WO2004042022A2 - Kinases et phosphatases - Google Patents

Kinases et phosphatases Download PDF

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WO2004042022A2
WO2004042022A2 PCT/US2003/034809 US0334809W WO2004042022A2 WO 2004042022 A2 WO2004042022 A2 WO 2004042022A2 US 0334809 W US0334809 W US 0334809W WO 2004042022 A2 WO2004042022 A2 WO 2004042022A2
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seq
polynucleotide
polypeptide
amino acid
acid sequence
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WO2004042022A3 (fr
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April J. A. Hafalia
Soo-Yeun Lee
Jagi Murage
Anita Swarnakar
Narinder K. Chawla
Reena Khare
Vicki S. Elliott
Uyen K. Tran
Jayalaxmi Ramkumar
Rajagopal Gururajan
Mariah R. Baughn
Kimberly J. Gietzen
Yonghong G. Yang
David Chien
Jonathan T. Wang
Kristin D. Favero
Shanya D. Becha
Thomas W. Richardson
Pei Jin
Phillip R. Hawkins
Henry Yue
Ernestine A. Lee
Joseph P. Marquis
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Incyte Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • the invention relates to novel nucleic acids, kinases and phosphatases encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of cardiovascular diseases, immune system disorders, neurological disorders, disorders affecting growth and development, lipid disorders, cell proliferative disorders, and cancers.
  • the invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and kinases and phosphatases.
  • kinases Cascades of kinases occur, as well as kinases sensitive to second messenger molecules. This system allows for the amplification of weak signals (low abundance growth factor molecules, for example), as well as the synthesis of many weak signals into an aH-or-nothing response. Phosphatases, then, are essential in determining the extent of phosphorylation in the cell and, together with kinases, regulate key cellular processes such as metabolic enzyme activity, proliferation, cell growth and differentiation, cell adhesion, and cell cycle progression. KINASES
  • 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.
  • protein kinases There are two classes of protein kinases. One class, protein tyrosine kinases (PTKs), phosphorylates tyrosine residues, and the other class, protein serine/threonine kinases (STKs), phosphorylates serine and threonine residues. Some PTKs and STKs possess structural characteristics of both families and have dual specificity for both tyrosine and serme/threonine residues. Almost all kinases contain a conserved 250-300 amino acid catalytic domain containing specific residues and sequence motifs characteristic of the kinase family. The protein kinase catalytic domain can be further divided into 11 subdomains.
  • PTKs protein tyrosine kinases
  • STKs protein serine/threonine kinases
  • 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.
  • 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
  • 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.
  • Protein Tyrosine Kinases Protein tyrosine kinases (PTKs) maybe 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.
  • PTKs Protein tyrosine kinases
  • 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.
  • 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 N.K. Tonks (1992) Annu. Rev. Cell Biol. 8:463-493). Regulation of PTK activity may therefore be an important strategy in controlling some types of cancer. Protein Serine/Threonine Kinases
  • STKs Protein serine/threorjine kinases
  • 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.
  • MEK MAP ERK kinase
  • 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.
  • ERK Activation of ERK is normally transient, and cells possess dual specificity phosphatases that are responsible for its down- regulation. Also, numerous studies have shown that elevated ERK activity is associated with some cancers.
  • Other STKs include the second messenger dependent protein kinases such as the cyclic-AMP dependent protein kinases (PKA), calcium-calmodulin (CaM) dependent protein kinases, and the mitogen-activated protein kinases (MAP); the cyclin-dependent protein kinases; checkpoint and cell cycle kinases; Numb-associated kinase (Nak); human Fused (bFu); proliferation-related kinases; 5 -AMP-activated protein kinases; and kinases involved in apoptosis.
  • PKA cyclic-AMP dependent protein kinases
  • CaM calcium-calmodulin dependent protein kinases
  • MAP mitogen-activated protein kinases
  • the cyclin-dependent protein kinases
  • the second messenger dependent protein kinases primarily mediate the effects of second messengers such as cychc AMP (cAMP), cychc GMP, inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cychc ADP ribose, arachidonic acid, diacylglycerol and calcium-calmodulin.
  • cAMP cychc AMP
  • GMP inositol triphosphate
  • phosphatidylinositol 3,4,5-triphosphate
  • cychc ADP ribose arachidonic acid
  • diacylglycerol 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, KJ. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York NY, pp. 416-431, 1887).
  • the casein kinase I (CKI) gene family is another subfamily of serine/lhreonine protein kinases.
  • CKI enzymes are present in the membranes, nucleus, cytoplasm and cytoskeleton of eukaryotic cells, and on the mitotic spindles of mammalian cells (Fish, KJ. et al. (1995) J. Biol. Chem 270:14875-14883).
  • the CKI family members all have a short amino-terminal domain of 9-76 amino acids, a highly conserved kinase domain of 284 amino acids, and a variable carboxyl-terminal domain that ranges from 24 to over 200 amino acids in length (Cegielska, A. et al. (1998) J. Biol. Chem. 273:1357-1364).
  • the CKI family is comprised of highly related proteins, as seen by the identification of isoforms of casein kinase I from a variety of sources. There are at least five mammahan isoforms, ⁇ , ⁇ , ⁇ , and ⁇ .
  • Fish et al. identified CKI-epsilon from a human placenta cDNA library.
  • 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.
  • CKI-epsilon is able to interact with mammahan PERIOD proteins, while the mutant enzyme is deficient in its ability to phosphorylate PERIOD.
  • 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).
  • HIPKs Homeodomain-interacting protein kinases
  • HIPKs are serine/lhreonine kinases and novel members of the DYRK kinase subfamily (Hofmann, T.G. et al. (2000) Biochimie 82:1123-1127).
  • HIPKs contain a conserved protein kinase domain separated from a domain that interacts with homeoproteins.
  • HIPKs are nuclear kinases, and HIPK2 is highly expressed in neuronal tissue (Kim, Y.H. et al. (1998) J. Biol. Chem. 273:25875-25879; Wang, Y. et al. (2001) Biochim. Biophys. Acta 1518:168-172).
  • HIPKs act as corepressors for homeodomian transcription factors. This corepressor activity is seen in posttranslational .modifications such as ubiquitination and phosphorylation, each of which are important in the regulation of cellular protein function (Kim, Y.H. et al. (1999) Proc. Natl. Acad. Sci. USA 96:12350-12355).
  • the human h- warts protein a homolog of Drosophila warts tumor suppressor gene, maps to chromosome 6q24-25.1. It has a serine/threonine kinase domain and is locahzed to centrosomes in interphase cells. It is involved in mitosis and functions as a component of the mitotic apparatus (Nishiyama, Y. et al. (1999) FEBS Lett. 459:159-165).
  • 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 intraceUular 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 J. 14:3679- 3686).
  • CaM kinase II also phosphorylates synapsin at different sites and controls the synthesis of catecholamines in the brain through phosphorylation and activation of tyrosine hydroxylase.
  • CaM kinase II controls the synthesis of catecholamines and seratonin, through phosphorylation/activation of tyrosine hydroxylase and tryptophan hydroxylase, respectively (Fujisawa, H. (1990) BioEssays 12:27-29).
  • the mRNA encoding a calmoduhn-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.
  • MAP mitogen-activated protein kinases
  • STK mitogen-activated protein kinases
  • MAP MAPK
  • MAPK MAPK
  • MAPK MAPK kinase
  • MKK MKK kinase
  • MAP3K MAPKKK
  • MEKK MEKK
  • EGF epidermal growth factor
  • LPS endotoxic lipopolysaccharide
  • JNK c-Jun N-terminal kinase
  • SAPK stress-activated kinase
  • p38 kinase pathway p38 kinase pathway
  • cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).
  • TNF tumor necrosis factor
  • IL-1 interleukin-1
  • CDKs The cychn-dependent protein kinases
  • the entry and exit of a cell from mitosis are regulated by the synthesis and destruction of a family of activating proteins called cychns.
  • 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.
  • cell cycle checkpoints 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.
  • Chkl checkpoint kinase 1
  • Chkl 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 Cdc2. Cdc2 activation controls the entry of cells into mitosis (Peng, C.-Y. et al. (1997) Science 277:1501-1505). Thus, activation of Chkl prevents the damaged cell from entering mitosis. A 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. 271:19402-19408).
  • ProHferation-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 protein kinase
  • a ligand-activated STK protein kinase is 5 -AMP-activated protein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem 271:8675-8681).
  • AMPK 5 -AMP-activated protein kinase
  • 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.
  • 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 diseases, neurodegenerative disorders, and cancer. Various STKs play key roles in this process.
  • ZIP kinase is an STK containing a C-te ⁇ ninal leucine zipper domain in addition to its N-terminal protein kinase domain.
  • DRAK1 and DRAK2 are STKs that share homology with the death-associated protein kinases (DAP kinases), known to function in interferon- ⁇ induced apoptosis (Sanjo et al., supra).
  • DAP kinases 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-te ⁇ ninal "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., supra). Mitochondria! 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).
  • MPKs 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).
  • Lipid kinases phosphorylate hydroxyl residues on hpid 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.
  • PI phosphatidylinositol
  • PI 3-kinase 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, hpid moieties, and other cytosolic factors.
  • the catalytic subunit When the adapter subunit binds tyrosine phosphorylated targets, such as the insulin responsive substrate (IRS)-l , the catalytic subunit is activated and converts PI (4,5) bisphosphate (PIP 2 ) to PI (3,4,5) P 3 (PIP 3 ). PIP 3 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.
  • SPP sphingosine- 1 -phosphate
  • SPP levels are regulated by spbingosine kinases that specifically phosphorylate D-eirthro-sphingosine to SPP.
  • 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., supra).
  • PDGF platelet-derived growth factor
  • nerve growth factor nerve growth factor
  • protein kinase C protein kinase C
  • 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 metabohsm and are crucial to the synthesis and regulation of cellular levels of ATP and GTP, respectively.
  • ATP AMP phosphotransferase
  • GuK guanylate kinase
  • AdK AdK is found in almost ah 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 subceUular structures that are involved in energy production and utilization, respectively. Recent studies have demonstrated a major function for AdK in transferring high energy phosphoryls frommetabohc processes generating ATP to cellular components consuming ATP (Zeleznikar, R.J. et al. (1995) J. Biol. Chem 270:7311-7319).
  • AdK may have a pivotal role in maintaining energy production in cells, particularly those having a high rate of growth or metabohsm such as cancer cells, and may provide a target for suppression of its activity in order to treat certain cancers.
  • 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.
  • 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, cychc AMP.
  • GDP binding to G proteins inhibits these processes.
  • GDP and GTP levels also control the activity of certain oncogenic proteins such as p21 ras 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 ras 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 R.L. Miller (1980) J. Biol. Chem 255:7204-7207; Stenberg, K. et al. (1986) J. Biol. Chem 261:2134-2139). Increasing GuK activity in infected cells may provide a therapeutic strategy for augmenting the effectiveness of these drugs and possibly for reducing the necessary dosages of the drugs. Pyrimidine Kinases
  • the pyrimidine kinases are deoxycytidine kinase and thymidine kinase 1 and 2.
  • Deoxycytidine kinase is located in the nucleus, and Ihy idine kinase 1 and 2 are found in the cytosol (Johansson, M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11941-11945).
  • Phosphorylation of deoxyribonucleosides by pyrimidine kinases provides an alternative pathway for de novo synthesis of DNA precursors.
  • PHOSPHATASES Protein phosphatases are generally characterized as either serme/threonine- or tyrosine- specific based on their preferred phospho-amino acid substrate. However, some phosphatases (DSPs, for dual specificity phosphatases) can act on phosphorylated tyrosine, serine, or threonine residues.
  • PSPs protein ser e/threonine phosphatases
  • PTPs Protein tyrosine phosphatases
  • Anotlier family of phosphatases is the acid phosphatase or histidine acid phosphatase (HAP) family whose members hydrolyze phosphate esters at acidic pH conditions.
  • the PPM class consists of several closely related isoforms of PP2C and is evolutionarily unrelated to the PPP class.
  • PP1 dephosphorylates many of the proteins phosphorylated by cychc AMP-dependent protein kinase (PKA) and is an important regulator of many cAMP-mediated hormone responses in cells.
  • PKA cychc AMP-dependent protein kinase
  • a number of isoforms have been identified, with the alpha and beta forms being produced by alternative splicing of the same gene.
  • Both ubiquitous and tissue-specific targeting proteins for PP1 have been identified.
  • DARPP-32 adenosine 3 ',5 - monophosphate-regulated phosphoprotein of 32kDa
  • PP2A is the main serine/threonine phosphatase.
  • the core PP2A enzyme consists of a single 36 kDa catalytic subunit (C) associated with a 65 kDa scaffold subunit (A), whose role is to recruit additional regulatory subunits (B).
  • C catalytic subunit
  • A 65 kDa scaffold subunit
  • B additional regulatory subunits
  • Three gene families encoding B subunits are known (PR55, PR61, and PR72), each of which contain multiple isoforms, and additional famuies may exist (Millward, T.A et al. (1999) Trends Biosci. 24:186-191).
  • B-type subunits are cell type- and tissue- specific and determine the substrate specificity, enzymatic activity, and subcellular localization of the holoenzyme.
  • PR55 The PR55 family is highly conserved and bears a conserved motif (PROSITE PDOC00785).
  • PR55 increases PP2A activity toward mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK).
  • MAPK mitogen-activated protein kinase
  • MEK MAPK kinase
  • PP2A dephosphorylates the MAPK active site, inhibiting the cell's entry into mitosis.
  • proteins can compete with PR55 for PP2A core enzyme binding, including the CKII kinase catalytic subunit, polyomavirus middle and small T antigens, and SV40 small t antigen. Viruses may use this mechanism to commandeer PP2A and stimulate progression of the cell through the cell cycle (Pallas, D.C. et al. (1992) J.
  • MAP kinase expression is also implicated in a variety of disease conditions including cancer, inflammation, immune disorders, and disorders affecting growth and development.
  • PP2A in fact, can dephosphorylate and modulate the activities of more than 30 protein kinases in vitro, and other evidence suggests that the same is true in vivo for such kinases as PKB, PKC, the calmodulin-dependent kinases, ERK family MAP kinases, cyclin-dependent kinases, and the I ⁇ B kinases (reviewed in Millward et al., supra).
  • PP2A is itself a substrate for CKI and CKII kinases, and can be stimulated by polycationic macromolecules.
  • a PP2A- like phosphatase is necessary to maintain the Gl phase destruction of mammahan cyclins A and B (Bastians, H. et al. (1999) Mol. Biol. Cell 10:3927-3941).
  • PP2A is a major activity in the brain and is implicated in regulating neurofilament stability and normal neural function, particularly the phosphorylation of the microtubule-associated protein tau. Hyperphosphorylation of tau has been proposed to lead to the neuronal degeneration seen in Alzheimer's disease (reviewed in Price and Mumby, supra).
  • PP2B or calcineurin
  • calcineurin is a Ca 2+ -activated dimeric phosphatase and is particularly abundant in the brain. It consists of catalytic and regulatory subunits, and is activated by the binding of the calcium/calmodulin complex. Calcineurin is the target of the hr-munosuppressant drugs cyclosporine and FK506. Along with other cellular factors, these drugs interact with calcineurin and inhibit phosphatase activity. In T cells, this blocks the calcium dependent activation of the NF-AT family of transcription factors, leading to immunosuppression. This family is widely distributed, and it is likely that calcineurin regulates gene expression in other tissues as well. In neurons, calcineurin modulates functions which range from the inhibition of neurotransmitter release to desensitization of postsynaptic NMDA-receptor coupled calcium channels to long term memory (reviewed in Price and Mumby, supra).
  • PP5 contains regulatory domains with tetratricopeptide repeats. It can be activated by polyunsaturated fatty acids and anionic phospholipids in vitro and appears to be involved in a number of signaling pathways, including those controlled by atrial natriuretic peptide or steroid hormones (reviewed in Andreeva, AN. and M.A. Kutuzov (1999) Cell Signal. 11:555-562).
  • PP2C is a ⁇ 42kDa monomer with broad substrate specificity and is dependent on divalent cations (mainly Mn 2+ or Mg 2+ ) for its activity.
  • PP2C proteins share a conserved ⁇ -terminal region with an invariant DGH motif, which contains an aspartate residue involved in cation binding (PROSITE PDOC00792). Targeting proteins and mechanisms regulating PP2C activity have not been identified.
  • PP2C has been shown to inhibit the stress-responsive p38 and Jun kinase (J ⁇ K) pathways (Takekawa, M. et al. (1998) EMBO J. 17:4744-4752).
  • tyrosine-specific phosphatases are generally monomeric proteins of very diverse size (from 20kDa to greater than lOOkDa) and structure that function primarily in the transduction of signals across the plasma membrane. PTPs are categorized as either soluble phosphatases or transmembrane receptor proteins that contain a phosphatase domain. All PTPs share a conserved catalytic domain of about 300 amino acids which contains the active site.
  • the active site consensus sequence includes a cysteine residue which executes a nucleophilic attack on the phosphate moiety during catalysis ( ⁇ eel, B.G. and ⁇ .K. Tonks (1997) Curr. Opin. Cell Biol.
  • Receptor PTPs are made up of an ⁇ -terminal extracellular domain of variable length, a transmembrane region, and a cytoplasmic region that generally contains two copies of the catalytic domain. Although only the first copy seems to have enzymatic activity, the second copy apparently affects the substrate specificity of the first.
  • the extracehular domains of some receptor PTPs contain fibronectin-like repeats, immunoglobulin-like domains, MAM domains (an extracellular motif likely to have an adhesive function), or carbonic anhydrase-like domains (PROSITE PDOC 00323). This wide variety of structural motifs accounts for the diversity in size and specificity of PTPs.
  • Soluble PTPs containing Src-homology-2 domains have been identified (SHPs), suggesting that these molecules might interact with receptor tyrosine kinases.
  • SHP-1 regulates cytokine receptor signaling by controlling the Janus family PTKs in hematopoietic cells, as well as signaling by the T-cell receptor and c-Kit (reviewed in Neel and Tonks, supra).
  • M-phase inducer phosphatase plays a key role in the induction of mitosis by dephosphorylating and activating the PTK CDC2, leading to cell division (Sadhu, K. et al. (1990) Proc. Natl. Acad. Sci. USA 87:5139-5143).
  • the genes encoding at least eight PTPs have been mapped to chromosomal regions that are translocated or rearranged in various neoplastic conditions, including lymphoma, small cell lung carcinoma, leukemia, adenocarcinoma, and neuroblastoma (reviewed in Charbonneau, H. and N.K. Tonks (1992) Annu. Rev. Cell Biol. 8:463- 493).
  • the PTP enzyme active site comprises the consensus sequence of the MTM1 gene family.
  • the MTM1 gene is responsible for X-liriked recessive myotubular myopathy, a congenital muscle disorder that has been linked to Xq28 (Kioschis, P.
  • PTKs are encoded by oncogenes, and it is well known that oncogenesis is often accompanied by increased tyrosine phosphorylation activity. It is therefore possible that PTPs may serve to prevent or reverse cell transformation and the growth of various cancers by controlling the levels of tyrosine phosphorylation in cells. This is supported by studies showing that overexpression of PTP can suppress transformation in cells and that specific inhibition of PTP can enhance cell transformation (Charbonneau and Tonks, supra).
  • Dual specificity phosphatases are structurally more similar to the PTPs than the PSPs. DSPs bear an extended PTP active site motif with an additional 7 amino acid residues. DSPs are primarily associated with cell prohferation and include the cell cycle regulators cdc25A, B, and C.
  • the phosphatases DUSP1 and DUSP2 inactivate the MAPK family members ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38 on both tyrosine and threonine residues (PROSITE PDOC 00323, supra).
  • HAP Histidine acid phosphatase
  • EXPASY EC 3.1.3.2 also known as acid phosphatase
  • HAPs share two regions of conserved sequences, each centered around a histidine residue which is involved in catalytic activity.
  • Members of the HAP family include lysosomal acid phosphatase (LAP) and prostatic acid phosphatase (PAP), both sensitive to inhibition by L-tartrate (PROSITE PDOC00538).
  • EPS 15 This binding is mediated by the C-terminal region of synaptojanin-170, which has 3 Asp-Pro- Phe amino acid repeats. Further, this 3 residue repeat had been found to be the binding site for the EH domains of EPS15 (Haffner, C. et al. (1997) FEBS Lett. 419:175-180). Additionally, synaptojanin may potentially regulate interactions of endocytic proteins with the plasma membrane, and be involved in synaptic vesicle recycling (Brodin, L. et al. (2000) Curr. Opin. Neurobiol. 10:312- 320).
  • Microarrays are analytical tools used inbioanalysis.
  • a microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a sohd support.
  • Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
  • array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
  • Lung cancer is the leading cause of cancer death for men and the second leading cause of cancer death for women in the U.S. Lung cancers are divided into four histopathologicaUy distinct groups. Three groups (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) are classified as non-small cell lung cancers (NSCLCs). The fourth group of cancers is referred to as small cell lung cancer (SCLC). Deletions on chromosome 3 are common in this disease and are thought to indicate the presence of a tumor suppressor gene in this region. Activating mutations in K- ras are commonly found in lung cancer and are the basis of one of the mouse models for the disease. Ovarian Cancer
  • Ovarian cancer is the leading cause of death from a gynecologic cancer.
  • the majority of ovarian cancers are derived from epithehal cells, and 70% of patients with epithehal ovarian cancers present with late-stage disease. As a result, the long-term survival rate for this disease is very low. Identification of early-stage markers for ovarian cancer would significantly increase the survival rate. Genetic variations involved in ovarian cancer development include mutation of p53 and microsatelhte instability. Gene expression patterns likely vary when normal ovary is compared to ovarian tumors.
  • BRCA1 and BRCA2 Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a woman to breast cancer and may be passed on from parents to children (Gish, supra).
  • this type of hereditary breast cancer accounts for only about 5% to 9% of breast cancers, while the vast majority of breast cancer is due to noninherited mutations that occur in breast epithehal cells.
  • a good deal is already known about the expression of specific genes associated with breast cancer. For example, the relationship between expression of epidermal growth factor (EGF) and its receptor, EGFR, to human mammary carcinoma has been particularly well studied.
  • EGF epidermal growth factor
  • erbB receptors such as HER-2/neu, HER-3, and HER- 4
  • their ligands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S.S. et al. (1994) Am. J. Clin. Pathol. 102:S 13-S24).
  • Estrogen stimulation plays a critical role in the development of normal rnammary epithelium
  • Estradiol has a direct mitogenic effect on breast cancer cells, causing them to divide more rapidly by shortening their cell cycle.
  • estradiol induces a large number of enzymes and other proteins involved in nucleic acid synthesis in isolated breast cancer cell lines.
  • Estradiol may increase the expression of the EGF receptor in response to TGF- ⁇ and EGF.
  • estrogens may promote proliferation of tumor cells by inducing the synthesis of TGF- ⁇ and EGF, and may block growth factors that would normally inhibit tumor cell growth.
  • Estrogen receptor (ER) has been investigated extensively as a prognostic marker in breast cancer. Patients whose tumors display high levels of estrogen receptor have a significantly better prognosis than patients with receptor-negative tumors. When ER is lost or cells expressing the ER are selected against by therapeutic treatments, the tumor becomes more aggressive.
  • Cell hnes derived from human mammary epithehal cells at various stages of breast cancer provide a useful model to study the process of malignant transformation and tumor progression as it has been shown that these cell hnes retain many of the properties of their parental tumors for lengthy culture periods (Wistuba II et al. (1998) Clin Cancer Res 4:2931-2938). Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epithehal cells at various stages of malignant transformation.
  • Tumor cells stimulate the formation of stroma that secretes various mediators, such as growth factors, cytokines, and proteases, which are critical for tumor growth.
  • various mediators such as growth factors, cytokines, and proteases, which are critical for tumor growth.
  • TNF- ⁇ serum tumor necrosis factor alpha
  • IFN- ⁇ Interferon-gamma
  • TNF- ⁇ also called cachectin, is produced by neutrophils, activated lymphocytes, macrophages, NK cells, LAK cells, astrocytes, endothelial cells, smooth muscle cells, and some transformed cells.
  • TNF- ⁇ occurs as a secreted, soluble form and as a membrane-anchored form, both of which are biologically active.
  • Two types of receptors for TNF- ⁇ have been described and virtually ah cell types studied show the presence of one or both of these receptor types.
  • TNF- ⁇ and TNF- ⁇ are extremely pleiotropic factors due to the ubiquity of their receptors, to their ability to activate multiple signal transduction path-ways and to their ability to induce or suppress the expression of a wide number of genes.
  • TNF- ⁇ and TNF- ⁇ play a critical role in mediation of the inflammatory response and in mediation of resistance to infections and tumor growth.
  • Atherosclerosis Blood vessel walls are composed of two tissue layers: an endothelial cell (EC) layer which comprises the lumenal surface of the vessel, and an underlying vascular smooth muscle cell (VSMC) layer.
  • EC endothelial cell
  • VSMC vascular smooth muscle cell
  • the inflammatory response is a complex vascular reaction mediated by numerous cytokines, chemokines, growth factors, and other signaling molecules expressed by activated ECs, VSMCs and leukocytes. Inflammation protects the organism during trauma and infection, but can also lead to pathological conditions such as atherosclerosis.
  • the pro-inflammatory cytokines, interleukin (IL)-1 and tumor necrosis factor (TNF) are secreted by a small number of activated macrophages or other cells and can set off a cascade of vascular changes, largely through their ability to alter gene expression patterns in ECs and VSMCs.
  • vascular changes include vasodilation and increased permeability of microvasculature, edema, and leukocyte extravasation and transmigration across the vessel wall.
  • leukocytes particularly neutrophils and monocytes/macrophages, accumulate in the extravascular space, where they remove injurious agents by phagocytosis and oxidative killing, a process accompanied by release of toxic factors, such as proteases and reactive oxygen species.
  • IL-1 and TNF induce pro-inflammatory, thrombotic, and anti-apoptotic changes in gene expression by signaling through receptors on the surface of ECs and VSMCs; these receptors activate transcription factors such as NF£B as weh as AP-1 , IRF-1 , and NF-GMa, leading to alterations in gene expression.
  • Genes known to be differentially regulated in EC by IL-1 and TNF include E selectin, VCAM-1, ICAM-1, PAF, IkB , IAP-1, MCP-1, eotaxin, ENA-78, G-CSF, A20, ICE, and complement C3 component.
  • a key event in inflammation, adhesion and transmigration of blood leukocytes across the vascular endothehum for example, is mediated by increased expression of E selectin, P selectin, ICAM-1, and VCAM-1 on activated endothehum.
  • HAECs Human aortic endothelial cells
  • HAECs Human aortic endothelial cells
  • Activation of the vascular endothehum is considered to be a central event in a wide range of both physiological and pathophysiological processes, such as vascular tone regulation coagulation and thrombosis, atherosclerosis, and inflammation.
  • Atherosclerosis is a pathological condition characterized by a chronic local inflammatory response within the vessel wall of major arteries. Disease progression results in the formation of atherosclerotic lesions, unstable plaques which occasionally rupture, precipitating a catastrophic thrombotic occlusion of the vessel lumen. Atherosclerosis and the associated coronary artery disease and cerebral stroke represent the most common causes of death in industrialized nations. Although certain key risk factors have been identified, a full molecular characterization that elucidates the causes and identifies ah potential therapeutic targets for this complex disease has not been achieved. Molecular characterization of atherosclerosis requires identification of the genes that contribute to lesion growth, stability, dissolution, rupture and induction of occlusive vessel thrombi.
  • Atherosclerosis is a pathological condition characterized by a chronic local inflammatory response within the vessel wall of major arteries. Disease progression results in the formation of atherosclerotic lesions, unstable plaques which occasionally rupture, precipitating a catastrophic thrombotic occlusion of the vessel lumen. Atherosclerosis and the associated coronary artery disease and cerebral stroke represent the most common causes of death in industrialized nations. Although certain key risk factors have been identified, a full molecular characterization that elucidates the causes and identifies ah potential therapeutic targets for this complex disease has not been achieved.
  • Tumor necrosis factor-alpha mediates immune regulation and inflammatory responses through various intermediates, including protein kinases, protein phosphatases, reactive oxygen intermediates, phosphohpases, proteases, sphingomyelinases and transcription factors.
  • TNF- ⁇ -related cytokines generate cellular responses including differentiation, prohferation, cell death, and activation of nuclear factor- ⁇ B (NF- ⁇ B) (Smith, CA. et al. (1994) Ceh 76:959-962), through their interaction with distinct ceh surface receptors (TNFRs).
  • NF- ⁇ B is a transcription factor that induces genes involved in physiological processes such as response to injury and infection.
  • IL-l ⁇ and TNF- ⁇ induce pro-inflammatory, thrombotic, and anti-apoptotic changes in gene expression by signaling through receptors on the surface of ECs and VSMCs; these receptors activate several transcription factors, leading to alterations in gene expression.
  • Genes known to be differentially regulated in EC by IL-l ⁇ and TNF- ⁇ include E-selectin, VCAM-1, ICAM-1, PAF, I/cB ⁇ , IAP-1, MCP-1, eotaxin, ENA-78, G-CSF, A20, ICE, and complement C3 component.
  • TNF- ⁇ is upregulated when the endothehum is physically disrupted or functionally perturbed by events such as postischemic reperfusion, acute and chronic inflammation, atherosclerosis, diabetes and chronic arterial hypertension. Inflammatory stimulation sets the stage for later tissue repair. Elevated TNF- ⁇ initiahy increases, and then inhibits, the activity of a number of key enzymes including protein-tyrosine kinase (PTKase) and protein-tyrosine phosphatase (Holden, R. J. et al. (1999) Med. Hypotheses 52:319-23).
  • PTKase protein-tyrosine kinase
  • Holden, R. J. et al. (1999) Med. Hypotheses 52:319-23 protein-tyrosine phosphatase
  • LDL low-density hpoprotein
  • macrophages produce cytokines including TNF- ⁇ , as weh as interleukin-1 and growth factors (e.g. M-CSF, VEGF, and PDGF-BB), that ehcit further events in atherogenesis such as smooth muscle ceh prohferation and production of extracehular matrix by vascular endothehum.
  • These macrophages may also activate genes in endothehum and smooth muscle tissue involved in inflammation and tissue differentiation, including superoxide dismutatse (SOD), IL-8, and ICAM-1.
  • SOD superoxide dismutatse
  • Non-atherosclerotic vascular endothehum not only mediates vascular dilatation but prevents platelet adhesion and activation, blocks thrombin formation, mitigates fibrin deposition, and attenuates adhesion and transmigration of inflammatory leukocytes.
  • the endothehum is physically disrupted, or perturbed by events such as postischemic reperfusion, acute and chronic inflammation, atherosclerosis, diabetes and chronic arterial hypertension, it acts in the opposite manner.
  • the perturbed or proinflammatory state is characterised by vaso-constriction, platelet and leukocyte activation and adhesion (involving externahsation, expression and upregulation of, for example, von Willebrand factor, platelet activating factor, P-selectin, ICAM-1, IL-8, MCP-1, and TNF- ⁇ ), promotion of thrombin formation, coagulation and deposition of fibrin at the vascular wall (expression of tissue factor, PAI-1, and phosphatidyl serine) and, in platelet-leukocyte coaggregates, additional inflammatory interactions via attachment of platelet CD40-hgand to endothehal, monocyte and B-ceU CD40.
  • Thrombin formation and inflammatory stimulation set the stage for later tissue repair, but limiting procoagulatory, prothrombotic actions of a dysfunctional vascular endothehum may be the goal of clinical interventions (for review, see Becker et al. (2000) Z Kardiol 89:160-167).
  • Several investigators have examined changes in vascular ceh gene expression associated with various inflammatory diseases or model systems. Examining human umbilical vein endothehal cells (HUVEC) activated by recombinant TNF- ⁇ or conditioned medium from activated human primary monocytes, Horrevoets et al. (1999; Blood 93:3418-3431) identified 106 differentially regulated genes. In a similar approach, deVries et al. (2000; J.
  • Biol. Chem 275:23939-23947) identified 40 differentially regulated genes in umbilical cord artery-derived smooth muscle cells activated by conditioned media from cultured macrophages after stimulation with oxidized LDL particles. In both studies, many of the identified genes were already known to be involved in inflammation. Comparing expression profiles from inflammatory diseased tissues, cultured macrophages, chondrocyte ceh lines, primary chondrocytes, and synoviocytes, Heller et al. (1997; Proc. Natl. Acad. Sci. USA 94:2150- 2155) identifed candidate genes involved in inflammatory responses, including TNF, IL-1 IL-6, IL-8 G-CSF, RANTES, and V-CAM.
  • tissue inhibitor of metahoproteinase 1, ferritin light chain, and manganese superoxide dismutase were found to be differentially expressed in rheumatoid arthritis (RA) relative to inflammatory bowel disease (IBD). Further, IL-3, chemokine Gro ⁇ , and metahoproteinase matrix metaho-elastase were expressed in both RA and IBD.
  • RA rheumatoid arthritis
  • IBD inflammatory bowel disease
  • IL-3, chemokine Gro ⁇ , and metahoproteinase matrix metaho-elastase were expressed in both RA and IBD.
  • Familial adenomatous polyposis is caused by mutations in the adenomatous polyposis coh gene (APC), resulting in truncated or inactive forms of the protein.
  • APC adenomatous polyposis coh gene
  • This tumor suppressor gene has been mapped to chromosome 5q.
  • Hereditary nonpolyposis colorectal cancer is caused by mutations in mis-match repair genes.
  • HDL plays a cardio-protective role in reverse cholesterol transport, the flux of cholesterol from peripheral cehs such as tissue macrophages through plasma lipoproteins to the hver.
  • the HDL protein, apolipoprotein A-I plays a major role in this process, interacting with the ceh surface to remove excess cholesterol and phosphohpids. This pathway is severely impaired in TD and the defect lies in a specific gene, the ABCl transporter. This gene is a member of the family of ATP-binding cassette transporters, which utilize ATP hydrolysis to transport a variety of substrates across membranes.
  • Steroids are a class of hpid-soluble molecules, including cholesterol, bile acids, vitamin D, and hormones, that share a common four-ring structure based on cyclopentanoperhydrophenanthrene and that carrry out a wide variety of functions.
  • Cholesterol for example, is a component of ceh membranes that controls membrane fluidity. It is also a precursor for bile acids which solubihze lipids and facilitate absorption in the small intestine during digestion. Vitamin D regulates the absorption of calcium in the small intestine and controls the concentration of calcium in plasma.
  • Steroid hormones produced by the adrenal cortex, ovaries, and testes, include glucocorticoids, mineralocorticoids, androgens, and estrogens.
  • Glucocorticoids for example, increase blood glucose concentrations by regulation of gluconeogenesis in the hver, increase blood concentrations of fatty acids by promoting hpolysis in adipose tissues, modulate sensitivity to catcholamines in the central nervous system, and reduce i- ⁇ flammation.
  • the principal mineralocorticoid, aldosterone, is produced by the adrenal cortex and acts on cehs of the distal tubules of the kidney to enhance sodium ion reabsorption.
  • Progesterone a naturaUy occurring progestin
  • Endogenous progesterone is responsible for inducing secretory activity in the endometrium of the estrogen-primed uterus in preparation for the implantation of a fertilized egg and for the maintenance of pregnancy. It is secreted from the corpus luteum in response to luteinizing hormone (LH).
  • LH luteinizing hormone
  • progestins diffuse freely into target cehs and bind to the progesterone receptor.
  • Target ceUs include the female reproductive tract, the mammary gland, the hypothalamus, and the pituitary. Once bound to the receptor, progestins slow the frequency of release of gonadotropin releasing hormone from the hypothalamus and blunt the pre-ovulatory LH surge, thereby preventing foUicular maturation and ovulation.
  • Progesterone has minimal estrogenic and androgenic activity. Progesterone is metabohzed hepatically to pregnanediol and conjugated with glucuronic acid.
  • MAH Medroxyprogesterone
  • 6 ⁇ -methyl-17-hydroxyprogesterone is a synthetic progestin with a pharmacological activity about 15 times greater than progesterone.
  • MAH is used for the treatment of renal and endometrial carcinomas, amenorrhea, abnormal uterine bleeding, and endometriosis associated with hormonal imbalance.
  • MAH has a stimulatory effect on respiratory centers and has been used in cases of low blood oxygenation caused by sleep apnea, chronic obstructive pulmonary disease, or hypercapnia.
  • Mifepristone also known as RU-486, is an antiprogesterone drug that blocks receptors of progesterone. It counteracts the effects of progesterone, which is needed to sustain pregnancy. Mifepristone induces spontaneous abortion when administered in early pregnancy followed by treatment with the prostaglandin misoprostol. Further studies show that mifepristone at a substantiaUy lower dose can be highly effective as a postcoital contraceptive when administered within five days after unprotected intercourse, thus providing women with a "morning-after piU" in case of contraceptive failure or sexual assault. Mifepristone also has potential uses in the treatment of breast and ovarian cancers in cases in which tumors are progesterone-dependent.
  • Mifepristone binds to glucocorticoid receptors and interferes with cortisol binding. Mifepristone also may act as an anti-glucocorticoid and be effective for treating conditions where cortisol levels are elevated such as AIDS, anorexia nervosa, ulcers, diabetes, Parkinson's disease, multiple sclerosis, and Alzheimer's disease.
  • Danazol is a synthetic steroid derived from ethinyl testosterone. Danazol indirectly reduces estrogen production by lowering pituitary synthesis of fo cle-stirnulating hormone and LH. Danazol also binds to sex hormone receptors in target tissues, thereby exhibiting anabohc, antiestrognic, and weakly androgenic activity. Danazol does not possess any progestogenic activity, and does not suppress normal pituitary release of corticotropin or release of cortisol by the adrenal glands. Danazol is used in the treatment of endometriosis to relieve pain and inhibit endometrial ceU growth. It is also used to treat fibrocystic breast disease and hereditary angioedema.
  • Corticosteroids are used to reheve inflammation and to suppress the immune response. They inhibit eosinopbil, basophil, and airway epithehal cell function by regulation of cytokines that mediate the inflammatory response. They inhibit leukocyte infiltration at the site of inflammation, interfere in the function of mediators of the inflammatory response, and suppress the humoral immune response. Corticosteroids are used to treat aUergies, asthma, arthritis, and skin conditions. Beclomethasone is a synthetic glucocorticoid that is used to treat steroid-dependent asthma, to reheve symptoms associated with aUergic or nonaUergic (vasomotor) rhinitis, or to prevent recurrent nasal polyps foUowing surgical removal.
  • intranasal beclomethasone is 5000 times greater than those produced by hydrocortisone.
  • Budesonide is a corticosteroid used to control symptoms associated with aUergic rhinitis or asthma.
  • Budesonide has high topical anti-inflammatory activity but low systemic activity.
  • Dexamethasone is a synthetic glucocorticoid used in anti-inflammatory or immunosuppressive compositions. It is also used in inhalants to prevent symptoms of asthma. Due to its greater ability to reach the central nervous system, dexamethasone is usuaUy the treatment of choice to control cerebral edema.
  • Betamethasone is a synthetic glucocorticoid with anti-inflammatory and immunosuppressive activity and is used to treat psoriasis and fungal infections, such as athlete's foot and ringwor
  • the anti-inflammatory actions of corticosteroids are thought to involve phospholipase A 2 inhibitory proteins, coUectively caUed hpocortins.
  • Lipocortins in turn, control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic acid.
  • Proposed mechanisms of action include decreased IgE synthesis, increased number of ⁇ -adrenergic receptors on leukocytes, and decreased arachidonic acid metabohsm.
  • allergens bridge the IgE antibodies on the surface of mast ceUs, which triggers these ceUs to release chemotactic substances.
  • Mast ceU influx and activation therefore, is partially responsible for the inflammation and hyperirritabihty of the oral mucosa in asthmatic patients. This inflammation can be retarded by administration of corticosteroids.
  • the metabohsm of many drug substances, such as acetaminophen, in the hver leads to the generation of catechol (ortho-hydroxy benzene) containing intermediates.
  • Catechol-containing intermediates may themselves be hepatotoxic by generating reactive intermediates or they may be further metabolized for example via catechol-o-mefhyl transferase to generate reactive methylating agents.
  • catechol-containing compounds to ceU toxicity is of interest.
  • the effects upon hver metabohsm and hormone clearance mechanisms are important to understand the pharmacodynamics of a drug.
  • the human C3A ceU line is a clonal derivative of HepG2/C3 (hepatoma ceU line, isolated from a 15-year-old male with hver tumor), which was selected for strong contact inhibition of growth.
  • the use of a clonal population enhances the reproducibhity of the ceUs.
  • C3A ceUs have many characteristics of primary human hepatocytes in culture: i) expression of insulin receptor and insulin-like growth factor II receptor; ii) secretion of a high ratio of serum albumin compared with ⁇ -fetoprotein; hi) conversion of ammonia to urea and glutamine; iv) metabohsm of aromatic amino acids; and v) proliferation in glucose-free and insulin- free medium.
  • Gemfibrizol is an anti-hpemic agent that lowers serum triglycerides and produces favorable changes in lipoproteins.
  • GemfibrozU is effective in reducing the risk of coronary heart disease in men (Frick, M.H., et al. (1987) New Engl. J. Med. 317:1237-1245).
  • the compound can inhibit peripheral hpolysis and decrease hepatic extraction of free fatty acids, which decreases hepatic triglyceride production.
  • Gemfibrozil also inhibits the synthesis and increases the clearance of apolipoprotein B, a carrier molecule for VLDL.
  • GemfibrozU has variable effects on LDL cholesterol.
  • GemfibrozU causes peroxisome prohferation and hepatocarcinogenesis in rats, which is a cause for concern generaUy for fibric acid derivative drugs.
  • fibric acid derivatives are known to increase the risk of gaU bladder disease although gemfibrozil is better tolerated than other fibrates.
  • the relative safety of gemfibrozU in humans compared to rodent species including rats may be attributed to differences in metabohsm and clearance of the compound in different species (Dix, K.J. et al. (1999) Drug Metab. Distrib. 27:138-146; Thomas, B.F. et al. (1999) Drug Metab. Distrib. 27:147-157).
  • Fibric acid compounds are usuaUy weU tolerated. Side effects may occur in 5-10% of patients but most often are not sufficient to cause discontinuation of the drug. Gastrointestinal side effects are most common (up to 5% of patients). Other side effects are reported infrequently and include rash, urticaria, hair loss, myalgias, fatigue, headache, impotence, and anemia. Minor increases in hver transaminases and decreases in alkaline phosphatase have been reported.
  • Gemfibrozil is used to treat the hyperhpidemia of renal fahure, but it should be used for this application with caution and at a reduced dosage. These agents should not be used during pregnancy or in children.
  • Clofibrate is available for oral administration. The usual dose is 2 g/day in divided doses. This compound is rarely used today, but it may be useful in patients with dysbetahpoproteinemia who do not respond to gemfibrozU.
  • compositions including nucleic acids and proteins, for the diagnosis, prevention, and treatment of cardiovascular diseases, immune system disorders, neurological disorders, disorders affecting growth and development, hpid disorders, ceU prohferative disorders, and cancers.
  • Various embodiments of the invention provide purified polypeptides, kinases and phosphatases, referred to coUectively as 'KPP' and individuaUy as 'KPP-1,' 'KPP-2,' 'KPP-3,' 'KPP-4,' 'KPP-5,' 'KPP-6,' 'KPP-7,' 'KPP-8,' 'KPP-9,' 'KPP-10,' 'KPP-11,' 'KPP-12,' 'KPP-13,' 'KPP-14,' 'KPP-15,' 'KPP-16,' 'KPP-17,' 'KPP-18,' 'KPP-19,' 'KPP-20,' 'KPP-21,' 'KPP-22,' 'KPP-23,' 'KPP-24,' 'KPP-25,' 'KPP-26,' 'KPP-27,' 'KPP-28,' 'KPP-29,'
  • Embodiments also provide methods for utilizing the purified kinases and phosphatases and/or their encoding polynucleotides for facUitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology.
  • Related embodiments provide methods for utilizing the purified kinases and phosphatases and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
  • An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l- 56, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-56.
  • Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:l-56.
  • Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-56.
  • polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:l-56. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:57-l 12.
  • StiU another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-56.
  • Another embodiment provides a ceh transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-56.
  • the method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said ceU is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • Yet another embodiment provides an isolated antibody which specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-56.
  • 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 specificaUy 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.
  • the method can include detecting the amount of the hybridization complex.
  • the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof.
  • the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.
  • compositions comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, and a pharmaceutically acceptable excipient
  • the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:l-56.
  • Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional KPP, comprising administering to a patient in need of such treatment the composition.
  • Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56.
  • StiU yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-56.
  • the method comprises a) contacting a sample comprising the polypeptide with a compound, and b) detecting antagonist activity in the sample.
  • Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional KPP, comprising administering to a patient in need of such treatment the composition.
  • Another embodiment provides a method of screening for a compound that specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56.
  • the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
  • Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid isequence selected from the group consisting of SEQ ID NO:l-56, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56.
  • 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.
  • StiU yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:57-112, the method comprising a) contacting a sample comprising the target polynucleotide with a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
  • Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:57-112, ii) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:57-112, hi) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-
  • Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:57-l 12, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO.57-112, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • a target polynucleotide selected from the group consisting of i) a polynu
  • the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
  • Table 2 shows the GenBarik identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention.
  • the probabihty scores for the matches between each polypeptide and its homolog(s) are also shown.
  • Table 5 shows representative cDN A libraries for polynucleotide embodiments.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA hbraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.
  • Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations.
  • a host ceU includes a plurality of such host ceUs
  • an antibody is a reference to one or more antibodies and equivalents thereof known to those skiUed in the art, and so forth.
  • aU technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skiU in the art to which this invention belongs.
  • agonist refers to a molecule which intensifies or mimics the biological activity of KPP.
  • Agonists may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of KPP either by directly interacting with KPP or by acting on components of the biological pathway in which KPP participates.
  • altered nucleic acid sequences encoding KPP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as KPP or a polypeptide with at least one functional characteristic of KPP. Included within this definition are polymorphisms which may or may not be readUy detectable using a particular oligonucleotide probe of the polynucleotide encoding KPP, and improper or unexpected hybridization to aUehc variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding KPP.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a sUent change and result in a functionaUy equivalent KPP.
  • Deliberate amino acid substitutions may be made on the basis of one or more simUarities in polarity, charge, solubility, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of KPP is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having simUar hydrophihcity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having simUar hydrophihcity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence can refer to an ohgopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturaUy occurring or synthetic molecules.
  • amino acid sequence is recited to refer to a sequence of a naturaUy 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. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies weU known in the art.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of KPP.
  • Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of KPP either by directly interacting with KPP or by acting on components of the biological pathway in which KPP participates.
  • antibody refers to intact immunoglobulin molecules as weU as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind KPP polypeptides can be prepared using intact polypeptides or using fragments containing smaU peptides of interest as the immunizing antigen.
  • the polypeptide or ohgopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • Commonly used carriers that are chemicaUy coupled to peptides include bovine serum albumin, fhyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • 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 specificaUy 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.
  • aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
  • Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory syste
  • Aptamers may be specificaUy cross-linked to their cognate hgands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
  • RNA aptamer refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturaUy occurring enzymes, which normaUy act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a polynucleotide having a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); ohgonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, orbenzylphosphonates; ohgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracU, or 7-deaza-2'- deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a ceU, the complementary antisense molecule base-pairs with a naturaUy occuning nucleic acid sequence produced by the ceU to form duplexes which block either transcription or translation.
  • the designation "negative” or “udinus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologicalcaUy active refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule.
  • immunologicalaUy active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic KPP, or of any ohgopeptide thereof, to induce a specific immune response in appropriate animals or ceUs 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'.
  • composition comprising a given polynucleotide and a “composition comprising a given polypeptide” can refer to any composition containing the given polynucleotide or polypeptide.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotides encoding KPP or fragments of KPP 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.
  • 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.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate; SDS
  • 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 uncaUed bases, extended using the XL-PCR kit (Apphed Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (Accelrys,
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especiaUy 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.
  • Trp Phe Tyr Tyr His, Phe, Trp Val He, Leu, Thr
  • Conservative amino acid substitutions generaUy maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha hehcal conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemicaUy modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus aUowing acceleration of the evolution of new protein functions.
  • a "fragment” is a unique portion of KPP or a polynucleotide encoding KPP which can be identical in sequence to, but shorter in length than, the parent sequence. A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from about 5 to about 1000 contiguous nucleotides or amino acid residues.
  • a fragment of SEQ ID NO:57-112 can comprise a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:57-112, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO:57-l 12 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amphfication technologies and in analogous methods that distinguish SEQ ID NO:57-l 12 from related polynucleotides.
  • the precise length of a fragment of SEQ ID NO:57-l 12 and the region of SEQ ID NO:57-l 12 to which the fragment corresponds are routinely determinable by one of ordinary skiU in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ID NO:l-56 is encoded by a fragment of SEQ ID NO:57-l 12.
  • a fragment of SEQ ID NO: 1-56 can comprise a region of unique amino acid sequence that specificaUy identifies SEQ ID NO:l-56.
  • a fragment of SEQ ID NO:l-56 can be used as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ ID NO:l-56.
  • the precise length of a fragment of SEQ ID NO:l-56 and the region of SEQ ID NO:l-56 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
  • a “fuU length” polynucleotide is one containing at least a translation initiation codon (e.g., methionine) foUowed by an open reading frame and a translation termination codon.
  • a “full length” polynucleotide sequence encodes a "fuU length” polypeptide sequence.
  • Homology refers to sequence simUarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of identical nucleotide matches between at least two polynucleotide sequences ahgned 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 ahgnment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEG ALIGN version 3.12e sequence ahgnment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Ahgnment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Ahgnment Search Tool
  • 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.
  • BLAST 2 Sequences 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 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example: Matrix: BLOSUM62 Reward for match: 1
  • 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 simUar 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 aU encode substantiaUy the same protein.
  • percent identity and % identity refer to the percentage of identical residue matches between at least two polypeptide sequences ahgned using a standardized algorithm.
  • Methods of polypeptide sequence ahgnment are weU-known. Some ahgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detaU above, generaUy preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • percent simUarity and % simUarity refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences ahgned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity maybe measured.
  • Human artificial chromosomes are linear rmcrochromosom.es which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain aU of the elements required for chromosome replication, segregation and maintenance.
  • 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 abUity.
  • 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 step(s) is particularly important in deterrnining the stringency of the hybridization process, with more stringent conditions ahowing less non-specific binding, i.e., binding between pahs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skUl in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.
  • Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ .g/ml sheared, denatured salmon sperm DNA.
  • GeneraUy stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point C for the specific sequence at a defined ionic strength and pH.
  • the T m 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 m and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. and D.W. RusseU (2001 ; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY, ch. 9).
  • High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS , for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C ma be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
  • hybridization complex refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a sohd support (e.g., paper, membranes, filters, chips, pins or glass shdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
  • insertion and “addition” refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
  • Immuno 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 ceUular and systemic defense systems.
  • an “immunogenic fragment” is a polypeptide or ohgopeptide fragment of KPP 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 ohgopeptide fragment of KPP which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • element and “array element” refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of KPP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of KPP.
  • 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.
  • 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.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an ohgonucleotide 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 hfespan in the ceh.
  • "Post-translational modification" of an KPP may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wUl vary by ceU type depending on the enzymatic milieu of KPP.
  • Probe refers to nucleic acids encoding KPP, then complements, or fragments thereof, which are used to detect identical, aUelic or related nucleic acids.
  • Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, hgands, chemUuminescent agents, and enzymes.
  • Primmers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pahs can be used for amphfication (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Probes and primers as used in the present invention typicaUy 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.
  • 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).
  • Ohgonucleotides 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 ohgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kUobases. SimUar primer selection programs have incorporated additional features for expanded capabUities.
  • the PrimOU primer selection program (avaUable to the pubhc from the Genome Center at University of Texas South West Medical Center, DaUas 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 (avaUable to the pubhc from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows the user to input a "n- spriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of ohgonucleotides 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 (avaUable to the pubhc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence ahgnments, 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.
  • 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 partiaUy complementary polynucleotides in a sample of nucleic acids. Methods of ohgonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and RusseU (supra).
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • 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 ceU.
  • 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 liiammal.
  • a "regulatory element” refers to a nucleic acid sequence usuaUy 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.
  • RNA equivalent in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occurrences of the nitrogenous base thymine are replaced with uracU, and the sugar backbone is composed of ribose instead of deoxyribose.
  • substantiallyUy purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturaUy associated.
  • 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, shdes, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient ceU. Transformation may occur under natural or artificial conditions according to various methods weU 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 ceU. The method for transformation is selected based on the type of host ceU being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, hpofection, and particle bombardment.
  • transformed ceUs includes stably transformed cells in which the inserted DNA is capable of rephcation either as an autonomously replicating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs 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.
  • 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 2.0.9 (May-07-1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an "aUehc" (as defined above), "splice,” “species,” or “polymorphic” variant.
  • a sphce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate splicing during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotides that vary from one species to another. The resulting polypeptides wiU generaUy 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.
  • SNPs single nucleotide polymorphisms
  • Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity or sequence simUarity over a certain defined length of one of the polypeptides.
  • Various embodiments of the invention include new human kinases and phosphatases (KPP), the polynucleotides encoding KPP, and the use of these compositions for the diagnosis, treatment, or prevention of cardiovascular diseases, immune system disorders, neurological disorders, disorders affecting growth and development, hpid disorders, ceU prohferative disorders, and cancers.
  • KPP human kinases and phosphatases
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project 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.
  • Column 6 shows the Incyte ID numbers of physical, fuU length clones corresponding to the polypeptide and polynucleotide sequences of the invention.
  • the full length clones encode polypeptides which have at least 95% sequence identity to the polypeptide sequences shown in column 3.
  • Table 2 shows sequences with homology to polypeptide embodiments of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the conesponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID NO:) of the nearest PROTEOME database homologs.
  • Column 4 shows the probabihty scores for the matches between each polypeptide and its homolog(s).
  • Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where apphcable, aU of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention.
  • Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the conesponding 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 amino acid residues comprising signature sequences, domains, motifs, potential phosphorylation sites, and potential glycosylation sites.
  • Column 5 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were apphed.
  • Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are kinases and phosphatases.
  • SEQ ID NO:3 is 97% identical, fromresidue L85 to residue C198, to rat protein tyrosine phosphatase (GenBank ID g409023) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
  • the BLAST probabihty score is 2.4e-94, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance.
  • SEQ ID NO:3 has homology to dual specificity phosphatase 1 , which is a stress factor-induced, non-membrane spanning protein phosphatase that inactivates members of the mitogen activated protein kinase famUy, is involved in chemotaxis, cell proliferation, and stress responses, and may possibly be involved in apopotosis and heat shock, as determined by BLAST analysis using the PROTEOME database.
  • dual specificity phosphatase 1 is a stress factor-induced, non-membrane spanning protein phosphatase that inactivates members of the mitogen activated protein kinase famUy, is involved in chemotaxis, cell proliferation, and stress responses, and may possibly be involved in apopotosis and heat shock, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID N0:3 also contains a dual specificity phosphatase, catalytic domain and a rhodanese-hke homology domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)- based PF AM/SMART databases of conserved protein fam ies/domains.
  • HMM hidden Markov model
  • SEQ ID NO:26 is 98% identical, from residue H207 to residue W902, to human leucine zipper bearing kinase (GenBank ID g2879898) as determined by BLAST. The BLAST probabihty score is 0.0. SEQ ID NO:26 also has homology to proteins that are locahzed to the cytoplasm, nucleus or golgi, have kinase activity, and are protein kinases, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:26 also contains a serme/threonine protein kinase catalytic domain, a tyrosine kinase catalytic domain, and a protein kinase domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based SMART and PFAM databases of conserved protein famihes/domains.
  • HMM hidden Markov model
  • SEQ ID NO:35 is 99% identical, from residue Ml to residue V552, to human protein kinase C-theta (GenBank ID g558099) as determined by BLAST.
  • the BLAST probabihty score is 0.0.
  • SEQ ID NO:35 also has homology to protein kinase C-theta which is involved in T ceU activation and protection from apoptosis, may play a role in insulin and multidrag resistance, and may regulate mitosis, preadipocyte differentiation, and spermatogenesis; further, rat Pkcq may play a role inhyperglycemia and hypertriglyceridemia, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:35 also contains protein kinase C conserved region 1 (Cl), extension to Ser/Thr-type protein kinases, serine/threonine protein kinases catalytic, phorbol esters/diacylglycerol binding, protein kinase, and protein kinase C terminal domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM/SMART databases of conserved protein families/domains.
  • HMM hidden Markov model
  • SEQ ID NO:35 is a protein kinase C- theta.
  • SEQ ID NO:49 is 98% identical, from residue Ml to residue
  • SEQ ID NO:49 also has homology to proteins that are related to the Ste20 famUy of kinases, that bind Nek and MEKK1 , that activate the JNK signaling pathway, that may be involved in TNF- ⁇ signaling, and that, if mutated, prevent development of somites or hindgut, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:49 also contains a domain found in NIKl-like kinases, mouse citron and yeast ROM1 and ROM2, and also a serine/threonine kinase catalytic domain, as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM and SMART database of conserved protein famihes/domains. (See Table 3.) Data from MOTIFS and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further conoborative evidence that SEQ ID NO:49 is a serine/threonine kinase.
  • HMM hidden Markov model
  • SEQ ID NO.1-2, SEQ ID NO:4-25, SEQ ID NO:27-34, SEQ ID NO:36-48, and SEQ ID NO:50-56 were analyzed and annotated in a simUar manner.
  • the algorithms and parameters for the analysis of SEQ ID NO:l-56 are described in Table 7.
  • the fuU length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Column 1 hsts the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the conesponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence inbasepairs.
  • Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the full length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO:57-l 12 or that distinguish between SEQ ID NO:57-l 12 and related polynucleotides.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA hbraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides.
  • the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (Le., those sequences including the designation "ENST").
  • a polynucleotide sequence identified as FL_XXXXX_N j _N 2 _YYYY_N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was apphed, and YYYYY is the number of the prediction generated by the algorithm, and N ;2, -... > if present, represent specific exons that may have been manuaUy edited during analysis (See Example V).
  • the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
  • a polynucleotide sequence identified as FLXXXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was apphed, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
  • a RefSeq identifier (denoted by " ⁇ M,” “ ⁇ P,” or “NT”) may be used in place of the GenBank identifier (i. e. , gBBBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
  • Table hsts examples of component sequence prefixes and conesponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA libraries for those fuU length polynucleotides which were assembled using Incyte cDNA sequences.
  • the representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides.
  • the tissues and vectors which were used to construct the cDNA hbraries shown in Table 5 are described in Table 6.
  • Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations.
  • Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ID NO:) and the corresponding Incyte project identification number (PID) for polynucleotides of the invention.
  • Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST ID), and column 4 shows the identification number for the SNP (SNP ID).
  • Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the full- length polynucleotide sequence (CB 1 SNP).
  • Column 7 shows the allele found in the EST sequence.
  • Columns 8 and 9 show the two aUeles found at the SNP site.
  • Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the allele found in the EST.
  • Columns 11- 14 show the frequency of allele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of aUele 1 in the population was too low to be detected, while n/a (not available) indicates that the allele frequency was not determined for the population.
  • KPP variants can have at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity to the KPP amino acid sequence, and can contain at least one functional or structural characteristic of KPP.
  • Various embodiments also encompass polynucleotides which encode KPP.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:57-l 12, which encodes KPP.
  • polynucleotide sequences of SEQ ID NO:57-l 12, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occunences of the nitrogenous base thymine are replaced with uracU, and the sugar backbone is composed of ribose instead of deoxyribose.
  • the invention also encompasses variants of a polynucleotide encoding KPP.
  • a variant polynucleotide wiU have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding KPP.
  • a particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:57-l 12 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 NO:57-l 12.
  • a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding KPP.
  • a sphce variant may have portions which have significant sequence identity to a polynucleotide encoding KPP, but wiU generaUy have a greater or lesser number of nucleotides due to additions or deletions of blocks of sequence arising from alternate sphcing during mRNA processing.
  • a sphce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding KPP over its entire length; however, portions of the sphce variant wiU have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding KPP.
  • a polynucleotide comprising a sequence of SEQ ID NO:76, a polynucleotide comprising a sequence of SEQ ID NO:92, and a polynucleotide comprising a sequence of SEQ ID NO:96 are sphce variants of each other;
  • a polynucleotide comprising a sequence of SEQ ID NO:79 and a polynucleotide comprising a sequence of SEQ ID NO:80 are sphce variants of each other;
  • a polynucleotide comprising a sequence of SEQ ID NO: 84 and a polynucleotide comprising a sequence of SEQ ID NO:94 are sphce variants of each other;
  • a polynucleotide comprising a sequence of SEQ ID NO:88 and a polynucleotide comprising a sequence of SEQ ID NO:99 are sphce variants of each other;
  • any one of the sphce variants described above can encode a polypeptide which contains at least one functional or structural characteristic of KPP. It wiU 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 KPP, some bearing minimal simUarity to the polynucleotide sequences of any known and naturally occuning gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as apphed to the polynucleotide sequence of naturaUy occurring KPP, and aU such variations are to be considered as being specificaUy disclosed.
  • polynucleotides which encode KPP and its variants are generaUy capable of hybridizing to polynucleotides encoding naturaUy occuning KPP under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding KPP or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • the invention also encompasses production of polynucleotides which encode KPP and KPP derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic polynucleotide may be inserted into any of the many avaUable expression vectors and ceU systems using reagents weU known in the art.
  • synthetic chemistry may be used to introduce mutations into a polynucleotide encoding KPP or any fragment thereof.
  • Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ID NO:57-l 12 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511). Hybridization conditions, including annealing and wash conditions, are described in "Definitions.”
  • Methods for DNA sequencing are weU known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Apphed Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amphfication system (Invitrogen, Carlsbad CA).
  • sequence preparation is automated with machines such as the MICROLAB 2200 hquid transfer system (HamUton, Reno NV), PTC200 thermal cycler (MJ Research, WatertownMA) and ABI CATALYST 800 thermal cycler (Apphed Biosystems). Sequencing is then canied out using either the ABI 373 or 377 DNA sequencing system (Apphed Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are weU known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Whey VCH, New York NY, pp. 856-853).
  • machines such as the MICROLAB 2200 hquid transfer system (HamUton, Reno NV), PTC200 thermal cycler (MJ Research, WatertownMA) and ABI CATALYST 800 thermal cycler (Apphed Biosystem
  • the nucleic acids encoding KPP 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.
  • various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods Apphc. 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 (Trigha, T. et al.
  • a third method involves PCR amplification of DNA fragments adjacent to known sequences inhuman and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Apphc. 1:111-119).
  • multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
  • primers may be designed using commerciaUy avaUable 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.
  • hbraries that have been size-selected to include larger cDNAs.
  • random-primed hbraries 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 fuU-length cDNA.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commerciaUy avaUable may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capUlary 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, Apphed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controUed.
  • CapUlary electrophoresis is especially preferable for sequencing smaU DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotides or fragments thereof which encode KPP may be cloned in recombinant DNA molecules that direct expression of KPP, or fragments or functional equivalents thereof, in appropriate host cehs. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantiaUy the same or a functionaUy equivalent polypeptides may be produced and used to express KPP.
  • the polynucleotides of the invention can be engineered using methods generaUy known in the art in order to alter KPP-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 ohgonucleotides may be used to engineer the nucleotide sequences.
  • ohgonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce sphce variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of KPP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
  • MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. e
  • 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 prefened variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
  • polynucleotides encoding KPP may be synthesized, in whole or in part, using one or more chemical methods weU known in the art (Caruthers, M.H. et al. (1980)
  • KPP itself or a fragment thereof may be synthesized using chemical methods known in the art.
  • peptide synthesis can be performed using various solution-phase or sohd-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60; Roberge, J.Y. et al. (1995) Science 269:202-204). Automated synthesis maybe achieved using the ABI 431 A peptide synthesizer (Applied Biosystems).
  • the polynucleotides encoding KPP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotides encoding KPP. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding KPP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • Methods which are weU known to those skUled in the art may be used to construct expression vectors containing polynucleotides encoding KPP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook and RusseU, supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1, 3, and 15). A variety of expression vector/host systems may be utilized to contain and express polynucleotides encoding KPP.
  • 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 ceU 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 ceU systems (Sambrook and Russell, supra; Ausubel et al., supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem.
  • 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 ceU systems transformed with viral expression vectors
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350- 356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; Buller, R.M. et al. (1985)
  • a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding KPP.
  • routine cloning, subcloning, and propagation of polynucleotides encoding KPP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Invitrogen).
  • PBLUESCRIPT Stratagene, La JoUa CA
  • PSPORT1 plasmid Invitrogen.
  • 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 (NanHeeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509).
  • vectors which direct high level expression of KPP may be used.
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of KPP.
  • a number of vectors containing constitutive or inducible promoters may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters
  • such vectors direct either the secretion or intraceUular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, CA. et al. (1994) Bio/Technology 12:181-184).
  • Plant systems may also be used for expression of KPP. Transcription of polynucleotides encoding KPP maybe driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence fromTMV (Takamatsu, ⁇ . (1987) EMBO J. 3:1631). Alternatively, plant promoters such as the smaU subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) : Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructs can be introduced into plant ceUs by direct D ⁇ A transformation or pathogen-mediated transfection (The McGraw HUl Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196).
  • a number of viral-based expression systems may be utilized.
  • polynucleotides encoding KPP may be hgated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses KPP in host ceUs (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammahan host ceUs.
  • SV40 or EBV-based vectors may also be used for high-level protein expression.
  • KPP stable expression of KPP in ceU lines
  • polynucleotides encoding KPP can be transformed into ceU 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.
  • cehs may be aUowed 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 aUows growth and recovery of ceUs which successfuUy express the introduced sequences.
  • Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type.
  • any number of selection systems may be used to recover transformed ceU lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk ' and apr ceUs, respectively (Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823). Also, antimetabohte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively
  • trpB and hisD which alter ceUular requirements for metabolites
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; BD Clontech), ⁇ -glucuronidase and its substrate ⁇ -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 (Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131).
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding KPP is inserted within a marker gene sequence
  • transformed ceUs containing polynucleotides encoding KPP can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding KPP under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaUy indicates expression of the tandem gene as well.
  • host cehs that contain the polynucleotide encoding KPP and that express KPP may be identified by a variety of procedures known to those of skill in the art.
  • DNA-DNA or DNA-RNA hybridizations 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 KPP using either specific polyclonal or monoclonal antibodies include enzyme-linked immunosorbent assays (ELISAs), radiohnmunoassays (RIAs), and fluorescence activated ceU sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radiohnmunoassays
  • FACS fluorescence activated ceU sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on KPP is preferred, but a competitive binding assay may be employed.
  • assays are weU known in the art (Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and WUey-Interscience, New York NY; Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ).
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding KPP include oligolabeling, nick translation, end-labeling, or PCR amphfication using a labeled nucleotide.
  • polynucleotides encoding KPP, 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 commerciaUy avaUable, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commerciaUy avaUable kits, such as those provided by Amersham Biosciences, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuchdes, enzymes, fluorescent, chemUuminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host ceUs transformed with polynucleotides encoding KPP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed ceU may be secreted or retained intraceUularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode KPP maybte designed to contain signal sequences which direct secretion of KPP through a prokaryotic or eukaryotic cell membrane.
  • a host ceU strain may be chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
  • 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 ceUs which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are avaUable from the American Type Culture CoUection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture CoUection
  • Manassas VA American Type Culture CoUection
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), fhioredoxin (Trx), cahnoduhn binding peptide (CBP), 6-His, FLAG, c-myc, and heiTOgglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, cahnoduhn, and metal- chelate resins, respectively.
  • a variant of KPP can be used to screen for compounds that bind to a variant of KPP, but not to KPP having the exact sequence of a sequence of SEQ ID NO: 1-56.
  • KPP variants used to perform such screening can have a range of about 50% to about 99% sequence identity to KPP, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
  • a compound identified in a screen for specific binding to KPP can be closely related to the natural receptor to which KPP binds, at least a fragment of the receptor, or a fragment of the receptor including aU or a portion of the ligand binding site or binding pocket.
  • the compound may be a receptor for KPP which is capable of propagating a signal, or a decoy receptor for KPP which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Cun. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328- 336).
  • the compound can be rationally designed using known techniques.
  • Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgGi (Taylor, P.C et al. (2001) Cun. Opin. Immunol. 13:611-616).
  • TNF tumor necrosis factor
  • two or more antibodies having simUar or, alternatively, different specificities can be screened for specific binding to KPP, fragments of KPP, or variants of KPP. The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of KPP.
  • an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of KPP. In another embodiment, an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of KPP.
  • anticalins can be screened for specific binding to KPP, fragments of KPP, or variants of KPP.
  • Anticalins are ligand-binding proteins that have been constructed based on a hpocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177-R184; Skena, A. (2001) J. Biotechnol. 74:257-275).
  • the protein architecture of lipocalins can include a beta-banel having eight antiparallel beta-strands, which supports four loops at its open end.
  • loops form the natural hgand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities.
  • the amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
  • screening for compounds which specifically bind to, stimulate, or inhibit KPP involves producing appropriate cells which express KPP, either as a secreted protein or on the cell membrane.
  • Prefened cells can include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing KPP or cell membrane fractions which contain KPP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either KPP or the compound is analyzed.
  • An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors.
  • examples of such assays include radio-labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No. 6,372,724.
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands (Matthews, DJ. and J.A. Wells. (1994) Chem. Biol. 1:25-30).
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (Cunningham, B.C. and J.A. Wells (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-10988).
  • a polypeptide compound such as a ligand
  • KPP, fragments of KPP, or variants of KPP may be used to screen for compounds that modulate the activity of KPP.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for KPP activity, wherein KPP is combined with at least one test compound, and the activity of KPP in the presence of a test compound is compared with the activity of KPP in the absence of the test compound. A change in the activity of KPP in the presence of the test compound is indicative of a compound that modulates the activity of KPP.
  • a test compound is combined with an in vitro or ceU-free system comprising KPP under conditions suitable for KPP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of KPP 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.
  • polynucleotides encoding KPP or their mammahan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs.
  • ES embryonic stem
  • Such techniques are weU known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337).
  • mouse ES ceUs such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture.
  • the ES ceUs 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.
  • 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 ceU 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 KPP may also be manipulated in vitro in ES cehs 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 ceU lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding KPP can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
  • knockin technology a region of a polynucleotide encoding KPP is injected into animal ES cells, and the injected sequence integrates into the animal ceU genome.
  • Transformed ceUs 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.
  • a mammal inbred to overexpress KPP e.g., by secreting KPP 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 e.g., by secreting KPP in its milk.
  • KPP appears to play a role in cardiovascular diseases, immune system disorders, neurological disorders, disorders affecting growth and development, hpid disorders, ceU proliferative disorders, and cancers.
  • disorders associated with increased KPP expression or activity it is desirable to decrease the expression or activity of KPP.
  • disorders associated with decreased KPP expression or activity it is desirable to increase the expression or activity of KPP.
  • KPP 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 KPP.
  • disorders include, but are not limited to, a cardiovascular disease such as arteriovenous fistula, atherosclerosis, hypertension, vascuhtis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and comphcations of thrombolysis, baUoon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart faUure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse
  • a cardiovascular disease
  • a vector capable of expressing KPP 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 KPP including, but not limited to, those described above.
  • composition comprising a substantiaUy purified KPP 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 KPP including, but not limited to, those provided above.
  • an agonist which modulates the activity of KPP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of KPP including, but not limited to, those hsted above.
  • a vector expressing the complement of the polynucleotide encoding KPP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of KPP including, but not limited to, those described above.
  • any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skUl in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergisticaUy 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.
  • Single chain antibodies may be potent enzyme inhibitors and may have application in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).
  • various hosts including goats, rabbits, rats, mice, camels, dromedaries, Uamas, humans, and others may be immunized by injection with KPP or with any fragment or ohgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • 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, oU emulsions, KLH, and dinitrophenol.
  • BCG BacUli Calmette-Guerin
  • Coiynebacterium pan m are especiaUy preferable.
  • the oligopeptides, peptides, or fragments used to induce antibodies to KPP have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are substantiaUy identical to a portion of the amino acid sequence of the natural protein. Short stretches of KPP 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 KPP may be prepared using any technique which provides for the production of antibody molecules by continuous ceU lines in culture.
  • chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).
  • techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce KPP-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin hbraries (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 hbraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for KPP may also be generated.
  • 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 disuifide bridges of the F(ab')2 fragments.
  • Fab expression hbraries may be constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al. (1989) Science 246:1275-1281).
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity.
  • K a is defined as the molar concentration of KPP-antibody complex divided by the molar concentrations of free antigen and free antibody under equUibrium conditions.
  • K a association constant
  • the K- determined for a preparation of monoclonal antibodies, which are monospecific for a particular KPP epitope, represents a true measure of affinity.
  • Hi ⁇ -affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are prefened for use in immunoassays in which the KPP-antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are prefened for use in immunopurification and simUar procedures which ultimately require dissociation of KPP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; LiddeU, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John WUey & Sons, New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generally employed in procedures requiring precipitation of KPP-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various apphcations, are generaUy avaUable (Catty, supra; Cohgan et al., supra).
  • polynucleotides encoding KPP may be used for therapeutic purposes.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified ohgonucleotides) to the coding or regulatory regions of the gene encoding KPP.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified ohgonucleotides
  • antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding KPP (Agrawal, S., ed. (1996) Antisense Therapeutics. Humana Press, Totawa NJ).
  • Antisense sequences can be delivered intraceUularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein (Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102:469-475; Scanlon, KJ. et al. (1995) FASEB J. 9: 1288- 1296).
  • Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A.D.
  • polynucleotides encoding KPP may be used for somatic or genriline gene therapy.
  • Gene therapy may be performed to (i) conect a genetic deficiency (e.g., in the cases of severe combined i-rnmunodeficiency (SCID)-Xl disease characterized by X- linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al.
  • SCID severe combined i-rnmunodeficiency
  • ADA adenosine deaminase
  • hepatitis B or C virus HBV, HCV
  • fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
  • protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi.
  • the expression of KPP from an appropriate population of transduced ceUs may aUeviate the clinical manifestations caused by the genetic deficiency.
  • diseases or disorders caused by deficiencies in KPP are treated by constructing i-mmmahan expression vectors encoding KPP and introducing these vectors by mechanical means into KPP-deficient ceUs.
  • Mechanical transfer technologies for use with cehs in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (ii) baUistic gold particle dehvery, (iii) hposome-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 KPP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (BD Clontech, Palo Alto CA).
  • KPP may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -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.N. and H.M. Blau (1998) Curr. Opin. Biotechnol.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
  • CommerciaUy avaUable hposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, avaUable from Invitrogen
  • aUow one with ordinary skill in the art to dehver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters.
  • transformation is performed using the calcium phosphate method (Graham, F.L. and AJ. 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 ceUs requires modification of these standardized mammahan transfection protocols.
  • diseases or disorders caused by genetic defects with respect to KPP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding KPP under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (hi) a Rev-responsive element (RRE) along with additional retrovirus cw-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commerciaUy available (Stratagene) and are based on pubhshed data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
  • the vector is propagated in an appropriate vector producing ceU line (VPCL) that expresses an envelope gene with a tropism for receptors on the target ceUs 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. MUler (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol. 72:8463-8471 ; Zufferey, R. et al.
  • VSVg vector producing ceU line
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging ceU lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of ceUs (e.g., CD4 + T- ceUs), and the return of transduced ceUs to a patient are procedures well known to persons skilled in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Virol. 71:7020- 7029; Bauer, G. et al.
  • an adenovirus-based gene therapy dehvery system is used to dehver polynucleotides encoding KPP to ceUs which have one or more genetic abnormalities with respect to the expression of KPP.
  • the construction and packaging of adenovirus-based vectors are weU known to those with ordinary skUl in the art.
  • Replication defective adenovirus vectors have proven to be versatUe for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268).
  • PotentiaUy useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference.
  • Adenovirus vectors for gene therapy For adenoviral vectors, see also Antinozzi, P. A. et al. (1999;
  • a herpes-based, gene therapy dehvery system is used to dehver polynucleotides encoding KPP to target cells which have one or more genetic abnormalities with respect to the expression of KPP.
  • the use of herpes simplex virus (HS V)-based vectors may be especiaUy valuable for introducing KPP to ceUs of the central nervous system, for which HS V has a tropis
  • the construction and packaging of herpes-based vectors are weU known to those with ordinary skUl in the art.
  • a rephcation-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.
  • HSV-1 virus vector has also been disclosed in detaU in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
  • U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a ceU under the control of the appropriate promoter for purposes including human gene therapy. Also tauglit by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
  • HSV vectors see also Goins, W.F. et al. (1999; J. Virol. 73:519-532) and Xu, H. et al. (1994; Dev. Biol. 163:152-161).
  • the manipulation of cloned herpesviras sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesviras genomes, the growth and propagation of herpesviras, and the infection of ceUs with herpesviras are tecliniques weU known to those of ordinary skUl in the art.
  • an alphaviras (positive, single-stranded RNA virus) vector is used to dehver polynucleotides encoding KPP to target ceUs.
  • SFV Semliki Forest Virus
  • SFV Semliki Forest Virus
  • 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).
  • enzymatic activity e.g., protease and polymerase.
  • WhUe alphaviras infection is typicaUy associated with ceU lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cehs (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 apphcation (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the specific transduction of a subset of ceUs in a population may require the sorting of ceUs prior to transduction.
  • the methods of manipulating infectious cDNA clones of alphaviruses, performing alphaviras cDNA and RNA transfections, and performing alphaviras infections, are weU known to those with ordinary skUl in the art.
  • Ohgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
  • inhibition can be achieved using triple hehx base-pairing methodology.
  • Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Can, Molecular and fmmunologic 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, foUowed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze t endonucleolytic cleavage of RNA molecules encoding KPP.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the foUowing sequences: GUA, GUU, and GUC.
  • RNA sequences of between 15 and 20 ribonucleotides 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 ohgonucleotides using ribonuclease protection assays.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA molecules encoding KPP. 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 ceU lines, ceUs, or tissues.
  • RNA molecules may be modified to increase intraceUular 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-mefhyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • This concept is inherent in the production of PNAs and can be extended in aU of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and simUarly modified forms of adenine, cytosine, guanine, thymine, and uracU which are not as easUy recognized by endogenous endonucleases.
  • nontraditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and simUarly modified forms of adenine, cytosine, guanine, thymine, and uracU which are not as easUy recognized by endogenous endonucleases.
  • RNAi RNA interference
  • PTGS post-transcriptional gene silencing
  • RNAi is a post-transcriptional mode of gene sUencing in which double-stranded RNA (dsRNA) introduced into a targeted cell specificaUy suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantiaUy reduces the expression of the targeted gene.
  • dsRNA double-stranded RNA
  • PTGS can also be accomplished by use of DNA or DNA fragments as well.
  • RNAi methods are described by Fire, A. et al. (1998; Nature 391 :806-811) and Gura, T. (2000; Nature 404:804-808).
  • PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene dehvery and/or viral vector dehvery methods described herein or known in the art.
  • RNAi can be induced in mammahan ceUs by the use of smaU interfering RNA also known as siRNA.
  • siRNA are shorter segments of dsRNA (typicaUy about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease.
  • siRNA appear to be the mediators of the RNAi effect in mammals. The most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3' overhangs.
  • the use of siRNA for inducing RNAi in mammahan ceUs is described by Elbasbir, S.M. et al.
  • siRNA can be generated indirectly by introduction of dsRNA into the targeted ceU.
  • siRNA can be synthesized directly and introduced into a ceU by transfection methods and agents described herein or known in the art (such as hposome-mediated transfection, viral vector methods, or other polynucleotide dehvery/introductory methods).
  • Suitable siRNAs can be selected by examining a transcript of the target polynucleotide (e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occurrence of each nucleotide and the 3 ' adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being preferred. Regions to be avoided for target siRNA sites include the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex.
  • UTRs untranslated regions
  • the selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be eliminated from consideration.
  • the selected siRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commerciaUy avaUable methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin TX).
  • long-term gene sUencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA.
  • This can be accomplished using expression vectors that are engineered to express hairpin RNAs (shRNAs) using methods known in the art (see, e.g., Brammelkamp, T.R. et al. (2002) Science 296:550-553; and Paddison, P.J. et al. (2002) Genes Dev. 16:948-958).
  • shRNAs can be delivered to target ceUs using expression vectors known in the art.
  • siRNA An example of a suitable expression vector for delivery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion).
  • PSILENCER1.0-U6 circular plasmid
  • shRNAs are processed in vivo into siRNA-like molecules capable of carrying out gene-specific silencing.
  • the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis.
  • Expression levels of the mRNA of a targeted gene can be determined, for example, by northern analysis methods using the NORTHERNMAX-GLY kit (Ambion); by microarray methods; by PCR methods; by real time PCR methods; and by other RNA/polynucleotide assays known in the art or described herein.
  • Expression levels of the protein encoded by the targeted gene can be determined, for example, by microarray methods; by polyacrylamide gel electrophoresis; and by Western analysis using standard techniques known in the art.
  • a compound which specifically inhibits expression of the polynucleotide encoding KPP may be therapeutically useful, and in the treatment of disorders associated with decreased KPP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding KPP may be therapeutically useful.
  • 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.
  • a particular embodiment of the present invention involves screening a combinatorial library of ohgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides) for antisense activity against a specific polynucleotide sequence (Braice, 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 ceUs or tissues are avaUable and equaUy suitable for use in vivo, in vitro, and ex vivo.
  • ohgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides
  • vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. Dehvery by transfection, by hposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art (Goldman, CK. et al. (1997) Nat. Biotechnol. 15:462-466).
  • compositions which generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
  • Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins.
  • formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
  • Such compositions may consist of KPP, antibodies to KPP, and mimetics, agonists, antagonists, or inhibitors of KPP.
  • compositions described herein may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • Compositions for pulmonary administration may be prepared in hquid or dry powder form
  • compositions may be prepared for direct intraceUular dehvery of macromolecules comprising KPP or fragments thereof.
  • hposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular delivery of the macromolecule.
  • KPP 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 ceUs of aU tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • a therapeuticaUy effective dose refers to that amount of active ingredient, for example KPP or fragments thereof, antibodies of KPP, and agonists, antagonists or inhibitors of KPP, which amehorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in ceU cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD 50 (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 LD 50 /ED 50 ratio. Compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from ceU 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 ED 50 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, drag combinations), 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-hfe and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, 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 generaUy available to practitioners in the art. Those skUled in the art wUl employ different formulations for nucleotides than for proteins or their inhibitors. SimUarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS
  • polynucleotides encoding KPP may be used for diagnostic purposes.
  • the polynucleotides which may be used include ohgonucleotides, 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 KPP may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of KPP, and to monitor regulation of KPP levels during therapeutic intervention.
  • Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the KPP encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:57-l 12 or from genomic sequences including promoters, enhancers, and introns of the KPP gene.
  • Means for producing specific hybridization probes for polynucleotides encoding KPP include the cloning of polynucleotides encoding KPP or KPP derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, are commerciaUy 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 radionuchdes such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotides encoding KPP may be used for the diagnosis of disorders associated with expression of KPP.
  • disorders include, but are not limited to, a cardiovascular disease such as arteriovenous fistula, atherosclerosis, hypertension, vascuhtis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebofhrombosis, vascular tumors, and comphcations of thrombolysis, bahoon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart faUure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitaUy bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and
  • Polynucleotides encoding KPP 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 KPP expression. Such qualitative or quantitative methods are weU known in the art.
  • polynucleotides encoding KPP may be used in assays that detect the presence of associated disorders, particularly those mentioned above.
  • Polynucleotides complementary to sequences encoding KPP 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.
  • the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding KPP 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 KPP, a normal or standard profile for expression is estabhshed.
  • 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 estabhsh the presence of a disorder.
  • 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.
  • 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 aUow health professionals to employ preventative measures or aggressive treatment earher, thereby preventing the development or further progression of the cancer.
  • Additional diagnostic uses for ohgonucleotides designed from the sequences encoding KPP may involve the use of PCR. These ohgomers may be chemicaUy synthesized, generated enzymatically, or produced in vitro.
  • Ohgomers will preferably contain a fragment of a polynucleotide encoding KPP, or a fragment of a polynucleotide complementary to the polynucleotide encoding KPP, and wUl be employed under optimized conditions for identification of a specific gene or condition. Ohgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • ohgonucleotide primers derived from polynucleotides encoding KPP 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.
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • ohgonucleotide primers derived from polynucleotides encoding KPP are used to amplify DNA using the polymerase chain reaction (PCR).
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
  • SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the oligonucleotide primers are fluorescently labeled, which aUows detection of the amplimers in gh-throughput equipment such as DNA sequencing machines.
  • Additionahy, sequence database analysis methods, termed in sUico SNP (isSNP) are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA). SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes meUitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceU anemia, or chronic granulomatous disease.
  • variants in the mannose-binding lectin, MBL2 have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis.
  • SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as hfe-threatening toxicity.
  • a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid
  • wlhle a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drag that targets the 5-hpoxygenase pathway.
  • Methods which may also be used to quantify the expression of KPP include radiolabehng or biotinylating nucleotides, coamphfication of a control nucleic acid, and interpolating results from standard curves (Melby, P.C et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem 212:229-236).
  • the speed of quantitation of multiple samples maybe accelerated by ranning the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dUutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • ohgonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray.
  • the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microanay 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.
  • 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.
  • therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • KPP, fragments of KPP, or antibodies specific for KPP may be used as elements on a microarray.
  • the microarray may be used to monitor or measure protein-protein interactions, drag-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 ceU type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or ceU type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions
  • 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 ceU type.
  • the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurahty of elements on a microarray.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images maybe generated using transcripts isolated from tissues, ceU 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 weU as toxicological testing of industrial and naturaUy-occurring envhonmental compounds.
  • AU 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.
  • test compound has a signature similar to that of a compound with known toxicity, it is hkely to share those toxic properties.
  • 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 quahty signature. Even genes whose expression is not altered by any tested compounds are important as weU, 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.
  • the toxicity of a test compound can be assessed by treating a biological sample containing nucleic acids with the test compound.
  • Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels conesponding 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.
  • proteome refers to the global pattern of protein expression in a particular tissue or ceU type.
  • 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 ceU's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU type.
  • the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
  • the proteins are visuahzed in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or sUver or fluorescent stains.
  • the optical density of each protein spot is generaUy proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment.
  • the proteins in the spots are partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage foUowed by mass spectrometry.
  • the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data may be obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for KPP to quantify the levels of KPP expression.
  • the antibodies are used as elements on a microarray, and protein expression levels are quantified by contacting the microarray with the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each anay element.
  • Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in paraUel 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. SeUhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more rehable and informative in such cases.
  • 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.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Microarrays may be prepared, used, and analyzed using methods known in the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; BaldeschweUer et al. (1995) PCT apphcation W095/25116; Shalon, D. et al. (1995) PCT apphcation WO95/35505; HeUer, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; Heller, MJ. et al. (1997) U.S.
  • nucleic acid sequences encoding KPP may be used to generate hybridization probes useful in mapping the naturaUy 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.
  • 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 PI constructions, or single chromosome cDNA hbraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, B J. (1991) Trends Genet. 7:149-154).
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA hbraries
  • the nucleic acid sequences may be used to develop genetic linkage maps, for example, which conelate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357).
  • Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data (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 Mendehan Inheritance in Man (OMIM) World Wide Web site. Conelation between the location of the gene encoding KPP 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.
  • RFLP restriction fragment length polymorphism
  • KPP its catalytic or immunogenic fragments, or ohgopeptides thereof can be used for screening hbraries 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 sohd support, borne on a cell surface, or located intraceUularly. The formation of binding complexes between KPP and the agent being tested may be measured.
  • Another technique for drag screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al. (1984) PCT apphcation WO84/03564).
  • large numbers of different smaU test compounds are synthesized on a sohd substrate.
  • the test compounds are reacted with KPP, or fragments thereof, and washed.
  • Bound KPP is then detected by methods weU known in the art.
  • Purified KPP can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobihze it on a sohd support.
  • nucleotide sequences which encode KPP 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 hmited to, such properties as the triplet genetic code and specific base pair interactions.
  • Incyte cDNAs are derived from cDNA hbraries described in the LIFESEQ database (Incyte, Palo Alto CA). Some tissues are homogenized and lysed in guanidinium isothiocyanate, while others are homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates are centrifuged over CsCl cushions or extracted with chloroform. RNA is precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • TRIZOL Invitrogen
  • RNA is treated with DNase.
  • poly(A)+ RNA is isolated using ohgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • RNA is isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
  • the cDNA is size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
  • cDNAs are hgated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen, Carlsbad CA), PCDNA2.1 plasmid (Invitrogen), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte, Palo Alto CA), pRARE (Incyte), or pINCY (Incyte), or derivatives thereof.
  • Recombinant plasmids are transformed into competent E. coli cehs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Invitrogen.
  • Plasmids obtained as described in Example I are recovered fromhost ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU lysis. Plasmids are 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. FoUowing precipitation, plasmids are resuspended in 0.1 ml of distUled water and stored, with or without lyophUization, at 4°C
  • plasmid DNA is amplified fromhost cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Bioche 216:1-14). Host ceU lysis and thermal cycling steps are carried out in a single reaction mixture. S mples are processed and stored in 384- well plates, and the concentration of amplified plasmid DNA is quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
  • PICOGREEN dye Molecular Probes, Eugene OR
  • FLUOROSKAN II fluorescence scanner Labsystems Oy, Helsinki, Finland.
  • Incyte cDNA recovered in plasmids as described in Example II are sequenced as follows. Sequencing reactions are processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Apphed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (HamUton) hquid transfer system. cDNA sequencing reactions are prepared using reagents provided by Amersham Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems).
  • Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides are carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Apphed 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 are identified using standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences are selected for extension using the techniques disclosed in Example VIII.
  • Polynucleotide sequences derived from Incyte cDNAs are validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic progran-tming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof are then queried against a selection of pubhc databases such as the GenBank primate, rodent, mammahan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte, Palo Alto CA); hidden Markov model (HMM)-based protein famUy databases such as PFAM, INCY, and TIGRFAM (Haft, D.H.
  • HMM hidden Markov model
  • HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244).
  • HMM is a probabilistic approach which analyzes consensus primary structures of gene families; see, for example, Eddy, S.R. (1996) Cun. Opin. Struct. Biol. 6:361-365.
  • the queries are performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
  • the Incyte cDNA sequences are assembled to produce full length polynucleotide sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences are used to extend Incyte cDNA assemblages to full length. Assembly is performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages are screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
  • the full length polynucleotide sequences are translated to derive the corresponding full length polypeptide sequences.
  • a polypeptide may begin at any of the rrethionine residues of the full length translated polypeptide.
  • FuU length polypeptide sequences are subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, bidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART.
  • GenBank protein databases Genpept
  • PROTEOME databases
  • BLOCKS BLOCKS
  • PRINTS DOMO
  • PRODOM Prosite
  • Prosite bidden Markov model
  • Prosite bidden Markov model
  • HMM-based protein family databases such as PFAM, INCY, and TIGRFAM
  • HMM-based protein domain databases such as SMART.
  • Polynucleotide and polypeptide sequence ahgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence ahgnment program (DNASTAR), which also calculates the percent identity between ahgned sequences.
  • Table 7 summarizes tools, programs, and dgorithms used for the analysis and assembly of Incyte cDNA and fuU length sequences and provides apphcable 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, aU of which are incorporated by reference herein in their entirety, and the fourth column presents, where apphcable, the scores, probabihty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabihty value, the greater the identity between two sequences).
  • Putative kinases and phosphatases are initiaUy identified by ranning the Genscan gene identification program against pubhc genomic sequence databases (e.g., gbpri and gbhtg).
  • Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
  • Genscan is a FASTA database of polynucleotide and polypeptide sequences.
  • the maximum range of sequence for Genscan to analyze at once is set to 30 kb.
  • the encoded polypeptides are analyzed by querying against PFAM models for kinases and phosphatases. Potential kinases and phosphatases are also identified by homology to Incyte cDNA sequences that have been annotated as kinases and phosphatases. These selected Genscan-predicted sequences are then compared by BLAST analysis to the genpept and gbpri pubhc databases.
  • Genscan-predicted sequences are then edited by comparison to the top BLAST hit from genpept to conect errors in the sequence predicted by Genscan, such as extra or omitted exons.
  • BLAST analysis is also used to find any Incyte cDNA or pubhc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.
  • Incyte cDNA coverage is avaUable, this information is used to conect or confirm the Genscan predicted sequence.
  • FuU length polynucleotide sequences are obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubhc cDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences are derived entirely from edited or unedited Genscan-predicted coding sequences.
  • Partial cDNA sequences are extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III are mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster is analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that are subsequently confirmed, edited, or extended to create a fuU length sequence. Sequence intervals in which the entire length of the interval is present on more than one sequence in the cluster are identified, and intervals thus identified are considered to be equivalent by transitivity.
  • Partial DNA sequences are extended to fuU length with an algorithm based on BLAST analysis.
  • First, partial cDNAs assembled as described in Example III are queried against pubhc databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program
  • the nearest GenBank protein homolog is then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV.
  • a chimeric protein is 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.
  • HSPs high-scoring segment pairs
  • GenBank protein homolog the cWmeric protein, or both are used as probes to search for homologous genomic sequences from the pubhc human genome databases. Partial DNA sequences are therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences are examined to detercnine whether they contain a complete gene.
  • sequences used to assemble SEQ ID NO:57-l 12 are 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:57-l 12 are 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 are used to determine if any of the clustered sequences have been previously mapped. Inclusion of a mapped sequence in a cluster results in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
  • SHGC Stanford Human Genome Center
  • WIGR Whitehead Institute for Genome Research
  • Map locations are represented by ranges, or intervals, of human chromosomes.
  • the map position of an interval, in centiMorgans is measured relative to the te ⁇ riinus of the chromosome's p- arm.
  • centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
  • the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters.
  • 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 frorn a particular ceU type or tissue have been bound (Sambrook and RusseU, supra, ch. 7; Ausubel et al., supra, ch. 4).
  • Analogous computer techniques applying BLAST are used to search for identical or related molecules in databases such as GenBank or LIFESEQ (Incyte). This analysis is much faster than multiple membrane-based hybridizations.
  • the sensitivity of the computer search can be modified to detennine whether any particular match is categorized as exact or simUar.
  • the basis of the search is the product score, which is defined as:
  • Each cDNA sequence is derived from a cDNA hbrary constructed from a human tissue.
  • Each human tissue is classified into one of the foUowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitaha, female; genitaha, male; germ ceUs; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of hbraries in each category is counted and divided by the total number of hbraries across aU categories.
  • Selected human cDNA hbraries are used to extend the sequence. If more than one extension is necessary or desired, additional or nested sets of primers are designed. High fidelity amphfication is obtained by PCR using methods weU known in the art. PCR is performed in 96-weU plates using the PTC-200 thermal cycler (MJ Research, Inc.).
  • the reaction mix contains DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the foUowing parameters for primer pah PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C
  • the parameters for primer pair T7 and SK+ are as foUows: 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
  • the concentration of DNA in each weU is determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowrng the DNA to bind to the reagent.
  • the plate is 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 ⁇ l to 10 ⁇ l aliquot of the reaction mixmre is analyzed by electrophoresis on a 1 % agarose gel to determine which reactions are successful in extending the sequence.
  • the extended nucleotides are desalted and concentrated, transferred to 384-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WT), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Biosciences).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WT
  • sonicated or sheared prior to religation into pUC 18 vector
  • the digested nucleotides are separated on low concentration (0.6 to 0.8%) agarose gels, fragments are 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 Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli ceUs. Transformed ceUs are selected on antibiotic-containing media, and individual colonies are picked and cultured overnight at 37 °C in 384-weU plates in LB/2x carb hquid media.
  • the ceUs are lysed, and DNA is amplified by PCR using Taq DNA polymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the foUowing 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 is quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries are reamplified using the same conditions as described above.
  • Samples are dUuted with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems).
  • Certain SNPs are selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations.
  • the Caucasian population comprises 92 individuals (46 male, 46 female), including 83 from Utah, four French, three deciualan, and two Amish individuals.
  • the African population comprises 194 individuals (97 male, 97 female), all African Americans.
  • the Hispanic population comprises 324 individuals (162 male, 162 female), all Mexican Hispanic.
  • the Asian population comprises 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. Allele frequencies are first analyzed in the Caucasian population; in some cases those SNPs which show no allelic variance in this population are not further tested in the other three populations.
  • Hybridization probes derived from SEQ ID NO.57-112 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of ohgonucleotides, consisting of about 20 base pahs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments.
  • Ohgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 p ol of each ohgomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont).
  • the labeled ohgonucleotides are substantiaUy purified using a SEPHADEX G- 25 superfine size exclusion dextran bead column (Amersham Biosciences). An ahquot containing 10 7 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 foUowing endonucleases: Ase I, Bgl II, Eco Rl, 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 NYTRAN PLUS nylon membranes (Schleicher & SchueU, 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.
  • the linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing; see, e.g., Baldeschweiler et al., supra), mechanical microspotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and sohd with a non-porous surface (Schena, M., ed. (1999) DNA Microarrays: A Practical Approach, Oxford University Press, London). Suggested substrates include silicon, silica, glass shdes, glass chips, and silicon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using avaUable methods and machines weU known to those of ordinary skill in the art and may contain any appropriate number of elements (Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; MarshaU, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).
  • Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or ohgomers thereof may comprise the elements of the microanay. Fragments or ohgomers suitable for hybridization can be selected using software weU known in the art such as LASERGENE software (DNASTAR).
  • the anay elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • 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.
  • microarray preparation and usage is described in detaU below.
  • 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/ ⁇ l oligo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMB RIGHT kits (Incyte).
  • Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA.
  • Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (BD Clontech, Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ⁇ l 5X SSC/0.2% SDS.
  • SpeedVAC SpeedVAC
  • Sequences of the present invention are used to generate anay elements.
  • Each anay element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
  • PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. Amplified anay elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
  • Purified anay 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-Aldrich, St. Louis MO) in 95% ethanol. Coated shdes are cured in a 110 C C oven.
  • Array elements are apphed to the coated glass substrate using a procedure described in U.S. Patent No. 5,807,522, incorporated herein by reference.
  • 1 ⁇ l of the anay element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capUlary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of anay element sample per shde.
  • Microanays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microanays are washed at room temperature once in 0.2% SDS and three times in distUled water. Non-specific binding sites are blocked by incubation of microanays 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.
  • PBS phosphate buffered saline
  • the chamber containing the anays is incubated for about 6.5 hours at 60°C
  • the arrays are washed for 10 min at 45°C in a first wash buffer (IX SSC, 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 Cy5.
  • the excitation laser light is focused on the anay using a 20X microscope objective (Nikon, Inc., MelviUe NY).
  • the slide containing the anay 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 anay used in the present example is scanned with a resolution of 20 micrometers.
  • a mixed gas multiline laser excites the two fluorophores sequentiaUy. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals.
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each anay 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 typicaUy 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 anay contains a complementary DNA sequence, allowing the intensity of the signal at that location to be conelated with a weight ratio of hybridizing species of 1 : 100,000.
  • the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first conected 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 conesponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). Array elements that exhibit at least about a two-fold change in expression, a signal-to-background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentially expressed.
  • SEQ ID NO:62, SEQ ID NO:93, SEQ ID NO:95, and SEQ ID NO:100 showed differential expression in colon cancer samples, as determined by microarray expression analysis.
  • Expression of SEQ ID NO:62 was upregulated by at least two-fold in matched normal versus tumorous colon tissues in one out of thirteen donors tested.
  • Expression of SEQ ID NO:93 was upregulated by at least two-fold in matched mmorous versus normal colon tissues in two out of six donors tested and expression of SEQ ID NO:95 was upregulated by at least two-fold in matched tumorous versus normal colon tissues in one out of fourteen donors tested.
  • Expression of SEQ ID NO:100 showed differential expression in colon cancer samples, as determined by microarray expression analysis.
  • SEQ ID NO:62, SEQ ID NO:93, SEQ ID NO:95, and SEQ ID NO:100 can be used for one or more of the foUowing: i) monitoring treatment of colon cancer, ii) diagnostic assays for colon cancer, and hi) developing therapeutics and/or other treatments for colon cancer.
  • SEQ ID NO:62-63, SEQ ID NO:82-83, SEQ ID NO:89, and SEQ ID NO:97 showed differential expression in lung cancer samples, as determined by microanay expression analysis.
  • Expression of SEQ ID NO:62 was upregulated by at least two-fold in matched normal versus tumorous lung tissues in five out of ten donors tested and downregulated by at least two-fold in matched normal versus tumorous lung tissues in one out of ten donors tested. Different donors could have different primary lesions and the differential regulation of the expression of SEQ ID NO: 62 in the tumorous tissues from different donors might be an indicator of this. Further, expression of SEQ ID NO:63 was upregulated by at least two-fold in matched normal versus tumorous lung tissues in one out of six donors tested, as determined by microarray analysis.
  • expression of SEQ ID NO: 82 was down-regulated in lung tumor tissue versus matched normal lung tissue as determined by microarray analysis. Expression of SEQ ID NO:82 was decreased at least two-fold in lung tumor tissue as compared to normal tissue from the same donor in one out of ten donors. In a simUar experiment, expression of SEQ ID NO:83 was up- regulated in lung tumor tissue versus matched normal lung tissue as determined by microarray analysis. Expression of SEQ ID NO:83 was decreased at least two-fold in lung tumor tissue as compared to normal tissue from the same donor in three out of four donors.
  • SEQ ID NO:89 and SEQ ID NO:97 were found to be downregulated by at least two-fold in matched tumorous versus normal lung tissues in one out of ten donors tested. They were both downregulated in the same donor in independently done experiments. Therefore, in various embodiments, SEQ ID NO:62-63, SEQ ID NO:82-83, SEQ ID NO:89, and/or SEQ ID NO:97 can be used for one or more of the foUowing: i) monitoring treatment of lung cancer, ii) diagnostic assays for lung cancer, and hi) developing therapeutics and/or other treatments for lung cancer.
  • expression of SEQ ID NO:62 was upregulated by at least two-fold in matched normal versus tumorous ovarian tissues in one donor tested in two different experiments, as determined by microarray analysis. Further, expression of SEQ ID NO:l 11 was differentiaUy expressed in ovarian tumor samples, as determined by microarray expression analysis. Tissue from a normal ovary from a 79 year-old female donor was compared to an ovarian tumor tissue from the same donor (Huntsman Cancer Institute, Salt Lake City, UT). Expression of SEQ ID NO:l 11 was increased at least 2-fold in the tumor tissue versus the normal tissue.
  • SEQ ID NO:62 and SEQ ID NO: 111 can be used for one or more of the foUowing: i) monitoring treatment of ovarian cancer, h) diagnostic assays for ovarian cancer, and iii) developing therapeutics and/or other treatments for ovarian cancer.
  • BT-20 is a breast carcinoma ceU line derived in vitro from the cells emigrating out of thin shces of the tumor mass isolated from a 74-year-old female.
  • BT-20 ceUs were treated with ⁇ - estradiol for 4, 8, 14, 24, 36, and 48 hours. These treated cells were compared to untreated BT-20 ceUs kept in culture for the same amount of time.
  • SEQ ID NO:88 was found to be downregulated by at least two-fold in the treated ceUs after four hours of treatment and continued to be downregulated by at least two-fold untU 36 hours of treatment.
  • MDA-mb-231 is a breast tumor ceh line isolated from the pleural effusion of a 51- year old female.
  • SEQ ID NO:94 can be used for one or more of the following: i) monitoring treatment of immune disorders and related diseases and conditions, ii) diagnostic assays for immune disorders and related diseases and conditions, and hi) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.
  • SEQ ID NO: 100 showed differential expression in activated vascular endothehum, as determined by microarray expression analysis.
  • the antisense ohgonucleotide was an 18mer shown to specificaUy target the degradation of the WSB-1 mRNA (Incyte gene ID 423565, GenBank g454669) under the transfection conditions employed.
  • SEQ ID NO:100 can be used for one or more of the foUowing: i) monitoring treatment of diseases or disorders involving activated vascular endothehum, ii) diagnostic assays for diseases or disorders involving activated vascular endothehum, and hi) developing therapeutics and/or other treatments for diseases or disorders involving activated vascular endothehum.
  • SEQ ID NO:93 and SEQ ID NO:100 showed differential expression in Tangier disease-derived fibroblasts, as determined by microarray expression analysis.
  • Human fibroblasts were obtained from skin explants from both normal subjects and two patients with homozygous Tangier disease.
  • Cell lines were immortalized by transfection with human papUlomavirus 16 genes E6 and E7 and a neomycin resistance selectable marker.
  • both types of ceUs were cultured in the presence of cholesterol and compared with the same cell type cultured in the absence of cholesterol.
  • TD derived cells are shown to be deficient in an assay of apoA-I mediated tritiated cholesterol efflux.
  • SEQ ID NO:93 and SEQ ID NO:100 showed increased expression of at least 2-fold in the TD-derived ceUs, when compared to normal fibroblasts. The same result was seen irrespective of treatment with cholesterol. Therefore, in various embodiments, SEQ ID NO:93 and SEQ ID NO:100 can be used for one or more of the foUowing: i) monitoring treatment of Tangier disease, ii) diagnostic assays for Tangier disease, and hi) developing therapeutics and/or other treatments for Tangier disease.
  • SEQ ID NO:l 11 expression increased at least 2-fold in treated C3A ceUs, compared to untreated C3A ceUs, at various time points and concentrations: beclomethasone - 10 ⁇ M for 3 h; MAH - 1 ⁇ M for 6 h, 10 ⁇ M for 1, 3, or 6 h, and 100 ⁇ M for 1 h; budesonide - 10 ⁇ M for 1 or 3 h and 100 ⁇ M for 1 h; Dex - 100 ⁇ M for 6 h; betamethasone - 1 ⁇ M for 1 or 6 h, 10 ⁇ M for 6 h, and 100 ⁇ M for 1, 3, or 6 h; and catechol - 10 ⁇ M for 3 h. Therefore, in various embodiments, SEQ ID NO:l 11 can be used for monitoring treatment with steroids or other potentiaUy toxic compounds.
  • SEQ ID NO:62 and SEQ ID NO:71 showed tissue-specific expression as determined by microanay analysis.
  • RNA samples isolated from a variety of normal human tissues were compared to a common reference sample. Tissues contributing to the reference sample were selected for their abihty to provide a complete distribution of RNA in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), small intestine (9%), spleen (3%), stomach (6%), testis (9%), and uterus (5%).
  • the normal tissues assayed were obtained from at least three different donors. RNA from each donor was separately isolated and individuaUy hybridized to the microarray. Since these hybridization experiments were conducted using a common reference sample, differential expression values are directly comparable from one tissue to another.
  • SEQ ID NO:62, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79-80, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95-97 showed tissue-specific expression as determined by microarray analysis.
  • RNA samples isolated from a variety of normal human tissues were compared to a common reference sample. Tissues contributing to the reference sample were selected for their ability to provide a complete distribution of RNA in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), smaU intestine (9%), spleen (3%), stomach (6%), testis (9%), and uterus (5%).
  • the normal tissues assayed were obtained from at least three different donors. RNA from each donor was separately isolated and individuaUy hybridized to the microarray. Since these hybridization experiments were conducted using a common reference sample, differential expression values are directly comparable from one tissue to another.
  • the expression of SEQ ID NO:62 was increased by at least two-fold in lung and spleen tissues as compared to the reference sample. Therefore, SEQ ID NO:62 can be used as a tissue marker for lung and spleen tissues.
  • the expression of SEQ ID NO:71 was increased by at least two-fold in heart ventricular tissue as compared to the reference sample. Therefore, SEQ ID NO:71 can be used as a tissue marker for heart ventricular tissue.
  • SEQ ID NO:89 and SEQ ID NO:97 can be used as tissue markers for blood leukocytes.
  • the expression of SEQ ID NO:73 was increased by at least two-fold in leukocytes, fhymus, spleen, and tonsU as compared to the reference sample. Therefore, SEQ ID NO:73 can be used as a tissue marker for leukocytes, thymus, spleen, and tonsU.
  • the expression of SEQ ID NO:87 was increased by at least two-fold in tonsU tissue as compared to the reference sample. Therefore, SEQ ID NO:87 can be used as a tissue marker for tonsU tissue.
  • the expression of SEQ ID NO:95 was increased by at least two-fold in omentum tissue as compared to the reference sample. Therefore, SEQ ID NO:95 can be used as a tissue marker for omentum tissue.
  • Sequences complementary to the KPP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring KPP.
  • ohgonucleotides comprising from about 15 to 30 base pahs is described, essentiahy the same procedure is used with smaUer or with larger sequence fragments.
  • Appropriate ohgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of KPP.
  • a complementary ohgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary ohgonucleotide is designed to prevent ribosomal binding to the KPP-encoding transcript.
  • KPP KPP expression and purification of KPP is achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not limited to, the tip-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express KPP upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
  • KPP in eukaryotic ceUs is achieved by infecting insect or mammahan ceU lines with recombinant Autographica califomica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographica califomica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding KPP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases.
  • GST a 26- I lodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences). FoUowing purification, the GST moiety can be proteolyticaUy cleaved from KPP at specificaUy engineered sites.
  • FLAG an 8-amino acid peptide, enables irnmunoaffinity purification using commerciaUy avaUable monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN).
  • KPP function is assessed by expressing the sequences encoding KPP at physiologically elevated levels in mammalian ceU culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cyto episcopovirus promoter. 5-10 g of recombinant vector are transiently transfected into a human cell hne, for example, an endothelial or hematopoietic ceU line, using either hposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected ceUs from nontransfected cells and is a rehable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; BD Clontech), CD64, or a CD64-GFP fusion protein.
  • Flow cytometry (FCM) an automated, laser optics-based technique, is used to identify transfected cehs expressing GFP or CD64-GFP and to evaluate the apoptotic state of the ceUs and other ceUular properties.
  • the influence of KPP on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encoding KPP and either CD64 or CD64-GFP.
  • CD64 and CD64- GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected ceUs are efficiently separated fromnontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the ceUs using methods weU known by those of skUl in the art. Expression of mRNA encoding KPP and other genes of interest can be analyzed by northern analysis or microanay techniques. l
  • the KPP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a conesponding ohgopeptide is synthesized and used to raise antibodies by means known to those of skUl in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophihc regions are weU described in the art (Ausubel et al., supra, ch. 11).
  • ohgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Apphed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysucciiiimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the ohgopeptide-KLH complex in complete Freund's adjuvant.
  • ABI 431 A peptide synthesizer Apphed Biosystems
  • KLH Sigma- Aldrich, St. Louis MO
  • MBS N-maleimidobenzoyl-N-hydroxysucciiiimide ester
  • Resulting antisera are tested for antipeptide and anti-KPP activity by, for example, binding the peptide or KPP to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • Media containing KPP are passed over the in-munoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of KPP (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/KPP 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 KPP is coUected.
  • Candidate molecules previously anayed in the weUs of a multi-weU plate are incubated with the labeled KPP, washed, and any weUs with labeled KPP complex are assayed. Data obtained using different concentrations of KPP are used to calculate values for the number, affinity, and association of KPP with the candidate molecules.
  • KPP molecules interacting with KPP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commercially avaUable kits based on the two-hybrid system, such as the MATCHMAKER system (BD Clontech). KPP 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 ah interactions between the proteins encoded by two large hbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • protein kinase activity is measured by quantifying the phosphorylation of a protein substrate by KPP in the presence of [ ⁇ - 32 P]ATP.
  • KPP is incubated with the protein substrate, 32 P-ATP, and an appropriate kinase buffer.
  • the 32 P incorporated into the substrate is separated from free 32 P-ATP by electrophoresis and the incorporated 32 P is counted using a radioisotope counter.
  • the amount of incorporated 32 P is proportional to the activity of KPP.
  • a determination of the specific amino acid residue phosphorylated is made by phosphoamino acid analysis of the hydrolyzed protein.
  • 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.
  • ATP adenosine triphosphate
  • the reaction occurs between a protein kinase sample with a biotinylated peptide substrate and gamma 32 P-ATP. FoUowing the reaction, free avidin in solution is added for binding to the biotinylated 32 P-peptide product.
  • the binding sample then undergoes a centrifugal ulfrafUtration process with a membrane which wiU retain the product-avidin complex and aUow passage of free gamma 3 P-ATP.
  • Suggested substrates and their respective enzymes include but are not limited to: Histone HI (Sigma) and p34 cdc kinase, 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 Enzymol. 200:62-81).
  • protein kinase activity of KPP is demonstrated in an assay containing
  • KPP 50 ⁇ l of kinase buffer, 1 ⁇ g substrate, such as myelin basic protein (MBP) or synthetic peptide substrates, 1 mM DTT, 10 ⁇ g ATP, and 0.5 ⁇ Ci [ ⁇ - 32 P]ATP.
  • substrate such as myelin basic protein (MBP) or synthetic peptide substrates
  • DTT 10 ⁇ g ATP
  • 0.5 ⁇ Ci [ ⁇ - 32 P]ATP 0.5 ⁇ Ci [ ⁇ - 32 P]ATP.
  • the reaction is incubated at 30 °C for 30 minutes and stopped by pipetting onto P81 paper.
  • the unincorporated [ ⁇ - 32 P]ATP is removed by washing and the incorporated radioactivity is measured using a scintillation counter.
  • the reaction is stopped by heating to 100 °C in the presence of SDS loading buffer and resolved on a 12% SDS polyacrylamide gel foUowed by autoradiography.
  • the amount of incorporated 32 P is proportional to the activity of KPP.
  • adenylate kinase or guanylate kinase activity of KPP may be measured by the incorporation of 32 P from [ ⁇ - 32 P]ATP into ADP or GDP using a gamma radioisotope counter.
  • KPP in a kinase buffer, is incubated together with the appropriate nucleotide mono-phosphate substrate (AMP or GMP) and 32 P-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 excised and counted. The radioactivity recovered is proportional to the activity of KPP.
  • KPP scintillation proximity assays
  • useful substrates include recombinant proteins tagged with glutathione transferase, or synthetic peptide substrates tagged with biotin.
  • Inhibitors of KPP activity such as smaU organic molecules, proteins or peptides, maybe identified by such assays.
  • phosphatase activity of KPP is measured by the hydrolysis of para- nitrophenyl phosphate (PNPP).
  • PNPP para- nitrophenyl phosphate
  • KPP is incubated together with PNPP in HEPES buffer pH 7.5, in the presence of 0.1 % ⁇ -mercaptoefhanol at 37 °C for 60 min.
  • the reaction is stopped by the addition of 6 ml of 10 N NaOH (Diamond, R.H. et al. (1994) Mol. Cell. Biol. 14:3752-62).
  • acid phosphatase activity of KPP is demonstrated by incubating KPP-containing extract with 100 ⁇ l of 10 mM PNPP in 0.1 M sodium citrate, pH 4.5, and 50 ⁇ l of 40 mM NaCl at 37 ° C for 20 min. The reaction is stopped by the addition of 0.5 ml of 0.4 M glycine/NaOH, pH 10.4 (Saftig, P. et al. (1997) J. Biol. Chem. 272:18628-18635). The increase in light absorbance at 410 nmresulting from the hydrolysis of PNPP is measured using a spectrophotometer. The increase in light absorbance is proportional to the activity of KPP in the assay.
  • KPP activity is dete ⁇ nined by measuring the amount of phosphate removed from a phosphorylated protein substrate. Reactions are performed with 2 or 4 nM KPP in a final volume of 30 ⁇ l containing 60 mM Tris, pH 7.6, 1 mM EDTA, 1 mM EGTA, 0.1% ⁇ -mercaptoethanol and 10 ⁇ M substrate, 32 P-labeled on serine/threonine or tyrosine, as appropriate. Reactions are initiated with substrate and incubated at 30° C for 10-15 min.
  • Binding of KPP to a FLAG-CD44 cyt fusion protein can be determined by incubating KPP with anti-KPP-conjugated immunoaffinity beads foUowed by incubating portions of the beads (having 10-20 ng of protein) with 0.5 ml of a binding buffer (20 mM Tris-HCL (pH 7.4), 150 mM NaCl, 0.1 % bovine serum albumin, and 0.05% Triton X-100) in the presence of 125 I-labeled FLAG- CD44cyt fusion protein (5,000 cpm/ng protein ) at 4 °C for 5 hours.
  • a binding buffer (20 mM Tris-HCL (pH 7.4), 150 mM NaCl, 0.1 % bovine serum albumin, and 0.05% Triton X-100
  • KPP activity is measured for each well and the abihty of each compound to inhibit KPP activity can be determined, as well as the dose-response kinetics. This assay could also be used to identify molecules which enhance KPP activity.
  • KPP "substrate-trapping" assay takes advantage of the increased substrate affinity that may be conferred by certain mutations in the PTP signature sequence of protein tyrosine phosphatases. KPP bearing these mutations form a stable complex with their substrate; this complex may be isolated biochemicaUy. Site-directed mutagenesis of invariant residues in the PTP signature sequence in a clone encoding the catalytic domain of KPP is performed using a method standard in the art or a commercial kit, such as the MUTA-GENE kit from BIO-RAD.
  • KPP mutants For expression of KPP mutants in Escherichia coli, DNA fragments containing the mutation are exchanged with the corresponding wUd-type sequence in an expression vector bearing the sequence encoding KPP or a glutathione S-transferase (GST)-KPP fusion protein. KPP mutants are expressed in E. coli and purified by chromatography.
  • the expression vector is transfected into COS 1 or 293 ceUs via calcium phosphate-mediated transfection with 20 ⁇ g of CsCl-purified DNA per 10-cm dish of ceUs or 8 ⁇ g per 6-cm dish.
  • ceUs are stimulated with 100 ng/ml epidermal growth factor to increase tyrosine phosphorylation in ceUs, as the tyrosine kinase EGFR is abundant in COS ceUs.
  • CeUs are lysed in 50 mM Tris-HCl, pH 7.5/5 mM EDTA/150 mM NaCl/1 % Triton X-100/5 mM iodoacetic acid/10 mM sodium phosphate/10 mM NaF/5 ⁇ g/ml leupeptin/5 ⁇ g/ml aprotinin/1 mM benzamidine (1 ml per 10-cm dish, 0.5 ml per 6-cm dish).
  • KPP is immunoprecipitated fromlysates with an appropriate antibody.
  • GST-KPP fusion proteins are precipitated with glutathione-Sepharose, 4 ⁇ g of mAb or 10 ⁇ l of beads respectively per mg of ceU lysate. Complexes can be visualized by PAGE or further purified to identify substrate molecules (Flint, A.J. et al. (1997) Proc. Natl. Acad. Sci. USA 94:1680-1685).
  • ABI FACTURA A program that removes vector sequences and masks Applied Biosystems, Foster City, CA. ambiguous bases in nucleic acid sequences.
  • ABI/PARACEL A Fast Data Finder useful in comparing and annotating Applied Biosystems, Foster City, CA; Mismatch ⁇ 50% FDF amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA.
  • BLAST includes five functions: blastp, Nucleic Acids Res. 25:3389-3402. Full Length sequences: Probabilit blastn, blastx, tblastn, and tblastx. valuer l.OE-10 or less
  • fastx score 100 or greater
  • Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8: 175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194.
  • TMHMMER A program that uses a hidden Markov model (7JMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelhgent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.

Abstract

On décrit divers modes de réalisation de l'invention qui mettent en oeuvre des kinases et des phosphatases humaines (KPP), ainsi que des polynucléotides qui identifient et codent KPP. Des modes de réalisation de l'invention mettent également en oeuvre des vecteurs, des cellules hôtes, des anticorps, des agonistes et des antagonistes. D'autres modes de réalisation se rapportent à des méthodes de diagnostic, de traitement ou de prévention de troubles associés à une expression aberrante de KPP.
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WO2006061130A2 (fr) * 2004-12-10 2006-06-15 Sanofi-Aventis Deutschland Gmbh Utilisation d'une kinase activee par le serum et les glucocorticoides
WO2006061130A3 (fr) * 2004-12-10 2007-04-26 Sanofi Aventis Deutschland Utilisation d'une kinase activee par le serum et les glucocorticoides
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EP1892248A1 (fr) * 2006-08-21 2008-02-27 Eidgenössische Technische Hochschule Zürich Protéines liantes spécifiques et de haute affinité comprenant des domaines SH3 de FYN kinase modifiés
KR101159903B1 (ko) * 2006-08-21 2012-06-26 아이제노에시쉐 테크니쉐 호히쉘 쮸리히 Ｆｙｎ 키나아제의 변형된 ｓｈ３ 도메인을 포함하는 특이적이며 친화력이 높은 결합 단백질
JP2013078316A (ja) * 2006-08-21 2013-05-02 Eidgenoessische Technische Hochschule Zuerich Ｆｙｎキナーゼの改変ｓｈ３ドメインを含む特異的かつ高親和性の結合タンパク質
US9513296B2 (en) 2006-08-21 2016-12-06 Eidgenoessische Technische Hochschule Zurich Specific and high affinity binding proteins comprising modified SH3 domains of Fyn kinase
US9689879B2 (en) 2006-08-21 2017-06-27 Eidgenoessische Technische Hochschule Zurich Specific and high affinity binding proteins comprising modified SH3 domains of Fyn kinase
US9989536B2 (en) 2006-08-21 2018-06-05 Eidgenoessische Technische Hochschule Zurich Specific and high affinity binding proteins comprising modified SH3 domains of FYN kinase
US10996226B2 (en) 2006-08-21 2021-05-04 Eidgenoessische Technische Hochschule Zurich Specific and high affinity binding proteins comprising modified SH3 domains of FYN kinase

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