttgcaaa tccaaaaaaa aaaaaaaagg 1530 <210> 25 <211> 3150 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 71636374CB1 <400> 25 attggcttat aggaaaaatt gatttataaa aagtggtaca ggttttcata gataaccatg 60 acaacatccc atatgaatgg gcatgttaca gaggaatcag acagcgaagt aaaaaatgtt 120 gatcttgcat caccagagga acatcagaag caccgagaga tggctgttga ctgccctgga 180 gatttgggca ccaggatgat gccaatacgt cgaagtgcac agttggagcg tattcggcaa 240 caacaggagg acatgaggcg taggagagag gaagaaggga aaaagcaaga acttgacctt 300 aattcttcca tgagacttaa gaaactagcc caaattcctc caaagaccgg aatagataac 360 cctatgtttg atacagagga aggaattgtc ttagaaagtc ctcattatgc tgtgaaaata 420 ttagaaatag aagacttgtt ttcttcactt aaacatatcc aacatacttt ggtagattct 480 cagagccagg aggatatttc actgctttta caacttgttc aaaataagga tttccagaat 540 gcatttaaga tacacaatgc catcacagta cacatgaaca aggccagtcc tccatttcct 600 cttatctcca acgcacaaga tcttgctcaa gaggtacaaa ctgttttgaa gccagttcat 660 cataaggaag gacaagaact aactgctttg ctgaatactc cacatattca ggcactttta 720 ctggcccacg ataaggttgc tgagcaggaa atgcagctag agcccattac agatgagaga 780 gtttatgaaa gtattggcca gtatggagga gaaactgtaa aaatagttcg tatagaaaag 840 gctcgtgata ttccgttggg tgctacagtt cgtaatgaaa tggactctgt catcattagc 900 cggatagtaa aagggggtgc tgcagagaaa agtggtctgt tgcatgaagg agatgaagtt 960 ctagagatta atggcattga aattcggggg aaagatgtca atgaggtttt tgacttgttg 1020 tctgatatgc atggtacttt gacttttgtc ctgattccca gtcaacagat caagccgcct 1080 cctgccaagg aaacagtaat ccatgtaaaa gctcattttg actatgaccc ctcagatgac 1140 ccttatgttc catgtcgaga gttaggtctg tcttttcaaa aaggtgatat acttcatgtg 1200 atcagtcaag aagatccaaa ctggtggcag gcctacaggg aaggggacga agataatcaa 1260 cctctagccg ggcttgttcc agggaaaagc tttcagcagc aaagggaagc catgaaacaa 1320 accatagaag aagataagga gccagaaaaa tcaggaaaac tgtggtgtgc aaagaagaat 1380 aaaaagaaga ggaaaaaggt tttatataat gccaataaaa atgatgatta tgacaacgag 1440 gagatcttaa cctatgagga aatgtcactt tatcatcagc cagcaaatag gaagagacct 1500 atcatcttga ttggtccaca gaactgtggc cagaatgaat tgcgtcagag gctcatgaac 1560 aaagaaaagg accgctttgc atctgcagtt cctcatacaa cccggagtag gcgagaccaa 1620 gaagtagccg gtagagatta ccactttgtt tcgcggcaag cattcgaggc agacatagca 1680 gctggaaagt tcattgagca tggtgaattt gagaagaatt tgtatggaac tagcatagat 1740 tctgtacggc aagtgatcaa ctctggcaaa atatgtcttt taagtcttcg tacacagtca 1800 ttgaagactc tccggaattc agatttgaaa ccatatatta tcttcattgc acccccttca 1860 caagaaagac ttcgggcatt attggccaaa gaaggcaaga atccaaagcc tgaagagttg 1920 agagaaatca ttgagaagac aagagagatg gagcagaaca atggccacta ctttgatacg 1980 gcaattgtga attccgatct tgataaagcc tatcaggaat tgcttaggtt aattaacaaa 2040 cttgatactg aacctcagtg ggtaccatcc acttggctga ggtgaaagaa acatccattc 2100 tgtggcatgt tggacttgat ctggcaaaaa ctgccaatag gaggactgcc cgacactgca 2160 gcaagattga ggataagatg gaaggcagca gtataagctg tagatctgtt cttagatctc 2220 ttgaattagt gagacgacag ttcccttagg cagtttgtgc atggcatcct ttattctcta 2280 tacatggctt tagcggttct tgcctcattt tgggattcta aatggaagct ttcaacagag 2340 cattccattt tgtcctgtta aaaccttttg ttttcaccta aaccctttct gcttagttgt 2400 atctctgtga aaaacttgta tacacaagcg tccatgtctc acacaaatat tgatgtgatt 2460 attcttaagt gttaaatcat taacacttaa atgacttcat tgggaatatt gagcagaggg 2520 actgtgcttc tatgcactgg gcaaggcagt atttgcttag gaaactaatt tagtcatcag 2580 agatactttc ctaaaaagga aaaataaaaa acaaaatggt gccactttgg gttgaagcta 2640 ctttgttagg cttgaattca tttatatgtc ttttgattct taaaaaaaca aaaaacattc 2700 cattagaagc accagttttt ttgctcagac tttgtggatc agactctaca ctcaacacac 2760 tctaatctac ttaaaggtat acaaaatatg ctgatctttt ttaaattatg atttcctgaa 2820 tttttttctt aagtcgtctc aactgattta ctcacttagc ttcccttccc tcatcagcat 2880 agtataatag aatgtatgtt acatttttat gaatggcagg tgttcattat aatctgtatt 2940 gacttaaaaa gtttcttcct catgatgcta atagtttttt gtatacatgg gaggatagca 3000 catttgacag tttttgcatt tttatgtatg agcacagtat cctatgactg tgctacgtat 3060 atataggtaa taaactggaa ttctgttgat gaatatagct gctgtactgt atattaatat 3120 ttaatagatc aacaaatggt cattgaaaac 3150 <210> 26 <211> 2901 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7480597CB1 <400> 26 atggcggaag gcaaggaagg gcaagtccca tcttacatgg atggcagcag gcaaagagag 60 aatgaggaag atgcaaaagc ggaaacccct gatgtaacca tcagatctta tgagatttat 120 tcactaccat ggaacagaca gcaaggccta tgtgaccatt ctctaaaata tttaagctcg 180 agaatcacag agcggaagct gcaaggctcc tggctgcctg ccagccgagg gaatctggag 240 aaaccattcc tggggccgcg tggccccgtc gtgcccttgt tctgccctcg gaatggcctt 300 cactcagcac atcctgagaa cagccctctg aagcccaggg tcgtgaccgt agtgaagctg 360 ggtgggcagc gcccccgaaa gatcactctg ctcctcaaca ggcgatcagt gcagacgttc 420 gagcagctct tagctgacat ctcagaagcc ttgggctctc ccagatggaa gaatgaccgt 480 gtgaggaaac tgtttaacct caagggcagg gaaatcagga gcgtctctga tttcttcagg 540 gaaggggatg ctttcatagc tatgggcaaa gaaccactga cactgaagag cattcaggtg 600 gctgtagaag aactgtaccc caacaaagcc cgggccctga cactggccca gcacagccgt 660 gccccttctc caaggctgag gagcaggctg tttagcaagg ctctgaaagg agaccaccgc 720 tgtggggaga ccgagacccc caagagctgc agcgaagttg caggatgcaa ggcagccatg 780 aggcaccagg ggaagatccc cgaggagctt tcactagatg acagagcgag gacccagaag 840 aagtggggga gggggaaatg ggagccagaa cccagtagca agccccccag ggaagccact 900 ctggaagaga ggcacgcaag gggagagaag catcttgggg tggagattga aaagacctcg 960 ggtgaaatta tcagatgcga gaagtgcaag agagagaggg agctccagca gagcctggag 1020 cgtgagaggc tttctctggg gaccagtgag ctggatatgg ggaagggccc aatgtatgat 1080 gtggagaagc tggtgaggac cagaagctgc aggaggtctc ccgaggcaaa tcctgcaagt 1140 ggggaggaag ggtggaaggg tgacagccac aggagcagcc ccaggaatcc cactcaagag 1200 ctgaggagac ccagcaagag catggacaag aaagaggaca gaggcccaga ggatcaagaa 1260 agccatgctc agggagcagc caaggccaag aaggaccttg tggaagttct tcctgtcaca 1320 gaggaggggc tgagggaggt gaagaaggac accaggccca tgagcaggag caaacatggt 1380 ggctggctcc tgagagagca ccaggcgggc tttgagaagc tccgcaggac ccgaggagaa 1440 gagaaggagg cagagaagga gaaaaagcca tgtatgtctg gaggcagaag gatgactctc 1500 agagatgacc aacctgcaaa gctagaaaag gagcccaaga cgaggccaga agagaacaag 1560 ccagagcggc ccagcggtcg gaagccacgg cccatgggca tcattgccgc caatgtggaa 1620 aagcattatg agactggccg ggtcattggg gatgggaact ttgctgtcgt gaaggagtgc 1680 agacaccgcg agaccaggca ggcctatgcg atgaagatca ttgacaagtc cagactcaag 1740 ggcaaggagg acatggtgga cagtgagatc ttgatcatcc agagcctctc tcaccccaac 1800 atcgtgaaat tgcatgaagt ctacgaaaca gacatggaaa tctacctgat cctggagtac 1860 gtgcagggag gagacctttt tgacgccatc atagaaagtg tgaagttccc ggagcccgat 1920 gctgccctca tgatcatgga cttatgcaaa gccctcgtcc acatgcacga caagagcatt 1980 gtccaccggg acctcaagcc ggaaaacctt ttggttcagc gaaatgagga caaatctact 2040 accttgaaat tggctgattt tggacttgca aagcatgtgg tgagacctat atttactgtg 2100 tgtgggaccc caacttacgt agctcccgaa attctttctg agaaaggtta tggactggag 2160 gtggacatgt gggctgctgg cgtgatcctc tatatcctgc tgtgtggctt tcccccattc 2220 cgcagccctg agagggacca ggacgagctc tttaacatca tccagctggg ccactttgag 2280 ttcctccccc cttactggga caatatctct gatgctgcta aagatctggt gagccggttg 2340 ctggtggtag accccaaaaa gcgctacaca gctcatcagg ttcttcagca cccctggatc 2400 gaaacagctg gcaagaccaa tacagtgaaa cgacagaagc aggtgtcccc cagcagcgag 2460 ggtcacttcc ggagccagca caagagggtt gtggagcagg tatcatagtc accaccttgg 2520 gaatctgtcc agcccccagt tctgctcaag gacagagaaa aggatagaag tttgagagaa 2580 aaacaatgaa agaggcttct tcacataatt ggtgaatcag agggagagac actgagtata 2640 ttttaaagca tattaaaaaa attaagtcaa tgttaaatgt cacaacatat ttttagattt 2700 gtatatttaa agcctttaat acatttttgg ggggtaagca ttgtcatcag tgaggaattt 2760 tggtaataat gatgtgtttt gcttcccctt tgtaaccaag tttattctgt actacaggag 2820 tggtgcttac cagggtctaa actccccctg tgagattaat aaggtgcatt gtggtctttc 2880 tgtgttaata aaatgtggtc c 2901 <210> 27 <211> 1671 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3227248CB1 <400> 27 atgaagctta taaatggcaa aaagcaaaca ttcccatggt ttggcatgga catcggtgga 60 acgctggtta aattggtgta tttcgagccg aaggatatta cagccgaaga ggagcaagag 120 gaagtggaga acctgaagag catccggaag tatttgactt ctaatactgc ttatgggaaa 180 actgggatcc gagacgtcca cctggaactg aaaaacctga ccatgtgtgg acgcaaaggg 240 aacctgcact tcatccgctt tcccagctgt gctatgcaca ggttcattca gatgggcagc 300 gagaagaact tctctagcct tcacaccacc ctctgtgcca caggaggcgg ggctttcaaa 360 ttcgaagagg acttcagaat gattgctgac ctgcagctgc ataaactgga tgaactggac 420 tgtctgattc agggcctgct ttatgtcgac tctgttggct tcaacggcaa gccagaatgt 480 tactattttg aaaatcccac aaatcctgaa ttgtgtcaaa aaaagccgta ctgccttgat 540 aacccatacc ctatgttgct ggttaacatg ggctcaggtg tcagcattct agccgtgtac 600 tccaaggaca actataaaag agttacaggg accagtcttg gaggtggaac attcctaggc 660 ctatgttgct tgctgactgg ttgtgagacc tttgaagaag ctctggaaat ggcagctaaa 720 ggcgacagca ccaatgttga taaactggtg aaggacattt acggaggaga ctatgaacga 780 tttggccttc aaggatctgc tgtagcatca agctttggca acatgatgag taaagaaaag 840 cgagattcca tcagcaagga agacctcgcc cgggccacat tggtcaccat caccaacaac 900 attggctcca ttgctcggat gtgtgcgttg aatgagaaca tagacagagt tgtgtttgtt 960 ggaaattttc tcagaatcaa tatggtctcc atgaagctgc tggcatatgc catggatttt 1020 tggtccaaag gacaactgaa agctctgttt ttggaacatg agggttattt tggagccgtt 1080 ggggcactgt tggaactgtt caaaatgact gatgataagt agagacgagc agtggaggaa 1140 acagcctccc aaaaggacag agaactaaaa aattgctgct ggagaaggtg aaagtcgctt 1200 tgggacggaa gccaagccat tatggcagat gaacctgctg gatttgtaaa taatttaaaa 1260 tccttccaga tgatctttta ctcttaggtt ttgagctaat gattcaaaac gggggaatat 1320 aaaaggtttt ttttctgtat actgtatttt tttaaaaaaa tggtgcagcg tggccaaacc 1380 taccaattgt atgcattaac tttgaaaagt tgtttgatgt ttaagaagga cctgatatgt 1440 aagcgctggt catttttctt ctggggttta ctgatcagtg tggtgatttt aacttcattt 1500 agtaattact ctaggagatt ttaccttgac ttatattttt catgacgttt catgatttgc 1560 tgttggtttc aaatgaaact acaaatctgg catgttttac tgtgaacact tttgttattt 1620 gttttgtacc ctttttgtct tgtttttctg ttttagttgt cttctgaaaa a 1671 <210> 28 <211> 2577 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 4207273CB1 <400> 28 atgccacaga tagcaaagaa gcaatcaact caccggactc agaaacctaa aaagcaatca 60 tttccttgca tctgtaaaaa tccaggaaca cagaagtcat gtgttcctct ctctgttcaa 120 ccgacagagc caagactaaa ttacctagat cttaagtata gtgatatgtt caaagaaatc 180 aattcaactg ctaatggacc tggaatctat gaaatgtttg ggacccctgt ttattgtcat 240 gtgcgagaga ctgaaaggga tgaaaacacg tattaccgtg agatatgttc ggctccatca 300 ggcagacgta tcaccaataa atgtcgatct tcacacagtg agaggaagag caatatcaga 360 acaagacttt ctcagaaaaa aacacatatg aaatgcccaa agacttcatt tggcattaaa 420 caagagcaca aagtcttaat ttctaaagaa aagagttcca aggctgtaca tagcaaccta 480 catgacattg aaaatggtga tggtatttca gaaccagact ggcagataaa gtcttcagga 540 aatgagtttc tatcttccaa agatgaaatt catcccatga acttggctca gacacctgag 600 cagtccatga aacagaatga attccctcct gtctcagatt tatccattgt tgaagaagtt 660 tctatggaag agtctactgg tgatagagac atttctaaca atcaaatact caccacaagc 720 ctcagagatc tgcaagaact tgaagagcta catcaccaga tcccatttat cccttcagaa 780 gacagctggg cagtgcccag tgagaagaat tctaacaagt atgtacagca agaaaagcag 840 aatacagcat ctcttagtaa agtaaatgcc agccgaattt taactaatga tctagagttt 900 gatagtgttt cagatcactc taaaacactt acaaatttct ctttccaagc aaaacaagaa 960 agtgcatctt cccagacata tcaatattgg gtacattatt tggatcatga tagtttagca 1020 aataagtcaa tcacatatca aatgtttgga aaaaccttaa gtggcacaaa ttcaatttcc 1080 caagaaatta tggactctgt aaataatgaa gaattgacag atgaactatt aggttgtcta 1140 gctgcagaat tattagctct tgatgagaaa gataacaact cttgccaaaa aatggcaaat 1200 gaaacagatc ctgaaaacct aaatcttgtc ctcagatgga gaggaagtac cccaaaagaa 1260 atgggcagag agacaacaaa agtcaaaata cagaggcata gtagtgggct caggatatat 1320 gacagggagg agaaatttct catctcaaat gaaaagaaga tattttctga aaatagttta 1380 aagtctgaag aacctatcct atggaccaag ggtgagattc ttggaaaggg agcctacggc 1440 acagtatact gtggtctcac tagtcaagga cagctaatag ctgtaaaaca ggtggctttg 1500 gatacctcta ataaattagc tgctgaaaag gaataccgga aactacagga agaagtagat 1560 ttgctcaaag cactgaaaca tgtcaacatt gtggcctatt tggggacatg cttgcaagag 1620 aacactgtga gcattttcat ggagtttgtt cctggtggct caatctctag tattataaac 1680 cgttttgggc cattgcctga gatggtgttc tgtaaatata cgaaacaaat acttcaaggt 1740 gttgcttatc tccatgagaa ctgtgtggta catcgcgata tcaaaggaaa taatgttatg 1800 ctcatgccaa ctggaataat aaagctgatt gactttggct gtgccaggcg tttggcctgg 1860 gcaggtttaa atggcaccca cagtgacatg cttaagtcca tgcatgggac tccatattgg 1920 atggccccag aagtcatcaa tgagtctggc tatggacgga aatcagatat ctggagcatt 1980 ggttgtactg tgtttgagat ggctacaggg aagcctccac tggcttccat ggacaggatg 2040 gccgccatgt tttacatcgg agcacaccga gggctgatgc ctcctttacc agaccacttc 2100 tcagaaaatg cagcagactt tgtgcgcatg tgcctgacca gggaccagca tgagcgacct 2160 tctgctctcc agctcctgaa gcactccttc ttggagagaa gtcactgaat atacatcaag 2220 actttcttcc cagttccact gcagatgctc ccttgcttaa ttgtggggaa tgatggctaa 2280 gggatctttg tttccccact gaaaattcag tctaacccag tttaagcaga tcctatggag 2340 tcattaactg aaagttgcag ttacatatta gcctcctcaa gtgtcagaca ttattactca 2400 tagtatcaga aaacatgttc ttaataacaa caaaaaacta tttcagtgtt tacagttttg 2460 attgtccagg aactacattc tctagtgttt tatatgacat ttctttttat ttttggcctg 2520 tcctgtcaat tttaatgttg ttagtttaaa ataaattgta aaaacaaaaa aaaaaaa 2577 <210> 29 <211> 2110 <212> DNA
<213> Homo sapiens <220>
<221> mist feature <223> Incyte ID No: 7483334CB1 <400> 29 ctagggtcgc cggggaagcg gtttgggaga gcccatggtg actgcgtgag tggagcccag 60 ctgtgtggat gccccagcat ggatgactac atggtcctga gaatgattgg ggagggctcc 120 ttcggcagag ctcttttggt tcagcttgaa agcagtaatc agatgtttgc catgaaagaa 180 ataaggcttc ccaagtcttt ctctaataca cagaattcta ggaaggaggc tgttctttta 240 gccaaaatga aacaccctaa tattgttgcc ttcaaagaat catttgaagc tgaaggacac 300 ttgtatattg tgatggaata ctgtgatgga ggggatctaa tgcaaaagat taaacagcag 360 aaaggaaagt tatttcctga agacatgata cttaattggt ttacccaaat gtgccttgga 420 gtaaatcaca ttcacaagaa acgtgtgcta cacagagata tcaagtccaa gaatatcttc 480 ctcactcaga atggaaaagt gaaattggga gactttggat ctgcccgtct tctctccaat 540 ccgatggcat ttgcttgtac ctatgtggga actccttatt atgtgcctcc agaaatttgg 600 gaaaacctgc cttataacaa taaaagtgac atctggtcct tgggttgcat cctgtatgaa 660 ctctgtaccc ttaagcatcc atttcaggca aatagttgga aaaatcttat cctcaaagta 720 tgtcaagggt gcatcagtcc actgccgtct cattactcct atgaacttca gttcctagtc 780 aagcagatgt ttaaaaggaa tccctcacat cgcccctcgg ctacaacgct tctctctcga 840 ggcatcgtag ctcggcttgt ccagaagtgc ttaccccccg agatcatcat ggaatatggt 900 gaggaagtat tagaagaaat aaaaaattcg aagcataaca caccaagaaa aaaaacaaac 960 cccagcagaa tcaggatagc tttgggaaat gaagcaagca cagtgcaaga ggaagaacaa 1020 gatagaaagg gtagccatac tgatttggaa agcattaatg aaaatttagt tgaaagtgca 1080 ttgagaagag taaacagaga agaaaaaggt aataagtcag tccatctgag gaaagccagt 1140 tcaccaaatc ttcatagacg acagtgggag aaaaatgtac ccaatacagc tcttacagct 1200 ttggaaaatg catccatact cacctccagt ttaacagcag aggacgatag aggtggttct 1260 gtaataaagt acagcaaaaa tactactcgt aagcagtggc tcaaagagac ccctgacact 1320 ttgttgaaca tccttaagaa tgctgatctc agcttggctt ttcaaacata cacaatatat 1380 agaccaggtt cagaagggtt cttgaaaggc cccctgtctg aagaaacaga agcatcggac 1440 agtgttgatg gaggtcacga ttctgtcatt ttggatccag agcgacttga gcctgggcta 1500 gatgaggagg acacggactt tgaggaggaa gatgacaacc ccgactgggt gtcagagctg 1560 aagaagcgag ctggatggca aggcctgtgc gacagataat gcctgaggaa atgttcctga 1620 gtcacgctga ggagagcctt cactcaggag ttcatgctga gatgatcatg agttcatgcg 1680 acgtatattt tcctttggaa acagaatgaa gcagaggaaa ctcttaatac ttaaaatcgt 1740 tcttgattag tatcgtgagt ttgaaaagtc tagaactcct gtaagttttt gaactcaagg 1800 gagaaggtat agtggaatga gtgtgagcat cgggctttgc agtcccatag aacagaaatg 1860 ggatgctagc gtgccactac ctacttgtgt gattgtggga aattacttaa cctcttcaag 1920 ccccaatttc ctcaaccata aaatgaagat aataatgcct acctcagagg gatgctgacc 1980 acagaccttt atagcagccc gtatgatatt attcacatta tgatatgtgt ttattattat 2040 gtgactcttt ttacatttcc taaaggtttg agaattaaat atatttaatt atgatttaaa 2100 aaaaaaaaaa 2110 <210> 30 <211> 7093 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7483337CB1 <400> 30 cgaggggacg cctcgcgacg gttcctggga gagctggcgg cggccttgct ctgcgcgctc 60 ttcgcgccgc cctccccgcc cgcccgcctc aggattgagg aagtgcgtct gggcccggcc 120 ccggcgcggg gggcagacgg cggtgggacg gccaggcccc ggccccgcca gtgtgtccgc 180 ccggccccgc gtcccggagg agtcagctgt gtgtccagaa cgtgccatgg agacgcttaa 240 cggtgccggg gacacgggcg gcaagccgtc cacgcggggc ggtgaccctg cagcgcggtc 300 ccgcaggacg gaaggcatcc gcgccgcgta caggcgggga gaccgcggcg gcgcccggga 360 cctgctggag gaggcctgcg accagtgcgc gtcccagctg gaaaagggcc agcttctgag 420 catcccggca gcctatgggg atctggagat ggtccgctac ctactcagca agagactggt 480 ggagctgccc accgagccca cggatgacaa cccagccgtg gtggcagcgt attttggaca 540 cacggcagtt gtgcaaaata cgctgcccac cgagcccacg gatgacaacc cagccgtggt 600 ggcagcgtat tttggacaca cggcagttgt gcaggaattg cttgagtcct taccaggtcc 660 ctgcagtccc cagcggcttc tgaactggat gctggccttg gcttgccagc gagggcacct 720 gggggttgtg aagctcctgg tcctgacgca cggggctgac ccggagagct acgctgtcag 780 gaagaatgag ttccctgtca-tcgtgcgctt gcccctgtat gcggccatca agtcagggaa 840 tgaagacatt gcaatattcc tgcttcggca tggggcctat ttctgttcct acatcttgct 900 ggatagtcct gaccccagca aacatctgct gagaaagtac ttcattgaag ccagtccctt 960 gcccagcagt tatccgggaa aaacagctct ccgtgtgaaa tggtcccatc tcagactgcc 1020 ctgggtagac ctagactggc tcatagacat ctcctgccag atcacggagc tcgacctttc 1080 tgccaactgc ctggcgaccc tcccctcggt tatcccctgg ggcctcatca atctccggaa 1140 gctgaacctc tccgacaacc acctggggga gctgcctggc gtgcagtcat cggacgaaat 1200 catctgttcc aggctacttg aaattgacat ttccagcaac aagttgtccc acctccctcc 1260 tggattcttg cacctctcaa aacttcaaaa actgacagct tcaaaaaatt gtttagaaaa 1320 attgttcgaa gaagaaaatg ccactaactg gataggttta cggaagctac aggaacttga 1380 tatatctgac aataaattga cagaactccc tgccctgttc cttcactctt tcaagtccct 1440 caattctctg aatgtctcca gaaacaacct gaaggtgttt ccagatccct gggcctgccc 1500 tttgaaatgt tgtaaagctt ccagaaatgc cctggaatgt ctgccagaca aaatggctgt 1560 cttttggaaa aatcacctga aggatgtgga tttctcagaa aacgcactca aagaagttcc 1620 cctgggactt ttccagcttg atgccctcat gttcttgagg ttacagggga accagctggc 1680 ggcacttcca cctcaagaga agtggacctg caggcagctc aaaaccctgg atctctccag 1740 aaaccaactt ggcaaaaatg aagatggact gaaaacgaag cgtattgcct ttttcaccac 1800 cagaggtcgc cagcgctccg ggactgaggc agagacaact atggagttca gtgcatctct 1860 ggtaaccatt gtgttcctgt ctaacaactg taacctctgt gcatacacat gtgcagcaag 1920 tgtgctggaa tttccggcct tcctaagtga gtctttggaa gtcctttgcc tgaacgacaa 1980 ccacctcgac acagtccctc cctcggtttg cctactgaag agcttatcag agctctactt 2040 gggaaacaac cctggcctcc gggagctccc tcctgagctg gggcagctgg gcaacctctg 2100 gcagctggac actgaagacc tgaccatcag caatgtgcct gcagaaatcc aaaaagaagg 2160 ccccaaagca atgctgtctt acctgcgtgc tcagctgcgg aaagcggaaa agtgcaagct 2220 gatgaagatg atcatcgtgg gtcccccgcg ccagggcaag tccaccctcc tggagatctt 2280 acagacgggg agggcccccc aggtggtgca tggagaggcc accatcagga ccaccaagtg 2340 ggagctccag aggccggctg gctcgagagc caaggtcaag gatggtctgc gtgcagagtc 2400 cctgtgggtt gagtccgtgg agttcaacgt ctgggacatc gggggaccgg ccagcatggc 2460 cactgtcaac cagtgcttct tcacggacaa ggccctgtac gtggtggtct ggaacctggc 2520 gctgggggag gaggccgtgg ccaacctcca gttctggctg ctcaacatcg aggccaaggc 2580 cccaaacgcc gtggtgctgg tggtcgggac gcacctggat ttaattgaag ccaagttccg 2640 tgtggaaagg attgcaacgc tgcgtgccta tgtgctggca ctctgccgct ccccctccgg 2700 ctccagggcc acaggcttcc cagacatcac cttcaaacac ttacatgaga tttcctgcaa 2760 gagcctggaa ggtcaggaag ggctgcgaca gctgattttc cacgtcacgt gcagcatgaa 2820 ggacgtcggc agcaccatcg gctgccagcg actggcaggg cggctgatcc ccaggagcta 2880 cctgagcctg caggaggccg tgctggcaga gcagcagcgc cgcagccggg acgacgacgt 2940 gcagtacctg acggacaggc agctggagca gctggtggag cagacgcccg acaacgacat 3000 caaggactac gaggacctgc agtcagccat cagcttcctc atagaaaccg gcaccctgct 3060 ccatttcccg gacaccagcc acggcctgag gaacctctac ttcctcgacc ctatttggct 3120 ctccgaatgt ctgcagagga tctttaatat taagggctct cggtcagtgg ccaagaatgg 3180 ggtgatcaga gcagaagacc tcaggatgct gctggtgggg actggcttca cgcagcagac 3240 ggaagagcag tacttccagt tcctggccaa gtttgagatc gccctgcccg tcgccaatga 3300 cagctacctc ctgccccatc tccttccatc taaacctggc ctggacaccc acggtatgcg 3360 gcaccccaca gccaacacca ttcagagggt atttaagatg agcttcgttc ccgttggctt 3420 ctggcaaagg tttatagcac ggatgctgat cagcctggcg gagatggacc tgcagctttt 3480 tgaaaacaag aagaatacta aaagcaggaa caggaaagtc accatttaca gttttacagg 3540 aaaccagaga aatcgctgta gcacattcag agtgaaaaga aatcagacca tctattggca 3600 ggaagggctc ctggtcactt ttgatggggg ctacctcagt gtggaatctt ccgacgtgaa 3660 ctggaaaaag aagaaaagcg gaggaatgaa aattgtttgc caatcagaag tgagggactt 3720 ctcagccatg gctttcatca cggaccacgt caattccttg attgatcagt ggtttcccgc 3780 cctgacagcc acagagagcg acgggacgcc actcatggag cagtacgtgc cctgcccggt 3840 ctgcgagaca gcctgggccc agcacacgga ccccagtgag aaatcagagg atgtgcagta 3900 cttcgacatg gaagactgtg tcctgacggc catcgagcgg gacttcatct cctgccccag 3960 acacccggac ctccccgtgc cgctgcagga gctggtccct gaactgttca tgaccgactt 4020 cccggccagg ctcttcctgg agaacagcaa gctggagcac agcgaggacg agggcagcgt 4080 cctgggccag ggcggcagtg gcaccgtcat ctaccgggcc cggtaccagg gccagcctgt 4140 ggccgtcaag cgcttccaca tcaaaaaatt caagaacttt gctaacgtac cggcagacac 4200 catgctgagg cacctgcggg ccaccgatgc catgaagaac ttctccgagt tccggcagga 4260 ggccagcatg ctgcacgcgc tgcagcaccc ctgcatcgtg gcgctcatcg gcatcagcat 4320 ccacccgctc tgcttcgccc tggagctcgc gccgctcagc agcctcaaca ccgtgctgtc 4380 cgagaacgcc agagattctt cctttatacc cctgggacac atgctcaccc aaaaaatagc 4440 ctaccagatc gcctcgggcc tggcctacct gcacaagaaa aacatcatct tctgtgacct 4500 gaagtcggac aacattctgg tgtggtccct tgacgtcaag gagcacatca acatcaagct 4560 atctgactac gggatttcga ggcagtcatt ccatgagggc gccctaggcg tggagggcac 4620 tcctggctac caggccccag agatcaggcc tcgcattgta tatgatgaga aggtagatat 4680 gttctcctat ggaatggtgc tctacgagtt gctgtcagga cagcgccctg cactgggcca 4740 ccaccagctc cagattgcca agaagctgtc caagggcatc cgcccggttc tggggcagcc 4800 ggaggaagtg cagttccggc gactgcaggc gctcatgatg gagtgctggg acactaagcc 4860 agagaagcga ccgctggccc tgtcggtggt gagccagatg aaggacccga cttttgccac 4920 cttcatgtat gaactgtgct gtgggaagca gacagccttc ttctcatccc agggccagga 4980 gtacaccgtg gtgttttggg atggaaaaga ggagtccagg aactacacgg tggtgaacac 5040 agagaagggc ctcatggagg tgcagaggat gtgctgccct gggatgaagg tgagctgcca 5100 gctccaggtc cagagatccc tgtggacagc caccgagaat tcctacctgg tcttagcggg 5160 cctcgccgat gggcttgtgg ctgtgtttcc cgtggtgcgg ggcaccccaa aggacagctg 5220 ctcctacctg tgctcacaca cagccaacag gtccaagttc agcatcgcgg atgaagacgc 5280 acggcagaac ccctacccag tgaaggccat ggaggtggtc aacagcggct ctgaggtctg 5340 gtacagcaat gggccgggcc tccttgtcat cgactgtgcc tccctggaga tctgcaggcg 5400 gctggagccc tacatggccc cctccatggt tacgtcagtc gtgtgcagct ctgagggcag 5460 aggggaggag gtcgtctggt gcctggatga caaggccaac tccttggtga tgtaccactc 5520 caccacctac cagctgtgtg cccggtactt ctgcggggtc cccagccccc tcagggacat 5580 gtttcccgtg cggcccttgg acacggaacc cccggcagcc agccacacgg ccaacccaaa 5640 ggtgcctgag ggggactcca tcgcggacgt gagcatcatg tacagtgagg agctgggcac 5700 gcagatcctg atccaccagg aatcactcac tgactactgc tccatgtcct cctactcctc 5760 atccccaccc cgccaggctg ccaggtcccc ctcaagcctc cccagctccc cagcaagttc 5820 ttccagtgtg cctttctcca ccgactgcga ggactcagac atgctacata cgcccggtgc 5880 tgcctccgac aggtctgagc atgacctgac ccccatggac ggggagacct tcagccagca 5940 cctgcaggcc gtgaagatcc tcgccgtcag agacctcatt tgggtcccca ggcgcggtgg 6000 agatgttatc gtcattggcc tggagaagga ttctggcgcc cagcggggcc gagtcattgc 6060 cgtcttaaaa gcccgagagc tgactccgca tggggtgctg gtggatgctg ccgtggtggc 6120 aaaggacact gttgtgtgca cctttgaaaa tgaaaacaca gagtggtgcc tggccgtctg 6180 gaggggctgg ggcgccaggg agttcgacat tttctaccag tcctacgagg agctgggccg 6240 gctggaggct tgcactcgca agagaaggta attcctgtgg aatgactgtc acacatcaga 6300 gctggctggc ccggggctgc agcctgactc ctctgccatc ggcctctagt tctccaagga 6360 cctagaagac agatggagtt ctcccctgaa ctccttgctg ctaagaagtg ctgagaagtt 6420 actcgcctgg cggtggctcc agggttctct ggttctctgg agcagagttc tctgaatacc 6480 ccatccccca actgctgatt ttacagcccc agggaagaca gtggtatcag gctgggagcg 6540 gcctcctctg gcctccccca tcagtttgca ggagcagggg tgcaggatcc tgttctgagc 6600 tgggtcaaac aaagcagggc cgggccttcc tgccatcccc aggtctcaga tggaattaca 6660 ctagaggccc tccgctggga agcacttgag gtagggcagg aggggggctg tgacccctgc 6720 cctttccccg ccagagacct caggctctca gcacattcca caggctcctg agtccccgag 6780 gcctgggcca gcttgggcaa gccaagatca gatgtctctg tgttcgggaa ggtctccgtg 6840 tgggaaagcc cttgggggat cccgggtgag gagtgttgcc ccatccagag aatgaatgag 6900 ttcctttaag tgccaccgcc agcaagccca gaggcacaca ttctgagtgc acccgcttag 6960 cctttacatt cctctccacc gacaaaagga aggggaaact caatcagcag gacttcagaa 7020 agggccttgt gtttatagct ttgtcaagta aatttggacg cagctggaaa cacaggcctg 7080 tttgttgcac ata 7093 <210> 31 <211> 1800 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 6035509CB1 <400> 31 gctgcagagt gctttacttt caacaagatg gagtcttgct ctgtttccca gcctgtagtg 60 cagtgacaca gtcttggctc actgtaacct ctgcctcctg ggttcaagtg attctcctgc 120 ctcagcctcc tgagtagctg ggattacagg aaacatctgt atggattatt tcactataat 180 cctatgatgc ttggacttga atcacttcca gatcccacag acacctggga aattata,gag 240 accattggta aaggcaccta tggcaaagtc tacaaggtaa ctaacaagag agatgggagc 300 ctggctgcag tgaaaattct ggatccagtc agtgatatgg atgaagaaat tgaggcagaa 360 tacaacattt tgcagttcct tcctaatcat cccaatgttg taaagtttta tgggatgttt 420 tacaaagcgg atcactgtgt agggggacag ctgtggctgg tcctggagct gtgtaatggg 480 ggctcagtca ctgagcttgt caaaggtcta ctcagatgtg gccagcggtt ggatgaagca 540 atgatctcat acatcttgta cggggccctc ttgggccttc agcatttgca caacaaccga 600 atcatccacc gtgatgtgaa ggggaataac attcttctga caacagaagg aggagttaag 660 ctcgttgact ttggtgtttc agctcaactc accagtacac gtctgcggag aaacacatct 720 gttggcaccc cgttctggat ggcccctgag gtcattgcct gtgagcagca gtatgactct 780 tcctatgacg ctcgctgtga cgtctggtcc ttggggatca cagctattga actgggggat 840 ggagaccctc ccctctttga catgcatcct gtgaaaacac tctttaagat tccaagaaat 900 cctccaccta ctttacttca tccagaaaaa tggtgtgaag aattcaacca ctttatttca 960 cagtgtctta ttaaggattt tgaaaggcga ccttccgtca cacatctcct tgaccaccca 1020 tttattaaag gagtacatgg aaaagttctg tttctgcaaa aacagctggc caaggttctc 1080 caagaccaga agcatcaaaa tcctgttgct aaaaccaggc atgagaggat gcataccaga 1140 agaccttatc atgtggaaga tgctgaaaaa tactgccttg aggatgattt ggtcaaccta 1200 gaggttctgg atgaggtact aaatatttag tagacaattc tcattgaaga catttgtttc 1260 atgtgaatgg tctgaacttt ctgttgtaga ccatgtcctc ctaaggtcat ttgaaaattt 1320 aattgtttgt gtagctatgg gatgaagttc agggagcatt cagttgctgt gactatgatc 1380 ctgtgctgtg tttatttaga tagcccctag aatgatgaag agaaaaggat ttggattttt 1440 gcaataaagc tctttatatt gtagccttaa tgatggatta tatcagctga aaatattttg 1500 tttgataaaa tttgataaaa tatttcaatt aacccttaag aagttgtttg ttcttcataa 1560 gaaagagctt catttaggga aatagtgaag ttaatatagc ttgaattcta aatttgaagt 1620 ctgtgataat ccccatttaa aatatgcatg tttaatagag ctgttaattg cactggacct 1680 gtttatgctg agtctaactc tggggattgt taccttcaat gtctaaatca ctaaagtgta 1740 atacaaagtg gttaattctg tatttatgcc acctaggttt taagtgcagt gctttgagaa 1800 <210> 32 <211> 6347 .
<212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7373485CB1 <400> 32 ggaagcgaga agccgcatca accatgtaag cagcttcgct tcctgccgca accgtccgcg 60 gcctgaggag cccaccgccg ctctcggggg ccgacttccg ggggctgagc cgttgaagcg 120 gaggctgggg cggggggcag ccggcgcggc cggggcagga ggcgcagact catgaaatgg 180 ccacagatga taagacgtcc ccaacactgg actctgctaa tgatttgcct cgatctccta 240 ctagtccttc tcatctcaca cactttaaac ctttgactcc tgatcaagat gagccccctt 300 ttaaatcagc ttatagttct tttgtaaatc tctttcgttt taacaaagag agagcagaag 360 gaggccaggg agaacagcag cctttgagtg gaagttggac cagccctcag ctcccttcga 420 ggacacagtc tgttaggtca cccacacctt ataaaaagca gcttaatgag gaactccagc 480 ggcgctcttc agcattagac acaagaagga aagcagaacc tacctttgga ggtcatgacc 540 ctcgtacagc tgttcagctt cgaagcctca gcacagtatt aaaacgcctc aaggaaatca 600 tggaggggaa aagccaggat agtgacctga aacaatactg gatgccagat agccaatgta 660 aagagtgcta tgactgtagt gagaaattta caacctttag gcgcagacac cattgccgac 720 tatgtgggca gattttctgc agtcgttgct gtaatcaaga aatccctgga aaatttatgg 780 gctatacagg agacctccga gcttgcacat attgtagaaa aatagcctta agttatgctc 840 attccacaga cagtaattct attggggaag acttgaatgc tctttcagat tctgcttgct 900 ctgtgtctgt gcttgatcca agtgaacccc gaacacctgt tgggagtagg aaagccagcc 960 gtaacatatt tttagaggat gatttggcct ggcaaagttt gattcatcca gattcctcaa 1020 atactcctct ttcaacaaga cttgtatctg tgcaagagga tgctgggaaa tctcctgctc'1080 gaaatagatc agccagcatt actaacctgt cactggatag atctggttct cctatggtac 1140 cttcatatga gacatctgtc agtccccagg ctaaccgaac atatgttagg acagagacca 1200 ctgaggatga acgcaaaatt cttctggaca gtgtgcagtt aaaagacctg tggaaaaaaa 1260 tctgccatca cagcagtgga atggagtttc aggatcaccg ctactggttg agaacgcatc 1320 ccaactgcat tgtaggaaag gaattagtca actggctaat ccgaaatggg catattgcca 1380 caagggcaca agctatagca attggacaag caatggttga tggacgttgg ctggattgtg 1440 ttagtcatca cgaccagctt ttcagagatg agtatgcgct gtatagacca ctgcagagta 1500 cagaattttc tgagacgcct tctcccgaca gtgactcagt gaactccgtg gaaggacact 1560 ctgagccatc ctggtttaaa gacataaagt ttgatgacag tgacacagaa cagatagctg 1620 aagaaggtga cgataatttg gctaagtatt tgatttctga cactggagga caacagctct 1680 caataagtga cgctttcatc aaagaatcct tatttaatcg ccgagtagag gaaaaatcca 1740 aagagctgcc tttcacacct ttgggctggc atcataacaa cctggagctc ctgagggagg 1800 agaatgggga gaaacaagcc atggagaggt tgctttcagc taatcataac cacatgatgg 1860 cactactcca gcagttgctc catagtgact cactgtcatc atcttggagg gacatcatcg 1920 tgtcattggt ctgccaggtt gttcagacag tccgacctga tgtcaagaac caggatgatg 1980 acatggatat ccgtcagttt gtccacatca aaaaaatccc aggtggaaag aagtttgatt 2040 ctgtggttgt caatggcttt gtttgtacca agaacattgc acataaaaag atgaattctt 2100 gtattaaaaa ccctaaaatt cttctgttga agtgttccat tgagtatctc tacagagaag 2160 aaactaagtt tacttgcatt gatcctattg tgcttcagga aagggaattc ttgaagaatt 2220 atgtccagcg aatagttgat gttcgaccca ccttggttct tgttgagaaa acagtgtctc 2280 ggattgccca ggacatgtta ttggaacatg gcattacttt ggtcattaat gtaaagtcac 2340 aagttttgga acgaatcagt cgaatgaccc aaggtgattt agtgatgtca atggaccagc 2400 tgcttacgaa accacgcctg ggcacttgtc acaaatttta tatgcagata tttcagttgc 2460 ctaatgaaca aaccaagaca ctgatgtttt ttgaaggttg tccacagcac ctaggctgta 2520 caatcaagct aagaggaggc tctgattatg agctggctcg agttaaggag atcctaatat 2580 ttatgatctg tgttgcttat cattctcaac tagaaatatc ctttctcatg gatgaatttg 2640 ctatgcctcc cacattaatg caaaaccctt cattccattc cctgattgag ggacgagggc 2700 atgagggggc tgtccaagag cagtacggtg gaggttccat cccctgggat cctgacatcc 2760 ctcctgagtc tctgccctgt gatgatagca gtttgctgga atcgaggatt gtgtttgaga 2820 agggtgagca ggaaaataaa aatcttccgc aggctgttgc ctctgtgaag catcaagaac 2880 atagcacaac agcttgcccg gcgggtctcc cttgtgcttt ctttgcacct gtaccggaat 2940 cattgttgcc actccctgtg gatgaccaac aagatgcttt aggcagcgag ctgccagaga 3000 gtttgcagca aacagttgtg ctgcaggatc ccaaaagcca gataagagcc tttagagacc 3060 ctctacagga tgacactgga ttatatgtta ctgaggaagt cacctcctct gaagataaac 3120 gaaagactta ttctttggcc tttaagcagg aattaaaaga tgtgatcctc tgtatctccc 3180 cagtaatcac attccgagaa ccctttcttt taactgaaaa ggggatgaga tgctctaccc 3240 gagattattt tgcagagcag gtttactggt ctcctctcct caataaagaa ttcaaagaaa 3300 tggagaacag gaggaagaaa cagctgctca gggatctctc tggacttcag ggcatgaatg 3360 gaagtattca ggccaagtct attcaagtct taccctcaca tgagctagtg agcactagaa 3420 ttgctgagca tctgggcgat agccagagct tgggtagaat gctggccgat tatcgagcca 3480 gaggaggaag aattcagccc aaaaattcag acccttttgc tcattcaaag gatgcatcaa 3540 gtacttcaag tggcaaatca ggaagcaaaa acgagggtga tgaagagaga gggcttattc 3600 tgagtgatgc tgtgtggtca acaaaggtgg actgtctgaa tcccattaat caccagagac 3660 tttgtgtgct cttcagcagc tcttctgccc agtccagcaa tgctcctagt gcctgtgtca 3720 gtccttggat tgtaacaatg gaattttatg gaaagaatga tcttacatta ggaatatttt 3780 tagagagata ctgtttcagg ccttcttatc agtgtccaag catgttctgt gataccccca 3840 tggtacatca tattcggcgc tttgttcatg gccaaggctg tgtgcagata atcctgaagg 3900 agttggattc tccagtacct ggatatcagc atacaattct tacatattcc tggtgtagaa 3960 tctgcaaaca ggtaacacca gttgttgctc tttccaatga gtcctggtct atgtcatttg 4020 caaaatacct tgaacttagg ttttatgggc accagtatac tcgcagagcc aacgctgagc 4080 cctgtggtca ctccatccat catgattatc accagtattt ctcctataac cagatggtgg 4140 cgtctttcag ttattctccc attcggcttc ttgaagtatg tgttccactc cccaaaatat 4200 tcattaagcg tcaggcccca ttaaaagtgt cccttcttca ggatctgaag gacttctttc 4260 aaaaagtttc acaggtatat gttgccattg atgaaagact tgcatctttg aaaactgata 4320 catttagtaa aacaagagag gaaaaaatgg aagatatttt tgcacagaaa gagatggaag 4380 aaggtgagtt caagaactgg attgagaaga tgcaagcaag gctcatgtct tcctctgtag 4440 atacccctca gcaactgcag tcggtctttg agtcactcat tgccaagaaa caaagtctct 4500 gtgaagtgct gcaagcttgg aataacaggt tgcaggacct tttccaacag gaaaagggta 4560 gaaagagacc ttcagttcct ccaagtcctg gaagactgag acaaggggaa gaaagcaaga 4620 taagtgcgat ggatgcatct ccacggaata tttctccagg acttcagaat ggagaaaaag 4680 aggatcgctt cttaacaact ttgtccagcc agagctccac cagttctact catctccaat 4740 tgcctacgcc acctgaagtc atgtctgaac agtcagtggg agggccccct gagctagata 4800 cagccagcag ttccgaagat gtgtttgatg ggcatttgct gggatccaca gacagccaag 4860 tgaaggaaaa gtcaaccatg aaagccatct ttgcaaattt gcttccagga aatagctata 4920 atcctattcc atttcctttt gatccagata aacactactt aatgtatgaa catgaacgag 4980 tgcccattgc agtctgcgag aaggaaccca gctccatcat tgcttttgct ctcagttgta 5040, aagaataccg aaatgcctta gaggaattgt ctaaagcgac tcagtggaac agtgccgaag 5100 aagggcttcc aacaaatagt acttcagata gcagaccaaa gagtagcagc cctatcagat 5160 tacctgaaat gagtggagga cagacaaatc gtacaacaga aacagaacca caaccaacca 5220 aaaaggcttc tggaatgctg tccttcttca gagggacagc agggaaaagc cccgatctct 5280 cttcccagaa gagagagacc ttacgtggag cagatagtgc ttactaccag gttgggcaga 5340 caggcaagga ggggaccgag aatcaaggcg ttgagcctca agatgaagta gatggaggag 5400 atacgcaaaa gaagcaactc ataaatcctc atgtggaact tcaattttca gatgctaatg 5460 ccaagtttta ctgtcggctc tactatgcgg gagagtttca taagatgcgt gaagtgattc 5520 tggacagcag tgaggaagat ttcattcgtt ccctctccca ctcatcaccc tggcaggccc 5580 ggggaggcaa atcaggagct gccttctatg caactgagga tgatagattt attttgaagc 5640 aaatgcctcg tctggaagtc cagtccttcc tcgactttgc accacattac ttcaattata 5700 ttacaaatgc tgttcaacaa aagaggccca cggcgttggc caaaattctt ggagtttaca 5760 gaattggtta taagaactct cagaacaaca ctgagaagaa gttagatctc cttgtcatgg 5820 aaaatctttt ctacgggaga aagatggcac aggtttttga tttgaagggc tctcttagga 5880 atcggaatgt aaaaactgac actggaaaag agagttgtga tgtggtcctg ctagatgaaa 5940 atctcctaaa gatggttcga gacaaccctc tatatattcg ttctcattcc aaagctgtgc 6000 tgagaacctc gatccatagt gactcccatt tcctttctag ccacctcatt atagattatt 6060 ctttgctggt tgggcgagat gatactagca atgagctagt agttggaatt atagattata 6120 ttcgaacatt tacatgggac aaaaagcttg agatggttgt gaaatcaaca ggaattttag 6180 gtggacaagg taaaatgcca acagtggtgt ctccggagtt gtacaggact aggttttgtg 6240 aggcaatgga caagtatttc ctaatggtac cagaccactg gacaggcttg ggtctgaatt 6300 gctgaaatca agacatattt gaaatggact gtgaggaaaa ggggaac 6347 <210> 33 <211> 1876 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5734965CB1 <400> 33 tggggttcgg cgcggctacg tgcagaatcc gtctagctaa aatgtaattt cagattggac 60 aagtactgtg gaggaactgc aatgtctggt ggagaacaga aaccagagag gtactatgtg 120 ggtgtggacg ttggaacagg cagtgtccgt gcagctctgg tggaccagag tggggtcctg 180 ttggcttttg cagaccagcc aattaagaat tgggagcccc agttcaacca ccatgagcag 240 tcctccgagg acatctgggc tgcgtgctgt gttgtcacaa agaaagttgt acaagggatt 300 gatttaaacc aaattcgagg acttgggttt gatgccacgt gttctctggt tgttttggat 36U
aagcagtttc acccattacc agtcaaccag gaaggggatt cccatcgaaa cgtcatcatg 420 tggctggacc atcgagcagt cagtcaagtt aacaggatca atgagaccaa gcacagtgtc 480 ctccagtacg tcgggggggt gatgtctgtg gaaatgcagg ccccgaaact tctgtggctg 540 aaagagaact tgagagagat ttgctgggat aaggcgggac atttctttga tctcccggac 600 ttcttatcgt ggaaggcaac aggtgtcaca gcacggtctc tctgctccct ggtgtgtaag 660 tggacatatt cagcagagaa aggctgggac gacagtttct ggaaaatgat tggtttggaa 720 gactttgttg cagataatta cagcaaaata ggaaaccaag tgctacctcc tggagcttct 780 cttggaaatg ggctcacacc agaggcagca agagaccttg gccttctccc tgggattgcg 840 gtcgcagctt cactcattga tgcccatgca ggaggactag gagtgattgg ggcagatgtg 900 agagggcacg gcctcatctg tgaggggcag ccagtgacgt cacggctggc tgtcatctgt 960 ggaacgtctt cttgtcacat ggggatcagc aaagacccga tttttgtacc aggcgtctgg 1020 gggccttatt tctcagccat ggtacctggg ttctggctga atgaaggtgg tcagagcgtt 1080 actggaaaat tgatagacca catggtacaa ggccatgctg cttttccaga actacaagta 1140 aaggccacag ccagatgcca gagtatatat gcatatttga acagtcacct ggatctgatt 1200 aagaaggctc agcctgtggg tttccttact gttgatttac atgtttggcc agatttccat 1260 ggcaaccggt ctcccttagc agatctgaca ctaaagggca tggtcaccgg attgaaactg 1320 tctcaggacc ttgatgatct tgccattctc tacctggcca cagttcaagc cattgctttg 1380 gggactcgct tcattataga agccatggag gcagcagggc actcaatcag tactcttttc 1440 ctatgtggag gcctcagcaa gaatcccctt tttgtgcaaa tgcatgcgga cattactggc 1500 atgcctgtgg tcctgtcgca agaggtggag tccgttcttg tgggtgctgc tgttctgggt 1560 gcctgtgcct caggggattt cgcttctgta caggaagcaa tggcaaaaat gagcaaagtt 1620 gggaaagttg tgttcccgag actacaggat aaaaaatact atgataagaa ataccaagta 1680 ttcctgaagc tggttgaaca ccagaaggag tatttggcga tcatgaatga tgactgaaca 1740 gggcttgcag gtgctgatgc cagaagcttc tgtgccattg cattaaagac ttctgtcatt 1800 tgatccatgt tcaagaccct tgaggtattg tttcatcatt tctgtattgt ctttcaataa 1860 agaatacaaa catgtg 1876 <210> 34 <211> 1487 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473788CB1 <400> 34 atgaggagtg gcgccgaacg caggggcagc agcgccgcgg cgtccccggg ctcgccgccc 60 cccggccgcg cgcgccccgc cggctccgac gcgccctcgg ccctgccgcc gcccgctgct 120 ggccagcccc gggcccggga ctcgggcgat gtccgctcgc agccgcgccc cctgtttcag 180 tggagcaagt ggaagaagag gatgggctcg tccatgtcgg cggccaccgc gcggaggccg 240 gtgtttgacg acaaggagga cgtgaacttc gaccacttcc agatccttcg ggccattggg 300 aagggcagct ttggcaaggt gtgcattgtg cagaagcggg acacggagaa gatgtacgcc 360 atgaagtaca tgaacaagca gcagtgcatc gagcgcgacg aggtccgcaa cgtcttccgg 420 gagctggaga tcctgcagga gatcgagcac gtcttcctgg tgaacctctg gtactccttc 480 caggacgagg aggacatgtt catggtcgtg gacctgctac tgggcgggga cctgcgctac 540 cacctgcagc agaacgtgca gttctccgag gacacggtga ggctgtacat ctgcgagatg 600 gcactggctc tggactacct gcgcggccag cacatcatcc acagagatgt caagcctgac 660 aacattctcc tggatgagag aggacatgca cacctgaccg acttcaacat tgccaccatc 720 atcaaggacg gggagcgggc gacggcatta gcaggcacca agccgtacat ggctccggag 780 atcttccact cttttgtcaa cggcgggacc ggctactcct tcgaggtgga ctggtggtcg 840 gtgggggtga tggcctatga gctgctgcga ggatggaggc cctatgacat ccactccagc 900 aacgccgtgg agtccctggt gcagctgttc agcaccgtga gcgtccagta tgtccccacg 960 tggtccaagg agatggtggc cttgctgcgg aagctcctca ctgtgaaccc cgagcaccgg 1020 ctctccagcc tccaggacgt gcaggcagcc ccggcgctgg ccggcgtgct gtgggaccac 1080 ctgagcgaga agagggtgga gccgggcttc gtgcccaaca aaggccgtct gcactgcgac 1140 cccacctttg agctggagga gatgatcctg gagtccaggc ccctgcacaa gaagaagaag 1200 cgcctggcca agaacaagtc ccgggacaac agcagggaca gctcccagtc cgagaatgac 1260 tatcttcaag actgcctcga tgccatccag caagacttcg tgatttttaa cagagaaaag 1320 ctgaagagga gccaggacct cccgagggag cctctccccg ccctgagtcc agggatgctg 1380 cggagcctgt ggaggacgag gcggacgctc cgcctgccca tgtgcggccc catttgcccc 1440 tcggccggga gcggctaggc cgggacgccc gtggtcctca ccccttg 1487 <210> 35 <211> 1884 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3107989CB1 <400> 35 gaggtgacca attttctctc caaaagagaa aggaagttga ttaaaaaaag aatccatgct 60 ccaaagcggc agccaaatcc atctatggcc cccaatgcat cacccagaaa ggggttccag 120 actctcctgc aaaaggccaa ctctacttcc cggctcccac ttcccctcct tcgccacagg 180 agggtggcga aggatttata acccacctct ttctttcagt tgccatggag acaagcccca 240 gtcctttcat tccttctggt acctctctct ccaacgcagg cggaaaggag gcggcttagc 300 ccaaacatgc tgggggaggg gctggcggcc tcgacggcag ctgcggaact aggccgaggg 360 acaaaggcta agtttttcca tggtttggac tggatatcgg tggaactctg gtcaagctgg 420 tatattttga acccaaagac atcactgctg aagaagaaga ggaagaagtg gaaagtctta 480 aaagcattcg gaagtacctg acctccaatg tggcttatgg gtctacaggc attcgggacg 540 tgcacctcga gctgaaggac ctgactctgt gtggacgcaa aggcaatctg cactttatac 600 gctttcccac tcatgacatg cctgctttta ttcaaatggg cagagataaa aacttctcga 660 gtctccacac tgtcttttgt gccactggag gtggagcgta caaatttgag caggattttc 720 tcacaatagg tgatcttcag ctttgcaaac tggatgaact agattgcttg atcaaaggaa 780 ttttatacat tgactcagtc ggattcaatg gacggtcaca gtgctattac tttgaaaacc 840 ctgctgattc tgaaaagtgt cagaagttac catttgattt gaaaaatccg tatcctctgc 900 ttctggtgaa cattggctca ggggttagca tcttagcagt atattccaaa gataattaca 960 aacgggtcac aggtactagt cttggaggag gaactttttt tggtctctgc tgtcttctta 1020 ctggctgtac cacttttgaa gaagctcttg aaatggcatc tcgtggagat agcaccaaag 1080 tggataaact agtacgagat atttatggag gggactatga gaggtttgga ctgccaggct 1140 gggctgtggc ttcaagcttt ggaaacatga tgagcaagga aaagcgagat tccatcagca 1200 aggaagacct cgcccgggcc acattggtca ccatcaccaa caacattggc tccattgctc 1260 ggatgtgtgc gttgaatgag aacatagaca gagttgtgtt tgttggaaat tttctcagaa 1320 tcaatatggt ctccatgaag ctgctggcat atgccatgga tttttggtcc aaaggacaac 1380 tgaaagctct gtttttggaa catgagggtt attttggagc cgttggggca ctgttggaac 1440 tgttcaaaat gactgatgac aagtagagac gagcagtgga ggaaacagcc tcccaaaagg 1500 acagagaact aaaaaattgc tgctggagaa ggtgaaagtc gctttgggac ggaagccaag 1560 ccattatggc agatgaacct gctggatttg taaataattt aaaatccttc cagatgatct 1620 tttactctta ggttttgagc taatgattca aaacggggga atataaaagg ttttttttct 1680 gtatactgta tttttttaaa aaaatggtgc agcgtggcca aacctaccaa ttgtatgcat 1740 taactttgaa aagttgtttg atgtttaaga aggacctgat atgtaagcgc tggtcatttt 1800 tcttctgggg tttactgatc agtgtggtga ttttaacttc atttagtaat tactctagga 1860 gattttacct tgacttatat tttc 1884 <210> 36 <211> 1070 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7482887CB1 <400> 36 gcaaatcaca cagcatggca gctcccagtc ctcctgcctc ttctgcattc cagacctgct 60 ctttaaaaac ctgggcattc cctccacaaa ttgaagagtg gaattttttt tcacctgctc 120 ttcctcttgc tggcacagat cataaagtct tgctctcttt ctatcacatc tcattattat 180 tttggcttct ttctacaagc aaggagcagc aggccctttt acattaccat tagtgaaggc 240 acttgagtta aatccgcaca acgaatctta ctcttgcctg taatcccagc actttggaaa 300 gccaaggcgg gtggatcacc tgaggtcagg agttcgagac cagcctggcc aatgtggtaa 360 aacctatctc tactaaaaat acaaaaaatt ggccaggtgt ggtggtgggg gcctgtaatc 420 tcagctactt gggaggctaa ggcaggagaa ttgcttgaat ctgggagaca gaggttgcgg 480 tgggccaaga tagcgccact gcactccagc cttagcaaca agagcacaac tccatctcaa 540 aataataata ataatttctt ggctccaagt ctcagctccc gcaccacctg acactgtcag 600 atcctcaggc catggccaac actgagagca tcattatcaa tccgagtgct gttcagcaca 660 gcctggtggg tgaaatcatc aaatactctg agcagaaggg attctacctg gtgaccatga 720 agttccttcg ggcctctgag aaacccctga agccgcacta cactaacctg aaagaccacc 780 cattcttccc ggaccttgtg aagtacatga actcagggca ggttgtggcc atggtcctgg 840 aggggctgaa tgtggcaaag acagggctaa ggatgcttgg ggagaccaat tcattgggct 900 ctatgctaga gactattatt cgcagggact tctgcgctaa aataggcggg aacgtcattg 960 gtggcagtga ttcattacaa agtgctgaaa aagaaatcag cctatggttt aagcccaaag 1020 aaccagttga ctacagatct tgtgcttatg actgggtcta tgcatgatag 1070 <210> 37 <211> 2890 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2963414CB1 <400> 37 gtgacccttc cctccccagg ccacggcagc ccggccctcc cgggcagacc tcccgcacca 60 gggctctggt gaacagcaaa tgctccacgc tgggacgggc cattgcctga tgcctgtaca 120 tggtgggcac tgagagacaa gattcctggg ccctgccttc catacactcc ccacgatctc 180 ggaggaagct ctgaggaccc cgctgagaac ccacagacag gaggacaact gcgctatgac 240 agcaataaag gccaagaagg agaaagttga ggaccgctga cagccccgtg tgctgttggg 300 agctgccctt tctacttcaa accttcctct agcagactgt gcagggaccc cccaccacca 360 ccatctgccg ccatggttgt gcaaaacagc gcagacgccg gggacatgag ggcaggcgtg 420 cagctggagc ccttcctgca ccaggtcggg gggcacatga gcgtgatgaa gtatgacgag 480 catacggtgt gcaagcccct cgtctcccgg gagcagaggt tctatgaatc cctgccgctg 540 gccatgaagc ggttcacccc acagtacaaa ggtaccgtca cagtgcacct ctggaaagac 600 agcacaggcc atctcagctt ggttgccaac ccagtgaagg agagccagga gcccttcaag 660 gtctccacag agtcggcggc ggtggccata tggcagacgc tccagcagac caccggcagc 720 aatggcagcg actgcaccct tgcccagtgg ccgcabgccc agctggcacg ctcacccaag 780 gagagcccgg ccaaggctct tctgaggtcc gagccccacc tcaacactcc agccttctcg 840 ctggtggaag acaccaacgg aaaccaggtt gagaggaaga gcttcaaccc gtggggcctg 900 caatgccacc aggcccacct gacccgcctg tgctccgagt acccagagaa caagcggcat 960 cggttcttgt tgctggaaaa tgtagtgtca cagtacacgc atccctgtgt cctggatctg 1020 aagatgggga cccggcagca cggcgatgat gcatcggagg agaagaaggc ccgccacatg 1080 aggaagtgtg cgcagagcac ctcagcctgc ctgggtgtgc gcatctgcgg catgcaggtt 1140 tatcaaacag ataagaagta ctttctctgc aaagacaagt actatggaag aaaactctca 1200 gtggaggggt tcagacaagc cctctatcag ttcctacata atggaagcca cctccggagg 1260 gagctcctgg agcccatcct gcaccagctc cgggccctcc tctccatcat taggagccag 1320 agttcatacc gcttctattc cagctctctc cttgtcatct atgatgggca ggaaccacca 1380 gaaagagccc caggcagccc gcatcctcac gaggctcccc aggcagccca cggtagctct 1440 cccggtggtc tcaccaaggt tgacatccgc atgattgact ttgctcatac cacatacaag 1500 ggctactgga atgagcacac cacctacgat ggaccagacc ctggctatat ttttggcctg 1560 gaaaacctca tcaggatcct gcaggatatc caagagggag aatgaaactt cctgggctta 1620 tctggattct tctgggctat agatctcaaa tagagacctg ttggttgcta gggtagtcca 1680 gacacccctt agatgtcttc ataatagtcc tatctacctt caaaaaccat ctctatatat 1740 ggcagactat attaacagct gctgaacaaa tcagctctgg aggtgattcc acatcccctg 1800 gcattatgct ctaatgctgc tcatcggaga acagacagcc aggataaagt ggcaccttct 1860 ggagtacact ggagggggca gcccaagtta gaggccagca ttgctgacat tctggaatat 1920 ttgcatctaa aaatgtttac tcgttgccat gctgcagtcc gcacaagctg tgaggcagaa 1980 aacttgactt gaagcagcct tgaagagtga gttcatgagc tcatggtttt tctccttgta 2040 tggactgctc gctccaaggg caggcagagc tcatgaatgc ctcttatctt cctaagcgga 2100 gttttaggtg acacaggatg aagcagaaga gatctaccca tctcacctgc tctgcaccca 2160 gcttctaagt ggacaaagcc aagcccaggc atgagctctg gcaaagcaag accccagatt 2220 ctccattttt gcctgtggaa aggagggtcc ctttacaggc ttttttttcc tttttttccc 2280 ccaaaatctc ttaaaatgag gaatctctta gcagactttg gagttcccca ttctgccaca 2340 ttctgaccat gagacgcggc ttgcagtggg ggtgaacgca cataaaaagg gaccactgac 2400 gtcctgctct actctctgct ttctatttat ttattttggg ggtgggttgg ggagtcagaa 2460 gaacctggag gacggaggaa accaggggca atgtttacaa gactggtgga caagtgtaaa 2520 tatggaataa gaacaaacag ttctaattaa ttccttcttc tgcagtacgg aaacctatta 2580 caatgccctt gagtcaagca ctgagatacg ttacccaatt agggaaataa atttgttaat 2640 aaaattgctg aggtcaccag tgattattgg tgtgccttat taccctttcc atttgtttat 2700 tctgatcaca ctgtgtggta gttccaattt atgagcgact agcatatacc acaagaacag 2760 ttcactgatt tcctacaatc cttcagggaa ctcgggtgga aatggtggct aataaaatat 2820 ttgcatgtat ctgcaaggga ggcaccagac ctgagaagtg gtccttttat ttgaatctca 2880 tacaatgtac 2890 <210> 38 <211> 5198 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477139CB1 <400> 38 cgacacggag cacccttcta gcttcttcgt ctccaggact gacgctcagg ctcctctctc 60 gccttagccc aacttgcttt cccgcctcgc aaactccggt ttccctccac tcccaactct 120 tttcactaca cgtttcccct cctctatctc ccacgccacg aaccccgatc cccagactcc 180 tctctcccgc cctcctcctt cctctctcct cccttcaact cttcatccgc ttccacctca 240 gactctgcgc gcacccaatt cagtcgcccg ctcccgttcg gctcctcgaa gccatggcgg 300 gacctggggg ctggagggac agggaggtca cggatctggg ccacctgccg gatccaactg 360 gaatattctc actagataaa accattggcc ttggtactta tggcagaatc tatttgggac 420 ttcatgagaa gactggtgca tttacagctg ttaaagtgat gaacgctcgt aagacccctt 480 tacctgaaat aggaaggcga gtgagagtga ataaatatca aaaatctgtt gggtggagat 540 acagtgatga ggaagaggat ctcaggactg aactcaacct tctgaggaag tactctttcc 600 acaaaaacat tgtgtccttc tatggagcat ttttcaagct gagtccccct ggtcagcggc 660 accaactttg gatggtgatg gagttatgtg cagcaggttc ggtcactgat gtagtgagaa 720 tgaccagtaa tcagagttta aaagaagatt ggattgctta tatctgccga gaaatccttc 780 agggcttagc tcaccttcac gcacaccgag taattcaccg ggacatcaaa ggtcagaatg 840 tgctgctgac tcataatgct gaagtaaaac tggttgattt tggagtgagt gcccaggtga 900 gcagaactaa tggaagaagg aatagtttca ttgggacacc atactggatg gcacctgagg 960 tgattgactg tgatgaggac ccaagacgct cctatgatta cagaagtgat gtgtggtctg 1020 tgggaattac tgccattgaa atggctgaag gagcccctcc tctgtgtaac cttcaaccct 1080 tggaagctct cttcgttatt ttgcgggaat ctgctcccac agtcaaatcc agcggatggt 1140 cccgtaagtt ccacaatttc atggaaaagt gtacgataaa aaatttcctg tttcgtccta 1200 cttctgcaaa catgcttcaa cacccatttg ttcgggatat aaaaaatgaa cgacatgttg 1260 ttgagtcatt aacaaggcat cttactggaa tcattaaaaa aagacagaaa aaaggaatac 1320 ctttgatctt tgaaagagaa gaagctatta aggaacagta caccgtgaga agattcagag 1380 gaccctcttg cactcacgag cttctgagat tgccaaccag cagcagatgc agaccactta 1440 gagtcctgca tggggaaccc tctcagccaa ggtggctacc tgatcgagaa gagccacagg 1500 tccaggcact tcagcagcta cagggagcag ccagggtatt catgccactg caggctctgg 1560 acagtgcacc taagcctcta aaggggcagg ctcaggcacc tcaacgacta caaggggcag 1620 ctcgggtgtt catgccacta caggctcagg tgaaggctaa agcctctaaa cctctacaaa 1680 tgcagattaa ggcacctcca cgactacgga gggcagccag ggtgctcatg ccactacagg 1740 cacaggttag ggcacctagg cttctgcagg tacagtccca ggtatccaaa aagcagcagg 1800 cccagaccca gacatcagaa ccacaagatt tggaccaggt accagaggaa tttcagggtc 1860 aagatcaggt acccgaacaa caaaggcagg gccaggcccc tgaacaacag cagaggcaca 1920 accaggtgcc tgaacaagag ctggagcaga accaggcacc tgaacagcca gaggtacagg 1980 aacaggctgc cgagcctgca caggcagaga ctgaggcaga ggaacctgag tcattacgag 2040 taaatgccca ggtatttctg cccctgctat cacaagatca ccatgtgctg ttgccactac 2100 atttggatac tcaggtgctc attccagtag aggggcaaac tgaaggatca cctcaggcac 2160 aggcttggac actagaaccc ccacaggcaa ttggctcagt tcaagcactg atagagggac 2220 tatcaagaga cttgcttcgg gcaccaaact caaataactc aaagccactt ggtccgttgc 2280 aaaccctgat ggaaaatctg tcatcaaata ggttttactc acaaccagaa caggcacggg 2340 agaaaaaatc aaaagtttct actctgaggc aagcactggc aaaaagacta tcaccaaaga 2400 ggttcagggc aaagtcatca tggagacctg aaaagcttga actctcggat ttagaagccc 2460 gcaggcaaag gcgccaacgc agatgggaag atatctttaa tcagcatgag gaagaattga 2520 gacaagttga taaaaccagt tggcgtcagt ggggtccttc agaccagttg attgacaata 2580 gtttcactgg tatgcaagac ctgaagaaat atctcaaagg aaaaacaaca tttcataatg 2640 ttcaagttgt tatctacaga gcagttaagg ggaatgatga tgttgcaaca aggtctaccg 2700 ttcctcagcg gtctcttttg gaacaagctc agaagcccat tgacatcaga caaaggagtt 2760 cgcaaaatcg tcaaaattgg ctggcagcat caggtgattc aaagcacaaa attttagcag 2820 gcaaaacaca gagctactgt ttaacaattt atatttcaga agtcaagaaa gaagaatttc 2880 aagaaggaat gaatcaaaag tgtcagggag cccaagtagg attaggacct gaaggccatt 2940 gtatttggca attgggtgaa tcttcttctg aggaagaaag tcctgtgact ggaaggaggt 3000 ctcagtcatc accaccttat tctactattg atcagaagtt gctggttgac atccatgttc 3060 cagatggatt taaagtagga aaaatatcac cccctgtata cttgacaaac gaatgggtag 3120 gctataatgc actctctgaa atcttccgga atgattggtt aactccggca cctgtcattc 3180 agccacctga agaggatggt gattatgttg aactctatga tgccagtgct gatactgatg 3240 gtgatgatga tgatgagtct aatgatactt ttgaagatac ctatgatcat gccaatggca 3300 atgatgactt ggataaccag gttgatcagg ctaatgatgt ttgtaaagac catgatgatg 3360 acaacaataa gtttgttgat gatgtaaata ataattatta tgaggcgcct agttgtccaa 3420 gcttgttgtc agggcaagct atggcagaga tggaagctgc aagcaagatg gttatgatgg 3480 aagtcgtgga aaagaggaag cctacagagg ctatggaagc catacagcca atagaagcca 3540 tggaggaagt gcagccagtg agggacaatg cagccattgg agatcaggaa gaacatgcag 3600 ccaatatagg cagtgaaaga agaggcagtg agggtgatgg aggtaaggga gtcgttcgaa 3660 ccagtgaaga gagtggagcc cttggactca atggagaaga aaattgctca gagacagatg 3720 gtccaggatt gaagagacct gcgtctcagg actttgaata tctacaggag gagccaggtg 3780 gtggaaatga ggcctcaaat gccattgact caggtgctgc accgtcagca cctgatcatg 3840 agagtgacaa taaggacata tcagaatcat caacacaatc agatttttct gccaatcact 3900 catctccttc caaaggttct gggatgtctg ctgatgctaa ctttgccagt gccatcttat 3960 acgctggatt cgtagaagta cctgaggaat cacctaagca accctctgaa gtcaatgtta 4020 acccactcta tgtctctcct gcatgtaaaa aaccactaat ccacatgtat gaaaaggagt 4080 tcacttctga gatctgctgt ggttctttgt ggggagtcaa tttgctgttg ggaacccgat 4140 ctaatctata tctgatggac agaagtggaa aggctgacat tactaaactt ataaggcgaa 4200 gaccattccg ccagattcaa gtcttagagc cactcaattt gctgattacc atctcaggtc 4260 ataagaacag acttcgggtg tatcatctga cctggttgag gaacaagatt ttgaataatg 4320 atccagaaag taaaagaagg caagaagaaa tgctgaagac agaggaagcc tgcaaagcta 4380 ttgataagtt aacaggctgt gaacacttca gtgtcctcca acatgaagaa acaacatata 4440 ttgcaattgc tttgaaatca tcaattcacc tttatgcatg ggcaccaaag tcctttgatg 4500 aaagcactgc tattaaagta tttccaacac ttgatcataa gccagtgaca gttgacctgg 4560 ctattggttc tgaaaaaaga ctaaagattt tcttcagctc agcagatgga tatcacctca 4620 tcgatgcaga atctgaggtt atgtctgatg tgaccctgcc aaagaataat atcatcattt 4680 tacctgattg cttgggaatt ggcatgatgc tcaccttcaa tgctgaagcc ctctctgtgg 4740 aagcaaatga acaactcttc aagaagatcc ttgaaatgtg gaaagacata ccatcttcta 4800 tagcttttga atgtacacag cgaaccacag gatggggcca aaaggccatt gaagtgcgct 4860 ctttgcaatc cagggttctg gaaagtgagc tgaagcgcag gtcaattaag aagctgagat 4920 tcctgtgcac ccggggtgac aagctgttct ttacctctac cctgcgcaat caccacagcc 4980 gggtttactt catgacactt ggaaaacttg aagagctcca aagcaattat gatgtctaaa 5040 agtttccagt gatttattac cacattataa acatcatgta taggcagtct gcatcttcag 5100 atttcagaga ttaaatgagt attcagtttt atttttagta aagattaaat ccaaaacttt 5160 acttttaatg tagcacagaa tagttttaat gagaaatg 5198 <210> 39 <211> 3969 <212> DNA

<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 55009053CB1 <400> 39 cttttttcct ttcagtgtgc ttcaaatgtc acgacacagg ttagctcagt cgacttgggg 60 ctgctgagct ctggtccctg ccagcctcac cgctcggacc cccccgatcc tccggactcc 120 gctggtcctg gccacgcgag gagcccacgc tagctccaaa gaatcccccg agggcacgtg 180 gaccgaggga gcccctgtga aggctgcgga agactccgcg cgtcccgagc tcccggactc 240 tgcagtgggc ccggggtcca gggagccgct aagggtccct gaagctgtgg ccctagagcg 300 gcggcgggag caggaagaaa aggaggacat ggagacccag gctgtggcaa cgtcccccga 360 tggccgatac ctcaagtttg acatcgagat tggacgtggc tccttcaaga cggtgtatcg 420 agggctagac accgacacca cagtggaggt ggcctggtgt gagctgcaga ctcggaaact 480 gtctagagct gagcggcagc gcttctcaga ggaggtggag atgctcaagg ggctgcagca 540 ccccaacatc gtccgcttct atgattcgtg gaagtcggtg ctgaggggcc aggtttgcat 600 cgtgctggtc accgaactca tgacctcggg cacgctcaag acgtacctga ggcggttccg 660 ggagatgaag ccgcgggtcc ttcagcgctg gagccgccaa atcctgcggg gacttcattt 720 cctacactcc cgggttcctc ccatcctgca ccgggatctc aagtgcgaca atgtctttat 780 cacgggacct tctggctctg tcaaaatcgg ggacctgggc ctggccacgc tcaagcgcgc 840 ctcctttgcc aagagtgtca tcgggacccc ggaattcatg gcccccgaga tgtacgagga 900 aaagtacgat gaggccgtgg acgtgtacgc gttcggcatg tgcatgctgg agatggccac 960 ctctgagtac ccgtactccg agtgccagaa tgccgcgcaa atctaccgca aggtcacttc 1020 gggcagaaag ccgaacagct tccacaaggt gaagataccc gaggtgaagg agatcattga 1080 aggctgcatc cgcacggata agaacgagag gttcaccatc caggacctcc tggcccacgc 1140 cttcttccgc gaggagcgcg gtgtgcacgt ggaactagcg gaggaggacg acggcgagaa 1200 gccgggcctc aagctctggc tgcgcatgga ggacgcgcgg cgcggggggc gcccacggga 1260 caaccaggcc atcgagttcc tgttccagct gggccgggac gcggccgagg aggtggcaca 1320 ggagatggtg gctctgggct tggtctgtga agccgattac cagccagtgg cccgtgcagt 1380 acgtgaacgg gttgctgcca tccagcgaaa gcgtgagaag ctgcgtaaag caagggaatt 1440 ggaggcactc ccaccagagc caggacctcc accagcaact gtgcccatgg cccccggtcc 1500 ccccagtgtc ttcccccctg agcctgagga gccagaggca gaccagcacc agcccttcct 1560 tttccgccac gccagctact catctaccac ttcggattgc gagactgatg gctacctcag 1620 ctcctccggc ttcctggatg cctcagaccc tgcccttcag ccccctgggg gggtgccatc 1680 cagcctggct gagtcccatc tctgcctgcc ctcggctttt gccctatcca ttccacgttc 1740 tggccctgga agtgactttt cccccgggga cagctatgcc tcagatgcag cttcaggcct 1800 tagcgatgtg ggagaaggga tgggacaaat gaggagaccc ccagggagga atctccggcg 1860 cagaccccga tcccggctgc gggtcactag tgtctcagac cagaatgaca gagtggttga 1920 gtgccagcta cagacccata acagcaagat ggtgaccttc cgatttgatc tggatgggga 1980 cagcccggaa gagattgcag ctgccatggt atataacgag ttcattctgc cttcggagcg 2040 agatggattt ctcagacgga ttcgggagat tatccagcga gtggagaccc tgttgaagag 2100 agacactggc cccatggagg ctgctgaaga caccctaagc ccccaggagg agccagcacc 2160 attacctgcc ctgcccgtcc ccctcccaga cccatccaat gaagagctcc agagcagcac 2220 ctccctggag cacaggagct ggacagcctt ctccacctcc tcatcttctc ctggaactcc 2280 tttgtctcct ggaaacccat tttcccctgg aacccccatt tccccaggtc ccatcttccc 2340 catcacttct cccccatgtc atcccagccc ctccccattc tcccccattt cttcccaggt 2400 ctcctcaaat ccctctccac accccaccag ctctccactt ccattctcct ccagcacacc 2460 cgagtttccg gtcccactct ctcagtgtcc ctggagttct ctccccacga cttctccacc 2520 tacgttctct cccacttgtt ctcaggtcac tcttagttcc cctttctttc ctccgtgccc 2580 ctccacttct tccttcccct ccaccacagc agcccctctc ctttctctgg ctagtgcctt 2640 ctcactggct gtgatgactg tggcccagtc cctgctgtcc ccctcacctg ggctcctttc 2700 ccagtctcct ccagcccctc ctagtcccct ccctagcctg ccccttcccc ctcccgttgc 2760 tcctggtggc caggaaagcc cttcacccca cacagctgag gtggagagtg aggcctcacc 2820 acctcctgct cggcccctcc caggggaagc caggctggcg cccatctctg aagagggaaa 2880 gccgcagctt gttgggcgtt tccaagtgac ttcatccaag gaaccggctg agcctcttcc 2940 cttgcagcca acatccccca ctctctc,tgg ttctccaaaa ccttcaaccc ctcagctcac 3000 ttcagagagc tcagatacag aggacagtgc tggaggcggg ccagagacca gggaagctct 3060 ggctgagagc gaccgtgcag ctgagggtct gggggctgga gttgaggagg aaggagatga 3120 tgggaaggaa ccccaagttg ggggcagccc ccaacccctg agccatccca gcccagtgtg 3180 gatgaactac tcctacagca gcctgtgttt gagcagcgag gagtcagaaa gcagtgggga 3240 agatgaggag ttctgggctg agctgcagag tcttcggcag aagcacttgt cagaggtgga 3300 aacactacag acactacaga aaaaagaaat tgaagatttg tacagccggc tggggaagca 3360 gcccccaccg ggtattgtgg ccccagctgc tatgctgtcc agccgccagc gccgcctctc 3420 caagggcagc ttccccacct cccgccgcaa cagcctacag cgctctgagc ccccaggccc 3480 tggcatcatg cgaaggaact ctctgagtgg cagcagcacc ggctcccagg agcagcgggc 3540 aagcaagggg gtgacattcg ccggggatgt tggcaggatg tgaattcaga acagaagcca 3600 tgtatctccc ccacaccagg gcccaccatg gagcttgtgt tctcagaatc tgatgctttc 3660 tgatcaacaa aactgagcaa ggaagatccc aacactgaag gggtagaagg ccaggggggc 3720 atggagagtg cagctccatt atagtgaaga gccaaacata tgtgaactgt ttgctgtgtg 3780 gaggtgttag ttctgctgcc taccatcttc atctctagca cctcccctgc caagagtcaa 3840 ccactaagca atcccaccca agcctggatg cttctagagg ggcccactcc cagctgggag 3900 agtgtagggg atatgctcac accacattag cagcaaccaa taaaaatgct ggaaacaaga 3960 aaaaaaaaa 3969 <210> 40 <211> 1803 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474648CB1 <400> 40 atgggtgaaa gtggaaacca tcattttcag caaactaaca caggaacaga aaaccaaaca 60 gcacatgttc tcactcataa gtgggagttg gacaatgaaa acatatgggc acagggaggg 120 gaacatcaca aactgggacc tgtcatgggt tggaaggcta ggagtgggaa aacattagga 180 gaaataccta acgtaggcac actcacactc ctcactggct atgggggatg ccagctgcca 240 tgctgcaagg acactcaggc agcctatgga gaaacccacg tggtgcggag tggaggcctt 300 ctgccaacag ccagctggga actgaggcct gctgacagtc acacggtgac cagcgatgat 360 ccaggcgtct cggtcgttag cgggtatcct gggggctgtc tccctgacca cgacccccca 420 gtggggtttc tttccgaggg tcccgcccct cgcagctgct ctttgataaa gggcggagga 480 acggggctgg ctgcttcccg agtccccagg tcccgcgagc ggcgggcgtg ttgcgggtat 540 ggggtgcggc gccagcagga aggtggtccc ggggccacca gcgctggctt gggccaagca 600 cgaaggtcaa aaccaagccg gcgtcggagg cgcggggcct gggcccgagg cggcggccca 660 ggcggcgcag aggatacagg tggctcgctt ccgagccaag ttcgaccccc gggtccttgc 720 cagtgcccag tacaatttct ctttgacatc tctgaacagg gagttcagag gatgggaaaa 780 aagagagcag gagcagcagc aaacaaggga aggaattcct atcttcggag atatgacatc 840 aaagctctta ttgggacagg cagtttcagc agggttgtca gggtagagca gaagaccacc 900 aagaaacctt ttgcaataaa agtgatggaa accagagaga gggaaggtag agaagcgtgc 960 gtgtctgagc tgagcgtcct gcggcgggtt agccatcgtt acattgtcca gctcatggag 1020 atctttgaga ctgaggatca agtttacatg gtaatggagc tggctaccgg aggggagctc 1080 tttgatcgac tcattgctca gggatccttt acagagcggg atgccgtcag gatcctccag 1140 atggttgctg atgggattag gtatttgcat gcgctgcaga taactcatag gaatctaaag 1200 cctgaaaacc tcttatacta tcatccaggt gaagagtcga aaattttaat tacagatttt 1260 ggtttggcat actccgggaa aaaaagtggt gactggacaa tgaagacact ctgtgggacc 1320 ccagagtaca tagctcctga ggttttgcta aggaagcctt ataccagtgc agtggacatg 1380 tgggctcttg gtgtgatcac atatgcttta cttagcggat tcctgccttt tgatgatgaa 1440 agccagacaa ggctttacag gaagattctg aaaggcaaat ataattatac aggagagcct 1500 tggccaagca tttcccactt ggcgaaggac tttatagaca aactactgat tttggaggct 1560 ggtcatcgca tgtcagctgg ccaggccctg gaccatccct gggtgatcac catggctgca 1620 gggtcttcca tgaagaatct ccagagggcc atatcccgaa acctcatgca gagggcctct 1680 ccccactctc agagtcctgg atctgcacag tcttctaagt cacattattc tcacaaatcc 1740 aggcatatgt ggagcaagag aaacttaagg atagtagaat cgccactgtc tgcgcttttg 1800 taa 1803 <210> 41 <211> 3472 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7483053CB1 <400> 41 atggcgaagg cgacgtccgg tgccgcgggg ctgcgtctgc tgttgctgct gctgctgccg 60 ctgctaggca aagtggcatt gggcctctac ttctcgaggg atgcttactg ggagaagctg 120 tatgtggacc aggcagccgg cacgcccttg ctgtacgtcc atgccctgcg ggacgcccct 180 gaggaggtgc ccagcttccg cctgggccag catctctacg gcacgtaccg aacacggctg 240 catgagaaca actggatctg catccaggag gacaccggcc tcctctacct taaccggagc 300 ctggaccata gctcctggga gaagctcagt gtccgcaacc gcggctttcc cctgctcacc 360 gtctacctca aggtcttcct gtcacccaca tcccttcgtg agggcgagtg ccagtggcca 420 ggctgtgccc gcgtatactt ctccttcttc aacacctcct ttccagcctg cagctccctc 480 aagccccggg agctctgctt cccagagaca aggccctcct tccgcattcg ggagaaccga 540 cccccaggca ccttccacca gttccgcctg ctgcctgtgc agttcttgtg ccccaacatc 600 agcgtggcct acaggctcct ggagggtgag ggtctgccct tccgctgcgc cccggacagc 660 ctggaggtga gcacgcgctg ggccctggac cgcgagcagc gggagaagta cgagctggtg 720 gccgtgtgca ccgtgcacgc cggcgcgcgc gaggaggtgg tgatggtgcc cttcccggtg 780 accgtgtacg acgaggacga ctcggcgccc accttccccg cgggcgtcga caccgccagc 840 gccgtggtgg agttcaagcg gaaggaggac accgtggtgg ccacgctgcg tgtcttcgat 900 gcagacgtgg tacctgcatc aggggagctg gtgaggcggt acacaagcac gctgctcccc 960 ggggacacct gggcccagca gaccttccgg gtggaacact ggcccaacga gacctcggtc 1020 caggccaacg gcagcttcgt gcgggcgacc gtacatgact ataggctggt tctcaaccgg 1080 aacctctcca tctcggagaa ccgcaccatg cagctggcgg tgctggtcaa tgactcagac 1140 ttccagggcc caggagcggg cgtcctcttg ctccacttca acgtgtcggt gctgccggtc 1200 agcctgcacc tgcccagtac ctactccctc tccgtgagca ggagggctcg ccgatttgcc 1260 cagatcggga aagtctgtgt ggaaaactgc caggcgttca gtggcatcaa cgtccagtac 1320 aagctgcatt cctctggtgc caactgcagc acgctagggg tggtcacctc agccgaggac 1380 acctcgggga tcctgtttgt gaatgacacc aaggccctgc ggcggcccaa gtgtgccgaa 1440 cttcactaca tggtggtggc caccgaccag cagacctcta ggcaggccca ggcccagctg 1500 cttgtaacag tggaggggtc atatgtggcc gaggaggcgg gctgccccct gtcctgtgca 1560 gtcagcaaga gacggctgga gtgtgaggag tgtggcggcc tgggctcccc aacaggcagg 1620 tgtgagtgga ggcaaggaga tggcaaaggg atcaccagga acttctccac ctgctctccc 1680 agcaccaaga cctgccccga cggccactgc gatgttgtgg agacccaaga catcaacatt 1740 tgccctcagg actgcctccg gggcagcatt gttgggggac acgagcctgg ggagccccgg 1800 gggattaaag ctggctatgg cacctgcaac tgcttccctg aggaggagaa gtgcttctgc 1860 gagcccgaag acatccagga tccactgtgc gacgagctgt gccgcacggt gatcgcagcc 1920 gctgtcctct tctccttcat cgtctcggtg ctgctgtctg ccttctgcat ccactgctac 1980 cacaagtttg cccacaagcc acccatctcc tcagctgaga tgaccttccg gaggcccgcc 2040 caggccttcc cggtcagcta ctcctcttcc agtgcccgcc ggccctcgct ggactccatg 2100 gagaaccagg tctccgtgga tgccttcaag atcctggagg atccaaagtg ggaattccct 2160 cggaagaact tggttcttgg aaaaactcta ggagaaggcg aatttggaaa agtggtcaag 2220 gcaacggcct tccatctgaa aggcagagca gggtacacca cggtggccgt gaagatgctg 2280 aaagagaacg cctccccgag tgagcttcga gacctgctgt cagagttcaa cgtcctgaag 2340 caggtcaacc acccacatgt catcaaattg tatggggcct gcagccagga tggcccgctc 2400 ctcctcatcg tggagtacgc caaatacggc tccctgcggg gcttcctccg cgagagccgc 2460 aaagtggggc ctggctacct gggcagtgga ggcagccgca actccagctc cctggaccac 2520 ccggatgagc gggccctcac catgggcgac ctcatctcat ttgcctggca gatctcacag 2580 gggatgcagt atctggccga gatgaagctc gttcatcggg acttggcagc cagaaacatc 2640 ctggtagctg aggggcggaa gatgaagatt tcggatttcg gcttgtcccg agatgtttat 2700 gaagaggatt cgtacgtgaa gaggagccag ggtcggattc cagttaaatg gatggcaatt 2760 gaatcccttt ttgatcatat ctacaccacg caaagtgatg tatggtcttt tggtgtcctg 2820 ctgtgggaga tcgtgaccct agggggaaac ccctatcctg ggattcctcc tgagcggctc 2880 ttcaaccttc tgaagaccgg ccaccggatg gagaggccag acaactgcag cgaggagatg 2940 taccgcctga tgctgcaatg ctggaagcag gagccggaca aaaggccggt gtttgcggac 3000 atcagcaaag acctggagaa gatgatggtt aagaggagag actacttgga ccttgcggcg 3060 tccactccat ctgactccct gatttatgac gacggcctct cagaggagga gacaccgctg 3120 gtggactgta ataatgcccc cctccctcga gccctccctt ccacatggat tgaaaacaaa 3180 ctctatggca tgtcagaccc gaactggcct ggagagagtc ctgtaccact cacgagagct 3240 gatggcacta acactgggtt tccaagatat ccaaatgata gtgtatatgc taactggatg 3300 ctttcaccct cagcggcaaa attaatggac acgtttgata gttaacattt ctttgtgaaa 3360 ggtaatggac tcacaagggg aagaaacatg ctgagaatgg aaagtctacc ggccctttct 3420 ttgtgaacgt cacattggcc gagccgtgtt cagttcccag gtggcagact cg 3472 <210> 42 <211> 1704 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7483117CB1 <400> 42 atggatgaca aagatattga caaagaacta aggcagaaat taaacttttc ctattgtgag 60 gagactgaga ttgaagggca gaagaaagta gaagaaagca gggaggcttc gagccaaacc 120 ccagagaagg gtgaagtgca ggattcagag gcaaagggta caccaccttg gactcccctt 180 agcaacgtgc atgagctcga cacatcttcg gaaaaagaca aagaaagtcc agatcagatt 240 ttgaggactc cagtgtcaca ccctctcaaa tgtcctgaga caccagccca accagacagc 300 aggagcaagc tgctgcccag tgacagcccc tctactccca aaaccatgct gagccggttg 360 gtgatttctc caacagggaa gcttccttcc agaggcccta agcatttgaa gctcacacct 420 gctcccctca aggatgagat gacctcattg gctctggtca atattaatcc cttcactcca 480 gagtcctata aaaaattatt tcttcaatct ggtggcaaga ggaaaataag aggagatctt 540 gaggaagctg gtccagagga aggcaaggga gggctgcctg ccaagagatg tgttttacga 600 gaaaccaaca tggcttcccg ctatgaaaaa gaattcttgg aggttgaaaa aattggggtt 660 ggcgaatttg gtacagtcta caagtgcatt aagaggctgg atggatgtgt ttatgcaata 720 aagcgctcta tgaaaacttt tacagaatta tcaaatgaga attcggcttt gcatgaagtt 780 tatgctcacg cagtgcttgg gcatcacccc catgtggtac gttactattc ctcatgggca 840 gaagatgacc acatgatcat tcagaatgaa tactgcaatg gtgggagttt gcaagctgct 900 atatctgaaa acactaagtc tggcaatcat tttgaagagc caaaactcaa ggacatcctt 960 ctacagattt cccttggcct taattacatc cacaactcta gcatggtaca cctggacatc 1020 aaacctagta atatattcat ttgtcacaag atgcaaagtg aatcctctgg agtcatagaa 1080 gaagttgaaa atgaagctga ttggtttctc tctgccaatg tgatgtataa aattggtgac 1140 ctgggccacg caacatcaat aaacaaaccc aaagtggaag aaggagatag tcgcttcctg 1200 gctaatgaga ttttgcaaga ggattaccgg caccttccca aagcagacat atttgccttg 1260 ggattaacaa ttgcagtggc tgcaggagca gagtcattgc ccaccaatgg tgctgcatgg 1320 caccatatcc gcaagggtaa ctttccggac gttcctcagg agctctcaga aagcttttcc 1380 agtctgctca agaacatgat ccaacctgat gccgaacaga gaccttctgc agcagctctg 1440 gccagaaata cagttctccg gccttccctg ggaaaaacag aagagctcca acagcagctg 1500 aatttggaaa agttcaagac tgccacactg gaaagggaac tgagagaagc ccagcaggcc 1560 cagtcacccc agggatatac ccatcatggt gacactgggg tctctgggac ccacacagga 1620 tcaagaagca caaaacgcct ggtgggagga aagagtgcaa ggtcttcaag ctttacctca 1680 ggagagcgtg agcctctgca ttaa 1704 <210> 43 <211> 6298 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7484498CB1 <400> 43 cgcggggcgg aacagatcgc agacctgggg gttcgcagag ccgccagtgg ggagatgttg 60 aagttcaaat atggagcgcg gaatcctttg gatgctggtg ctgctgaacc cattgccagc 120 cgggcctcca ggctgaatct gttcttccag gggaaaccac cctttatgac tcaacagcag 180 atgtctcctc tttcccgaga agggatatta gatgccctct ttgttctctt tgaagaatgc 240 agtcagcctg ctctgatgaa gattaagcac gtgagcaact ttgtccggaa gtattccgac 300 accatagctg agttacagga gctccagcct tcggcaaagg acttcgaagt cagaagtctt 360 gtaggttgtg gtcactttgc tgaagtgcag gtggtaagag agaaagcaac cggggacatc 420 tatgctatga aagtgatgaa gaagaaggct ttattggccc aggagcaggt ttcatttttt 480 gaggaagagc ggaacatatt atctcgaagc acaagcccgt ggatccccca attacagtat 540 gcctttcagg acaaaaatca cctttatctg gtcatggaat atcagcctgg aggggacttg 600 ctgtcacttt tgaatagata tgaggaccag ttagatgaaa acctgataca gttttaccta 660 gctgagctga ttttggctgt tcacagcgtt catctgatgg gatacgtgca tcgagacatc 720 aagcctgaga acattctcgt tgaccgcaca ggacacatca agctggtgga ttttggatct 780 gccgcgaaaa tgaattcaaa caagatggtg aatgccaaac tcccgattgg gaccccagat 840 tacatggctc ctgaagtgct gactgtgatg aacggggatg gaaaaggcac ctacggcctg 900 gactgtgact ggtggtcagt gggcgtgatt gcctatgaga tgatttatgg gagatccccc 960 ttcgcagagg gaacctctgc cagaaccttc aataacatta tgaatttcca gcggtttttg 1020 aaatttccag atgaccccaa agtgagcagt gactttcttg atctgattca aagcttgttg 1080 tgcggccaga aagagagact gaagtttgaa ggtctttgct gccatccttt cttctctaaa 1140 attgactgga acaacattcg taactctcct ccccccttcg ttcccaccct caagtctgac 1200 gatgacacct ccaattttga tgaaccagag aagaattcgt gggtttcatc ctctccgtgc 1260 cagctgagcc cctcaggctt ctcgggtgaa gaactgccgt ttgtggggtt ttcgtacagc 1320 aaggcactgg ggattcttgg tagatctgag tctgttgtgt cgggtctgga ctcccctgcc 1380 aagactagct ccatggaaaa gaaacttctc atcaaaagca aagagctaca agactctcag 1440 gacaagtgtc acaagatgga gcaggaaatg acccggttac atcggagagt gtcagaggtg 1500 gaggctgtgc ttagtcagaa ggaggtggag ctgaaggcct ctgagactca gagatccctc 1560 ctggagcagg accttgctac ctacatcaca gaatgcagta gcttaaagcg aagtttggag 1620 caagcacgga tggaggtgtc ccaggaggat gacaaagcac tgcagcttct ccatgatatc 1680 agagagcaga gccggaagct ccaagaaatc aaagagcagg agtaccaggc tcaagtggaa 1740 gaaatgaggt tgatgatgaa tcagttggaa gaggatcttg tctcagcaag aagacggagt 1800 gatctctacg aatctgagct gagagagtct cggcttgctg ctgaagaatt caagcggaaa 1860 gcgacagaat gtcagcataa actgttgaag gctaaggatc aagggaagcc tgaagtggga 1920 gaatatgcga aactggagaa gatcaatgct gagcagcagc tcaaaattca ggagctccaa 1980 gagaaactgg agaaggctgt aaaagccagc acggaggcca ccgagctgct gcagaatatc 2040 cgccaggcaa aggagcgagc cgagagggag ctggagaagc tgcagaaccg agaggattct 2100 tctgaaggca tcagaaagaa gctggtggaa gctgaggaac gccgccattc tctggagaac 2160 aaggtaaaga gactagagac catggagcgt agagaaaaca gactgaagga tgacatccag 2220 acaaaatccc aacagatcca gcagatggct gataaaattc tggagctcga agagaaacat 2280 cgggaggccc aagtctcagc ccagcaccta gaagtgcacc tgaaacagaa agagcagcac 2340 tatgaggaaa agattaaagt gttggacaat cagataaaga aagacctggc tgacaaggag 2400 acactggaga acatgatgca gagacacgag gaggaggccc atgagaaggg caaaattctc 2460 agcgaacaga aggcgatgat caatgctatg gattccaaga tcagatccct ggaacagagg 2520 attgtggaac tgtctgaagc caataaactt gcagcaaata gcagtctttt tacccaaagg 2580 aacatgaagg cccaagaaga gatgatttct gaactcaggc aacagaaatt ttacctggag 2640 acacaggctg ggaagttgga ggcccagaac cgaaaactgg aggagcagct ggagaagatc 2700 agccaccaag accacagtga caagaatcgg ctgctggaac tggagacaag attgcgggag 2760 gtcagtctag agcacgagga gcagaaactg gagctcaagc gccagctcac agagctacag 2820 ctctccctgc aggagcgcga gtcacagttg acagccctgc aggctgcacg ggcggccctg 2880 gagagccagc ttcgccaggc gaagacagag ctggaagaga ccacagcaga agctgaagag 2940 gagatccagg cactcacggc acatagagat gaaatccagc gcaaatttga tgctcttcgt 3000 aacagctgta ctgtaatcac agacctggag gagcagctaa accagctgac cgaggacaac 3060 gctgaactca acaaccaaaa cttctacttg tccaaacaac tcgatgaggc ttctggcgcc 3120 aacgacgaga ttgtacaact gcgaagtgaa gtggaccatc tccgccggga gatcacggaa 3180 cgagagatgc agcttaccag ccagaagcaa acgatggagg ctctgaagac cacgtgcacc 3240 atgctggagg aacaggtcat ggatttggag gccctaaacg atgagctgct agaaaaagag 3300 cggcagtggg aggcctggag gagcgtcctg ggtgatgaga aatcccagtt tgagtgtcgg 3360 gttcgagagc tgcagaggat gctggacacc gagaaacaga gcagggcgag agccgatcag 3420 cggatcaccg agtctcgcca ggtggtggag ctggcagtga aggagcacaa ggctgagatt 3480 ctcgctctgc agcaggctct caaagagcag aagctgaagg ccgagagcct ctctgacaag 3540 ctcaatgacc tggagaagaa gcatgctatg cttgaaatga atgcccgaag cttacagcag 3600 aagctggaga ctgaacgaga gctcaaacag aggcttctgg aagagcaagc caaattacag 3660 cagcagatgg acctgcagaa aaatcacatt ttccgtctga ctcaaggact gcaagaagct 3720 ctagatcggg ctgatctact gaagacagaa agaagtgact tggagtatca gctggaaaac 3780 attcaggttc tctattctca tgaaaaggtg aaaatggaag gcactatttc tcaacaaacc 3840 aaactcattg attttctgca agccaaaatg gaccaacctg ctaaaaagaa aaaggttcct 3900 ctgcagtaca atgagctgaa gctggccctg gagaaggaga aagctcgctg tgcagagcta 3960 gaggaagccc ttcagaagac ccgcatcgag ctccggtccg cccgggagga agctgcccac 4020 cgcaaagcaa cggaccaccc acacccatcc acgccagcca ccgcgaggca gcagatcgcc 4080 atgtccgcca tcgtgcggtc gccagagcac cagcccagtg ccatgagcct gctggccccg 4140 ccatccagcc gcagaaagga gtcttcaact ccagaggaat ttagtcggcg tcttaaggaa 4200 cgcatgcacc acaatattcc tcaccgattc aacgtaggac tgaacatgcg agccacaaag 4260 tgtgctgtgt gtctggatac cgtgcacttt ggacgccagg catccaaatg tctcgaatgt 4320 caggtgatgt gtcaccccaa gtgctccacg tgcttgccag ccacctgcgg cttgcctgct 4380 gaatatgcca cacacttcac cgaggccttc tgccgtgaca aaatgaactc cccaggtctc 4440 cagaccaagg agcccagcag cagcttgcac ctggaagggt ggatgaaggt gcccaggaat 4500 aacaaacgag gacagcaagg ctgggacagg aagtacattg tcctggaggg atcaaaagtc 4560 ctcatttatg acaatgaagc cagagaagct ggacagaggc cggtggaaga atttgagctg 4620 tgccttcccg acggggatgt atctattcat ggtgccgttg gtgcttccga actcgcaaat 4680 acagccaaag cagatgtccc atacatactg aagatggaat ctcacccgca caccacctgc 4740 tggcccggga gaaccctcta cttgctagct cccagcttcc ctgacaaaca gcgctgggtc 4800 accgccttag aatcagttgt cgcaggtggg agagtttcta gggaaaaagc agaagctgat 4860 gctaaactgc ttggaaactc cctgctgaaa ctggaaggtg atgaccgtct agacatgaac 4920 tgcacgctgc ccttcagtga ccaggtggtg ttggtgggca ccgaggaagg gctctacgcc 4980 ctgaatgtct tgaaaaactc cctaacccat gtcccaggaa ttggagcagt cttccaaatt 5040 tatattatca aggacctgga gaagctactc atgatagcag gagaagagcg ggcactgtgt 5100 cttgtggacg tgaagaaagt gaaacagtcc ctggcccagt cccacctgcc tgcccagccc 5160 gacatctcac ccaacatttt tgaagctgtc aagggctgcc acttgtttgg ggcaggcaag 5220 attgagaacg ggctctgcat ctgtgcagcc atgcccagca aagtcgtcat tctccgctac 5280 aacgaaaacc tcagcaaata ctgcatccgg aaagagatag agacctcaga gccctgcagc 5340 tgtatccact tcaccaatta cagtatcctc attggaacca ataaattcta cgaaatcgac 5400 atgaagcagt acacgctcga ggaattcctg gataagaatg accattcctt ggcacctgct 5460 gtgtttgccg cctcttccaa cagcttccct gtctcaatcg tgcaggtgaa cagcgcaggg 5520 cagcgagagg agtacttgct gtgtttccac gaatttggag tgttcgtgga ttcttacgga 5580 agacgtagcc gcacagacga tctcaagtgg agtcgcttac ctttggcctt tgcctacaga 5640 gaaccctatc tgtttgtgac ccacttcaac tcactcgaag taattgagat ccaggcacgc 5700 tcctcagcag ggacccctgc ccgagcgtac ctggacatcc cgaacccgcg ctacctgggc 5760 cctgccattt cctcaggagc gatttacttg gcgtcctcat accaggataa attaagggtc 5820 atttgctgca agggaaacct cgtgaaggag tccggcactg aacaccaccg gggcccgtcc 5880 acctcccgca gcagccccaa caagcgaggc ccacccacgt acaacgagca catcaccaag 5940 cgcgtggcct ccagcccagc gccgcccgaa ggccccagcc acccgcgaga gccaagcaca 6000 ccccaccgct accgcgaggg gcggaccgag ctgcgcaggg acaagtctcc tggccgcccc 6060 ctggagcgag agaagtcccc cggccggatg ctcagcacgc ggagagagcg gtcccccggg 6120 aggctgtttg aagacagcag caggggccgg ctgcctgcgg gagccgtgag gaccccgctg 6180 tcccaggtga acaaggtctg ggaccagtct tcagtataaa tctcagccag aaaaaccaac 6240 tcctcatctt gatctgcagg aaaacaccaa acacactatg gaactctgct gatgggga 6298 <210> 44 <211> 5454 <212> DNA
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 7638121CB1 <400> 44 cacgcacacc gcacgtacgg ggttgggccc agctgggtta taagcgtgat ccccatgccc 60 cctgcccagg ctggggggca tttgcacatc tgcaaaggcc tcccagcctg tcccagccct 120 gccccagcct gggaccccca cattctactc accgtgtctc ctcagagggg ccagaaccct 180 ccactgggga gaggcaagtg gcggtgaact tggtgtccat aggaccctgt ccctgagagc 240 gacagctgag ttagtgagct ccactggccc caccaactcc ttctgatcac ctggccagct 300 gaggtcagag tgggagaggc agtggttcca ttgaaggagt actcctaact gtcagaagcc 360 tgggcggtca ggatggggtg ctgtcgcttg ggctgcgggg ggtgttcagt tgcccacagt 420 gtatctcagg gtctcaccaa ccatccaagc atggtaggct gtggctggca cccagggttg 480 tgtggctggg gaggtggtct ccacagttcc ctccctgccc tcccagggcc cccatccatg 540 caggtaacca tcgaggatgt gcaggcacag acaggcggaa cggcccaatt cgaggctatc 600 attgagggcg acccacagcc ctcggtgacc tggtacaagg acagcgtcca gctggtggac 660 agcacccggc ttagccagca gcaagaaggc accacatact ccctggtgct gaggcatatg 720 gcctcgaagg atgccggcgt ttacacctgc ctggcccaaa acactggtgg ccaggtgctc 780 tgcaaggcag agctgctggt gcttgggggg gacaatgagc cggactcaga gaagcaaagc 840 caccggagga agctgcactc cttctatgag gtcaaggagg agattggaag gggcgtgttt 900 ggcttcgtaa aaagagtgca gcacaaagga aacaagatct tgtgcgctgc caagttcatc 960 cccctacgga gcagaactcg ggcccaggca tacagggagc gagacatcct ggccgcgctg 1020 agccacccgc tggtcacggg gctgctggac cagtttgaga cccgcaagac cctcatcctc 1080 atcctggagc tgtgctcatc cgaggagctg ctggaccgcc tgtacaggaa gggcgtggtg 1140 acggaggccg aggtcaaggt ctacatccag cagctggtgg aggggctgca ctacctgcac 1200 agccatggcg ttctccacct ggacataaag ccctctaaca tcctgatggt gcatcctgcc 1260 cgggaagaca ttaaaatctg cgactttggc tttgcccaga acatcacccc agcagagctg 1320 cagttcagcc agtacggctc ccctgagttc gtctcccccg agatcatcca gcagaaccct 1380 gtgagcgaag cctccgacat ttgggccatg ggtgtcatct cctacctcag cctgacctgc 1440 tcatccccat ttgccggcga gagtgaccgt gccaccctcc tgaacgtcct ggaggggcgc 1500 gtgtcatgga gcagccccat ggctgcccac ctcagcgaag acgccaaaga cttcatcaag 1560 gctacgctgc agagagcccc tcaggcccgg cctagtgcgg cccagtgcct ctcccacccc 1620 tggttcctga aatccatgcc tgcggaggag gcccacttca tcaacaccaa gcagctcaag 1680 ttcctcctgg cccgaagtcg ctggcagcgt tccctgatga gctacaagtc catcctggtg 1740 atgcgctcca tccctgagct gctgcggggc ccacccgaca gcccctccct cggcgtagcc 1800 cggcacctct gcagggacac tggtggctcc tccagttcct cctcctcctc tgacaacgag 1860 ctcgccccat ttgcccgggc taagtcactg ccaccctccc cggtgacaca ctcaccactg 1920 ctgcaccccc ggggcttcct gcggccctcg gccagcctgc ctgaggaagc cgaggccagt 1980 gagcgctcca ccgaggcccc agctccgcct gcatctcccg agggtgccgg gccaccggcc 2040 gcccagggct gcgtgccccg gcacagcgtc atccgcagcc tgttctacca ccaggcgggt 2100 gagagccctg agcacggggc cctggccccg gggagcaggc ggcacccggc ccggcggcgg 2160 cacctgctga agggcgggta cattgcgggg gcgctgccag gcctgcgcga gccactgatg 2220 gagcaccgcg tgctggagga ggaggccgcc agggaggagc aggccaccct cctggccaaa 2280 gccccctcat tcgagactgc cctccggctg cctgcctctg gcacccactt ggcccctggc 2340 cacagccact ccctggaaca tgactctccg agcacccccc gcccctcctc ggaggcctgc 2400 ggtgaggcac agcgactgcc ttcagccccc tccggggggg cccctatcag ggacatgggg 2460 caccctcagg gctccaagca gcttccatcc actggtggcc acccaggcac tgctcagcca 2520 gagaggccat ccccggacag cccttggggg cagccagccc ctttctgcca ccccaagcag 2580 ggttctgccc cccaggaggg ctgcagcccc cacccagcag ttgccccatg ccctcctggc 2640 tccttccctc caggatcttg caaagaggcc cccttagtac cctcaagccc cttcttggga 2700 cagccccagg caccccttgc ccctgccaaa gcaagccccc cattggactc taagatgggg 2760 cctggagaca tctctcttcc tgggaggcca aaacccggcc cctgcagttc cccagggtca 2820 gcctcccagg cgagctcttc ccaagtgagc tccctcaggg tgggctcctc ccaggtgggc 2880 acagagcctg gcccctccct ggatgcggag ggctggaccc aggaggctga ggatctgtcc 2940 gactccacac ccaccttgca gcggcctcag gaacaggtga ccatgcgcaa gttctccctg 3000 ggtggtcgcg ggggctacgc aggcgtggct ggctatggca cctttgcctt tggtggagat 3060 gcagggggca tgctggggca ggggcccatg tgggccagga tagcctgggc tgtgtcccag 3120 tcggaggagg aggagcagga ggaggccagg gctgagtccc agtcggagga gcagcaggag 3180 gccagggctg agagcccact gccccaggtc agtgcaaggc ctgtgcctga ggtcggcagg 3240 gctcccacca ggagctctcc agagcccacc ccatgggagg acatcgggca ggtctccctg 3300 gtgcagatcc gggacctgtc aggtgatgcg gaggcggccg acacaatatc cctggacatt 3360 tccgaggtgg accccgccta cctcaacctc tcagacctgt acgatatcaa gtacctccca 3420 ttcgagttta tgatcttcag gaaagtcccc aagtccgctc agccagagcc gccctccccc 3480 atggctgagg aggagctggc cgagttcccg gagcccacgt ggccctggcc aggtgaactg 3540 ggcccccacg caggcctgga gatcacagag gagtcagagg atgtggacgc gctgctggca 3600 gaggctgccg tgggcaggaa gcgcaagtgg tcctcgccgt cacgcagcct cttccacttc 3660 cctgggaggc acctgccgct ggacgagcct gcagagctgg ggctgcgtga gagagtgaag 3720 gcctccgtgg agcacatctc ccggatcctg aagggcaggc cggaaggtct ggagaaggag 3780 gggcccccca ggaagaagcc aggccttgct tccttccggc tctcaggtct gaagagctgg 3840 gaccgagcgc cgacattcct aagggagctc tcagatgaga ctgtggtcct gggccagtca 3900 gtgacactgg cctgccaggt gtcagcccag ccagctgccc aggccacctg gagcaaagac 3960 ggagcccccc tggagagcag cagccgtgtc ctcatctctg ccaccctcaa gaacttccag 4020 cttctgacca tcctggtggt ggtggctgag gacctgggtg tgtacacctg cagcgtgagc 4080 aatgcgctgg ggacagtgac caccacgggc gtcctccgga aggcagagcg cccctcatct 4140 tcgccatgcc cggatatcgg ggaggtgtac gcggatgggg tgctgctggt ctggaagccc 4200 gtggaatcct acggccctgt gacctacatt gtgcagtgca gcctagaagg cggcagctgg 4260 accacactgg cctccgacat ctttgactgc tgctacctga ccagcaagct ctcccggggt 4320 ggcacctaca ccttccgcac ggcatgtgtc agcaaggcag gaatgggtcc ctacagcagc 4380 ccctcggagc aagtcctcct gggagggccc agccacctgg cctctgagga ggagagccag 4440 gggcggtcag cccaacccct gcccagcaca aagaccttcg cattccagac acagatccag 4500 aggggccgct tcagcgtggt gcggcaatgc tgggagaagg ccagcgggcg ggcgctggcc 4560 gccaagatca tcccctacca ccccaaggac aagacagcag tgctgcgcga atacgaggcc 4620 ctcaagggcc tgcgccaccc gcacctggcc cagctgcacg cagcctacct cagcccccgg 4680 cacctggtgc tcatcttgga gctgtgctct gggcccgagc tgctcccctg cctggccgag 4740 agggcctcct actcagaatc cgaggtgaag gactacctgt ggcagatgtt gagtgccacc 4800 cagtacctgc acaaccagca catcctgcac ctggacctga ggtccgagaa catgatcatc 4860 accgaataca acctgctcaa ggtcgtggac ctgggcaatg cacagagcct cagccaggag 4920 aaggtgctgc cctcagacaa gttcaaggac tacctagaga ccatggctcc agagctcctg 4980 gagggccagg gggctgttcc acagacagac atctgggcca tcggtgtgac agccttcatc 5040 atgctgagcg ccgagtaccc ggtgagcagc gagggtgcac gcgacctgca gagaggactg 5100 cgcaaggggc tggtccggct gagccgctgc tacgcggggc tgtccggggg cgccgtggcc 5160 ttcctgcgca gcactctgtg cgcccagccc tggggccggc cctgcgcgtc cagctgcctg 5220 cagtgcccgt ggctaacaga ggagggcccg gcctgttcgc ggcccgcgcc cgtgaccttc 5280 cctaccgcgc ggctgcgcgt cttcgtgcgc aatcgcgaga agagacgcgc gctgctgtac 5340 aagaggcaca acctggccca ggtgcgctga gggtcgcccc ggccacaccc ttggtctccc 5400 cgctgggggt cgctgcagac gcgccaataa aaacgcacag ccgggcgaga aaaa 5454

Claims (99)

What is claimed is:

1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90%o identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22.

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

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

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

5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:23-44.

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

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

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

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

10. A method of claim 9, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:1-22.

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

12. An isolated polynucleotide selected from the group consisting of:
a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:23-44, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ
ID NO:23-44, c) a polynucleotide complementary to a polynucleotide of a), d) a polynucleotide complementary to a polynucleotide of b), and e) an RNA equivalent of a)-d).

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

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

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

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

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

18. A composition of claim 17, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:1-22.

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

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

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

39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising:
a) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide having an amino acid sequence selected from the group consisting of SEQ
ID NO:1-22.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

68. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
N0:13.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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