CA2395102A1 - Human kinases - Google Patents

Human kinases Download PDF

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CA2395102A1
CA2395102A1 CA002395102A CA2395102A CA2395102A1 CA 2395102 A1 CA2395102 A1 CA 2395102A1 CA 002395102 A CA002395102 A CA 002395102A CA 2395102 A CA2395102 A CA 2395102A CA 2395102 A1 CA2395102 A1 CA 2395102A1
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polynucleotide
polypeptide
pkin
sequence
sequences
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Junming Yang
Mariah R. Baughn
Neil Burford
Janice Au-Young
Dyung Aina M. Lu
Roopa Reddy
Henry Yue
Monique G. Yao
Preeti Lal
Farrah A. Khan
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Incyte Corp
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/12Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/06Antihyperlipidemics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system

Abstract

The invention provides human kinases (PKIN) and polynucleotides which identi fy and encode PKIN. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods f or 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.
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-calmodulin, 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 serineJthreonine 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. Almost all kinases contain a conserved 250-300 amino acid catalytic domain containng 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 II 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. Cell stimulation induces a signaling cascade leading to phosphorylation of MEK
(MAP/ERK 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; checlq~oint 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 serinelthreonine 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 CHI 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 isoforn~s of casein , kinase I from a variety of sources. There are at least five mammalian isoforms, a, (3, 'y, 8, and E. 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 arid 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 catecholanunes and seratonin, through phosphorylation/activation of tyrosine hydroxylase and tryptophan hydroxylase, respectively (Fujisawa, H. (1990) BioEssays 12:27-29). The mRNA encoding a calmodulin-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).
Mitogen-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 a 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 ).
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 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 to dephosphorylate and activate the cyclin-dependent kinase Cde2. Cdc2 activation controls the entry of cells into mitosis. (Peng, C-Y et al. (1997) Science 277:1501- 1505.) Thus, activation of Chk1 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.
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 tissue and may be a proto-oncogene whose deregulated expression in normal tissue leads to oncogenic transformation.
5'-AMP-activated~rotein 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 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 leucine 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. supra).
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.
supra). 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 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) su ra).
KINASES WITH NON-PROTEIN SUBSTRATES
Lipid 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) Curr. 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). P1P2 is then cleaved into inositol triphosphate (IP3) 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, 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, supra).
An example of lipid kinase phosphorylation activity is the phosphoryladon 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 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.
Pvrimidine Kinases The pyrimidine 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 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.
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," "PKIN-7,"
"PKIN-8," "PKIN-9," "PKIN-10," "PKIN-1 l," and "PKIN-12." In one aspect, the invention provides an isolated polypeptide comprising an anuno acid sequence selected fiom the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-12, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO:1-12.
The invention further provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:l-12. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID N0:13-24.
Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected fiom the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID
NO:l-12, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-12. 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 comprising an amino acid sequence selected from the group consisting of a) an anuno acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID
N0:1-12, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12. 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 comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:l-12, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, c) a biologically active fragment of an amino acid sequence selected fiom the group consisting of SEQ ID N0:1-12, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12.
The invention further provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to 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 comprising a polynucleotide sequence selected from the group consiseing of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to 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 comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerise chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID N0:1-12, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:l-12, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-12, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ 1D NO:1-12. The invention additionally 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 comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:l-12, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID N0:1-12, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12. 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 comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, c) a biologically active tiagment of an amino acid sequence selected from the group consisting of SEQ ID N0:1-12, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12. 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 PKIN, 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 comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID N0:1-12, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12. 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 comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an anuno acid sequence selected from the group consisting of SEQ ID NO:1-12, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID N0:1-12. 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 sequence selected from the group consisting of SEQ ID N0:13-24, 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 contaiW ng 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 comprising a polynucleotide sequence selected from the group consisting of i) a polynucleotide sequence selected from the group consisting of SEQ ID
N0:13-24, ii) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24, iii) a polynucleotide sequence complementary to i), iv) a polynucleotide sequence complementary to 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 comprising a polynucleotide sequence selected liom the group consisting of i) a polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24, ii) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24, iii) a polynucleotide sequence complementary to i), iv) a polynucleotide sequence complementary to 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 each polypeptide 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 each polypeptide sequence, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of each polypeptide.
Table 4 lists the cDNA and genomic DNA fragments which were used to assemble each polynucleotide sequence, along with selected fragments of the polynucleotide sequences.
Table 5 shows the representative cDNA library for each polynucleotide of the invention.
Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.

Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described.
All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"PKIN" refers to the amino acid sequences of substantially purified PHIN
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of PHIN. 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 PHIN or by acting on components of the biological pathway in which PHIN
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 IS include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine;
and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of 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 "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation "negative" or "minus" can refer to the antisense strand, and the designation "positive" or "plus" can refer to the sense strand of a reference DNA molecule.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic 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 anuno 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. Chenucal 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.
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 fiagment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ ID N0:13-24 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:13-24, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:13-24 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID N0:13-24 from related polynucleotide sequences. The precise length of a fragment of SEQ
ID N0:13-24 and the region of SEQ ID N0:13-24 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A fragment of SEQ ID NO:1-12 is encoded by a fragment of SEQ ID N0:13-24. A
fragment of SEQ ID NO:1-12 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:l-12. For example, a fragment of SEQ ID NO:l-12 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-12.
The precise length of a fragment of SEQ ID NO:l-12 and the region of SEQ ID NO:1-12 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a translation initiation colon (e.g., methionine) followed by an open reading frame and a translation termination colon. A "full length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G.
and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS 8:189-191.
For pairwise alignments of polynucleotide sequences, the default parameters are set as follows:
Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted"
residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off. 50 Expect: l0 Word Size: Il Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity"
between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example:

Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: I penalties Gap x drop-off:' S0 Expect: 10 Word Size: 3 Filter: on Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least S0, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1 % (w/v) SDS, and about 100 ~~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,n and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J.
et al. (1989) Molecular Cloning: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 pg/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, 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
polymerise enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerise chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2°d ed., vol. I-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et al. (1990) PCR
Protocols. A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU
primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead InstitutelMIT 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 artifcial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra. .The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (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 PHIN, 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 tree 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 cells"
includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. ( 1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% 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 alternative 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 95%, or at least 98%
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 kinases (PKIN), the polynucleotides encoding PHIN, 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 ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.

Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide 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 each of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains, and motifs. Column 7 shows analytical methods for protein structurelfunction analysis and in some cases, searchable databases to which the analytical methods were applied.
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 N0:13-24 or that distinguish between SEQ ID
N0:13-24 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 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, 2287966H1 is the identification number of an Incyte cDNA sequence, and BRA1NON01 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., 70166939V1). Alternatively, the identification numbers in column 5 may refer to GenBank cDNAs or ESTs (e.g., g2821547) which contributed to the assembly of the full length polynucleotide sequences. Alternatively, the identification numbers in column 5 may refer to coding regions predicted by Genscan analysis of genomic DNA. For example, g4454511.v113.gs_3.nt.edit is the identification number of a Genscan-predicted coding sequence, with g4454511 being the GenBank identification number of the sequence to which Genscan was applied. The Genscan-predicted coding sequences may have been edited prior to assembly. (See Example IV.) Alternatively, the identification numbers in column 5 may refer to assemblages of both eDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. (See Example V.) Alternatively, the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon-stretching" algorithm. (See Example V.) 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 eDNA 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 PK1N 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 N0:13-24, which encodes PKIN. The polynucleotide sequences of SEQ 1D N0:13-24, 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 PK1N. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID
N0:13-24 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID N0:13-24.
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 colon 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 colon usage, e.g., inclusion of non-naturally occurring colons. Colons 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 colons 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
N0:13-24 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 liagment of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise (Applied Biosystems), thermostable T7 polymerise (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerises and proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular Biology and Biotechnolo~y, 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 in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.
(1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and legations 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 PHIN.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter PHIN-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 Number 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 occurnng 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 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, supra, 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' untxanslated 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 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 Biolo~y, 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, supra; Ausubel, suQra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~v (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet..15:345-355.) Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding 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 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 antibodies, vectors which direct high level expression of PHIN 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 PHIN. 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 nastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, 1995, su ra;
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 PHIN. Transcription of sequences 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 liom 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 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 PHIN
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.
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 artiricial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355.) For long term production of recombinant proteins in mammalian systems, stable expression of PKIN in cell lines is preferred. For example, sequences encoding PKIN can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232;
Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J.
Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051.) Visible markers, e.g., anthoc;yanins, green fluorescent proteins (GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, or luciferase and its substrate luciferin may be used.
These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, C.A.
(1995) Methods Mol. Biol. 55:121-131.) Although the presencelabsence 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 PHIN 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 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 Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunoc;hemical 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 polymerise 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 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), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the PHIN 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 that specitlcally 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 identii-ied is closely related to the natural ligand of PKIN, e.g., a ligand or iiagment 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 Immunology 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 polypepdde, 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 compounds) 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 Number 5,175,383 and U.S. Patent Number 5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo;
Capecchi, M.R. (1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP
system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D.
(1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the 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 cancers, cell proliferation and cardiovascular diseases.
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, PHIN 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-ec;todermal 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, Sjogrcn'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, 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 neurotibromatosis, 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 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 palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM2 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 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 including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified PKIN 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 cancers, 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 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. For example, such fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al.
(1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between 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, supra).
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 1~ determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple PHIN
epitopes, represents the average affinity, or avidity, of the antibodies for PHIN. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular PHIN epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 10'2 L/mole are preferred for use in immunoassays in which the 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 specirc 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 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 Cli. 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, su ra; 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 (SCID)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and 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; Ivies, 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 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) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M. V.
and Blau, H.M. su ra)), 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 KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to 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.S. Patent Number 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 Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu.
Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding 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 Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent Number 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) Curr. Opin. Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for 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~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177.) A
complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding 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' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding 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 PKIN
expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding PKIN may be therapeutically useful, and in the treament of disorders associated with decreased PKIN 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 speciiuc 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-occurnng 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 specif c nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the 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 Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, 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 ReminQton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of PKIN, antibodies to PKIN, and mimetics, agonists, antagonists, or inhibitors of PKIN.
The compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry powder form.
These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g.
larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton. J.S. et al., U.S.
Patent No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
Compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended 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 ~g to 100,000 fig, 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 PHIN 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 PHIN 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 PHIN expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to PHIN 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 PHIN. 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 IS 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.
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 ID
N0:13-24 or liom genomic sequences including promoters, enhancers, and introns of the PKIN
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 immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a 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, mural annular calcification, mural valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of cardiac transplantation, congenital lung anomalies, atelectasis, pulmonary congestion and edema, pulmonary 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 noninllammatory 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 palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM2 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 PHIN 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 PK1N 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 PK.IN 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 PHIN, 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 fiom 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 PHIN, 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 PHIN 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 polymerise chain reaction (PCR). The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. Additionally, sequence database analysis methods, termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA liagments 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 microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
In another embodiment, PKIN, Iragments 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.
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 Number 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 pioteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type. In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, su ra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry.
The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for 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 (Luelcing, 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 tiom 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 PHIN
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP). (See, for example, 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-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding 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 PHIN 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., Geyser, 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 tiagments thereof, and washed. Bound PKIN is then detected by methods well known in the art. Purified PKIN 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.
The disclosures of all patents, applications and publications, mentioned above and below, in particular U.S. Ser. No. 60/172,066, U.S. Ser. No. 60/176,107, U.S. Ser. No.
60/177,731, and U.S.
Ser. No. 60/178,573, are expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. The Incyte cDNA shown for SEQ
ID N0:13 was derived from a eDNA library constructed from musculoskeletal tissue. The Incyte cDNA shown for SEQ ID N0:14 was derived from cDNA libraries constructed from prostate, brain and ovarian tissues, including tissues associated with brain, prostate and thyroid tumors. 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, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte Genomics, Palo Alto CA), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DHlOB, or ElectroMAX
DH10B from Life Technologies.
II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC
Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL
8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L.
PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN I1 fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows.
Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI
protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, 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 (HMM)-based protein family databases such as PFAM.
(HMM is a probabilistic approach which analyzes consensus primary structures of gene families.
See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA
sequences were assembled to produce full length polynucleotide sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV and V) were used to extend Incyte cDNA assemblages to full length.
Assembly was performed using programs based on Phred, Phrap, and Conned, 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 (HMM)-based protein family databases such as PFAM. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
The programs described above for the assembly and analysis of lull length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ ID
N0:13-24. 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 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 kinases. Potential human kinases were also identified by homology to Incyte cDNA
sequences that had been annotated as 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 lncyte eDNA or public eDNA 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" Se9uences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence.
Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by eDNA sequence. Intervals thus identified were then "stitched" together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants. Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.
"Stretched" Seguences Partial DNA sequences were extended to full length with an algorithm based on BLAST
analysis. First, partial cDNAs assembled as described in Example III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte eDNA 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 loom 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 N0:13-24 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID NO:l 3-24 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
Map locations are represented by ranges, or intervals, or 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 G~nethon 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.nebi.nlm.nih.gov/genemap~, can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, s-upra, ch. 7; Ausubel (1995) su ra, 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 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;
heroic and immune system; liver; musculoskeletal system; nervous system;
pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract. The number of libraries in each category is counted and divided by the total number of libraries across all categories.
Similarly, each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of eDNA
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+, (NH4)ZS04, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerise (Stratagene), with the following parameters for primer pair PCI A and PC1 B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~1 PICOGREEN
quantitation reagent (0.25% (v/v) P1COGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 ~1 of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 ~1 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 Plu DNA polymerise (Stratagene) to till-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 Garb 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 reamplilied 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 N0:13-24 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 carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
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 S analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470;
Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol.
16:27-31.) Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample are conjugated to a Iluorescent 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/i.~l oligo-(dT) primer (2lmer), 1X first strand buffer, 0.03 units/Nl RNase inhibitor, 500 ~M dATP, 500 ~M dGTP, 500 pM
dTTP, 40 NM
dCTP, 40 NM 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 (I 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 ~15X SSC/0.2% SDS.
Microarray Preparation Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 fig. 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 US
Patent No. 5,807,522 , incorporated herein by reference. 1 ~1 of the array element DNA, at an average concentration of 100 ng/~.xl, 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 Nl of sample mixture consisting of 0.2 ~g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65 ° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 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 p1 of 5X SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45 ° C in a first wash buffer (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, Ine., 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 NJ7 corresponding to the two Iluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fiuorophores used are 565 nm for Cy3 and 650 nm for CyS. Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A
specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fiuorophore, 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 fiuorophores 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 PHIN-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 smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of PKIN. 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 PHIN 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 eDNA 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 Spodoptera 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 iaponicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved liom 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 Examples XVI, XVII, and XVIII where applicable.
XIII. Functional 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 ~sg 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 transfeeted 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 PKIN 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.
S 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, supra, eh. 11.) Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431 A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma-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-KL,H 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 immunoaflinity chromatography using antibodies specific for PKIN. An immunoaftinity 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 immunoaftinity 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 PHIN is collected.
XVI. Identification of Molecules Which Interact with PKIN
PKIN, or biologically active fragments thereof, are labeled with'ZSI 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 PHIN 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 3zP-ATP. PKIN is incubated with the protein substrate, 32P-ATP, and an appropriate kinase buffer. The 32P incorporated into the substrate is separated from free 32P-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 32P-ATP. The reservoir of the centrifuged unit containing the 32P-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 H1 (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 PK1N is demonstrated in vitro in an assay containing PKIN, 501 of kinase buffer, lpg substrate, such as myelin basic protein (MBP) or synthetic peptide substrates, 1 mM DTT, 10 ~g ATP, and O.Sp.Ci [y-33P]ATP. The reaction is incubated at 30°C for 30 minutes and stopped by pipetting onto P81 paper. The unincorporated ['y-3~P]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 32P-labeled ATP as the phosphate donor. The 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.
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 those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

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<110> INCYTE GENOMICS, INC.
YANG, Junming BAUGHN, Mariah R.
BURFORD, Neil AU-YOUNG, Janice LU, Dyung Aina M.
REDDY, Roopa YUE, Henry YAO, Monique G.
LAL, Preeti KAHN, Farrah A.
<120> HUMAN KINASES
<130> PI-0002 PCT
<140> To Be Assigned <141> Herewith <150> 60/172,066; 60/176,107; 60/176,107; 60/177,731 <151> 1999-12-23; 2000-01-14; 2000-01-14; 2000-01-21 <160> 24 <170> PERL Program <210> 1 <211> 466 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 058860CD1 <400> 1 Met Glu Asp Gly Thr Pro Asn Glu His Phe Tyr Thr Pro Thr Glu Glu Arg Gly Ser Ala Tyr Glu Ile Trp Arg Ser Asp Ser Phe Gly Thr Pro Asn Glu Ala Ile Glu Pro Lys Asp Asn Glu Met Pro Pro Ser Phe Ile Glu Pro Leu Thr Lys Arg Lys Val Tyr Glu Asn Thr Thr Leu Gly Phe Ile Val Glu Val Glu Gly Leu Pro Val Pro Gly Val Lys Trp Tyr Arg Asn Lys Ser Leu Leu Glu Pro Asp Glu Arg Ile Lys Met Glu Arg Val Gly Asn Val Cys Ser Leu Glu Ile Ser Asn Ile Gln Lys Gly Glu Gly Gly Glu Tyr Met Cys His Ala Val Asn Ile Ile Gly Glu Ala Lys Ser Phe Ala Asn Val Asp Ile Met Pro Gln Glu Glu Arg Val Val Ala Leu Pro Pro Pro Val Thr His Gln His Val Met Glu Phe Asp Leu Glu His Thr Thr Ser Ser Arg Thr Pro Ser Pro Gln Glu Ile Val Leu Glu Val Glu Leu Ser Glu Lys Asp Val Lys Glu Phe Glu Lys Gln Val Lys Ile Val Thr Val Pro Glu Phe Thr Pro Asp His Lys Ser Met Ile Val Ser Leu Asp Val Leu Pro Phe Asn Phe Val Asp Pro Asn Met Asp Ser Arg Glu Gly Glu Asp Lys Glu Leu Lys Ile Asp Leu Glu Val Phe Glu Met Pro Pro Arg Phe Ile Met Pro Ile Cys Asp Phe Lys Ile Pro Glu Asn Ser Asp Ala Val Phe Lys Cys Ser Val Ile Gly Ile Pro Thr Pro Glu Val Lys Trp Tyr Lys Glu Tyr Met Cys Ile Glu Pro Asp Asn Ile Lys Tyr Val Ile Ser Glu Glu Lys Gly Ser His Thr Leu Lys Ile Arg Asn Val Cys Leu Ser Asp Ser Ala Thr Tyr Arg Cys Arg Ala Val Asn Cys Val Gly Glu Ala Ile Cys Arg Gly Phe Leu Thr Met Gly Asp Ser Glu Ile Phe Ala Val Ile Ala Lys Lys Ser Lys Val Thr Leu Ser Ser Leu Met Glu Glu Leu Val Leu Lys Ser Asn Tyr Thr Asp Ser Phe Phe Glu Phe Gln Val Val Glu Gly Pro Pro Arg Phe Ile Lys Gly Ile Ser Asp Cys Tyr Ala Pro Ile Gly Thr Ala Ala Tyr Phe Gln Cys Leu Val Arg Gly Ser Pro Arg Pro Thr Val Tyr Trp Tyr Lys Asp Gly Lys Leu Val Gln Gly Arg Arg Phe Thr Val Glu Glu Ser Gly Thr Gly Phe His Asn Leu Phe Ile Thr Ser Leu Val Lys Ser Asp Glu Gly Glu Tyr Arg Cys Val Ala Thr Asn Lys Ser Gly Met Ala Glu Ser Phe Ala Ala Leu Thr Leu Thr <210> 2 <211> 513 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2041716CD1 <400> 2 Met Glu Gly Gly Pro Ala Val Cys Cys Gln Asp Pro Arg Ala Glu Leu Val Glu Arg Val Ala Ala Ile Asp Val Thr His Leu Glu Glu Ala Asp Gly Gly Pro Glu Pro Thr Arg Asn Gly Val Asp Pro Pro Pro Arg Ala Arg Ala Ala Ser Val Ile Pro Gly Ser Thr Ser Arg Leu Leu Pro Ala Arg Pro Ser Leu Ser Ala Arg Lys Leu Ser Leu Gln Glu Arg Pro Ala Gly Ser Tyr Leu Glu Ala Gln Ala Gly Pro Tyr Ala Thr Gly Pro Ala Ser His Ile Ser Pro Arg Ala Trp Arg Arg Pro Thr Ile Glu Ser His His Val Ala Ile Ser Asp Ala Glu Asp Cys Val Gln Leu Asn Gln Tyr Lys Leu Gln Ser Glu Ile Gly Lys Val Gly Leu Thr Asp Ala Tyr Leu Gln Gly Ala Tyr Gly Val Val Arg Leu Ala Tyr Asn Glu Ser Glu Asp Arg His Tyr Ala Met Lys Val Leu Ser Lys Lys Lys Leu Leu Lys Gln Tyr Gly Phe Pro Arg Arg Pro Pro Pro Arg Gly Ser Gln Ala Ala Gln Gly Gly Pro Ala Lys Gln Leu Leu Pro Leu Glu Arg Val Tyr Gln Glu Ile Ala Ile Leu Lys Lys Leu Asp His Val Asn Val Val Lys Leu Ile Glu Val Leu Asp Asp Pro Ala Glu Asp Asn Leu Tyr Leu Val Asp Leu Leu Arg Lys Gly Pro Val Met Glu Vah Pro Cys Asp Lys Pro Phe Ser Glu Glu Gln Ala Arg Leu Tyr Leu Arg Asp Val Ile Leu Gly Leu Glu Tyr Leu His Cys Gln Lys Ile Val His Arg Asp Ile Lys Pro Ser Asn Leu Leu Leu Gly Asp Asp Gly His Val Lys Ile Ala Asp Phe Gly Val Ser Asn Gln Phe Glu Gly Asn Asp Ala Gln Leu Ser Ser Thr Ala Gly Thr Pro Ala Phe Met Ala Pro Glu Ala Ile Ser Asp Ser Gly Gln Ser Phe Ser Gly Lys Ala Leu Asp Val Trp Ala Thr Gly Val Thr Leu Tyr Cys Phe Val Tyr Gly Lys Cys Pro Phe Ile Asp Asp Phe Ile Leu Ala Leu His Arg Lys Ile Lys Asn Glu Pro Val Val Phe Pro Glu Glu Pro Glu Ile Ser Glu Glu Leu Lys Asp Leu Ile Leu Lys Met Leu Asp Lys Asn Pro Glu Thr Arg Ile Gly Val Pro Asp Ile Lys Leu His Pro Trp Val Thr Lys Asn Gly Glu Glu Pro Leu Pro Ser Glu Glu Glu His Cys Ser Val Val Glu Val Thr Glu Glu Glu Val Lys Asn Ser Val Arg Leu Ile Pro Ser Trp Thr Thr Val Ile Leu Val Lys Ser Met Leu Arg Lys Arg Ser Phe Gly Asn Pro Phe Glu Pro Gln Ala Arg Arg Glu Glu Arg Ser Met Ser Ala Pro Gly Asn Leu Leu Val Lys Glu Gly Phe Gly Glu Gly Gly Lys Ser Pro Glu Leu Pro Gly Val Gln Glu Asp Glu Ala Ala Ser <210> 3 <211> 1012 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472005CD1 <400> 3 Met Ala Pro Ala Arg Gly Arg Leu Pro Pro Ala Leu Trp Val Val Thr Ala Ala Ala Ala Ala Ala Thr Cys Val Ser Ala Ala Arg Gly Glu Val Asn Leu Leu Asp Thr Ser Thr Ile His Gly Asp Trp Gly Trp Leu Thr Tyr Pro Ala His Gly Trp Asp Ser Ile Asn Glu Val Asp Glu Ser Phe Gln Pro Ile His Thr Tyr Gln Val Cys Asn Val Met Ser Pro Asn Gln Asn Asn Trp Leu Arg Thr Ser Trp Val Pro Arg Asp Gly Ala Arg Arg Val Tyr Ala Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Met Pro Gly Val Leu Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Leu Glu Ser Asp Arg Asp Leu Gly Ala Ser Thr Gln Glu Ser Gln Phe Leu Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser Phe Thr Gly Ala Asp Leu Gly Val Arg Arg Leu Lys Leu Asn Thr Glu Val Arg Ser Val Gly Pro Leu Ser Lys Arg Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala Cys Leu Ala Ile Leu Ser Leu Arg Ile Tyr Tyr Lys Lys Cys Pro Ala Met Val Arg Asn Leu Ala Ala Phe Ser Glu Ala Val Thr Gly Ala Asp Ser Ser Ser Leu Val Glu Val Arg Gly Gln Cys Val Arg His Ser Glu Glu Arg Asp Thr Pro Lys Met Tyr Cys Ser Ala Glu Gly Glu Trp Leu Val Pro Ile Gly Lys Cys Val Cys Ser Ala Gly Tyr Glu Glu Arg Arg Asp Ala Cys Val Ala Cys Glu Leu Gly Phe Tyr Lys Ser Ala Pro Gly Asp Gln Leu Cys Ala Arg Cys Pro Pro His Ser His Ser Ala Ala Pro Ala Ala Gln Ala Cys His Cys Asp Leu Ser Tyr Tyr Arg Ala Ala Leu Asp Pro Pro Ser Ser Ala Cys Thr Arg Pro Pro Ser Ala Pro Val Asn Leu Ile Ser Ser Val Asn Gly Thr Ser Val Thr Leu Glu Trp Ala Pro Pro Leu Asp Pro Gly Gly Arg Ser Asp Ile Thr Tyr Asn Ala Val Cys Arg Arg Cys Pro Trp Ala Leu Ser Arg Cys Glu Ala Cys Gly Ser Gly Thr Arg Phe Val Pro Gln Gln Thr Ser Leu Val Gln Ala Ser Leu Leu Val Ala Asn Leu Leu Ala His Met Asn Tyr Ser Phe Trp Ile Glu Ala Val Asn Gly Val Ser Asp Leu Ser Pro Glu Pro Arg Arg Ala Ala Val Val Asn Ile Thr Thr Asn Gln Ala Ala Pro Ser Gln Val Val Val Ile Arg Gln Glu Arg Ala Gly Gln Thr Ser Val Ser Leu Leu Trp Gln Glu Pro Glu Gln Pro Asn Gly Ile Ile Leu Glu Tyr Glu Ile Lys Tyr Tyr Glu Lys Asp Lys Glu Met Gln Ser Tyr Ser Thr Leu Lys Ala Val Thr Thr Arg Ala Thr Val Ser Gly Leu Lys Pro Gly Thr Arg Tyr Val Phe Gln Val Arg Ala Arg Thr Ser Ala Gly Cys Gly Arg Phe Ser Gln Ala Met Glu Val Glu Thr Gly Lys Pro Arg Pro Arg Tyr Asp Thr Arg Thr Ile Val Trp Ile Cys Leu Thr Leu Ile Thr Gly Leu Val Val Leu Leu Leu Leu Leu Ile Cys Lys Lys Arg His Cys Gly Tyr Ser Lys Ala Phe Gln Asp Ser Asp Glu Glu Lys Met His Tyr Gln Asn Gly Gln Ala Pro Pro Pro Val Phe Leu Pro Leu His His Pro Pro Gly Lys Leu Pro Glu Pro Gln Phe Tyr Ala Glu Pro His Thr Tyr Glu Glu Pro Gly Arg Ala Gly Arg Ser Phe Thr Arg Glu Ile Glu Ala Ser Arg Ile His Ile Glu Lys Ile Ile Gly Ser Gly Asp Ser Gly Glu Val Cys Tyr Gly Arg Leu Arg Val Pro Gly Gln Arg Asp Val Pro Val Ala Ile Lys Ala Leu Lys Ala Gly Tyr Thr Glu Arg Gln Arg Arg Asp Phe Leu Ser Glu Ala Ser Ile Met Gly Gln Phe Asp His Pro Asn Ile Ile Arg Leu Glu Gly Val Val Thr Arg Gly Arg Leu Ala Met Ile Val Thr Glu Tyr Met Glu Asn Gly Ser Leu Asp Thr Phe Leu Arg Thr His Asp Gly Gln Phe Thr Ile Met Gln Leu Val Gly Met Leu Arg Gly Val Gly Ala Gly Met Arg Tyr Leu Ser Asp Leu Gly Tyr Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Asp Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro Asp Ala Ala Tyr Thr Thr Thr Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ala Phe Arg Thr Phe Ser Ser Ala Ser Asp Val Trp Ser Phe Gly Val Val Met Trp Glu Val Leu Ala Tyr Gly Glu Arg Pro Tyr Trp Asn Met Thr Asn Arg Asp Val Ser Ala Lys Pro Trp Gln Val Ile Ser Ser Val Glu Glu Gly Tyr Arg Leu Pro Ala Pro Met Gly Cys Pro His Ala Leu His Gln Leu Met Leu Asp Cys Trp His Lys Asp Arg Ala Gln Arg Pro Arg Phe Ser Gln Ile Val Ser Val Leu Asp Ala Leu Ile Arg Ser Pro Glu Ser Leu Arg Ala Thr Ala Thr Val Ser Arg Cys Pro Pro Pro Ala Phe Val Arg Ser Cys Phe Asp Leu Arg Gly Gly Ser Gly Gly Gly Gly Gly Leu Thr Val Gly Asp Trp Leu Asp Ser Ile Arg Met Gly Arg Tyr Arg Asp His Phe Ala Ala Gly Gly Tyr Ser Ser Leu Gly Met Val Leu Arg Met Asn Ala Gln Asp Val Arg Ala Leu Gly Ile Thr Leu Met Gly His Gln Lys Lys Ile Leu Gly Ser Ile Gln Thr Met Arg Ala Gln Leu Thr Ser Thr Gln Gly Pro Arg Arg His Leu <210> 4 <211> 367 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7472006CD1 <400> 4 Met Asp Asp Ala Ala Val Leu Lys Arg Arg Gly Tyr Leu Leu Gly Ile Asn Leu Gly Glu Gly Ser Tyr Ala Lys Val Lys Ser Ala Tyr Ser Glu Arg Leu Lys Phe Asn Val Ala ,Ile Lys Ile Ile Asp Arg Lys Lys Ala Pro Ala Asp Phe Leu Glu Lys Phe Leu Pro Arg Glu Ile Glu Ile Leu Ala Met Leu Asn His Cys Ser Ile Ile Lys Thr Tyr Glu Ile Phe Glu Thr Ser His Gly Lys Val Tyr Ile Val Met Glu Leu Ala Val Gln Gly Asp Leu Leu Glu Leu Ile Lys Thr Arg Gly Ala Leu His Glu Asp Glu Ala Arg Lys Lys Phe His Gln Leu Ser Leu Ala Ile Lys Tyr Cys His Asp Leu Asp Val Val His Arg Asp Leu Lys Cys Asp Asn Leu Leu Leu Asp Lys Asp Phe Asn Ile Lys Leu Ser Asp Phe Ser Phe Ser Lys Arg Cys Leu Arg Asp Asp Ser Gly Arg Met Ala Leu Ser Lys Thr Phe Cys Gly Ser Pro Ala 170 175 ' 180 Tyr Ala Ala Pro Glu Val Leu Gln Gly Ile Pro Tyr Gln Pro Lys Val Tyr Asp Ile Trp Ser Leu Gly Val Ile Leu Tyr Ile Met Val Cys Gly Ser Met Pro Tyr Asp Asp Ser Asn Ile Lys Lys Met Leu Arg Ile Gln Lys Glu His Arg Val Asn Phe Pro Arg Ser Lys His Leu Thr Gly Glu Cys Lys Asp Leu Ile Tyr His Met Leu Gln Pro Asp Val Asn Arg Arg Leu His Ile Asp Glu Ile Leu Ser His Cys Trp Met Gln Pro Lys Ala Arg Gly Ser Pro Ser Val Ala Ile Asn Lys Glu Gly Glu Ser Ser Arg Gly Thr Glu Pro Leu Trp Thr Pro Glu Pro Gly Ser Asp Lys Lys Ser Ala Thr Lys Leu Glu Pro Glu Gly Glu Ala Gln Pro Gln Ala Gln Pro Glu Thr Lys Pro Glu Gly Thr Ala Met Gln Met Ser Arg Gln Ser Glu Ile Leu Gly Phe Pro Ser Lys Pro Ser Thr Met Glu Thr Glu Glu Gly Pro Pro Gln Gln Pro Pro Glu Thr Arg Ala Gln <210> 5 <211> 798 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2902460CD1 <400> 5 Met Phe Glu Ala His Ile Gln Ala Gln Ser Ser Ala Ile Gln Ala Pro Arg Ser Pro Arg Leu Gly Arg Ala Arg Ser Pro Ser Pro Cys Pro Phe Arg Ser Ser Ser Gln Pro Pro Gly Arg Val Leu Val Gln Gly Ala Arg Ser Glu Glu Arg Arg Thr Lys Ser Trp Gly Glu Gln Cys Pro Glu Thr Ser Gly Thr Asp Ser Gly Arg Lys Gly Gly Pro Ser Leu Cys Ser Ser Gln Val Lys Lys Gly Met Pro Pro Leu Pro Gly Arg Ala Ala Pro Thr Gly Ser Glu Ala Gln Gly Pro Ser Ala Phe Val Arg Met Glu Lys Gly Ile Pro Ala Ser Pro Arg Cys Gly Ser Pro Thr Ala Met Glu Ile Asp Lys Arg Gly Ser Pro Thr Pro Gly Thr Arg Ser Cys Leu Ala Pro Ser Leu Gly Leu Phe Gly Ala Ser Leu Thr Met Ala Thr Glu Val Ala Ala Arg Val Thr Ser Thr Gly Pro His Arg Pro Gln Asp Leu Ala Leu Thr Glu Pro Ser Gly Arg Ala Arg Glu Leu Glu Asp Leu Gln Pro Pro Glu Ala Leu Val Glu Arg Gln Gly Gln Phe Leu Gly Ser Glu Thr Ser Pro Ala Pro Glu Arg Gly Gly Pro Arg Asp Gly Glu Pro Pro Gly Lys Met Gly Lys Gly Tyr Leu Pro Cys Gly Met Pro Gly Ser Gly Glu Pro Glu Val Gly Lys Arg Pro Glu Glu Thr Thr Val Ser Val Gln Ser Ala Glu Ser Ser Asp Ala Leu Ser Trp Ser Arg Leu Pro Arg Ala Leu Ala Ser Val Gly Pro Glu Glu Ala Arg Ser Gly Ala Pro Val Gly Gly Gly Arg Trp Gln Leu Ser Asp Arg Val Glu Gly Gly Ser Pro Thr Leu Gly Leu Leu Gly Gly Ser Pro Ser Ala Gln Pro Gly Thr Gly Asn Val Glu Ala Gly Ile Pro Ser Gly Arg Met Leu Glu Pro Leu Pro Cys Trp Asp Ala Ala Lys Asp Leu Lys Glu Pro Gln Cys Pro Pro Gly Asp Arg Val Gly Val Gln Pro Gly Asn Ser Arg Val Trp Gln Gly Thr Met Glu Lys Ala Gly Leu Ala Trp Thr Arg Gly Thr Gly Val Glri Ser Glu Gly Thr Trp Glu Ser Gln Arg Gln Asp Ser Asp Ala Leu Pro Ser Pro Glu Leu Leu Pro Gln Asp Gln Asp Lys Pro Phe Leu Arg Lys Ala Cys Ser Pro Ser Asn Ile Pro Ala Val Ile Ile Thr Asp Met Gly Thr Gln Glu Asp Gly Ala Leu Glu Glu Thr Gln Gly Ser Pro Arg Gly Asn Leu Pro Leu Arg Lys Leu Ser Ser Ser Ser Ala Ser Ser Thr Gly Phe Ser Ser Ser Tyr Glu Asp Ser Glu Glu Asp Ile Ser Ser Asp Pro Glu Arg Thr Leu Asp Pro Asn Ser Ala Phe Leu His Thr Leu Asp Gln Gln Lys Pro Arg Val Ser Lys Ser Trp Arg Lys Ile Lys Asn Met Val His Trp Ser Pro Phe Val Met Ser Phe Lys Lys Lys Tyr Pro Trp Ile Gln Leu Ala Gly His Ala Gly Ser Phe Lys Ala Ala Ala Asn Gly Arg Ile Leu Lys Lys His Cys Glu Ser Glu Gln Arg Cys Leu Asp Arg Leu Met Val Asp Val Leu Arg Pro Phe Val Pro Ala Tyr His Gly Asp Val Val Lys Asp Gly Glu Arg Tyr Asn Gln Met Asp Asp Leu Leu Ala Asp Phe Asp Ser Pro Cys Val Met Asp Cys Lys Met Gly Ile Arg Thr Tyr Leu Glu Glu Glu Leu Thr Lys Ala Arg Lys Lys Pro Ser Leu Arg Lys Asp Met Tyr Gln Lys Met Ile Glu Val Asp Pro Glu Ala Pro Thr Glu Glu Glu Lys Ala Gln Arg Ala Val Thr Lys Pro Arg Tyr Met Gln Trp Arg Glu Thr Ile Ser Ser Thr Ala Thr Leu Gly Phe Arg Ile Glu Gly Ile Lys Lys Glu Asp Gly Thr Val Asn Arg Asp Phe Lys Lys Thr Lys Thr Arg Glu Gln Val Thr Glu Ala Phe Arg Glu Phe Thr Lys Gly Asn His Asn Ile Leu Ile Ala Tyr Arg Asp Arg Leu Lys Ala Ile Arg Thr Thr Leu Glu Val Ser Pro Phe Phe Lys Cys His Glu Val Ile Gly Ser Ser Leu Leu Phe Ile His Asp Lys Lys Glu Gln Ala Lys Val Trp Met Ile Asp Phe Gly Lys Thr Thr Pro Leu Pro Glu Gly Gln Thr Leu Gln His Asp Val Pro Trp Gln Glu Gly Asn Arg Glu Asp Gly Tyr Leu Ser Gly Leu Asn Asn Leu Val Asp Ile Leu Thr Glu Met Ser Gln Asp Ala Pro Leu Ala <210> 6 <211> 358 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 6383934CD1 <400> 6 Met Asp Asp Ala Thr Val Leu Arg Lys Lys Gly Tyr Ile Val Gly Ile Asn Leu Gly Lys Gly Ser Tyr Ala Lys Val Lys Ser Ala Tyr Ser Glu Arg Leu Lys Phe Asn Val Ala Val Lys Ile Ile Asp Arg Lys Lys Thr Pro Thr Asp Phe Val Glu Arg Phe Leu Pro Arg Glu Met Asp Ile Leu Ala Thr Val Asn His Gly Ser Ile Ile Lys Thr Tyr Glu Ile Phe Glu Thr Ser Asp Gly Arg Ile Tyr Ile Ile Met Glu Leu Gly Val Gln Gly Asp Leu Leu Glu Phe Ile Lys Cys Gln Gly Ala Leu His Glu Asp Val Ala Arg Lys Met Phe Arg Gln Leu Ser Ser Ala Val Lys Tyr Cys His Asp Leu Asp Ile Val His Arg Asp Leu Lys Cys Glu Asn Leu Leu Leu Asp Lys Asp Phe Asn Ile Lys Leu Ser Asp Phe Gly Phe Ser Lys Arg Cys Leu Arg Asp Ser Asn Gly Arg Ile Ile Leu Ser Lys Thr Phe Cys Gly Ser Ala Ala Tyr Ala Ala Pro Glu Val Leu Gln Ser Ile Pro Tyr Gln Pro Lys Val Tyr Asp Ile Trp Ser Leu Gly Val Ile Leu Tyr Ile Met Val Cys Gly Ser Met Pro Tyr Asp Asp Ser Asp Ile Lys Lys Met Leu Arg Ile Gln Lys Glu His Arg Val Asn Phe Pro Arg Ser Lys His Leu Thr Cys Glu Cys Lys Asp Leu Ile Tyr His Met Leu Gln Pro Asp Val Ser Gln Arg Leu His Ile Asp Glu Ile Leu Ser His Ser Trp Leu Gln Pro Pro Lys Pro Lys Ala Thr Ser Ser Ala Ser Phe Lys Arg Glu Gly Glu Gly Lys Tyr Arg Ala Glu Cys Lys Leu Asp Thr Lys Thr Gly Leu Arg Pro Asp His Arg Pro Asp His Lys Leu Gly Ala Lys Thr Gln His Arg Leu Leu Val Val Pro Glu Asn Glu Asn Arg Met Glu Asp Arg Leu Ala Glu Thr Ser Arg Ala Lys Asp His His Ile Ser Gly Ala Glu Val Gly Lys Ala Ser Thr <210> 7 <211> 1049 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3210906CD1 <400> 7 Met Pro Ala Gly Gly Arg Ala Gly Ser Leu Lys Asp Pro Asp Val Ala Glu Leu Phe Phe Lys Asp Asp Pro Glu Lys Leu Phe Ser Asp Leu Arg Glu Ile Gly His Gly Ser Phe Gly Ala Val Tyr Phe Ala Arg Asp Val Arg Asn Ser Glu Val Val Ala Ile Lys Lys Met Ser Tyr Ser Gly Lys Gln Ser Asn Glu Lys Trp Gln Asp Ile Ile Lys Glu Val Arg Phe Leu Gln Lys Leu Arg His Pro Asn Thr Ile Gln Tyr Arg Gly Cys Tyr Leu Arg Glu His Thr Ala Trp Leu Val Met Glu Tyr Cys Leu Gly Ser Thr Ser Asp Leu Leu Glu Val His Lys Lys Pro Leu Gln Glu Val Glu Ile Ala Ala Val Thr His Gly Ala Leu Gln Gly Leu Ala Tyr Leu His Ser His Asn Met Ile His Arg Asp Val Lys Ala Gly Asn Ile Leu Leu Ser Glu Pro Gly Leu Val Lys Leu Gly Asp Phe Gly Ser Ala Ser Ile Met Ala Pro Ala Asn Ser Phe Val Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Ile Leu Ala Met Asp Glu Gly Gln Tyr Asp Gly Lys Val Asp Val Trp Ser Leu Gly Ile Thr Cys Ile Glu Leu Ala Glu Arg Lys Pro Pro Leu Phe Asn Met Asn Ala Met Ser Ala Leu Tyr His Ile Ala Gln Asn Glu Ser Pro Val Leu Gln Ser Gly His Trp Ser Glu Tyr Phe Arg Asn Phe Val Asp Ser Cys Leu Gln Lys Ile Pro Gln Asp Arg Pro Thr Ser Glu Val Leu Leu Lys His Arg Phe Val Leu Arg Glu Arg Pro Pro Thr Val Ile Met Asp Leu Ile Gln Arg Thr Lys Asp Ala Val Arg Glu Leu Asp Ser Leu Gln Tyr Arg Lys Met Lys Lys Ile Leu Phe Gln Glu Ala Pro Asn Gly Pro Gly Ala Glu Ala Pro Glu Glu Glu Glu Glu Ala Glu Pro Tyr Met His Leu Ala Gly Thr Leu Thr Ser Leu Glu Ser Ser His Ser Val Pro Ser Met Ser Ile Ser Ala Ser Ser Gln Ser Ser Ser Val Asn Ser Leu Ala Asp Ala Ser Asp Asn Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Gly Pro Glu Ala Arg Glu Met Ala Met Met Gln Glu Gly Glu His Thr Val Thr Ser His Ser Ser Ile Ile His Arg Leu Pro Gly Ser Asp Asn Leu Tyr Asp Asp Pro Tyr Gln Pro Glu Ile Thr 425 ~ 430 435 Pro Ser Pro Leu Gln Pro Pro Ala Ala Pro Ala Pro Thr Ser Thr Thr Ser Ser Ala Arg Arg Arg Ala Tyr Cys Arg Asn Arg Asp His Phe Ala Thr Ile Arg Thr Ala Ser Leu Val Ser Arg Gln Ile Gln Glu His Glu Gln Asp Ser Ala Leu Arg Glu Gln Leu Ser Gly Tyr Lys Arg Met Arg Arg Gln His Gln Lys Gln Leu Leu Ala Leu Glu Ser Arg Leu Arg Gly Glu Arg Glu Glu His Ser Ala Arg Leu Gln Arg Glu Leu Glu Ala Gln Arg Ala Gly Phe Gly Ala Glu Ala Glu Lys Leu Ala Arg Arg His Gln Ala Ile Gly Glu Lys Glu Ala Arg Ala Ala Gln Ala Glu Glu Arg Lys Phe Gln Gln His Ile Leu Gly Gln Gln Lys Lys Glu Leu Ala Ala Leu Leu Glu Ala Gln Lys Arg Thr Tyr Lys Leu Arg Lys Glu Gln Leu Lys Glu Glu Leu Gln Glu Asn Pro Ser Thr Pro Lys Arg Glu Lys Ala Glu Trp Leu Leu Arg Gln Lys Glu Gln Leu Gln Gln Cys Gln Ala Glu Glu Glu Ala Gly Leu Leu Arg Arg Gln Arg Gln Tyr Phe Glu Leu Gln Cys Arg Gln Tyr Lys Arg Lys Met Leu Leu Ala Arg His Ser Leu Asp Gln Asp Leu Leu Arg Glu Asp Leu Asn Lys Lys Gln Thr Gln Lys Asp Leu Glu Cys Ala Leu Leu Leu Arg Gln His Glu Ala Thr Arg Glu Leu Glu Leu Arg Gln Leu Gln Ala Val Gln Arg Thr Arg Ala Glu Leu Thr Arg Leu Gln His Gln Thr Glu Leu Gly Asn Gln Leu Glu Tyr Asn Lys Arg Arg Glu Gln Glu Leu Arg Gln Lys His Ala Ala Gln Val Arg Gln Gln Pro Lys Ser Leu Lys Ser Lys Glu Leu Gln Ile Lys Lys Gln Phe Gln Glu Thr Cys Lys Ile Gln Thr Arg Gln Tyr Lys Ala Leu Arg Ala His Leu Leu Glu Thr Thr Pro Lys Ala Gln His Lys Ser Leu Leu Lys Arg Leu Lys Glu Glu Gln Thr Arg Lys Leu Ala Ile Leu Ala Glu Gln Tyr Asp Gln Ser Ile Ser Glu Met Leu Ser Ser Gln Ala Leu Arg Leu Asp Glu Thr Gln Glu Ala Glu Phe Gln Ala Leu Arg Gln Gln Leu Gln Gln Glu Leu Glu Leu Leu Asn Ala Tyr Gln Ser Lys Ile Lys Ile Arg Thr Glu Ser Gln His Glu Arg Glu Leu Arg Glu Leu Glu Gln Arg Val Ala Leu Arg Arg Ala Leu Leu Glu Gln Arg Val Glu Glu Glu Leu Leu Ala Leu Gln Thr Gly Arg Ser Glu Arg Ile Arg Ser Leu Leu Glu Arg Gln Ala Arg Glu Ile Glu Ala Phe Asp Ala Glu Ser Met Arg Leu Gly Phe Ser Ser Met Ala Leu Gly Gly Ile Pro Ala Glu Ala Ala Ala Gln Gly Tyr Pro Ala Pro Pro Pro Ala Pro Ala Trp Pro Ser Arg Pro Val Pro Arg Ser Gly Ala His Trp Ser His Gly Pro Pro Pro Pro Gly Met Pro Pro Pro Ala Trp Arg Gln Pro Ser Leu Leu Ala Pro Pro Gly Pro Pro Asn Trp Leu Gly Pro Pro Thr Gln Ser Gly Thr Pro Arg Gly Gly Ala Leu Leu Leu Leu Arg Asn Ser Pro Gln Pro Leu Arg Arg Ala Ala Ser Gly Gly Ser Gly Ser Glu Asn Val Gly Pro Pro Ala Ala Ala Val Pro Gly Pro Leu Ser Arg Ser Thr Ser Val Ala Ser His Ile Leu Asn Gly Ser Ser His Phe Tyr Ser <210> 8 <211> 322 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3339024CD1 <400> 8 Met Pro Thr Phe Ser Ile Pro Gly Thr Leu Glu Ser Gly His Pro Arg Asn Leu Thr Cys Ser Val Pro Trp Ala Cys Glu Gln Gly Thr Pro Pro Thr Ile Thr Trp Met Gly Ala Ser Val Ser Ser Leu Asp Pro Thr Ile Thr Arg Ser Ser Met Leu Ser Leu Ile Pro Gln Pro Gln Asp His Gly Thr Ser Leu Thr Cys Gln Val Thr Leu Pro Gly Ala Gly Val Thr Met Thr Arg Ala Val Arg Leu Asn Ile Ser Tyr Pro Pro Gln Asn Leu Thr Met Thr Val Phe Gln Gly Asp Gly Thr Ala Ser Thr Thr Leu Arg Asn Gly Ser Ala Leu Ser Val Leu Glu Gly Gln Ser Leu His Leu Val Cys Ala Val Asp Ser Asn Pro Pro Ala Arg Leu Ser Trp Thr Trp Gly Ser Leu Thr Leu Ser Pro Ser Gln Ser Ser Asn Leu Gly Val Leu Glu Leu Pro Arg Val His Val Lys Asp Glu Gly Glu Phe Thr Cys Arg Ala Gln Asn Pro Leu Gly Ser Gln His Ile Ser Leu Ser Leu Ser Leu Gln Asn Glu Tyr Thr Gly Lys Met Arg Pro Ile Ser Gly Val Thr Leu Gly Ala Phe Gly Gly Ala Gly Ala Thr Ala Leu Val Phe Leu Tyr Phe Cys Ile Ile Phe Val Val Val Arg Ser Cys Arg Lys Lys Ser Ala Arg Pro Ala Val Gly Val Gly Asp Thr Gly Met Glu Asp Ala Asn Ala Val Trp Gly Ser Ala Ser Gln Gly Pro Leu Ile Glu Ser Pro Ala Asp Asp Ser Pro Pro His His Ala Pro Pro Ala Leu Ala Thr Pro Ser Pro Glu Glu Gly Glu Ile Gln Tyr Ala Ser Leu Ser Phe His Lys Ala Arg Pro Gln Tyr Pro Gln Glu Gln Glu Ala Ile Gly Tyr Glu Tyr Ser Glu Ile Asn Ile Pro Lys <210> 9 <211> 1212 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4436929CD1 <400> 9 Met Ala Asn Asp Ser Pro Ala Lys Ser Leu Val Asp Ile Asp Leu Ser Ser Leu Arg Asp Pro Ala Gly Ile Phe Glu Leu Val Glu Val Val Gly Asn Gly Thr Tyr Gly Gln Val Tyr Lys Gly Arg His Val Lys Thr Gly Gln Leu Ala Ala Ile Lys Val Met Asp Val Thr Glu Asp Glu Glu Glu Glu Ile Lys Leu Glu Ile Asn Met Leu Lys Lys Tyr Ser His His Arg Asn Ile Ala Thr Tyr Tyr Gly Ala Phe Ile Lys Lys Ser Pro Pro Gly His Asp Asp Gln Leu Trp Leu Val Met Glu Phe Cys Gly Ala Gly Ser Ile Thr Asp Leu Val Lys Asn Thr Lys Gly Asn Thr Leu Lys Glu Asp Trp Ile Ala Tyr Ile Ser Arg Glu Ile Leu Arg Gly Leu Ala His Leu His Ile His His Val Ile His Arg Asp Ile Lys Gly Gln Asn Val Leu Leu Thr Glu Asn Ala Glu Val Lys Leu Val Asp Phe Gly Val Ser Ala Gln Leu Asp Arg Thr Val Gly Arg Arg Asn Thr Phe Ile Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Ile Ala Cys Asp Glu Asn Pro Asp Ala Thr Tyr Asp Tyr Arg Ser Asp Leu Trp Ser Cys Gly Ile Thr Ala Ile Glu Met Ala Glu Gly Ala Pro Pro Leu Cys Asp Met His Pro Met Arg Ala Leu Phe Leu Ile Pro Arg Asn Pro Pro Pro Arg Leu Lys Ser Lys Lys Trp Ser Lys Lys Phe Phe Ser Phe Ile Glu Gly Cys Leu Val Lys Asn Tyr Met Gln Arg Pro Ser Thr Glu Gln Leu Leu Lys His Pro Phe Ile Arg Asp Gln Pro Asn Glu Arg Gln Val Arg Ile Gln Leu Lys Asp His Ile Asp Arg Thr Arg Lys Lys Arg Gly Glu Lys Asp Glu Thr Glu Tyr Glu Tyr Ser Gly Ser Glu Glu Glu Glu Glu Glu Val Pro Glu Gln Glu Gly Glu Pro Ser Ser Ile Val Asn Val Pro Gly Glu Ser Thr Leu Arg Arg Asp Phe Leu Arg Leu Gln Gln Glu Asn Lys Glu Arg Ser Glu Ala Leu Arg Arg Gln Gln Leu Leu Gln Glu Gln Gln Leu Arg Glu Gln Glu Glu Tyr Lys Arg Gln Leu Leu Ala Glu Arg Gln Lys Arg Ile Glu Gln Gln Lys Glu Gln Arg Arg Arg Leu Glu Glu Gln Gln Arg Arg Glu Arg Glu Ala Arg Arg Gln Gln Glu Arg Glu Gln Arg Arg Arg Glu Gln Glu Glu Lys Arg Arg Leu Glu Glu Leu Glu Arg Arg Arg Lys Glu Glu Glu Glu Arg Arg Arg Ala Glu Glu Glu Lys Arg Arg Val Glu Arg Glu Gln Glu Tyr Ile Arg Arg Gln Leu Glu Glu Glu Gln Arg His Leu Glu Val Leu Gln Gln Gln Leu Leu Gln Glu Gln Ala Met Leu Leu His Asp His Arg Arg Pro His Pro Gln His Ser Gln Gln Pro Pro Pro Pro Gln Gln Glu Arg Ser Lys Pro Ser Phe His Ala Pro Glu Pro Lys Ala His Tyr Glu Pro Ala Asp Arg Ala Arg Glu Val Glu Asp Arg Phe Arg Lys Thr Asn His Ser Ser Pro Glu Ala Gln Ser Lys Gln Thr Gly Arg Val Leu Glu Pro Pro Val Pro Ser Arg Ser Glu Ser Phe Ser Asn Gly Asn Ser Glu Ser Val His Pro Ala Leu Gln Arg Pro Ala Glu Pro Gln Val Pro Val Arg Thr Thr Ser Arg Ser Pro Val Leu Ser Arg Arg Asp Ser Pro Leu Gln Gly Ser Gly Gln Gln Asn Ser Gln Ala Gly Gln Arg Asn Ser Thr Ser Ser Ile Glu Pro Arg Leu Leu Trp Glu Arg Val Glu Lys Leu Val Pro Arg Pro Gly Ser Gly Ser Ser Ser Gly Ser Ser Asn Ser Gly Ser Gln Pro Gly Ser His Pro Gly Ser Gln Ser Gly Ser Gly Glu Arg Phe Arg Val Arg Ser Ser Ser Lys Ser Glu Gly Ser Pro Ser Gln Arg Leu Glu Asn Ala Val Lys Lys Pro Glu Asp Lys Lys Glu Val Phe Arg Pro Leu Lys Pro Ala Gly Glu Val Asp Leu Thr Ala Leu Ala Lys Glu Leu Arg Ala Val Glu Asp Val Arg Pro Pro His Lys Val Thr Asp Tyr Ser Ser Ser Ser Glu Glu Ser Gly Thr Thr Asp Glu Glu Asp Asp Asp Val Glu Gln Glu Gly Ala Asp Glu Ser Thr Ser Gly Pro Glu Asp Thr Arg Ala Ala Ser Ser Leu Asn Leu Ser Asn Gly Glu Thr Glu Ser Val Lys Thr Met Ile Val His Asp Asp Val Glu Ser Glu Pro Ala Met Thr Pro Ser Lys Glu Gly Thr Leu Ile Val Arg Gln Thr Gln Ser Ala Ser Ser Thr Leu Gln Lys His Lys Ser Ser Ser Ser Phe Thr Pro Phe Ile Asp Pro Arg Leu Leu Gln Ile Ser Pro Ser Ser Gly Thr Thr Val Thr Ser Val Val Gly Phe Ser Cys Asp Gly Met Arg Pro Glu Ala Ile Arg Gln Asp Pro Thr Arg Lys Gly Ser Val Val Asn Val Asn Pro Thr Asn Thr Arg Pro Gln Ser Asp Thr Pro Glu Ile Arg Lys Tyr Lys Lys Arg Phe Asn Ser Glu Ile Leu Cys Ala Ala Leu Trp Gly Val Asn Leu Leu Val Gly Thr Glu Ser Gly Leu Met Leu Leu Asp Arg Ser Gly Gln Gly Lys Val Tyr Pro Leu Ile Asn Arg Arg Arg Phe Gln Gln Met Asp Val Leu Glu Gly Leu Asn Val Leu Val Thr Ile Ser Gly Lys Lys Asp Lys Leu Arg Val Tyr Tyr Leu Ser Trp Leu Arg Asn Lys Ile Leu His Asn Asp Pro Glu Val Glu Lys Lys Gln Gly Trp Thr Thr Val Gly Asp Leu Glu Gly Cys Val His Tyr Lys Val Val Lys Tyr Glu Arg Ile Lys Phe Leu Val Ile Ala Leu Lys Ser Ser Val Glu Val Tyr Ala Trp Ala Pro Lys Pro Tyr His Lys Phe Met Ala Phe Lys Ser Phe Gly Glu Leu Val His Lys Pro Leu Leu Val Asp Leu Thr Val Glu Glu Gly Gln Arg Leu Lys Val Ile Tyr Gly Ser Cys Ala Gly Phe His Ala Val Asp Val Asp Ser Gly Ser Val Tyr Asp Ile Tyr Leu Pro Thr His Ile Gln Cys Ser Ile Lys Pro His Ala Ile Ile Ile Leu Pro Asn Thr Asp Gly Met Glu Leu Leu Val Cys Tyr Glu Asp Glu Gly Val Tyr Val Asn Thr Tyr Gly Arg Ile Thr Lys Asp Val Val Leu Gln Trp Gly Glu Met Pro Thr Ser Val Ala Tyr Ile Arg Ser Asn Gln Thr Met Gly Trp Gly Glu Lys Ala Ile Glu Ile Arg Ser Val Glu Thr Gly His Leu Asp Gly Val Phe Met His Lys Arg Ala Gln Arg Leu Lys Phe Leu Cys Glu Arg Asn Asp Lys Val Phe Phe Ala Ser Val Arg Ser Gly Gly Ser Ser Gln Val Tyr Phe Met Thr Leu Gly Arg Thr Ser Leu Leu Ser Trp <210> 10 <211> 280 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 5046791CD1 <400> 10 Met Gln Pro Leu Arg Val Asn Ser Gln Pro Gly Pro Gln Lys Arg Cys Leu Phe Val Cys Arg His Gly Glu Arg Met Asp Val Val Phe Gly Lys Tyr Trp Leu Ser Gln Cys Phe Asp Ala Lys Gly Arg Tyr Ile Arg Thr Asn Leu Asn Met Pro His Ser Leu Pro Gln Arg Ser Gly Gly Phe Arg Asp Tyr Glu Lys Asp Ala Pro Ile Thr Val Phe Gly Cys Met Gln Ala Arg Leu Val Gly Glu Ala Leu Leu Glu Ser Asn Thr Ile Ile Asp His Val Tyr Cys Ser Pro Ser Leu Arg Cys Val Gln Thr Ala His Asn Ile Leu Lys Gly Leu Gln Gln Glu Asn His Leu Lys Ile Arg Val Glu Pro Gly Leu Phe Glu Trp Thr Lys Trp Val Ala Gly Ser Thr Leu Pro Ala Trp Ile Pro Pro Ser Glu Leu Ala Ala Ala Asn Leu Ser Val Asp Thr Thr Tyr Arg Pro His Ile Pro Ile Ser Lys Leu Val Val Ser Glu Ser Tyr Asp Thr Tyr Ile Ser Arg Ser Phe Gln Val Thr Lys Glu Ile Ile Ser Glu Cys Lys Ser Lys Gly Asn Asn Ile Leu Ile Val Ala His Ala Ser Ser Leu Glu Ala Cys Thr Cys Gln Leu Gln Gly Leu Ser Pro Gln Asn Ser Lys Asp Phe Val Gln Met Val Arg Lys Ile Pro Tyr Leu Gly Phe Cys Ser Cys Glu Glu Leu Gly Glu Thr Gly Ile Trp Gln Leu Thr Asp Pro Pro Ile Leu Pro Leu Thr His Gly Pro Thr Gly Gly Phe Asn Trp Arg Glu Thr Leu Leu Gln Glu <210> 11 <211> 114 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1416174CD1 <400> 11 Met Leu Ala Ile Ser Pro Ser His Leu Gly Ala Asp Leu Val Ala Ala Pro His Ala Arg Phe Asp Asp Gly Leu Val His Leu Cys Trp Val Arg Thr Gly Ile Ser Arg Ala Ala Leu Leu Arg Leu Phe Leu Ala Met Glu Arg Gly Ser His Phe Ser Leu Gly Cys Pro Gln Leu Gly Tyr Ala Ala Ala Arg Ala Phe Arg Leu Glu Pro Leu Thr Pro Arg Gly Val Leu Thr Val Asp Gly Glu Gln Val Glu Tyr Gly Pro Leu Gln Ala Gln Met His Pro Gly Ile Gly Thr Leu Leu Thr Gly Pro Pro Gly Cys Pro Gly Arg Glu Pro <210> 12 <211> 375 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3244919CD1 <400> 12 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 Ala Pro Arg Ser Lys Ser Lys Pro Ser Thr Gln Arg Gln Gly Ser Trp Ala Leu Ala Ser Ser Gly Leu Gly Glu <210> 13 <211> 1859 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 058860CB1 <400> 13 gagagatact ccacaccccc aggagagact ctagagagat attccacacc cccaggagag 60 actctggaga gatactccac acccccagga gagactctag agcgatattc cacaccccca 120 ggggaggcac tagagagata ttctattcct actggaggac caaaccccac tggtactttt 180 aaaacatatc catcaaaaat agaaatggaa gacggtacac caaatgagca tttctacaca 240 cctacagaag agaggggttc agcttatgaa atatggcgtt ccgattcatt tggtacaccc 300 aatgaagcca ttgagccaaa agacaatgaa atgcctccat cttttattga acctctgacc 360 aaaaggaagg tatatgaaaa cacaacacta ggcttcattg ttgaagttga aggtcttcca 420 gttcctggtg tgaaatggta tcgaaataaa tctttactag agccagatga aagaatcaaa 480 atggaaagag tgggtaatgt gtgttcactg gaaatttcta acattcaaaa aggagaaggg 540 ggagagtaca tgtgtcatgc tgtaaacatc ataggggaag caaagagctt tgcaaatgta 600 gacataatgc cccaggaaga aagagtggtg gcactaccac ctccagtaac acatcagcat 660 gtcatggagt ttgatttgga acacaccaca tcatcaagaa caccttctcc tcaagaaatt 720 gtcctggaag ttgaattaag tgaaaaagac gttaaagaat ttgagaagca ggtgaaaata 780 gtgacagttc ccgaatttac tcctgaccat aaaagtatga ttgtgagtct agatgttctt 840 ccatttaatt ttgtagatcc aaatatggat tcaagggagg gagaagacaa agaactaaaa 900 attgatttag aagtatttga aatgcctcct cgctttataa tgcctatttg tgattttaaa 960 attccagaaa attcagatgc tgtattcaaa tgttcagtca tagggatccc gactcccgaa 1020 gttaagtggt ataaagaata tatgtgtatt gagccagata atattaaata cgtgattagc 1080 gaggagaagg gaagtcacac tcttaaaatt cgaaatgtct gtctttctga tagtgcaaca 1140 tacaggtgca gagctgtgaa ttgtgtagga gaggctatct gtcggggatt cctcaccatg 1200 ggagattctg aaatatttgc tgtgatagca aagaaaagca aagtgacttt aagcagttta 1260 atggaagaat tggtcttaaa gagcaactac acagacagtt tttttgaatt tcaggtggtg 1320 gaagggcctc ccaggtttat caaaggtatt tctgactgtt atgcaccaat aggtacagca 1380 gcatattttc agtgcttagt tcgtggctct ccaagaccca cggtttactg gtacaaagat 1440 ggaaaattag tccaaggaag aaggttcact gttgaggaaa gtggcacagg gttccataac 1500 ctgtttataa caagcttagt aaagagtgat gaaggagagt ataggtgtgt agctacaaac 1560 aaatcaggaa tggctgagag ctttgcagca ctcaccttaa cttaaaatgt aatgttttag 1620 tgcctcagta attattagca ttgatctgag tgctttcata ttttccaaat tatgtggatc 1680 taataaactt ccaaacaggt ccaccatatt tgaattcatt accttggaga cccctaaaga 1740 aataatctct atgtagaaat ctcatctttg taatacatgt aaatattttg ttatctgaac 1800 tgtggaatca tcacttgtgt caatcatgct gtgtaatatc aaacacaatt aaatctctc 1859 <210> 14 <211> 3501 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2041716CB1 <400> 14 gtggtgtggc tgcagtggag agttcccaac aaggctacgc agaagaaccc ccttgactga 60 agcaatggag gggggtccag ctgtctgctg ccaggatcct cgggcagagc tggtagaacg 120 ggtggcagcc atcgatgtga ctcacttgga ggaggcagat ggtggcccag agcctactag 180 aaacggtgtg gaccccccac cacgggccag agctgcctct gtgatccctg gcagtacttc 240 aagactgctc ccagcccggc ctagcctctc agccaggaag ctttccctac aggagcggcc 300 agcaggaagc tatctggagg cgcaggctgg gccttatgcc acggggcctg ccagccacat 360 ctccccccgg gcctggcgga ggcccaccat cgagtcccac cacgtggcca tctcagatgc 420 agaggactgc gtgcagctga accagtacaa gctgcagagt gagattggca aggtggggct 480 gactgatgcc tatctgcagg gtgcctacgg tgtggtgagg ctggcctaca acgaaagtga 540 agacagacac tatgcaatga aagtcctttc caaaaagaag ttactgaagc agtatggctt 600 tccacgtcgc cctcccccga gagggtccca ggctgcccag,ggaggaccag ccaagcagct 660 gctgcccctg gagcgggtgt accaggagat tgccatcctg aagaagctgg accacgtgaa 720 tgtggtcaaa ctgatcgagg tcctggatga cccagctgag gacaacctct atttggttga 780 cctcctgaga aaggggcccg tcatggaagt gccctgtgac aagcccttct cggaggagca 840 agctcgcctc tacctgcggg acgtcatcct gggcctcgag tacttgcact gccagaagat 900 cgtccacagg gacatcaagc catccaacct gctcctgggg gatgatgggc acgtgaagat 960 cgccgacttt ggcgtcagca accagtttga ggggaacgac gctcagctgt ccagcacggc 1020 gggaacccca gcattcatgg cccccgaggc catttctgat tccggccaga gcttcagtgg 1080 gaaggccttg gatgtatggg ccactggcgt cacgttgtac tgctttgtct atgggaagtg 1140 cccgttcatc gacgatttca tcctggccct ccacaggaag atcaagaatg agcccgtggt 1200 gtttcctgag gagccagaaa tcagcgagga gctcaaggac ctgatcctga agatgttaga 1260 caagaatccc gagacgagaa ttggggtgcc agacatcaag ttgcaccctt gggtgaccaa 1320 gaacggggag gagccccttc cttcggagga ggagcactgc agcgtggtgg aggtgacaga 1380 ggaggaggtt aagaactcag tcaggctcat ccccagctgg accacggtga tcctggtgaa 1440 gtccatgctg aggaagcgtt cctttgggaa cccgtttgag ccccaagcac ggagggaaga 1500 gcgatccatg tctgctccag gaaacctact ggtgaaagaa gggtttggtg aagggggcaa 1560 gagcccagag ctccccggcg tccaggaaga cgaggctgca tcctgagccc ctgcatgcac 1620 ccagggccac ccggcagcac actcatcccg cgcctccaga ggcccacccc tcatgcaaca 1680 gccgcccccg caggcagggg gctggggact gcagccccac tcccgcccct cccccatcgt 1740 gctgcatgac ctccacgcac gcacgtccag ggacagactg gaatgtatgt catttggggt 1800 cttgggggca gggctcccac gaggccatcc tcctcttctt ggacctcctt ggcctgaccc 1860 attctgtggg gaaaccgggt gcccatggag cctcagaaat gccacccggc tggttggcat 1920 ggcctggggc aggaggcaga ggcaggagac caagatggca ggtggaggcc aggcttacca 1980 caacggaaga gacctcccgc tggggccggg caggcctggc tcagctgcca caggcatatg 2040 gtggagaggg gggtaccctg cccaccttgg ggtggtggca ccagagctct tgtctattca 2100 gacgctggta tgggggctcg gacccctcac tggggacagg gccagtgttg gagaattctg 2160 attccttttt tgttgtcttt tacttttgtt tttaacctgg gggttcgggg agaggccctg 2220 cttgggaaca tctcacgagc tttcctacat cttccgtggt tcccagcaca gcccaagatt 2280 atttggcagc caagtggatg gaactaactt tcctggactg tgtttcgcat tcggcgttat 2340 ctggaaagtg gactgaacgg aatcaagctc tgagcagagg cctgaagcgg aagcaccaca 2400 tcgtccctgc ccatctcact ctctcccttg atgatgcccc tagagctgag gctggagaag 2460.
acaccagggc tgactttgac cgagggccat ggacgcgaca ggcctgtggc cctgcgcatg 2520 ctgaaataac tggaacccag cctctcctcc tacaccggcc tacccatctg ggcccaagag 2580 ctgcactcac actcctacaa cgaaggacaa actgtccagg tcggagggat cacgagacac 2640 agaacctgga ggggtgtgca cgctggcagg tggcctctgc ggcaattgcc tcaccctgag 2700 gacatcagca gtcagcctgc tcagagcggg ggtgctggag cgcgtgcaga cacagctctt 2760 ccggagcagc cttcaccttc tctctgggat cagtgtccgg ctggccgacg tggcatttgc 2820 tgaccgaatg ctcatagagg ttgaccccca cagggtcacg caggactcgg acactgccct 2880 ggaaacatgg atggacaagg gcttttggcc acaggtgtgg gtgtcctgtt ggaggagggc 2940 ttgtttggag aagggaggct ggctggggga gaaacccgga tcccgctgca tctccgcgcc 3000 tgtgggtgca tgtcgcgtgc tcatctgttg cacacagctc actcgtatgt cctgcactgg 3060 tacatgcatc tgtaatacag tttctacgtc tatttaaggc taggagccga atgtgcccca 3120 ttgtcagtgg gtccacgttt ctccccggct cctctgggct aaggcagtgt ggcccgaggc 3180 ttaaaaagtt actcggtact gtttttaaga acacttttat agagttagtg gaaggcaagt 3240 taagagccaa tcactgatcc ccaagtgttt cttgagcatc tggtctgggg ggaccacttt 3300 gatcggaccc acccttggaa agctcagggg taggcccagg tgggatgctc accctgtcac 3360 tgagggtttt ggttggcatc gttgtttttg aatgtagcac aagcgatgag caaactctat 3420 aagagtgttt taaaaattaa cttcccagga agtgagttaa aaacaataaa agccctttct 3480 tgagttaaaa agaaaaaaaa a 3501 <210> 15 <211> 3039 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472005CB1 <400> 15 atggcccccg cccggggccg cctgccccct gcgctctggg tcgtcacggc cgcggcggcg 60 gcggccacct gcgtgtccgc ggcgcgcggc gaagtgaatt tgctggacac gtcgaccatc 120 cacggggact ggggctggct cacgtatccg gctcatgggt gggactccat caacgaggtg 180 gacgagtcct tccagcccat ccacacgtac caggtttgca acgtcatgag ccccaaccag 240 aacaactggc tgcgcacgag ctgggtcccc cgagacggcg cccggcgcgt ctatgctgag 300 atcaagttta ccctgcgcga ctgcaacagc atgcctggtg tgctgggcac ctgcaaggag 360 accttcaacc tctactacct ggagtcggac cgcgacctgg gggccagcac acaagaaagc 420 cagttcctca aaatcgacac cattgcggcc gacgagagct tcacaggtgc cgaccttggt 480 gtgcggcgtc tcaagctcaa cacggaggtg cgcagtgtgg gtcccctcag caagcgcggc 540 ttctacctgg ccttccagga cataggtgcc tgcctggcca tcctctctct ccgcatctac 600 tataagaagt gccctgccat ggtgcgcaat ctggctgcct tctcggaggc agtgacgggg 660 gccgactcgt cctcactggt ggaggtgagg ggccagtgcg tgcggcactc agaggagcgg 720 gacacaccca agatgtactg cagcgcggag ggcgagtggc tcgtgcccat cggcaaatgc 780 gtgtgcagtg ccggctacga ggagcggcgg gatgcctgtg tggcctgtga gctgggcttc 840 tacaagtcag cccctgggga ccagctgtgt gcccgctgcc ctccccacag ccactccgca 900 gctccagccg cccaagcctg ccactgtgac ctcagctact accgtgcagc cctggacccg 960 ccgtcctcag cctgcacccg gccaccctcg gcaccagtga acctgatctc cagtgtgaat 1020 gggacatcag tgactctgga gtgggcccct cccctggacc caggtggccg cagtgacatc 1080 acctacaatg ccgtgtgccg ccgctgcccc tgggcactga gccgctgcga ggcatgtggg 1140 agcggcaccc gctttgtgcc ccagcagaca agcctggtgc aggccagcct gctggtggcc 1200 aacctgctgg cccacatgaa ctactccttc tggatcgagg ccgtcaatgg cgtgtccgac 1260 ctgagccccg agccccgccg ggccgctgtg gtcaacatca ccacgaacca ggcagccccg 1320 tcccaggtgg tggtgatccg tcaagagcgg gcggggcaga ccagcgtctc gctgctgtgg 1380 caggagcccg agcagccgaa cggcatcatc ctggagtatg agatcaagta ctacgagaag 1440 gacaaggaga tgcagagcta ctccaccctc aaggccgtca ccaccagagc caccgtctcc 1500 ggcctcaagc cgggcacccg ctacgtgttc caggtccgag cccgcacctc agcaggctgt 1560 ggccgcttca gccaggccat ggaggtggag accgggaaac cccggccccg ctatgacacc 1620 aggaccattg tctggatctg cctgacgctc atcacgggcc tggtggtgct tctgctcctg 1680 ctcatctgca agaagaggca ctgtggctac agcaaggcct tccaggactc ggacgaggag 1740 aagatgcact atcagaatgg acaggcaccc ccacctgtct tcctgcctct gcatcacccc 1800 ccgggaaagc tcccagagcc ccagttctat gcggaacccc acacctacga ggagccaggc 1860 cgggcgggcc gcagtttcac tcgggagatc gaggcctcta ggatccacat cgagaaaatc 1920 atcggctctg gagactccgg ggaagtctgc tacgggaggc tgcgggtgcc agggcagcgg 1980 gatgtgcccg tggccatcaa ggccctcaaa gccggctaca cggagagaca gaggcgggac 2040 ttcctgagcg aggcgtccat catggggcaa ttcgaccatc ccaacatcat ccgcctcgag 2100 ggtgtcgtca cccgtggccg cctggcaatg attgtgactg agtacatgga gaacggctct 2160 ctggacacct tcctgaggac ccacgacggg cagttcacca tcatgcagct ggtgggcatg 2220 ctgagaggag tgggtgccgg catgcgctac ctctcagacc tgggctatgt ccaccgagac 2280 ctggccgccc gcaacgtcct ggttgacagc aacctggtct gcaaggtgtc tgacttcggg 2340 ctctcacggg tgctggagga cgacccggat gctgcctaca ccaccacggg cgggaagatc 2400 cccatccgct ggacggcccc agaggccatc gccttccgca ccttctcctc ggccagcgac 2460 gtgtggagct tcggcgtggt catgtgggag gtgctggcct atggggagcg gccctactgg 2520 aacatgacca accgggatgt gagtgccaag ccctggcagg tcatcagctc tgtggaggag 2580 gggtaccgcc tgcccgcacc catgggctgc ccccacgccc tgcaccagct catgctcgac 2640 tgttggcaca aggaccgggc gcagcggcct cgcttctccc agattgtcag tgtcctcgat 2700 gcgctcatcc gcagccctga gagtctcagg gccaccgcca cagtcagcag gtgcccaccc 2760 cctgccttcg tccggagctg ctttgacctc cgagggggca gcggtggcgg tgggggcctc 2820 accgtggggg actggctgga ctccatccgc atgggccggt accgagacca cttcgctgcg 2880 ggcggatact cctctctggg catggtgcta cgcatgaacg cccaggacgt gcgcgccctg 2940 ggcatcaccc tcatgggcca ccagaagaag atcctgggca gcattcagac catgcgggcc 3000 cagctgacca gcacccaggg gccccgccgg cacctctga 3039 <210> 16 <211> 1104 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472006CB1 <400> 16 atggatgacg ctgctgtcct caagcgacga ggctacctcc tggggataaa tttaggagag 60 ggctcctatg caaaagtaaa atctgcttac tctgagcgcc tgaagttcaa tgtggcgatc 120 aagatcatcg accgcaagaa ggcccccgca gacttcttgg agaaattcct tccccgggaa 180 attgagattc tggccatgtt aaaccactgc tccatcatta agacctacga gatctttgag 240 acatcacatg gcaaggtcta catcgtcatg gagctcgcag tccagggcga cctcctcgag 300 ttaatcaaaa cccggggagc cctgcatgag gacgaagctc gcaagaagtt ccaccagctt 360 tccttggcca tcaagtactg ccacgacctg gacgtcgtcc accgggacct caagtgtgac 420 aaccttctcc ttgacaagga cttcaacatc aagctgtccg acttcagctt ctccaagcgc 480 tgcctgcggg atgacagtgg tcgaatggcc ttaagcaaga ccttctgtgg gtcaccagcg 540 tatgcggccc cagaggtgct gcagggcatt ccctaccagc ccaaggtgta cgacatctgg 600 agcctaggcg tgatcctcta catcatggtc tgcggctcca tgccctacga cgactccaac 660 atcaagaaga tgctgcgtat ccagaaggag caccgcgtca acttcccacg ctccaagcac 720 ctgacaggcg agtgcaagga cctcatctac cacatgctgc ag.cccgacgt caaccggcgg 780 ctccacatcg acgagatcct cagccactgc tggatgcagc ccaaggcacg gggatctccc 840 tctgtggcca tcaacaagga gggggagagt tcccggggaa ctgaaccctt gtggaccccc 900 gaacctggct ctgacaagaa gtctgccacc aagctggagc ctgagggaga ggcacagccc 960 caggcacagc ctgagacaaa acccgagggg acagcaatgc aaatgtccag gcagtcggag 1020 atcctgggtt tccccagcaa gccgtcgact atggagacag aggaagggcc cccccaacag 1080 cctccagaga cgcgggccca gtga 1104 <210> 17 <211> 3939 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2902460CB1 <400> 17 ccgcagtgtg ctggaaaggc agctgcggca gtagcgtgag cagcccaagt tgggctggtc 60 gcctgcgagg ggaccggcag caggtggtgg cagccggtac cctctccccg ccaggccgga 120 ggaggccaag aggaagctgc ggatcttgca gcgcgagttg cagaacgtgc aggtgaacca 180 gaaagtgggc atgtttgagg cgcacatcca ggcacagagc tccgccattc aagcgccccg 240 cagcccgcgt ttgggcaggg ctcgctcgcc ctccccgtgc cccttccgca gcagcagtca 300 gccccctgga agggtcctgg ttcagggcgc ccggagcgag gaacggagga caaagtcctg 360 gggggagcaa tgtccagaga cttcaggaac cgactccggg aggaaaggag ggcccagcct 420 atgctcctcg caggtgaaga aaggaatgcc acctcttccc ggccgggctg cccctacagg 480 atcagaggct cagggtccat ccgcttttgt aaggatggag aagggtatcc ctgccagtcc 540 ccgctgtggc tcacccacag ctatggaaat tgacaaaagg ggctctccta ccccgggaac 600 tcggagctgc ctagctccct cattggggct gttcggagct agcttaacga tggccacgga 660 agtggcagcg agagttacat ccactgggcc acaccgtcca caggatcttg ccctcactga 720 gccgtctggg agagcccgtg agcttgagga cctgcagccc ccagaggccc tggtggagag 780 gcaggggcag tttctgggca gtgagacaag cccagcccca gaaaggggcg ggccccgcga 840 tggagaaccc cctgggaaga tggggaaagg atatctgccc tgtggcatgc cgggctctgg 900 ggagcctgaa gtgggcaaaa ggccagagga gacgactgtg agcgtgcaaa gcgcagagtc 960 ctctgatgcc ctgagctggt ccaggctgcc cagggccctg gcctccgtag gccctgagga 1020 ggcccgaagt ggggcccccg tgggcggggg gcgttggcag ctctccgaca gagtggaggg 1080 agggtcccca acgctgggct tgcttggggg cagcccctca gcacagccgg ggaccgggaa 1140 tgtggaggcg ggaattcctt ctggcagaat gctggagcct ttgccctgtt gggacgctgc 1200 gaaagatctg aaagaacctc agtgccctcc tggggacagg gtgggtgtgc agcctgggaa 1260 ctccagggtt tggcagggca ccatggagaa agccggtttg gcttggacgc gtggcacagg 1320 ggtgcaatca gaggggactt gggaaagcca gcggcaggac agtgatgccc tcccaagtcc 1380 ggagctgcta ccccaagatc aggacaagcc tttcctgagg aaggcctgca gccccagcaa 1440 catacctgct gtcatcatta cagacatggg cacccaggag gatggggcct tggaggagac 1500 gcagggaagc cctcggggca acctgcccct gaggaaactg tcctcttcct cggcctcctc 1560 cacgggcttc tcctcatcct acgaagactc agaggaggac atctccagtg accctgagcg 1620 caccctggac cccaactcag ccttcctgca taccctggac cagcagaaac ctagagtgag 1680 caaatcatgg aggaagataa aaaacatggt gcactggtct cccttcgtca tgtccttcaa 1740 gaagaagtac ccctggatcc agctggcagg acacgcaggg agtttcaagg cagctgccaa 1800 tggcaggatc ctgaagaagc actgtgagtc agagcagcgc tgcctggacc ggctgatggt 1860 ggatgtgctg aggcccttcg tacctgccta ccatggggat gtggtgaagg acggggagcg 1920 ctacaaccag atggacgacc tgctggccga cttcgactcg ccctgtgtga tggactgcaa 1980 gatgggaatc aggacctacc tggaggagga gctcacgaag gcccggaaga agcccagcct 2040 gcggaaggac atgtaccaga agatgatcga ggtggacccc gaggccccca ccgaggagga 2100 aaaagcacag cgggctgtga ccaagccacg gtacatgcag tggcgggaga ccatcagctc 2160 cacggccacc ctggggttca ggatcgaggg aatcaagaaa gaagacggca ccgtgaaccg 2220 ggacttcaag aagaccaaaa cgagggagca ggtcaccgag gccttcagag agttcactaa 2280 aggaaaccat aacatcctga tcgcctatcg ggaccggctg aaggccattc gaaccactct 2340 agaagtttct cccttcttca agtgccacga ggtcattggc agctccctcc tcttcatcca 2400 cgacaagaag gaacaggcca aagtgtggat gatcgacttt gggaaaacca cgcccctgcc 2460 tgagggccag accctgcagc atgacgtccc ctggcaggag gggaaccggg aggatggcta 2520 cctctcgggg ctcaataacc tcgtcgacat cctgaccgag atgtcccagg atgccccact 2580 cgcctgagct gcccacgccc tccctggccc ccgcctgggc ctcctttcct cctcctgtgc 2640 ttcctttctc gttcctaact tttccttcac ttacacctga ctgaccctcc tgaactgcac 2700 tacaagacac tttgtagaag aggagatgag agtttctagt cattttccta acttcagggc 2760 ttggaggtgg tgtttgcact gctttttgta gagagggtca cctactagaa gagaaatgcc 2820 cagtcttaga ggtgggtcag gtgtagagct ggagggggtc cctggctgct gaggggaccc 2880 taccagatga gccctgcctc tgggagcccc ctaggaagca ccagcctgga cctaccacct 2940 gcggaggcct gctgccccct ggcggccagt gctgttagag tgctgccaag cacagcctta 3000 tttctgccgg ggcctcccca ccggagagcc cagggggccg gccgggttcc tggtccctgg 3060 ctgggagcag ggctttctgg tagttggggc acaaaaccat cggggaacca catgttgact 3120 gtgagcaaag tgtcttccga ttagcagcct cagggatgcc ctggtggcct ctccagggct 3180 gctcaggcaa ggccccccac ccatctggta tggaaacctg ccggctccag gccagaccca 3240 ggagccaaga gaaggctgaa gccagcttgg ctgtgttctc tgatctaggc cttcccagag 3300 gaggcgagca gaagctgtgc cacttggaat tgcaacccat gagttcagaa ggcacactct 3360 gccatgctga gctccaaggg tgctaccagg ggaagatggg atctatagag tctctgggcc 3420 ctggccccag ggaggagcac atttttcttg accctcacct acctggtgct agttggtcaa 3480 ccctgcctgc atacatgggc tcctgtcatg gggcccagag tcccttgcag atatagaaat 3540 aggggaggag ctcaggtctg cgccaggcag gaagaaggca ggcttctggc ttccagaggt 3600 gccgcggtgg cctcctggca tcatttgtta ttgcctctga aacaagcctt actgcctgga 3660 gggcttagat tcctgcttct ccaatgtagt gtgggtatct tgtagggtat gtggtggatg 3720 ccagggcgtg ctccaggcac ctcttcctga agtctctgca tttggagatt cgtggagaac 3780 ctatttaagc ccaattttaa ctgaaagcca gtgagtctga tatggaaggg aatgtaaaat 3840 ttgcctgact tcttaagaac aaaaccccca gctctgtgcc ccatgctcct tggggcttgc 3900 cacccactcc tttgctgtca gaggtacagg agctgggag 3939 <210> 18 <211> 1381 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 6383934CB1 <400> 18 atgaggacaa tgcctgctgg cccacatgac ggggggatgt agacggcagc ggcgccagtc 60 gctcctggca ccatggacga tgccacagtc ctaaggaaga agggttacat cgtaggcatc 120 aatcttggca agggttccta cgcaaaagtc aaatctgcct actctgagcg cctcaagttc 180 aatgtggctg tcaagatcat cgaccgcaag aaaacaccta ctgactttgt ggagagattc 240 cttcctcggg agatggacat cctggcaact gtcaaccacg gctccatcat caagacttac 300 gagatctttg agacctctga cggacggatc tacatcatca tggagcttgg cgtccagggc 360 gacctcctcg agttcatcaa gtgccaggga gccctgcatg aggacgtggc acgcaagatg 420 ttccgacagc tctcctccgc cgtcaagtac tgccacgacc tggacatcgt ccaccgggac 480 ctcaagtgcg agaaccttct cctcgacaag gacttcaaca tcaagctgtc tgactttggc 540 ttctccaagc gctgcctgcg ggacagcaat gggcgcatca tcctcagcaa gaccttctgc 600 gggtcggcag catatgcagc ccccgaggtg ctgcagagca tcccctacca gcccaaggtg 660 tatgacatct ggagcctagg cgtgatcctc tacatcatgg tctgcggctc catgccctac 720 gacgactccg acatcaagaa gatgctgcgt atccagaagg agcaccgcgt caacttccca 780 cgctccaagc acctgacctg cgagtgcaag gacctcatct accacatgct gcagcccgac 840 gtcagccagc ggctccacat cgatgagatc ctcagccact cgtggctgca gccccccaag 900 cccaaagcca cgtcttctgc ctccttcaag agggaggggg agggcaagta ccgcgctgag 960 tgcaaactgg acaccaagac aggcttgagg cccgaccacc ggcccgacca caagcttgga 1020 gccaaaaccc agcaccggct gctggtggtg cccgagaacg agaacaggat ggaggacagg 1080 ctggccgaga cctccagggc caaagaccat cacatctccg gagctgaggt ggggaaagca 1140 agcacctagc atgacaatgg ccccgttgtg tgtggtgggg gtcggggttg gggggcatgg 1200 tgcagtcggc cttcacgtaa actaagtagg caggtaggat ctgaagaagg cacaggtgca 1260 agtaaaattc gtcaattaaa ccactatttt gattacgttc cattagcttt cttccactta 1320 gcagcaaaga cgttccttac tgaccaccaa ataaaccaca gggtgtgtgc aagcatcaaa 1380 a 1381 <210> 19 <211> 3904 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 3210906CB1 <400> 19 tattcggggt tcagacccca caatcagaaa tccggaattc ggcagctgtc gccctcgacg 60 agggggagga ctggaccgcg aggtcagatt aggttgtcac cccctcccct ccaggggagg 120 cttcccgggc ccgcccctca ggaagggcga aagccgagga agaggtggca aggggaaagg 180 tctccttgcc cctctccctg acttggcaga gccgctggag gaccccaggc ggaagcggag 240 gcgctggggc accatagtga,cccctaccag gccaggcccc actctcaggg cccccagggg 300 ccaccatgcc agctgggggc cgggccggga gcctgaagga cccagatgtg gctgagctct 360 tcttcaagga tgacccagaa aagctcttct ctgacctccg ggaaattggc catggcagct 420 ttggagccgt atactttgcc cgggatgtcc ggaatagtga ggtggtggcc atcaagaaga 480 tgtcctacag tgggaagcag tccaatgaga aatggcaaga catcatcaag gaggtgcggt 540 tcttacagaa gctccggcat cccaacacca ttcagtaccg gggctgttac ctgagggagc 600 acacggcttg gctggtaatg gagtattgcc tgggctcaac ttctgacctt ctagaagtgc 660 acaagaaacc ccttcaggag gtagagatcg cagctgtgac ccacggggcg cttcagggcc 720 tggcatatct gcactcccac aacatgatcc atagggatgt gaaggctgga aacatcctgc 780 tgtcagagcc agggttagtg aagctagggg actttggttc tgcgtccatc atggcacctg 840 ccaactcctt cgtgggcacc ccatactgga tggcacccga ggtgatcctg gccatggatg 900 aggggcagta cgatggcaaa gtggacgtct ggtccttggg gataacctgc atcgagctgg 960 ctgaacggaa accaccgctc tttaacatga atgcgatgag tgccttatac cacattgcac 1020 agaacgaatc ccccgtgctc cagtcaggac actggtctga gtacttccgg aattttgtcg 1080 actcctgtct tcagaaaatc cctcaagaca gaccaacctc agaggttctc ctgaagcacc 1140 gctttgtgct ccgggagcgg ccacccacag tcatcatgga cctgatccag aggaccaagg 1200 atgccgtgcg ggagctggac agcctgcagt accgcaagat gaagaagatc ctgttccaag 1260 aggcacccaa cggccctggt gccgaggccc cagaggagga agaggaggcc gagccctaca 1320 tgcacctggc cgggactctg accagcctcg agagtagcca ctcagtgccc agcatgtcca 1380 tcagcgcctc cagccagagc agctccgtca acagcctagc agatgcctca gacaacgagg 1440 aagaggagga ggaggaggag gaagaggagg aggaggaaga aggccctgaa gcccgggaga 1500 tggccatgat gcaggagggg gagcacacag tcacctctca cagctccatt atccaccggc 1560 tgccgggctc tgacaaccta tatgatgacc cctaccagcc agagataacc cccagccctc 1620 tccagccgcc tgcagcccca gctcccactt ccaccacctc ttccgcccgc cgccgggcct 1680 actgccgtaa ccgagaccac tttgccacca tccgaaccgc ctccctggtc agccgtcaga 1740 tccaggagca tgagcaggac tctgcgctgc gggagcagct gagcggctat aagcggatgc 1800 gacgacagca ccagaagcag ctgctggccc tggagtcacg gctgaggggt gaacgggagg 1860 agcacagtgc acggctgcag cgggagcttg aggcgcagcg ggctggcttt ggggcagagg 1920 cagaaaagct ggcccggcgg caccaggcca taggtgagaa ggaggcacga gctgcccagg 1980 ccgaggagcg gaagttccag cagcacatcc ttgggcagca gaagaaggag ctggctgccc 2040 tgctggaggc acagaagcgg acctacaaac ttcgcaagga acagctgaag gaggagctcc 2100.
aggagaaccc cagcactccc aagcgggaga aggccgagtg gctgctgcgg cagaaggagc 2160 agctccagca gtgccaggcg gaggaggaag cagggctgct gcggcggcag cgccagtact 2220 ttgagctgca gtgtcgccag tacaagcgca agatgttgct ggctcggcac agcctggacc 2280 aggacctgct gcgggaggac ctgaacaaga agcagaccca gaaggacttg gagtgtgcac 2340 tgctgcttcg gcagcacgag gccacgcggg agctggagct gcggcagctc caggccgtgc 2400 agcgcacgcg ggctgagctc acccgcctgc agcaccagac ggagctgggc aaccagctgg 2460 agtacaacaa gcggcgtgag caagagttgc ggcagaagca tgcggcccag gttcgccagc 2520 agcccaagag cctcaaatct aaggagctgc agatcaagaa gcagttccag gagacgtgta 2580 agatccagac tcggcagtac aaggctctgc gagcacactt gctggagacc acgcccaaag 2640 ctcagcacaa gagcctcctt aagcggctca aggaagagca gacccgcaag ctggcgatct 2700 tggcggagca gtatgaccag tccatctcag agatgctcag ctcacaggcg ctgcggcttg 2760 atgagaccca ggaggcagag ttccaggccc ttcggcagca gcttcaacag gagctggagc 2820 tgctcaacgc ttaccagagc aagatcaaga tccgcacaga gagccagcac gagagggagc 2880 tgcgggagct ggagcagagg gtcgcgctgc ggcgggcact gctggagcag cgggtggaag 2940 aggagctgct ggccctgcag acaggacgct ccgagcgaat ccgcagtctg cttgagcggc 3000 aggcccgtga gatcgaggcc ttcgatgcgg aaagcatgag gctgggcttc tccagcatgg 3060 ctctgggggg catcccggct gaagctgctg cccagggcta tcctgctcca ccccctgccc 3120 cagcctggcc ctcccgtccc gttccccgtt ctggggcaca ctggagccat ggccctcctc 3180 caccaggcat gccccctcca gcctggcgtc agccgtctct gctggctccc ccaggccccc 3240 caaactggct ggggcccccc acacaaagtg ggacaccccg tggcggagcc ctgctgctgc 3300 taagaaacag cccccagccc ctgcggcggg cagcctcggg gggcagtggc agtgagaatg 3360 tgggcccccc tgctgccgcg gtgcccgggc ccctgagccg cagcaccagt gtcgcttccc 3420 acatcctcaa tggttcttcc cacttctatt cctgaggtgc agcggggagg agcagatgag 3480 ctgggcaggg caggggtggg tggagcctga ccctggaggg cactgagctg gaggcccctg 3540 caagggtagg ggacaagatg taggctccag ctcccctcag acctcctcat ctcatgagct 3600 tcttggggct ggccagtggc ccagggccag cttggcgata gatgcctcaa ggctgcctgg 3660 gagccccgcc tccctaccat ggtgccaggg gtctccctcc gccacctagg aaaggaggga 3720 gatgtgcgtg tcaaatattc atctagtccc ctgggggagg ggaagggtgg gtctagacat 3780 actatattca gagaactata ctaccctcac agtgaggccc tcagacctgc cacagggcag 3840 agcaggtctg gggcctgagg cagggagaat gagaggccac ttactggcag gaaggatcag 3900 gatg 3904 <210> 20 <211> 1987 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3339024CB1 <400> 20 gaagaaccct gaggaacaga cttacctcag caaccctggc acctccaacc cgacacatgc 60 tactgctgct gctactgctg ccacccctgc tctgtgggag agtgggggct aaggaacaga 120 aggattacct gctgacaatg tagaagtccg tgacggtgca ggagggcctg tgtgtctctg 180 tgctttgctc cttctcctac ccccaaaatg gctggactgc ctccgatcca gttcatggct 240 actggttccg ggcaggggac catgtaagcc ggaacattcc agtggccaca aacaacccag 300 ctcgagcagt gcaggaggag actcgggacc gattccacct ccttggggac ccacagaaca 360 aggattgtac cctgagcatc agagacacca gagagagtga tgcagggaca tacgtctttt 420 gtgtagagag aggaaatatg aaatggaatt ataaatatga ccagctctct gtgaatgtga 480 cagcgtccca ggacctactg tcaagataca ggctggaggt gccagagtcg gtgactgtgc 540 aggagggtct gtgtgtctct gtgccctgca gtgtccttta cccccattac aactggactg 600 cctctagccc tgtttatgga tcctggttca aggaaggggc cgatatacca tgggatattc 660 cagtggccac aaacacccca agtggaaaag tgcaagagga tacccacggt cgattcctcc 720 tccttgggga cccacagacc aacaactgct ccctgagcat cagagatgcc aggaaggggg 780 attcagggaa gtactacttc caggtggaga gaggaagcag gaaatggaac tacatatatg 840 acaagctctc tgtgcatgtg acagccctga ctcacatgcc caccttctcc atcccgggga 900 ccctggagtc tggccacccc aggaacctga cctgctctgt gccctgggcc tgtgaacagg 960 ggacgccccc cacgatcacc tggatggggg cctccgtgtc ctccctggac cccactatca 1020 ctcgctcctc gatgctcagc ctcatcccac agccccagga ccatggcacc agcctcacct 1080 gtcaggtgac cttgcctggg gccggcgtga ccatgaccag ggctgtccga ctcaacatat 1140 cctatcctcc tcagaacttg accatgactg tcttccaagg agatggcaca gcatccacaa 1200 ccttgaggaa tggctcggcc ctttcagtcc tggagggcca gtccctgcac cttgtctgtg 1260 ctgtcgacag caatccccct gccaggctga gctggacctg ggggagcctg accctgagcc 1320 cctcacagtc ctcgaacctt ggggtgctgg agctgcctcg agtgcatgtg aaggatgaag 1380 gggaattcac ctgccgagct cagaaccctc taggctccca gcacatttcc ctgagcctct 1440 ccctgcaaaa cgagtacaca ggcaaaatga ggcctatatc aggagtgacg ctaggggcat 1500 tcgggggagc tggagccaca gccctggtct tcctgtactt ctgcatcatc ttcgttgtag 1560 tgaggtcctg caggaagaaa tcggcaaggc cagcagtggg cgtgggggat acaggcatgg 1620 aggacgcaaa cgctgtctgg ggctcagcct ctcagggacc cctgattgaa tccccggcag 1680 atgacagccc cccacaccat gctccgccag ccctggccac cccctcccca gaggaaggag 1740 agatccagta tgcatccctc agcttccaca aagcgaggcc tcagtaccca caggaacagg 1800 aggccatcgg ctatgagtac tccgagatca acatccccaa gtgagaaact gcagagactc 1860 aggcctgttt gagggctcac gacccctcca gcaaagaagc ccgagactga ttcctttaga 1920 attaaaagcc ctccatgctg tgcaacgggg gatccactag ttaagagcgg cgcacccgcg 1980 tgcccct 1987 <210> 21 <211> 3925 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4436929CB1 <400> 21 ccgtcctcga ggcgaggaga gtaccgggcc ggcccggctg ccgcgcgagg agcgcggtcg 60 gcggcctggt ctgcggctga gatacacaga gcgacagaga catttattgt tatttgtttt 120 ttggtggcaa aaagggaaaa tggcgaacga ctcccctgca aaaagtctgg tggacatcga 180 cctctcctcc ctgcgggatc ctgctgggat ttttgagctg gtggaagtgg ttggaaatgg 240 cacctatgga caagtctata agggtcgaca tgttaaaacg ggtcagttgg cagccatcaa 300 agttatggat gtcactgagg atgaagagga agaaatcaaa ctggagataa atatgctaaa 360 gaaatactct catcacagaa acattgcaac atattatggt gctttcatca aaaagagccc 420 tccaggacat gatgaccaac tctggcttgt tatggagttc tgtggggctg ggtccattac 480 agaccttgtg aagaacacca aagggaacac actcaaagaa gactggatcg cttacatctc 540 cagagaaatc ctgaggggac tggcacatct tcacattcat catgtgattc accgggatat 600 caagggccag aatgtgttgc tgactgagaa tgcagaggtg aaacttgttg actttggtgt 660 gagtgctcag ctggacagga ctgtggggcg gagaaatacg ttcataggca ctccctactg 720 gatggctcct gaggtcatcg cctgtgatga gaacccagat gccacctatg attacagaag 780 tgatctttgg tcttgtggca ttacagccat tgagatggca gaaggtgctc cccctctctg 840 tgacatgcat ccaatgagag cactgtttct cattcccaga aaccctcctc cccggctgaa 900 gtcaaaaaaa tggtcgaaga agttttttag ttttatagaa gggtgcctgg tgaagaatta 960 catgcagcgg ccc,tctacag agcagctttt gaaacatcct tttataaggg atcagccaaa 1020 tgaaaggcaa gttagaatcc agcttaagga tcatatagat cgtaccagga agaagagagg 1080 cgagaaagat gaaactgagt atgagtacag tgggagtgag gaagaagagg aggaagtgcc 1140 tgaacaggaa ggagagccaa gttccattgt gaacgtgcct ggtgagtcta ctcttcgccg 1200 agatttcctg agactgcagc aggagaacaa ggaacgttcc gaggctcttc ggagacaaca 1260 gttactacag gagcaacagc tccgggagca ggaagaatat aaaaggcaac tgctggcaga 1320 gagacagaag cggattgagc agcagaaaga acagaggcga cggctagaag agcaacaaag 1380 gagagagcgg gaagctagaa ggcagcagga acgtgaacag cgaaggagag aacaagaaga 1440 aaagaggcgt ctagaggagt tggagagaag gcgcaaagaa gaagaggaga ggagacgggc 1500 agaagaagaa aagaggagag ttgaaagaga acaggagtat atcaggcgac agctagaaga 1560 ggagcagcgg cacttggaag tccttcagca gcagctgctc caggagcagg ccatgttact 1620 gcatgaccat aggaggccgc acccgcagca ctcgcagcag ccgccaccac cgcagcagga 1680 aaggagcaag ccaagcttcc atgctcccga gcccaaagcc cactacgagc ctgctgaccg 1740 agcgcgagag gtggaagata gatttaggaa aactaaccac agctcccctg aagcccagtc 1800 taagcagaca ggcagagtat tggagccacc agtgccttcc cgatcagagt ctttttccaa 1860 tggcaactcc gagtctgtgc atcccgccct gcagagacca gcggagccac aggttcctgt 1920 gagaacaaca tctcgctccc ctgttctgtc ccgtcgagat tccccactgc agggcagtgg 1980 gcagcagaat agccaggcag gacagagaaa ctccaccagc agtattgagc ccaggcttct 2040 gtgggagaga gtggagaagc tggtgcccag acctggcagt ggcagctcct cagggtccag 2100 caactcagga tcccagcccg ggtctcaccc tgggtctcag agtggctccg gggaacgctt 2160 cagagtgaga tcatcatcca agtctgaagg ctctccatct cagcgcctgg aaaatgcagt 2220 gaaaaaacct gaagataaaa aggaagtttt cagacccctc aagcctgctg gcgaagtgga 2280 tctgaccgca ctggccaaag agcttcgagc agtggaagat gtacggccac ctcacaaagt 2340 aacggactac tcctcatcca gtgaggagtc ggggacgacg gatgaggagg acgacgatgt 2400 ggagcaggaa ggggctgacg agtccacctc aggaccagag gacaccagag cagcgtcatc 2460 tctgaatttg agcaatggtg aaacggaatc tgtgaaaacc atgattgtcc atgatgatgt 2520 agaaagtgag ccggccatga ccccatccaa ggagggcact ctaatcgtcc gccagactca 2580 gtccgctagt agcacactcc agaaacacaa atcttcctcc tcctttacac cttttataga 2640 ccccagatta ctacagattt ctccatctag cggaacaaca gtgacatctg tggtgggatt 2700 ttcctgtgat gggatgagac cagaagccat aaggcaagat cctacccgga aaggctcagt 2760 ggtcaatgtg aatcctacca acactaggcc acagagtgac accccggaga ttcgtaaata 2820 caagaagagg tttaactctg agattctgtg tgctgcctta tggggagtga atttgctagt 2880 gggtacagag agtggcctga tgctgctgga cagaagtggc caagggaagg tctatcctct 2940 tatcaaccga agacgatttc aacaaatgga cgtacttgag ggcttgaatg tcttggtgac 3000 aatatctggc aaaaaggata agttacgtgt ctactatttg tcctggttaa gaaataaaat 3060 acttcacaat gatccagaag ttgagaagaa gcagggatgg acaaccgtag gggatttgga 3120 aggatgtgta cattataaag ttgtaaaata tgaaagaatc aaatttctgg tgattgcttt 3180 gaagagttct gtggaagtct atgcgtgggc accaaagcca tatcacaaat ttatggcctt 3240 taagtcattt ggagaattgg tacataagcc attactggtg gatctcactg ttgaggaagg 3300 ccagaggttg aaagtgatct atggatcctg tgctggattc catgctgttg atgtggattc 3360 aggatcagtc tatgacattt atctaccaac acatatccag tgtagcatca aaccccatgc 3420 aatcatcatc ctccccaata cagatggaat ggagcttctg gtgtgctatg aagatgaggg 3480 ggtttatgta aacacatatg gaaggatcac caaggatgta gttctacagt ggggagagat 3540 gcctacatca gtagcatata ttcgatccaa tcagacaatg ggctggggag agaaggccat 3600 agagatccga tctgtggaaa ctggtcactt ggatggtgtg ttcatgcaca aaagggctca 3660 aagactaaaa ttcttgtgtg aacgcaatga caaggtgttc tttgcctctg ttcggtctgg 3720 tggcagcagt caggtttatt tcatgacctt aggcaggact tctcttctga gctggtagaa 3780 gcagtgtgat ccagggatta ctggcctcca gagtcttcaa gatcctgaga acttggaatt 3840 ccttgtaact ggagctcgga gctgcaccga gggcaaccag gacagctgtg tgtgcagacc 3900 tcatgtgttg ggttctctcc cctcc 3925 <210> 22 <211> 1210 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 5046791CB1 <400> 22 ttacaggtca tctaccccta taccccacaa aatgacgatg agctggagct ggtccccggg 60 gacttcatct tcatgtctcc aatggagcag accagcacca gcgagggttg gatctatggc 120 acgtccttaa ccaccggctg ctctggactc ctcctgagaa ttacattacc aaggctgatg 180 aatgcagcac ctggatattt catggttctt attcaatctt aaatacatcg tcatccaact 240 ctctcacgtt tggggatgga gtattggaga ggcggcctta tgaggaccag gggctcgggg 300 agacgactcc tcttactatc atctgccagc ccatgcagcc gctgagggtc aacagccagc 360 ccggccccca, gaagcgatgc ctttttgtgt gtcggcatgg tgagaggatg,gatgttgtgt 420 ttgggaagta ctggctgtcc cagtgcttcg atgccaaagg ccgctacata cgcaccaacc 480 tgaacatgcc tcatagttta cctcagcgga gtggtggttt ccgagattac gagaaagatg 540 ctcccatcac tgtgtttgga tgcatgcaag caagactagt gggtgaagcc ttattagaga 600 gcaataccat tatcgatcat gtctattgct ccccgtccct tcgctgcgtt cagactgcac 660 acaatatctt gaaaggttta caacaagaaa atcacttgaa gatccgtgta gagcccggct 720 tatttgagtg gacaaaatgg gttgctggga gcacattacc tgcatggata cctccatcag 780 agttagctgc agccaacctg agtgttgata caacctacag acctcacatt ccaatcagca 840 aattagttgt ttcagaatcc tatgatactt atatcagtag aagtttccaa gtaacaaaag 900 aaataataag tgaatgtaaa agtaaaggaa ataacatcct gattgtggcc cacgcatctt 960 cccttgaagc gtgtacctgc caacttcagg gcctgtcacc tcagaactcc aaggacttcg 1020 tacaaatggt ccgaaagatc ccatatctgg gattttgttc ctgtgaagaa ttaggagaaa 1080 ctggaatatg gcagctgaca gatccaccaa tccttcctct tacccatgga ccaactgggg 1140 gcttcaactg gagagagacc ttgcttcaag aataaaccat accagtgaac aagaaggaaa 1200 aaaaaaaaaa 1210 <210> 23 <211> 1521 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1416174CB1 <400> 23 ggcacggtgc tgggcctcgc cacactgcac acctaccgcg gacgcctctc ctacctcccc 60 gccactgtgg aacctgcctc gcccacccct gcccatagcc tgcctcgtgc caagtcggag 120 ctgaccctaa ccccagaccc agccccgccc atggcccact cacccctgca tcgttctgtg 180 tctgacctgc ctcttcccct gccccagcct gccctggcct ctcctggctc gccagaaccc 240 ctgcccatcc tgtccctcaa cggtgggggc ccagagctgg ctggggactg gggtggggct 300 ggggatgctc cgctgtcccc ggacccactg ctgtcttcac ctcctggctc tcccaaggca 360 gctctacact cacccgtctc cgaagggccc ccgtaattcc cccatcctct gggctcccac 420 ttcccacccc tgatgcccgg gtaggggcct ccacctgcgg cccgcccgac cacctgctgc 480 ctccgctggg caccccgctg cccccagact gggtgacgct ggagggggac tttgtgctca 540 tgttggccat ctcgcccagc cacctaggcg ctgacctggt ggcagctccg catgcgcgct 600 tcgacgacgg cctggtgcac ctgtgctggg tgcgtacggg catctcgcgg gctgcgctgc 660 tgcgcctttt cttggccatg gagcgtggta gccacttcag cctgggctgt ccgcagctgg 720 gctacgccgc ggcccgtgcc ttccgcctag agccgctcac accacgcggc gtgctcacag 780 tggacgggga gcaggtggag tatgggccgc tacaggcaca gatgcaccct ggcatcggta 840 cactgctcac tgggcctcct ggctgcccgg ggcgggagcc ctgaaactaa acaagcttgg 900 tacccgccgg gggcggggcc tacattccaa tggggcggag ctgagctagg gggtgtggcc 960 tggctgctag agttgtggtg gcaggggccc tggccccgtc tcaggattgc gctcgctttc 1020 atgggaccag acgtgatgct ggaaggtggg cgtcgtcacg gttaaagaga aatgggctcg 1080 tcccgagggt agtgcctgat caatgagggc ggggcctggc gtctgatctg gggccgccct 1140 tacggggcag ggctcagtcc tgacgcttgc cacctgctcc tacccggcca ggatggctga 1200 gggcggagtc tattttacgc gtcgcccaat gacaggacct ggaatgtact ggctggggta 1260 ggcctcagtg agtcggccgg tcagggcccg cagcctcgcc ccatccactc cggtgcctcc 1320 atttagctgg ccaatcagcc caggaggggc aggttccccg gggccggcgc taggatttgc 1380 actaatgttc ctctccccgc gggtgggggc ggggaaattc atatcccctg ttcgtctcat 1440 gcgcgtcctc cgtccccaat ctaaaaagca attgaaaagg tctatgcaat aaaggcagtc 1500 gcttcattcc tctcaaaaaa a 1521 <210> 24 <211> 1640 <212> DNA
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 3244919CB1 <400> 24 gcagcgccgc ggcgtccccg ggctcgccgc cccccggccg cgcgcgcccc gccggctccg 60 acgcgccctc ggccctgccg ccgcccgctg ctggccagcc ccgggcccgg gactcgggcg 120 atgtccgctc gcagccgcgc cccctgtttc agtggagcaa gtggaagaag aggatgggct 180 cgtccatgtc ggcggccacc gcgcggaggc cggtgtttga cgacaaggag gacgtgaact 240 tcgaccactt ccagatcctt cgggccattg ggaagggcag ctttggcaag gtgtgcattg 300 tgcagaagcg ggacacggag aagatgtacg ccatgaagta catgaacaag cagcagtgca 360 tcgagcgcga cgaggtccgc aacgtcttcc gggagctgga gatcctgcag gagatcgagc 420 acgtcttcct ggtgaacctc tggtactcct tccaggacga ggaggacatg ttcatggtcg 480 tggacctgct actgggcggg gacctgcgct accacctgca gcagaacgtg cagttctccg 540 aggacacggt gaggctgtac atctgcgaga tggcactggc tctggactac ctgcgcggcc 600 agcacatcat ccacagagat gtcaagcctg acaacattct cctggatgag agaggacatg 660 cacacctgac cgacttcaac attgccacca tcatcaagga cggggagcgg gcgacggcat 720 tagcaggcac caagccgtac atggctccgg agatcttcca ctcttttgtc aacggcggga 780 ccggctactc cttcgaggtg gactggtggt cggtgggggt gatggcctat gagctgctgc 840 gaggatggag gccctatgac atccactcca gcaacgccgt ggagtccctg gtgcagctgt 900 tcagcaccgt gagcgtccag tatgtcccca cgtggtccaa ggagatggtg gccttgctgc 960 ggaagctcct cactgtgaac cccgagcacc ggctctccag cctccaggac gtgcaggcag 1020 ccccggcgct ggccggcgtg ctgtgggacc acctgagcga gaagagggtg gagccgggct 1080 tcgtgcccaa caaaggccgt ctgcactgcg accccacctt tgagctggag gagatgatcc 1140 tggagtccag gcccctgcac aagaagaaga agcgcctggc caagaacaag tcccgggaca 1200 acagcaggga cagctcccag tccgccccac ggagcaagtc caagccatcc acccagaggc 1260 aagggagctg ggccttggca tcctcgggct tgggagaatg actatcttca agactgcctc 1320 gatgccatcc agcaagactt cgtgattttt aacagagaaa agctgaagag gagccaggac 1380 ctcccgaggg agcctctccc cgcccctgag tccagggatg ctgcggagcc tgtggaggac 1440 gaggcggaac gctccgccct gcccatgtgc ggccccattt gcccctcggc cgggagcggc 1500 taggccggga cgcccgtggt cctcacccct tgagctgctt tggagactcg gctgccagag 1560 ggagggccat gggccgaggc ctggcattca cgttcccacc cagcctggct ggcggtgccc 1620 acagtgcccc ggacacattt 1640

Claims (28)

What is claimed is:
1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12.
2. An isolated polypeptide of claim 1 selected from the group consisting of SEQ ID NO:1-12.
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 selected from the group consisting of SEQ ID
NO:13-24.
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 for 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. An isolated antibody which specifically binds to a polypeptide of claim 1.
11. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:

a) a polynucleotide sequence selected from the group consisting of SEQ ID
NO:13-24, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO:13-24, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d).
12. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 11.
13. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, 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.
14. A method of claim 13, wherein the probe comprises at least 60 contiguous nucleotides.
15. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, 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.
16. A composition comprising an effective amount of a polypeptide of claim 1 and a pharmaceutically acceptable excipient.
17. A composition of claim 16, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-12.
18. 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 16.
19. A method for 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.
20. A composition comprising an agonist compound identified by a method of claim 19 and a pharmaceutically acceptable excipient.
21. 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 20.
22. A method for 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.
23. A composition comprising an antagonist compound identified by a method of claim 22 and a pharmaceutically acceptable excipient.
24. 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 23.
25. A method of screening for a compound that specifically binds to the polypeptide of claim 1, said method comprising the steps of:

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.
26. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, said 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.
27. A method for 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.
28. 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 of claim 11 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 11 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.
CA002395102A 1999-12-23 2000-12-20 Human kinases Abandoned CA2395102A1 (en)

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US17206699P 1999-12-23 1999-12-23
US60/172,066 1999-12-23
US17610700P 2000-01-14 2000-01-14
US60/176,107 2000-01-14
US17773100P 2000-01-21 2000-01-21
US60/177,731 2000-01-21
US17857300P 2000-01-28 2000-01-28
US60/178,573 2000-01-28
PCT/US2000/035304 WO2001046397A2 (en) 1999-12-23 2000-12-20 Human kinases

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Families Citing this family (13)

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Publication number Priority date Publication date Assignee Title
US20040018199A1 (en) * 1999-10-15 2004-01-29 Raj Bandaru Compositions and methods of use for an ephrin receptor
WO2001096544A2 (en) * 2000-06-15 2001-12-20 Millennium Pharmaceuticals, Inc. 53070, a novel human protein kinase family member and uses thereof
EP1294896A2 (en) * 2000-04-25 2003-03-26 Millennium Pharmaceuticals, Inc. 13295 novel protein kinase molecules and uses therefor
EP1278866A2 (en) * 2000-05-05 2003-01-29 MERCK PATENT GmbH Serine-threonine kinase
US6482935B1 (en) 2000-05-24 2002-11-19 Applera Corporation Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof
US7094587B2 (en) 2000-06-27 2006-08-22 Millennium Pharmaceuticals, Inc. 16002 Molecules and uses therefor
US6555352B2 (en) 2000-08-31 2003-04-29 Applera Corporation Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof
AU2002220630A1 (en) * 2000-10-16 2002-04-29 Bayer Aktiengesellschaft Regulation of human serine-threonine protein kinase
DE60142984D1 (en) * 2000-11-09 2010-10-14 Univ Virginia HUMAN SPECIMENS SERIN / THREONINE KINASE
WO2002059324A2 (en) * 2001-01-12 2002-08-01 Pioneer Hi-Bred International, Inc. Novel inositol polyphosphate kinase genes and uses thereof
US6686176B2 (en) * 2001-01-23 2004-02-03 Applera Corporation Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof
JP2003259878A (en) * 2002-03-11 2003-09-16 Toyota Central Res & Dev Lab Inc Dna encoding lactate dehydrogenase and utilization of the same
CA2580478A1 (en) * 2004-01-28 2005-08-11 Exelixis, Inc. Man2as as modifiers of the igfr pathway and methods of use

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