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  • 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
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  • 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.
• Reversible protein phosphorylation is the ubiquitous strategy used to control many of the intracellular events in eukaryotic cells. It is estimated that more than ten percent of proteins active in a typical mammalian cell are phosphorylated.
• Kinases catalyze the transfer of high-energy phosphate groups from adenosine triphosphate (ATP) to target proteins on the hydroxyamino acid residues serine, threonine, or tyrosine. Phosphatases, in contrast, remove these phosphate groups.
• Extracellular signals including hormones, neurotransmitters, and growth and differentiation factors can activate kinases, which can occur as cell surface receptors or as the activator of the final effector protein, as well as other locations along the signal transduction pathway.
• 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 all-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
• Kinases comprise the largest known enzyme superfamily and vary widely in their target molecules. Kinases catalyze the transfer of high energy phosphate groups from a phosphate donor to a phosphate acceptor. Nucleotides usually serve as the phosphate donor in these reactions, with most kinases utilizing adenosine triphosphate (ATP).
• the phosphate acceptor can be any of a variety of molecules, including nucleosides, nucleotides, lipids, carbohydrates, and proteins. Proteins are phosphorylated on hydroxyamino acids. Addition of a phosphate group alters the local charge on the acceptor molecule, causing internal conformational changes and potentially influencing intermolecular contacts.
• Reversible protein phosphorylation is the primary method for regulating protein activity in eukaryotic cells.
• proteins are activated by phosphorylation in response to extracellular signals such as hormones, neurotransmitters, and growth and differentiation factors.
• the activated proteins initiate the cell's intracellular response by way of intracellular signaling pathways and second messenger molecules such as cyclic nucleotides, calcium-calmodulin, inositol, and various mitogens, that regulate protein phosphorylation.
• Kinases are involved in all aspects of a cell's function, from basic metabolic processes, such as glycolysis, to cell-cycle regulation, differentiation, and communication with the extracellular environment through signal transduction cascades. Inappropriate phosphorylation of proteins in cells has been linked to changes in cell cycle progression and cell differentiation. Changes in the cell cycle have been linked to induction of apoptosis or cancer. Changes in cell differentiation have been linked to diseases and disorders of the reproductive system, immune system, and skeletal muscle.
• 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 serine/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 VE which may confer hydroxyamino acid specificity.
• kinases may also be classified by additional amino acid sequences, generally between 5 and 100 residues, which either flank or occur within the kinase domain. These additional amino acid sequences regulate kinase activity and determine substrate specificity. (Reviewed in Hardie, G. and S. Hanks (1995) The Protein Kinase Facts Book, Vol I, pp. 17-20 Academic Press, San Diego CA.). In particular, two protein kinase signature sequences have been identified in the kinase domain, the first containing an active site lysine residue involved in ATP binding, -and the second containing an aspartate residue important for catalytic activity.
• Protein Tyrosine Kinases Protein tyrosine kinases (PTKs) may be classified as either transmembrane, receptor PTKs or nontransmembrane, nonreceptor PTK proteins. Transmembrane tyrosine kinases function as receptors for most growth factors. Growth factors bind to the receptor tyrosine kinase (RTK), which causes the receptor to phosphorylate itself (autophosphorylation) and specific intracellular second messenger proteins.
• RTK receptor tyrosine kinase
• 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/t-hreonine 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.
• MAP/ERK kinase phosphorylation of MEK
• 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 (hFu); 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
• ERK 7 One member of the ERK family of MAP kinases, ERK 7, is a novel 61-kDa protein that has motif similarities to ERKl and ERK2, but is not activated by extracellular stimuli as are ERKl and ERK2 nor by the common activators, c-Jun N-terminal kinase (JNK) and p38 kinase.
• JNK c-Jun N-terminal kinase
• ERK7 regulates its nuclear localization and inhibition of growth through its C-terminal tail, not through the kinase domain as is typical with other MAP kinases (Abe, M.K. (1999) Mol. Cell. Biol. 19:1301-1312).
• the second messenger dependent protein kinases primarily mediate the effects of second messengers such as cyclic AMP (cAMP), cyclic GMP, inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cyclic ADP ribose, arachidonic acid, diacylglycerol and calcium-calmodulin.
• cAMP cyclic AMP
• GMP cyclic GMP
• inositol triphosphate phosphatidylinositol
• 3,4,5-triphosphate cyclic ADP ribose
• arachidonic acid diacylglycerol
• calcium-calmodulin calcium-calmodulin.
• PKAs cyclic AMP
• the PKAs are involved in mediating hormone-induced cellular responses and are activated by cAMP produced within the cell in response to hormone stimulation.
• cAMP is an intracellular mediator of hormone action in all animal cells that have been studied.
• Hormone-induced cellular responses include thyroid hormone secretion, cortisol secretion, progesterone secretion, glycogen breakdown, bone resorption, and regulation of heart rate and force of heart muscle contraction.
• PKA is found in all animal cells and is thought to account for the effects of cAMP in most of these cells. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, K.J. et al. (1994) Harrison's Principles of Internal Medicine. McGraw-Hill, New York NY, pp. 416-431, 1887).
• CKI casein kinase I
• CKI casein kinase I
• CKI enzymes are present in the membranes, nucleus, cytoplasm and cytoskeleton of eukaryotic cells, and on the mitotic spindles of mammalian cells (Fish, K.J. et al. (1995) J. Biol. Chem. 270:14875-14883).
• the CKI family members all have a short amino-terminal domain of 9-76 amino acids, a highly conserved kinase domain of 284 amino acids, and a variable carboxyl-terminal domain that ranges from 24 to over 200 amino acids in length (Cegielska, A. et al. (1998) J. Biol. Chem. 273:1357-1364).
• the CKI family is comprised of highly related proteins, as seen by the identification of isoforms of casein kinase I from a variety of sources. There are at least five mammalian isoforms, ⁇ , ⁇ , ⁇ , and ⁇ . Fish et al. identified CKI-epsilon from a human placenta cDNA library.
• 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/threonine 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 localized 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 intracellular calcium receptor, in response to the concentration of free calcium in the cell. Many CaM kinases are also activated by phosphorylation. Some CaM kinases are also activated by autophosphorylation or by other regulatory kinases.
• CaM kinase I phosphorylates a variety of substrates including the neurotransmitter-related proteins synapsin I and ⁇ , 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 calmodulin-binding protein kinase-like protein was found to be enriched in mammalian forebrain. This protein is associated with vesicles in both axons and dendrites and accumulates largely postnatally.
• the amino acid sequence of this protein is similar to CaM-dependent STKs, and the protein binds calmodulin in the presence of calcium (Godbout, M. et al. (1994) J. Neurosci. 14:1-13).
• MAP mitogen-activated protein kinases
• the extracellular-regulated kinase (ERK) pathway is activated by growth factors and mitogens, for example, epidermal growth factor (EGF), ultraviolet light, hyperosmolar medium, heat shock, or endotoxic lipopolysaccharide (LPS).
• EGF epidermal growth factor
• LPS endotoxic lipopolysaccharide
• JNK c-Jun N-terminal kinase
• SAPK stress-activated kinase
• p38 kinase pathway are activated by stress stimuli and proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin- 1 (IL-1).
• MAP kinase expression is implicated in a variety of disease conditions including cancer, inflammation, immune disorders, and disorders affecting growth and development.
• MAP kinase signaling pathways are present in mammalian cells as well as in yeast.
• Cvclin-Dependent Protein Kinases are present in mammalian cells as well as in yeast.
• CDKs The cyclin-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 cyclins.
• Cyclins are small regulatory proteins that bind to and activate CDKs, which then phosphorylate and activate selected proteins involved in the mitotic process.
• CDKs are unique in that they require multiple inputs to become activated. In addition to cyclin binding, CDK activation requires the phosphorylation of a specific threonine residue and the dephosphorylation of a specific tyrosine residue on the CDK.
• NIMA severe in mitosis
• Neks NIMA-related kinases
• 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 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).
• Proliferation-related kinase is related to the polo (derived from Drosophila polo gene) family of STKs implicated in cell division.
• Proliferation-related kinase is downregulated in lung tumor tissue and may be a proto-oncogene whose deregulated expression in normal tissue leads to oncogenic transformation.
• S'-AMP-activated 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-terminal 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 NTH3T3 cells (Sanjo et al., supra). However, deletion of either the N-terminal kinase catalytic domain or the C-terminal domain of these proteins abolishes apoptosis activity, indicating that in addition to the kinase activity, activity in the C-terminal domain is also necessary for apoptosis, possibly as an interacting domain with a regulator or a specific substrate.
• RICK is another STK recently identified as mediating a specific apoptotic pathway involving the death receptor, CD95 (Inohara, N. et al. (1998) J. Biol. Chem. 273:12296-12300).
• CD95 is a member of the tumor necrosis factor receptor superfamily and plays a critical role in the regulation and homeostasis of the immune system (Nagata, S. (1997) Cell 88:355-365).
• the CD95 receptor signaling pathway involves recruitment of various intracellular molecules to a receptor complex following ligand binding. This process includes recruitment of the cysteine protease caspase-8 which, in turn, activates a caspase cascade leading to cell death.
• RICK is composed of an N-terminal kinase catalytic domain and a C-terminal "caspase-recruitment" domain that interacts with caspase-like domains, indicating that RICK plays a role in the recruitment of caspase-8. This interpretation is supported by the fact that the expression of RICK in human 293T cells promotes activation of caspase-8 and potentiates the induction of apoptosis by various proteins involved in the CD95 apoptosis pathway (Inohara et al., supra). Mitochondrial Protein Kinases
• a novel class of eukaryotic kinases related by sequence to prokaryotic histidine protein kinases, are the mitochondrial protein kinases (MPKs) which seem to have no sequence similarity with other eukaryotic protein kinases. These protein kinases are located exclusively in the mitochondrial matrix space and may have evolved from genes originally present in respiration-dependent bacteria which were endocytosed by primitive eukaryotic cells. MPKs are responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase complexes (Harris, R.A. et al. (1995) Adv. Enzyme Regul. 34:147-162).
• 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 lipid head groups.
• a family of kinases involved in phosphorylation of phosphatidylinositol (PI) has been described, each member phosphorylating a specific carbon on the inositol ring (Leevers, S.J. et al. (1999) Curr. Opin. Cell. Biol. 11 :219-225).
• the phosphorylation of phosphatidylinositol is involved in activation of the protein kinase C signaling pathway.
• the inositol phospholipids (phosphoinositides) intracellular signaling pathway begins with binding of a signaling molecule to a G-protein linked receptor in the plasma membrane.
• PIP 2 phosphatidylinositol
• JP_ inositol triphosphate
• JP_ diacylglycerol
• 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, lipid 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. et al.
• SPP sphingosine-1-phosphate
• SPP levels are regulated by sphingosine kinases that specifically phosphorylate D-erythro-sphingosine to SPP.
• the importance of sphingosine kinase in cell signaling is indicated by the fact that various stimuli, including platelet-derived growth factor (PDGF), nerve growth factor, and activation of protein kinase C, increase cellular levels of SPP by activation of sphingosine kinase, and the fact that competitive inhibitors of the enzyme selectively inhibit cell proliferation induced by PDGF (Kohama et al., 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 metabolism and are crucial to the synthesis and regulation of cellular levels of ATP and GTP, respectively.
• ATP AMP phosphotransferase
• GuK guanylate kinase
• AdK ATP synthesis and utilization such as skeletal muscle.
• AdK is physically associated with mitochondria and myofibrils, the subcellular structures that are involved in energy production and utilization, respectively.
• Recent studies have demonstrated a major function for AdK in transferring high energy phosphoryls from metabolic processes generating ATP to cellular components consuming ATP (Zeleznikar, RJ. 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 metabolism 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. Specifically, GTP binding to membrane associated G proteins mediates the activation of cell receptors, subsequent intracellular activation of adenyl cyclase, and production of the second messenger, cyclic AMP. GDP binding to G proteins inhibits these processes. GDP and GTP levels also control the activity of certain oncogenic proteins such as p21 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 ⁇ 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.
• GTP binding to membrane associated G proteins mediates the activation of cell receptors, subsequent intracellular activation
• 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.
• ll kinase is located in the nucleus, and thymidine kinase 1 and 2 are found in the cytosol (Johansson, M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11941-11945).
• Phosphorylation of deoxyribonucleosides by pyrimidine kinases provides an alternative pathway for de novo synthesis of DNA precursors.
• the role of pyrimidine kinases, like purine kinases, in phosphorylation is critical to the activation of several chemotherapeutically important nucleoside analogues (Arner E.S. and S. Eriksson (1995) Pharmacol. Ther. 67:155-186).
• PHOSPHATASES PHOSPHATASES
• Protein phosphatases are generally characterized as either serine/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.
• DSPs protein serine/threonine phosphatases
• PSPs protein serine/threonine phosphatases
• PSPs Protein tyrosine phosphatases
• PSPs are found in the cytosol, nucleus, and mitochondria and in association with cytoskeletal and membranous structures in most tissues, especially the brain. Some PSPs require divalent cations, such as Ca 2+ or Mn 2+ , for activity. PSPs play important roles in glycogen metabolism, muscle contraction, protein synthesis, T cell function, neuronal activity, oocyte maturation, and hepatic metabolism (reviewed in Cohen, P. (1989) Annu. Rev. Biochem. 58:453-508). PSPs can be separated into two classes.
• the PPP class includes PPl, PP2A, PP2B/calcineurin, PP4, PP5, PP6, and PP7.
• Members of this class are composed of a homologous catalytic subunit bearing a very highly conserved signature sequence, coupled with one or more regulatory subunits (PROSITE PDOC00115). Further interactions with scaffold and anchoring molecules determine the intracellular localization of PSPs and substrate specificity.
• the PPM class consists of several closely related isoforms of PP2C and is evolutionarily unrelated to the PPP class.
• PPl dephosphorylates many of the proteins phosphorylated by cyclic AMP-dependent protein kinase (PKA) and is an important regulator of many cAMP-mediated hormone responses in cells.
• PKA cyclic 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 PPl 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 families 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).
• PP2A dephosphorylates the MAPK active site, inhibiting the cell's entry into mitosis.
• Several proteins can compete with PR55 for PP2A core enzyme binding, including the CKH 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 CKQ kinases, and can be stimulated by polycationic macromolecules.
• a PP2A-like phosphatase is necessary to maintain the Gl phase destruction of mammalian 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 immunosuppressant drags 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.
• 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).
• Other members of the PPP class have recently been identified (Cohen, P.T. (1997) Trends
• 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. 9:193-204).
• 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 extracellular domains of some receptor PTPs contain fibronectin-like repeats, i munoglobulin-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.
• PTPs play important roles in biological processes such as cell adhesion, lymphocyte activation, and cell proliferation.
• PTPs ⁇ and K are involved in cell-cell contacts, perhaps regulating cadherin/catenin function.
• a number of PTPs affect cell spreading, focal adhesions, and cell motility, most of them via the integrin/tyrosine kinase signaling pathway (reviewed in Neel and Tonks, supra).
• CD45 phosphatases regulate signal transduction and lymphocyte activation (Ledbetter, J.A. et al. (1988) Proc. Natl. Acad. Sci. USA 85:8628-8632).
• 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-linked recessive myotubular myopathy, a congenital muscle disorder that has been linked to Xq28 (Kioschis, P. et al., (1998) Genomics 54:256-266).
• 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 proliferation 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).
• Synaptojanin a polyphosphoinositide phosphatase, dephosphorylates phosphoinositides at positions 3, 4 and 5 of the inositol ring.
• Synaptojanin is a major presynaptic protein found at clathrin- coated endocytic intermediates in nerve terminals, and binds the clathrin coat-associated protein,
• 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 in bioanalysis.
• a microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid 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. When the expression of a single gene is examined, 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.
• 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, lipid disorders, cell proliferative disorders, and cancers.
• Various embodiments of the invention provide purified polypeptides, kinases and phosphatases, referred to collectively as 'KPP' and individually 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,' 'KPP-30,'
• Embodiments also provide methods for utilizing the purified kinases and phosphatases and/or their encoding polynucleotides for facilitating the drag 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- 42, 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: 1-42, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-42, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-42.
• Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-42.
• 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: 1-42, 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: 1-42, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-42, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-42.
• polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-42. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:43-84.
• Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42, 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:l-42, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-42, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42.
• Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42, 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: 1-42, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42.
• the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
• Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42, 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:l-42, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42.
• Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:43-84, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:43-84, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
• the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
• Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:43-84, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:43-84, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
• a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleot
• the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex.
• 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.
• Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:43-84, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:43-84, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
• a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleo
• 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-42, 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: 1-42, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42, and a pharmaceutically acceptable excipient.
• the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:l-42.
• 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-42, 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:l-42, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-42.
• the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
• Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
• Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional KPP, comprising administering to a patient in need of such treatment the composition.
• Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42, 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: 1-42, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42.
• the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
• Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient.
• Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional KPP, comprising administering to a patient in need of such treatment the composition.
• Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-42, 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:l-42, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-42, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-42.
• 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: 1-42, 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: 1-42, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-42.
• 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.
• Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consistmg of SEQ ID NO:43-84, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
• Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:43-84, 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:43-84, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of
• 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:43-84, 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:43-84, 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).
• 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 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.
• Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
• Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
• Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
• Table 5 shows representative cDNA libraries for polynucleotide embodiments.
• Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
• Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.
• Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with allele frequencies in different human populations.
• KPP refers to the amino acid sequences of substantially purified KPP obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
• agonist refers to a molecule which intensifies or mimics the biological activity of
• KPP KPP.
• Agonists may include proteins, nucleic acids, carbohydrates, small 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.
• allelic variant is an alternative form of the gene encoding KPP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
• altered nucleic acid sequences encoding 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 readily detectable using a particular oligonucleotide probe of the polynucleotide encoding KPP, and improper or unexpected hybridization to allelic 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 silent change and result in a functionally equivalent KPP.
• Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of 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 similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
• Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
• 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, small 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 well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
• Antibodies that bind KPP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
• the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
• an animal e.g., a mouse, a rat, or a rabbit
• Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
• 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 specifically to antigenic determinants (particular regions or three-dimensional structures on the protein).
• An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
• 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 maybe 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 system.
• Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
• introduction 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 naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
• antisense refers to any composition capable of base-pairing with the "sense"
• Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'- deoxyguanosine.
• Antisense molecules may be produced by any method including chemical synthesis or transcription.
• the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
• the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
• biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
• immunologically active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic KPP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
• “Complementary” describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
• 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 uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVTEW fragment assembly system (Accelrys, Burlington MA) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
• 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 especially the function of the protein is conserved and not significantly changed by such substitutions.
• the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
• Trp Phe Tyr Tyr His, Phe, Trp
• Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
• a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
• derivative refers to a chemically modified polynucleotide or polypeptide.
• Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
• a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
• a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
• a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
• “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, dete ⁇ nined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
• Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
• a “fragment” is a unique portion of 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.
• a fragment may comprise from about 5 to about 1000 contiguous nucleotides or amino acid residues.
• a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
• a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
• a fragment of SEQ ID NO:43-84 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:43-84, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
• a fragment of SEQ ID NO:43-84 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:43-84 from related polynucleotides.
• the precise length of a fragment of SEQ ID NO:43-84 and the region of SEQ ID NO:43-84 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
• a fragment of SEQ ID NO:l-42 is encoded by a fragment of SEQ ID NO:43-84.
• a fragment of SEQ ID NO: 1-42 can comprise a region of unique amino acid sequence that specifically identifies SEQ ID NO:l-42.
• a fragment of SEQ ID NO:l-42 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:l-42.
• the precise length of a fragment of SEQ ID NO:l-42 and the region of SEQ ID NO:l-42 to which the fragment conesponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
• a “full length” polynucleotide is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
• a “full length” polynucleotide sequence encodes a "full length” polypeptide sequence.
• “Homology” refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
• the terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of identical residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein.
• NCBI National Center for Biotechnology Information
• BLAST Basic Local AUgnment Search Tool
• NCBI National Center for Biotechnology Information
• BLAST Basic Local AUgnment Search Tool
• the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to aUgn 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 maybe, for example: Matrix: BLOSUM62
• Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that aU encode substantiaUy the same protein.
• percent identity and % identity refer to the percentage of identical residue matches between at least two polypeptide sequences aUgned using a standardized algorithm.
• Methods of polypeptide sequence aUgnment are weU-known. Some aUgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generaUy preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
• percent similarity and % similarity refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aUgned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.
• NCBI BLAST software suite may be used.
• BLAST 2 Sequences Version 2.0.12 (April-21-2000) with blastp set at default parameters.
• Such default parameters may be, for example:
• Gap x drop-off 50
• Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• HACs Human artificial chromosomes
• HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain aU of the elements required for chromosome repUcation, 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 stiU retains its original binding abiUty.
• 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 determining the stringency of the hybridization process, with more stringent conditions aUowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
• Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skiU 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 typically selected to be about 5°C to 20°C lower than the thermal melting point (T- for the specific sequence at a defined ionic strength and pH.
• 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 may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
• blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
• Organic solvent such as formamide at a concentration of about 35-50% v/v
• Organic solvent such as formamide at a concentration of about 35-50% v/v
• Useful variations on these wash conditions wiU be readily apparent to those of ordinary skill in the art.
• Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
• 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 maybe formed in solution (e.g., C 0 t or Rot analysis) or formed between one nucleic acid present in solution and another nucleic acid immobiUzed on a soUd support (e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
• soUd support e.g., paper, membranes, filters, chips, pins or glass sUdes, 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 cellular and systemic defense systems.
• factors e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
• an "immunogenic fragment” is a polypeptide or oUgopeptide fragment of KPP which is capable of eUciting an immune response when introduced into a Uving organism, for example, a mammal.
• the term “immunogenic fragment” also includes any polypeptide or oUgopeptide fragment of KPP which is useful in any of the antibody production methods disclosed herein or known in the art.
• microanay refers to an anangement of a pluraUty of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
• element and “anay element” refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microanay.
• 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, oUgonucleotide, 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-Uke 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 oUgonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubiUty to the composition.
• PNAs preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their Ufespan in the ceU.
• Post-translational modification of an KPP may involve Upidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications will vary by cell type depending on the enzymatic miUeu of KPP.
• Probe refers to nucleic acids encoding KPP, their complements, or fragments thereof, which are used to detect identical, aUeUc or related nucleic acids.
• Probes are isolated oUgonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, Ugands, chemiluminescent agents, and enzymes.
• Primmers are short nucleic acids, usually DNA oUgonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for ampUf ⁇ cation (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. Methods for preparing and using probes and primers are described in, for example, Sambrook,
• 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).
• OUgonucleotides 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 oUgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabiUties. For example, the PrimOU primer selection program (available to the pubUc 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.
• Primer3 primer selection program (available to the pubUc from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows the user to input a "n spriming Ubrary," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oUgonucleotides for microanays.
• the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
• the PrimeGen program (available to the pubUc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence aUgnments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of aUgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oUgonucleotides and polynucleotide fragments.
• oUgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oUgonucleotide selection are not limited to those described above.
• a "recombinant nucleic acid” is a nucleic acid that is not naturaUy 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 accompUshed by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and Russell (supra).
• the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
• 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 mammal.
• 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 stabiUty.
• Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionucUdes; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
• RNA equivalent in reference to a DNA molecule, is composed of the same Unear sequence of nucleotides as the reference DNA molecule with the exception that aU occunences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
• sample is used in its broadest sense.
• a sample suspected of containing KPP, nucleic acids encoding KPP, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organeUe, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
• specific binding and “specificaUy binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition.
• the interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic dete-rminant or epitope, recognized by the binding molecule.
• a particular structure of the protein e.g., the antigenic dete-rminant or epitope
• the binding molecule e.g., the antigenic dete-rminant or epitope
• an antibody is specific for epitope "A”
• the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A in a reaction containing free labeled A and the antibody wiU reduce the amount of labeled A that binds to the antibody.
• 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, sUdes, 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 cell 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 well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not -limited to, bacteriophage or viral infection, electroporation, heat shock, Upofection, and particle bombardment.
• transformed cells includes stably transformed cells in which the inserted DNA is capable of repUcation either as an autonomously repUcating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for Umited periods of time.
• a "transgenic organism,” as used herein, is any organism, including but not Umited to animals and plants, in which one or more of the ceUs 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 ceU, directly or indirectly by introduction into a precursor of the cell, by way of dehberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
• the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872).
• the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
• the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
• the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and RusseU (supra).
• a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 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 "aUeUc” (as defined above), “spUce,” “species,” or “polymorphic” variant.
• a spUce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate spUcing of exons during mRNA processing.
• the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
• Species variants are polynucleotides that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other.
• a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
• Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
• SNPs single nucleotide polymorphisms
• a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity or sequence similarity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters.
• Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity or sequence similarity over a certain defined length of one of the polypeptides.
• 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, Upid disorders, cell proUferative disorders, and cancers.
• KPP human kinases and phosphatases
• Table 1 summarizes the nomenclature for the fuU length polynucleotide and polypeptide embodiments of the invention.
• Each polynucleotide and its corresponding polypeptide are conelated 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 conesponding to the polypeptide and polynucleotide sequences of the invention.
• the fuU 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 corresponding Incyte polypeptide sequence number (Incyte Polypeptide DD) 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 probabiUty 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 appUcable, 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
• Table 3 shows the number of amino acid residues in each polypeptide.
• Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (AcceUys, Burlington MA).
• Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
• Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were appUed.
• Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties estabUsh that the claimed polypeptides are kinases and phosphatases.
• SEQ DD NO:4 is 100% identical, from residue A26 to residue A279, to human SIRP-betal (GenBank DD g2052058) as determined by the Basic Local AUgnment Search Tool (BLAST).
• BLAST Basic Local AUgnment Search Tool
• the BLAST probabiUty score is 6.0e-136, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
• SEQ DD NO:4 also has homology to signal regulatory protein beta-1, as determined by BLAST analysis using the PROTEOME database.
• SEQ DD NO:4 also contains a immunoglobulin domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
• HMM hidden Markov model
• PROFDLESCAN analyses provide further conoborative evidence that SEQ ID NO:4 is a signal regulatory protein beta-1.
• SEQ DD NO:ll is 98% identical, from residue Ml to residue G341, to human MNK1 (GenBank DD g2077825) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 2.1E-181, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. As determined by BLAST analysis using the PROTEOME database, SEQ ID NO: 11 also has homology to MAP kinase-interacting serine/threonine kinase 1, a kinase which is activated by the ERK and p38 MAP kinase signaling pathways.
• BLAST Basic Local AUgnment Search Tool
• SEQ ID NO:l 1 also contains a protein kinase domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, PROFILESCAN and additional BLAST analyses provide further corroborative evidence that SEQ DD NO: 11 is a MAP kinase-activated protein kinase.
• SEQ DD NO:12 is 99% identical, from residue Ml to residue R498, to human clk2 kinase (GenBank DD g2564911) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 2.6e-277, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
• SEQ ID NO: 12 also has homology to proteins that have kinase activity and are members of the LAMMER family of dual specificity protein kinases, as determined by BLAST analysis using the PROTEOME database.
• SEQ ID NO: 12 also contains a protein kinase domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, BLAST, MOTIFS, and PROFILESCAN analyses provide further conoborative evidence that SEQ DD NO: 12 is a member of the kinase family.
• HMM hidden Markov model
• SEQ DD NO: 15 is 92% identical, from residue S89 to residue L893, to human homolog of Drosophila discs large protein (GenBank ID g558436) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 0.0, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
• SEQ DD NO: 15 also has homology to proteins that are locaUzed to the plasma membrane, have kinase activity, and are a member of the membrane-associated guanylate kinase (MAGUK) family, as determined by BLAST analysis using the PROTEOME database.
• MAGUK membrane-associated guanylate kinase
• SEQ ID NO: 15 also contains a guanylate kinase domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and additional BLAST analyses provide further conoborative evidence that SEQ ID NO: 15 is a kinase.
• HMM hidden Markov model
• SEQ ID NO:25 is 92% identical, from residue VI 1 to residue K323, and 99% identical, from residue M362 to residue Q495, to rat calmodulin-dependent protein kinase D gamma subunit (GenBank DD g206152) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 2.9e-227, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ DD NO:25 also has homology to proteins that have protein kinase activity, and are calcium/calmoduUn-dependent protein kinase D, as determined by BLAST analysis using the PROTEOME database.
• SEQ ID NO:25 also contains a protein kinase domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains and a serine/threonine protein kinase domain as dete ⁇ nined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based SMRT database of conserved protein family domains.
• HMM hidden Markov model
• HMM hidden Markov model
• SEQ ID NO:l-3 SEQ DD NO:5-10, SEQ DD NO:13-14, SEQ DD NO:16-24, and SEQ DD NO:26-42 were analyzed and annotated in a similar manner.
• the algorithms and parameters for the analysis of SEQ ID NO: 1-42 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 Usts the polynucleotide sequence identification number (Polynucleotide SEQ DD NO:), the conesponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
• Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the fuU length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or ampUfication technologies that identify SEQ ID NO:43-84 or that distinguish between SEQ ID NO:43-84 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 Ubraries or from pooled cDNA Ubraries.
• the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the fuU length polynucleotides.
• the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST").
• the polynucleotide fragments described in column 2 maybe derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP”).
• the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
• a polynucleotide sequence identified as ⁇ L_XXXXX_N 1 _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 appUed, and YYYYY is the number of the prediction generated by the algorithm, and N j 23 ..., 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 appUed, 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).
• RefSeq identifier (denoted by " ⁇ M,” “ ⁇ P,” or “NT”) maybe 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.
• 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 Ubraries for those full length polynucleotides which were assembled using Incyte cDNA sequences.
• the representative cDNA Ubrary is the Incyte cDNA Ubrary 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 Ubraries 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 allele frequencies in different human populations.
• Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ID NO:) and the conesponding Incyte project identification number (PDD) 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 fuU- length polynucleotide sequence (CB1 SNP).
• Column 7 shows the aUele 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 aUele found in the EST.
• Columns 11-14 show the frequency of aUele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of allele 1 in the population was too low to be detected, while n/a (not available) indicates that the aUele 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 DD NO:43-84, which encodes KPP.
• polynucleotide sequences of SEQ DD NO:43-84 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occunences of the nitrogenous base thymine are replaced with uracil, 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 DD NO:43-84 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 DD NO:43-84.
• Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of KPP.
• a polynucleotide variant of the invention is a spUce variant of a polynucleotide encoding KPP.
• a spUce variant may have portions which have significant sequence identity to a polynucleotide encoding KPP, but will generaUy have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate spUcing of exons during mRNA processing.
• a spUce 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 spUce 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 DD NO:50 and a polynucleotide comprising a sequence of SEQ DD NO:51 are spUce variants of each other;
• a polynucleotide comprising a sequence of SEQ DD NO:62, a polynucleotide comprising a sequence of SEQ DD NO:63 a polynucleotide comprising a sequence of SEQ ID NO:64, and a polynucleotide comprising a sequence of SEQ ID NO: 65 are spUce variants of each other;
• a polynucleotide comprising a sequence of SEQ DD NO:71 and a polynucleotide comprising a sequence of SEQ ID NO:72 are spUce variants of each other;
• polynucleotides which encode KPP and its variants are generally capable of hybridizing to polynucleotides encoding naturally 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 utiUzed by the host.
• RNA transcripts having more desirable properties such as a greater half-Ufe, than transcripts produced from the naturaUy occurring sequence.
• 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 available 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 DD NO:43-84 and fragments thereof, under various conditions of stringency (Wahl, GM. 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 (AppUed Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE ampUfication system (Invitrogen, Carlsbad CA).
• sequence preparation is automated with machines such as the MICROLAB 2200 Uquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (AppUed Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (AppUed 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. Wiley VCH, New York NY, pp.
• the nucleic acids encoding KPP may be extended utiUzing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
• 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 ampUfy unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods AppUc. 2:318-322).
• Another method, inverse PCR uses primers that extend in divergent directions to ampUfy unknown sequence from a circularized template.
• the template is derived from restriction fragments comprising a known genomic locus and sunounding sequences (TrigUa, T.
• a third method involves PCR ampUfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods AppUc. 1:111-119).
• multiple restriction enzyme digestions and Ugations 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 available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72 °C.
• Genomic Ubraries does not yield a fuU-length cDNA. Genomic Ubraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
• CapiUary electrophoresis systems which are commerciaUy available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
• capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
• Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppUed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controUed.
• CapiUary electrophoresis is especially preferable for sequencing smaU DNA fragments which may be present in Umited 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 ceUs. 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 oUgonucleotides may be used to engineer the nucleotide sequences.
• oUgonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce spUce 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 abiUty 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.
• DNA shuffling is a process by which a Ubrary of gene variants is produced using PCR-mediated recombination of gene fragments. The Ubrary is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
• 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.
• 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 naturaUy occuning genes in a directed and controUable 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) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).
• 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 soUd-phase techniques (Creighton, T. (1984) Proteins. Structures and Molecular Properties. WH Freeman, New York NY, pp.
• the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
• 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.
• no additional transcriptional or translational control signals may be needed.
• exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
• Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used (Scharf, D. et al. (1994) Results Probl. CeU Differ. 20:125-162).
• Methods which are well known to those skiUed 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 utiUzed to contain and express polynucleotides encoding KPP. These include, but are not Umited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceU systems transformed with viral expression vectors (e.g., cauUflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems (Sambrook and RusseU, supra; Ausubel et al., supra; Van Heeke, G.
• microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
• yeast transformed with yeast expression vectors e
• Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids maybe used for deUvery 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; BuUer, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31:219-226; Verma, I.M. and N. Somia (1997) Nature 389:239-242).
• the invention is not -limited by the host ceU employed.
• cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding KPP. For example, 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
• vectors which direct high level expression of KPP may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
• Yeast expression systems may be used for production of KPP.
• a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
• such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, CA. et al. (1994) Bio/Technology 12:181-184).
• Plant systems may also be used for expression of KPP. Transcription of polynucleotides encoding KPP may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:1631). Alternatively, plant promoters such as the smaU subunit of RUBISCO or heat shock promoters may be used (Corazzi, G. et al. (1984) EMBO J. 3:1671-1680; BrogUe, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. CeU Differ. 17:85-105). These constructs can be introduced into plant ceUs by direct DNA transformation or pathogen-mediated transfection (The McGraw HiU Yearbook of Science and Technology (1992) McGraw Hffl, New York NY, pp. 191-196).
• a number of viral-based expression systems may be utiUzed.
• polynucleotides encoding KPP may be Ugated 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 cells (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 mammaUan host ceUs.
• SV40 or EBV-based vectors may also be used for high-level protein expression.
• HACs Human artificial chromosomes
• HACs Human artificial chromosomes
• HACs Human artificial chromosomes
• plasmids may also be employed to deUver larger fragments of DNA than can be contained in and expressed from a plasmid.
• HACs of about 6 kb to 10 Mb are constructed and deUvered via conventional deUvery methods (Uposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355).
• deUvery methods Uposomes, polycationic amino polymers, or vesicles
• KPP in ceU lines is prefened.
• polynucleotides encoding KPP can be transformed into cell lines using expression vectors which may contain viral origins of repUcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. FoUowing the introduction of the vector, cells 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 allows growth and recovery of cells which successfully express the introduced sequences.
• Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the ceU type.
• any number of selection systems may be used to recover transformed ceU Unes. These include, but are not Umited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr ceUs, respectively (Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also, antimetaboUte, antibiotic, or herbicide resistance can be used as the basis for selection.
• dhfr confers resistance to methotrexate
• neo confers resistance to the aminoglycosides ne ⁇ mycin and G-418
• als and pat confer resistance to chlorsulfiiron and phosphinotricin acetyltransferase, respectively
• trpB and hisD Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabohtes (Hartman, S.C and R.C.
• Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; 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. For example, if the 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 weU.
• host ceUs that contain the polynucleotide encoding KPP and that express KPP may be identified by a variety of procedures known to those of skiU in the art. These procedures include, but are not Umited to, DNA-DNA or DNA-RNA hybridizations, PCR ampUfication, 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 are known in the art. Examples of such techniques include enzyrne-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
• ELISAs enzyrne-linked immunosorbent assays
• RIAs radioimmunoassays
• FACS fluorescence activated cell sorting
• Label and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays.
• Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding KPP include oUgolabeUng, nick translation, end-labeling, or PCR ampUfication using a labeled nucleotide.
• polynucleotides encoding KPP, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
• a vector for the production of an mRNA probe Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commerciaUy available kits, such as those provided by Amersham Biosciences, Promega (Madison WI), and US Biochemical.
• Suitable reporter molecules or labels which may be used for ease of detection include radionucUdes, enzymes, fluorescent, chemiluminescent, 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 ceU 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 may be 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 abiUty 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 Umited to, acetylation, carboxylation, glycosylation, phosphorylation, Upidation, and acylation.
• Post-translational processing which cleaves a "prepro” or "pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
• Different host cells which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture CoUection (ATCC, Manassas VA) and may be chosen to ensure the conect modification and processing of the foreign protein.
• ATCC American Type Culture CoUection
• Manassas VA American Type Culture CoUection
• natural, modified, or recombinant polynucleotides encoding KPP may be Ugated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
• a chimeric KPP protein containing a heterologous moiety that can be recognized by a commerciaUy available antibody may faciUtate the screening of peptide Ubraries for inhibitors of KPP activity.
• Heterologous protein and peptide moieties may also faciUtate purification of fusion proteins using commerciaUy available affinity matrices.
• Such moieties include, but are not Umited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
• GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobiUzed glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
• FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commerciaUy available monoclonal and polyclonal antibodies that specificaUy recognize these epitope tags.
• a fusion protein may also be engineered to contain a proteolytic cleavage site located between the KPP encoding sequence and the heterologous protein sequence, so that KPP may be cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16).
• a variety of commerciaUy available kits may also be used to faciUtate expression and purification of fusion proteins.
• synthesis of radiolabeled KPP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
• KPP, fragments of KPP, or variants of KPP may be used to screen for compounds that specificaUy bind to KPP.
• One or more test compounds may be screened for specific binding to KPP.
• 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to KPP.
• Examples of test compounds can include antibodies, anticalins, oUgonucleotides, proteins (e.g., Ugands or receptors), or small molecules.
• variants of KPP can be used to screen for binding of test compounds, such as antibodies, to KPP, a variant of KPP, or a combination of KPP and/or one or more variants KPP.
• 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 DD NO: 1-42.
• 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 Ugand of KPP, e.g., a Ugand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (CoUgan, J.E. et al. (1991) Cunent Protocols in Immunologv l(2):Chapter 5).
• the compound thus identified can be a natural Ugand of a receptor KPP (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132-140; Wise, A. et al. (2002) Drag Discovery Today 7:235-246).
• 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 Ugand binding site or binding pocket.
• the compound maybe 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. CeU Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336).
• the compound can be rationaUy designed using known techniques.
• Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgGj (Taylor, P.C et al. (2001) Cun. Opin. Immunol. 13:611-616).
• TNF tumor necrosis factor
• two or more antibodies having similar 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 aUows for preferential identification of specific fragments or variants of KPP.
• an antibody can be selected such that its binding specificity aUows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of KPP.
• anticaUns can be screened for specific binding to KPP, fragments of KPP, or variants of KPP.
• AnticaUns are Ugand-binding proteins that have been constructed based on a UpocaUn scaffold (Weiss, G.A. and KB. Lowman (2000) Chem. Biol. 7:R177-R184; Skerra, A. (2001) J. Biotechnol. 74:257-275).
• the protein architecture of UpocaUns can include a beta-banel having eight antiparallel beta-strands, which supports four loops at its open end.
• loops form the natural Ugand-binding site of the UpocaUns, 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 specificaUy bind to, stimulate, or inhibit KPP involves producing appropriate ceUs which express KPP, either as a secreted protein or on the ceU membrane.
• Prefened ceUs can include ceUs from mammals, yeast, Drosophila, or E. coli.
• CeUs expressing KPP or ceU 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 may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
• the assay may comprise the steps of combining at least one test compound with KPP, either in solution or affixed to a soUd support, and detecting the binding of KPP to the compound.
• the assay may detect or measure binding of a test compound in the presence of a labeled competitor. AdditionaUy, the assay may be carried out using ceU-free preparations, chemical Ubraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a soUd support.
• An assay can be used to assess the abiUty of a compound to bind to its natural Ugand and/or to inhibit the binding of its natural Ugand 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 abiUty to bind to its natural Ugands (Matthews, DJ. and J.A. WeUs. (1994) Chem. Biol. 1:25-30).
• one or more amino acid substitutions can be introduced into a polypeptide compound (such as a Ugand) to improve or alter its abiUty to bind to its natural receptors (Cunningham, B.C. and J.A. WeUs (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 Ugand
• 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 cell- 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 pluraUty of test compounds may be screened.
• polynucleotides encoding KPP or their mammaUan 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 ceU 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 disrapted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
• the vector integrates into the conesponding 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) CUn. 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 transfened 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 ceUs derived from human blastocysts. Human ES ceUs have the potential to differentiate into at least eight separate ceU lineages including endoderm, mesoderm, and ectodermal ceU types.
• ceU Uneages differentiate into, for example, neural ceUs, hematopoietic Uneages, 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. With 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 cells are injected into blastulae, and the blastulae are implanted as described above.
• Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
• 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).
• KPP appears to play a role in cardiovascular diseases, immune system disorders, neurological disorders, disorders affecting growth and development, Upid disorders, ceU proUferative 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 Umited to, a cardiovascular disease such as arteriovenous fistula, atherosclerosis, hypertension, vascuUtis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and compUcations of thrombolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitaUy bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, r
• 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 Umited to, those described above.
• compositions comprising a substantially 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 Umited 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 Umited to, those Usted above.
• an antagonist of KPP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of KPP.
• disorders include, but are not Umited to, those cardiovascular diseases, immune system disorders, neurological disorders, disorders affecting growth and development, Upid disorders, ceU proUferative disorders, and cancers described above.
• an antibody which specificaUy binds KPP may be used directly as an antagonist or indirectly as a targeting or deUvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express KPP.
• 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 Umited 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 skiU 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.
• An antagonist of KPP may be produced using methods which are generaUy known in the art.
• purified KPP may be used to produce antibodies or to screen Ubraries of pharmaceutical agents to identify those which specificaUy bind KPP.
• Antibodies to KPP may also be generated using methods that are weU known in the art.
• Such antibodies may include, but are not Umited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Ubrary.
• neutraUzing antibodies i.e., those which inhibit dimer formation
• Single chain antibodies may be potent enzyme inhibitors and may have appUcation 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 oUgopeptide thereof which has immunogenic properties.
• various adjuvants may be used to increase immunological response.
• Such adjuvants include, but are not Umited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
• BCG BacilU Calmette-Guerin
• Corynebacterium parvum are especiaUy preferable. It is prefened that the oUgopeptides, 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.
• these oUgopeptides, 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. These include, but are not Umited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, RJ. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole, SP. et al. (1984) Mol. CeU Biol. 62:109-120).
• chimeric antibodies such as the spUcing 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 maybe 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 Ubraries (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 immunoglobuUn Ubraries or panels of highly specific binding reagents as disclosed in the Uterature (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 Umited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
• Fab expression Ubraries 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).
• immunoassays may be used for screening to identify antibodies having the desired specificity.
• Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with estabUshed specificities are weU known in the art.
• Such immunoassays typicaUy involve the measurement of complex formation between KPP and its specific antibody.
• a two-site, monoclonal-based immunoassay utiUzing monoclonal antibodies reactive to two non-interfering KPP epitopes is generaUy used, but a competitive binding assay may also be employed (Pound, supra).
• K. association constant
• High-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 similar 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 Wiley & Sons, New York NY).
• polyclonal antibody preparations may be further evaluated to determine the quaUty and suitabiUty of such preparations for certain downstream appUcations.
• a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generaUy employed in procedures requiring precipitation of KPP-antibody complexes.
• Procedures for evaluating antibody specificity, titer, and avidity, and guideUnes for antibody quaUty and usage in various appUcations are generaUy available (Catty, supra; CoUgan 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 oUgonucleotides) to the coding or regulatory regions of the gene encoding KPP.
• complementary sequences or antisense molecules DNA, RNA, PNA, or modified oUgonucleotides
• antisense oUgonucleotides 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 deUvered intraceUularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the ceUular sequence encoding the target protein (Slater, J.E. et al. (1998) J. AUergy CUn. Immunol. 102:469-475; Scanlon, K.J. et al. (1995) 9:1288-1296).
• Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (MiUer, A.D. (1990) Blood 76:271; Ausubel et al., supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63:323-347).
• viral vectors such as retrovirus and adeno-associated virus vectors
• Other gene deUvery mechanisms include Uposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J.J. (1995) Br. Med. BuU. 51:217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87:1308-1315; Morris, M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736).
• polynucleotides encoding KPP may be used for somatic or ge ⁇ nline gene therapy.
• Gene therapy may be performed to (i) conect a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SC D)-Xl disease characterized by X- linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) CeU 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene
• conditionaUy lethal gene product e.g., in the case of cancers which result from unregulated cell proUferation
• a protein which affords protection against intraceUular parasites e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
• hepatitis B or C virus HBV, HCV
• fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
• protozoan parasites such as Plasmodium falciparum and Trypanosoma cruz ⁇ .
• diseases or disorders caused by deficiencies in KPP are treated by constructing mammaUan expression vectors encoding KPP and introducing these vectors by mechanical means into KPP-deficient ceUs.
• Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (n) ballistic gold particle deUvery, (in) Uposome-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) CeU 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
• 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), (n) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H.
• a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
• an inducible promoter e.g., the tetracycline-regulated promoter (Gossen, M. and H.
• Uposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen
• aUow one with ordinary skiU in the art to deUver polynucleotides to target ceUs in culture and require minimal effort to optimize experimental parameters.
• transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845).
• the introduction of DNA to primary ceUs requires modification of these standardized mammaUan 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 teirninal repeat (LTR) promoter, (n) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cts-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 pubUshed data (Riviere, I. et al. (1995) Proc. Natl.
• 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. MiUer (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol.
• VPCL vector producing ceU line
• U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retroviras packaging ceU Unes and is hereby incorporated by reference. Propagation of retroviras vectors, transduction of a population of ceUs (e.g., CD4 + T-ceUs), and the return of transduced ceUs to a patient are procedures weU known to persons skiUed in the art of gene therapy and have been weU documented (Ranga, U. et al.
• an adenovirus-based gene therapy deUvery system is used to deUver polynucleotides encoding KPP to ceUs which have one or more genetic abnormaUties with respect to the expression of KPP.
• the construction and packaging of adenovirus-based vectors are weU known to those with ordinary skill in the art.
• RepUcation defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent No.
• Adadenovirus vectors for gene therapy hereby incorporated by reference.
• adenoviral vectors see also Antinozzi, P.A. et al. (1999; Annu. Rev. Nutr. 19:511-544) and Verma, I.M. and N. Somia (1997; Nature 18:389:239-242).
• a herpes-based, gene therapy deUvery system is used to deUver polynucleotides encoding KPP to target ceUs which have one or more genetic abnormaUties with respect to the expression of KPP.
• the use of herpes simplex virus (HSV)-based vectors may be especiaUy valuable for introducing KPP to ceUs of the central nervous system, for which HSV has a tropism.
• the construction and packaging of herpes-based vectors are weU known to those with ordinary skiU in the art.
• a repUcation-competent herpes simplex virus (HSV) type 1 -based vector has been used to deUver a reporter gene to the eyes of primates (Liu, X.
• HSV-1 virus vector has also been disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
• U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transfened to a ceU under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
• HSV vectors see also Goins, W.F. et al. (1999; J. Virol. 73:519-532) and Xu, H. et al. (1994; Dev. Biol. 163:152-161).
• the manipulation of cloned herpesvirus sequences, the generation of recombinant virus foUowing the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of ceUs with herpesvirus are techniques weU known to those of ordinary skiU in the art.
• an alphavirus (positive, single-stranded RNA virus) vector is used to deUver polynucleotides encoding KPP to target ceUs.
• SFV Semliki Forest Virus
• This subgenomic RNA repUcates to higher levels than the fuU 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.
• inserting the coding sequence for KPP into the alphavirus genome in place of the capsid-coding region results in the production of a large number of KPP-coding RNAs and the synthesis of high levels of KPP in vector transduced ceUs.
• alphavirus infection is typicaUy associated with ceU lysis within a few days
• the abiUty to estabUsh a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic repUcation of alphavirases can be altered to suit the needs of the gene therapy appUcation (Dryga, S.A. et al. (1997) Virology 228:74-83).
• the wide host range of alphavirases will allow the introduction of KPP into a variety of ceU types.
• the specific transduction of a subset of ceUs in a population may require the sorting of ceUs prior to transduction.
• 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
• Ribozymes 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 endonucleolytic cleavage of RNA molecules encoding KPP.
• RNA sequences within any potential RNA target are initiaUy identified by scanning the target molecule for ribozyme cleavage sites, including the foUowing sequences: GUA, GUU, and GUC
• short RNA sequences of between 15 and 20 ribonucleotides, conesponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oUgonucleotide inoperable.
• the suitabiUty of candidate targets may also be evaluated by testing accessibiUty to hybridization with complementary oUgonucleotides 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 stabiUty and half- ⁇ fe. Possible modifications include, but are not Umited to, the addition of flanking sequences at the 5 ' and/or 3 ' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase -linkages within the backbone of the molecule.
• RNA interference RNA interference
• PTGS post-transcriptional gene silencing
• RNAi is a posttranscriptional mode of gene silencing in which double-stranded RNA (dsRNA) introduced into a targeted ceU 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 accompUshed by use of DNA or DNA fragments as weU. 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 deUvery and/or viral vector deUvery methods described herein or known in the art.
• RNAi can be induced in mammaUan 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 mammaUan ceUs is described by Elbashir, S.M. et al. (2001; Nature 411:494-498).
• SiRNA can either be generated indirectly by introduction of dsRNA into the targeted ceU, or directly by mammaUan transfection methods and agents described herein or known in the art (such as Uposome-mediated transfection, viral vector methods, or other polynucleotide deUvery/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 occunence of each nucleotide and the 3' adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being prefened.
• Regions to be avoided for target siRNA sites include the 5' and 3 'untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex.
• the selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be eUminated from consideration.
• the selected SiRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commerciaUy available methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin TX).
• long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA.
• This can be accompUshed 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 deUvered to target ceUs using expression vectors known in the art.
• siRNA An example of a suitable expression vector for deUvery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion). Once deUvered to the target tissue, 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 by northern analysis methods using, for example, the NORTHERNMAX-GLY kit (Ambion); by microanay 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 by Western analysis using standard techniques known in the art.
• An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding KPP.
• Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not Umited to, oUgonucleotides, antisense oUgonucleotides, triple heUx-forming oUgonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
• a compound which specificaUy 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 specificaUy promotes expression of the polynucleotide encoding KPP may be therapeuticaUy useful.
• one or more test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
• a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commerciaUy-available or proprietary Ubrary of naturaUy-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a Ubrary of chemical compounds created combinatoriaUy or randomly.
• a sample comprising a polynucleotide encoding KPP is exposed to at least one test compound thus obtained.
• the sample may comprise, for example, an intact or permeabiUzed ceU, or an in vitro ceU-free or reconstituted biochemical system.
• Alterations in the expression of a polynucleotide encoding KPP are assayed by any method commonly known in the art.
• TypicaUy the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding KPP.
• the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
• Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide.
• a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al.
• a particular embodiment of the present invention involves screening a combinatorial Ubrary of oUgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oUgonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Braice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
• oUgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oUgonucleotides
• vectors may be introduced into stem cells taken from the patient and clonaUy propagated for autologous transplant back into that same patient. DeUvery by transfection, by Uposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462- 466).
• any of the therapeutic methods described above may be appUed to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
• An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
• Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
• Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack PubUshing, 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 Uquid or dry powder form. These compositions are generaUy aerosoUzed immediately prior to inhalation by the patient. In the case of smaU molecules (e.g. traditional low molecular weight organic drugs), aerosol deUvery of fast- acting formulations is weU-known in the art.
• compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
• the determination of an effective dose is weU within the capabiUty of those skiUed in the art.
• SpeciaUzed forms of compositions may be prepared for direct intraceUular deUvery of macromolecules comprising KPP or fragments thereof.
• Uposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular deUvery 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).
• the therapeuticaUy effective dose can be estimated initially either in ceU culture assays, e.g., of neoplastic ceUs, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
• a 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 ameUorates 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 prefened.
• 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 Uttle or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route
• the exact dosage wiU be determined by the practitioner, in Ught 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 combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-Ufe 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 deUvery is provided in the Uterature and generaUy available to practitioners in the art. Those skiUed in the art wiU employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, deUvery of polynucleotides or polypeptides wiUbe specific to particular ceUs, conditions, locations, etc. DIAGNOSTICS
• antibodies which specificaUy bind KPP may be used for the diagnosis of disorders characterized by expression of KPP, or in assays to monitor patients being treated with KPP or agonists, antagonists, or inhibitors of KPP.
• Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for KPP include methods which utilize the antibody and a label to detect KPP in human body fluids or in extracts of ceUs or tissues.
• the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
• a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
• KPP KPP protein kinase
• ELISAs ELISAs
• RIAs RIAs
• FACS fluorescence-activated chromatography
• KPP expression normal or standard values for KPP expression are estabUshed by combining body fluids or ceU extracts taken from normal mammaUan subjects, for example, human subjects, with antibodies to KPP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of KPP expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values estabUshes the parameters for diagnosing disease.
• polynucleotides encoding KPP may be used for diagnostic purposes.
• the polynucleotides which maybe used include oUgonucleotides, 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 conelated 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.
• hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding KPP or closely related molecules may be used to identify nucleic acid sequences which encode KPP.
• the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or ampUfication wiU determine whether the probe identifies only naturaUy occurring sequences encoding KPP, alleUc variants, or related sequences.
• Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the KPP encoding sequences.
• the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ DD NO:43-84 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 commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
• Hybridization probes may be labeled by a variety of reporter groups, for example, by radionucUdes 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 Umited to, a cardiovascular disease such as arteriovenous fistula, atherosclerosis, hypertension, vascuUtis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and compUcations of thrombolysis, baUoon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitaUy bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheu
• Polynucleotides encoding KPP maybe 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 quaUtative 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. hi order to provide a basis for the diagnosis of a disorder associated with expression of KPP, a normal or standard profile for expression is estabUshed.
• 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 substantiaUy 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 estabUsh 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 allow health professionals to employ preventative measures or aggressive treatment eariier, thereby preventing the development or further progression of the cancer.
• oUgonucleotides designed from the sequences encoding KPP may involve the use of PCR. These oUgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro.
• OUgomers wiU preferably contain a fragment of a polynucleotide encoding KPP, or a fragment of a polynucleotide complementary to the polynucleotide encoding KPP, and wiU be employed under optimized conditions for identification of a specific gene or condition. OUgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
• oUgonucleotide 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 Umited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods.
• SSCP single-stranded conformation polymorphism
• fSSCP fluorescent SSCP
• oUgonucleotide primers derived from polynucleotides encoding KPP are used to ampUfy 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 oUgonucleotide primers are fluorescently labeled, which aUows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
• AdditionaUy sequence database analysis methods, termed in siUco 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. For example, variants in the mannose-binding lectin, MBL2, have been shown to be conelated 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 Ufe-threatening toxicity.
• N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drag isoniazid, while 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-Upoxygenase pathway.
• Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weU as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. and Z. Gu (1999) Mol. Med.
• the speed of quantitation of multiple samples may be accelerated by ranning the assay in a high-throughput format where the oUgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
• oUgonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microanay.
• the microanay 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 maybe 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 microanay.
• the microanay may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
• a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or 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 and at a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484; hereby expressly incorporated by reference herein).
• a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totaUty 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 pluraUty of elements on a microanay.
• the resultant transcript image would provide a profile of gene activity.
• Transcript images may be generated using transcripts isolated from tissues, cell Unes, 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 ceU 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 precUnical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and naturaUy-occurring environmental 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. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
• the toxicity of a test compound can be assessed by treating a biological sample containing nucleic acids with the test compound.
• Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention 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 cell 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 visuaUzed in the gel as discrete and uniquely positioned spots, typicaUy by staining the gel with an agent such as Coomassie Blue or silver 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 microanay, and protein expression levels are quantified by exposing the microanay to the sample and detecting the levels of protein bound to each anay element (Lueking, A. et al. (1999) Anal. Biochem. 270:103- 111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection maybe performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each 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 conelation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
• the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiUng may be more reUable 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 conesponding 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.
• Microanays may be prepared, used, and analyzed using methods known in the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT appUcation WO95/251116; Shalon, D. et al. (1995) PCT appUcation WO95/35505; HeUer, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; HeUer, M.J. et al. (1997) U.S. Patent No. 5,605,662).
• Various types of microanays are well known and thoroughly described in Schena, M., ed. (1999; DNA Microanays: A Practical Approach, Oxford University Press, London).
• 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 constractions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constractions, or single chromosome cDNA Ubraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, BJ. (1991) Trends Genet. 7:149-154).
• HACs human artificial chromosomes
• YACs yeast artificial chromosomes
• BACs bacterial artificial chromosomes
• PI constractions or single chromosome cDNA Ubraries
• nucleic acid sequences may be used to develop genetic Unkage 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).
• RFLP restriction fragment length polymorphism
• Fluorescent in situ hybridization 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 OnUne MendeUan 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.
• FISH Fluorescent in situ hybridization
• a gene on the chromosome of another mammaUan species may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques.
• the gene or genes responsible for a disease or syndrome have been crudely locaUzed by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to llq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation (Gatti, R.A. et al. (1988) Namre 336:577-580).
• the nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
• KPP its catalytic or immunogenic fragments, or oUgopeptides thereof can be used for screening Ubraries of compounds in any of a variety of drag screening techniques.
• the fragment employed in such screening may be free in solution, affixed to a soUd support, borne on a ceU 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 appUcation WO84/03564).
• This method large numbers of different small test compounds are synthesized on a soUd substrate. The test compounds are reacted with KPP, or fragments thereof, and washed.
• Bound KPP is then detected by methods well known in the art.
• Purified KPP can also be coated directly onto plates for use in the aforementioned drug screening techniques.
• non-neutraUzing antibodies can be used to capture the peptide and immobiUze it on a soUd 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 cunently known, including, but not Umited to, such properties as the triplet genetic code and specific base pair interactions.
• Incyte cDNAs were derived from cDNA Ubraries described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
• TRIZOL Invitrogen
• poly(A)+ RNA was isolated using oUgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
• Stratagene was provided with RNA and constructed the conesponding cDNA Ubraries. Otherwise, cDNA was synthesized and cDNA Ubraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al., supra, ch. 5). Reverse transcription was initiated using oUgo d(T) or random primers. Synthetic oUgonucleotide adapters were Ugated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
• the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
• cDNAs were Ugated into compatible restriction enzyme sites of the poly nker 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 Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof.
• Recombinant plasmids were transformed into competent E. coli ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Invitrogen.
• Plasmids obtained as described in Example I were recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU lysis. Plasmids were purified using at least one of the foUowing: 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 were resuspended in 0.1 ml of distiUed water and stored, with or without lyophiUzation, at 4°C
• plasmid DNA was ampUfied from host ceU lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-weU plates, and the concentration of ampUfied plasmid DNA was quantified fluorometricaUy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN D fluorescence scanner (Labsystems Oy, Helsinki, Finland).
• Incyte cDNA recovered in plasmids as described in Example D were sequenced as foUows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (AppUed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) Uquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or suppUed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppUed Biosystems).
• Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (AppUed Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VDI.
• the polynucleotide sequences derived from Incyte cDNAs were vaUdated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
• the Incyte cDNA sequences or translations thereof were then queried against a selection of pubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, ENCY, and ⁇ GRFAM (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 famiUes; see, for example, Eddy, S.R. (1996) Cun. Opin. Struct. Biol. 6:361-365.
• the queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
• the Incyte cDNA sequences were assembled to produce fuU length polynucleotide sequences.
• GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to fuU length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
• the fuU length polynucleotide sequences were translated to derive the conesponding fuU length polypeptide sequences.
• a polypeptide may begin at any of the methionine residues of the fuU length translated polypeptide.
• FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM, DSfCY, and ⁇ GRFAM; and HMM-based protein domain databases such as SMART.
• GenBank protein databases Genpept
• PROTEOME databases
• BLOCKS BLOCKS
• PRINTS DOMO
• PRODOM Prosite
• Prosite Prosite
• HMM-based protein family databases such as PFAM, DSfCY, and ⁇ GRFAM
• HMM-based protein domain databases such as SMART.
• FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda
• Polynucleotide and polypeptide sequence aUgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence aUgnment program (DNASTAR), which also calculates the percent identity between aUgned sequences.
• Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and fuU length sequences and provides appUcable 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 appUcable, the scores, probabiUty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabiUty value, the greater the identity between two sequences).
• Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C. and S. KarUn (1997) J. Mol. Biol. 268:78-94; Burge, C. and S. KarUn (1998) Cun. 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 was set to 30 kb.
• the encoded polypeptides were analyzed by querying against PFAM models for kinases and phosphatases. Potential kinases and phosphatases were also identified by homology to Incyte cDNA sequences that had been annotated as kinases and phosphatases. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubUc databases.
• Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct enors in. the sequence predicted by Genscan, such as extra or omitted exons.
• BLAST analysis was also used to find any Incyte cDNA or pubUc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.
• Incyte cDNA coverage was available, this information was used to conect or confirm the Genscan predicted sequence.
• FuU length polynucleotide sequences were obtained by assembUng Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubUc cDNA sequences using the assembly process described in Example ID.
• fuU length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
• Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example ID were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible spUce variants that were subsequently confirmed, edited, or extended to create a fuU length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
• Partial DNA sequences were extended to fuU length with an algorithm based on BLAST analysis.
• the nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV.
• a chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
• HSPs high-scoring segment pairs
• GenBank protein homolog The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubUc human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to dete-rmine whether it contained a complete gene.
• Map locations are represented by ranges, or intervals, of human chromosomes.
• the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm.
• centiMorgan cM
• 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.
• the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
• the product score is a normaUzed value between 0 and 100, and is calculated as foUows: the BLAST score is multipUed by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
• the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
• the product score represents a balance between fractional overlap and quaUty in a BLAST aUgnment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
• polynucleotides encoding KPP are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example ID). Each cDNA sequence is derived from a cDNA Ubrary 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; genitaUa, female; genitaUa, male; germ ceUs; hemic and immune system; Uver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
• the number of Ubraries in each category is counted and divided by the total number of Ubraries across aU categories.
• each human tissue is classified into one of the following disease/condition categories: cancer, ceU Une, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of Ubraries in each category is counted and divided by the total number of Ubraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding KPP.
• cDNA sequences and cDNA Ubrary/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII. Extension of KPP Encoding Polynucleotides
• FuU length polynucleotides are produced by extension of an appropriate fragment of the fuU length molecule using oUgonucleotide primers designed from this fragment.
• One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3 ' extension of the known fragment.
• the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided. Selected human cDNA Ubraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
• the concentration of DNA in each well was 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 weU of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing the DNA to bind to the reagent.
• the plate was scanned in a Fluoroskan D (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
• a 5 l to 10 ⁇ l aUquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
• the extended nucleotides were desalted and concentrated, transfened to 384-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to reUgation into pUC 18 vector (Amersham Biosciences).
• CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
• sonicated or sheared prior to reUgation into pUC 18 vector
• the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
• Extended clones were reUgated using T4 Ugase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fiU-in restriction site overhangs, and transfected into competent E. coli ceUs. Transformed ceUs were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-weU plates in LB/2x carb Uquid media.
• the ceUs were lysed, and DNA was ampUfied 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 was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reampUfied using the same conditions as described above.
• Clustering enor filters used statisticaUy generated algorithms to identify enors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed dupUcates and SNPs found in immunoglobulins or T-ceU receptors.
• Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze aUele frequencies at the SNP sites in four different human populations.
• the Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezuelan, and two A ish individuals.
• the African population comprised 194 individuals (97 male, 97 female), aU African Americans.
• the Hispanic population comprised 324 individuals (162 male, 162 female), aU Mexican Hispanic.
• the Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
• AUele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no aUeUc variance in this population were not further tested in the other three populations.
• Hybridization probes derived from SEQ DD NO:43-84 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oUgonucleotides, consisting of about 20 base pairs, is specifically described, essentiaUy the same procedure is used with larger nucleotide fragments. OUgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oUgomer, 250 ⁇ C ⁇ of [ ⁇ - 32 P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
• the labeled oUgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences). An aUquot 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 D, Eco RI, Pst I, Xba I, or Pvu D (DuPont NEN). The DNA from each digest is fractionated on a 0.7% agarose gel and transfened to nylon membranes (Nytran Plus, Schleicher & SchueU, Durham NH).
• Hybridization is carried out for 16 hours at 40 °C To remove nonspecific signals, blots are sequentiaUy 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 visuaUzed using autoradiography or an alternative imaging means and compared.
• microarrays The linkage or synthesis of anay elements upon a microanay can be achieved utilizing photoUthography, 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 soUd with a non-porous surface (Schena, M., ed. (1999) DNA Microanays: A Practical Approach, Oxford University Press, London). Suggested substrates include siUcon, siUca, glass sUdes, glass chips, and siUcon wafers.
• a procedure analogous to a dot or slot blot may also be used to anange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
• a typical anay may be produced using available 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 oUgomers thereof may comprise the elements of the microarray. Fragments or oUgomers 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 anay 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 microanay may be assessed.
• microanay preparation and usage is described in detail below.
• Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oUgo-(dT) ceUulose method.
• Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oUgo-(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 GEMBRIGHT kits (Incyte Genomics).
• Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA.
• Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech, 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 array elements.
• Each anay element is ampUfied from bacterial ceUs containing vectors with cloned cDNA inserts.
• PCR ampUfication uses primers complementary to the vector sequences flanking the cDNA insert.
• Anay elements are ampUfied in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. AmpUfied anay elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
• Purified anay elements are immobiUzed on polymer-coated glass sUdes.
• Glass microscope sUdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
• Glass sUdes are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol.
• Coated sUdes are cured in a 110°C oven.
• Array elements are appUed to the coated glass substrate using a procedure described in U.S. Patent No. 5,807,522, incorporated herein by reference.
• 1 ⁇ l of the array element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capiUary printing element by a high-speed robotic apparatus.
• the apparatus then deposits about 5 nl of anay element sample per sUde.
• 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 distilled 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 foUowed by washes in 0.2% SDS and distiUed water as before.
• Hybridization Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
• the sample mixture is heated to 65° C for 5 minutes and is aUquoted onto the microarray surface and covered with an 1.8 cm 2 coversUp.
• the anays are transfened to a waterproof chamber having a cavity just sUghtly larger than a microscope sUde.
• the chamber is kept at 100% humidity internaUy by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
• the chamber containing the anays is incubated for about 6.5 hours at 60°C
• the anays 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 Ught is focused on the array using a 20X microscope objective (Nikon, Inc., MelviUe NY).
• the sUde containing the array is placed on a computer-controUed 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 Ught is spUt, based on wavelength, into two photomultipUer tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) conesponding to the two fluorophores. Appropriate filters positioned between the anay and the photomultipUer 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 typicaUy 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 caUbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
• a specific location on the array contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be conelated with a weight ratio of hybridizing species of 1:100,000.
• the caUbration is done by labeling samples of the caUbrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
• the output of the photomultipUer tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) instaUed in an IBM-compatible PC computer.
• the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
• the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore 's emission spectrum.
• a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
• the fluorescence signal within each element is then integrated to obtain a numerical value conesponding to the average intensity of the signal.
• the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte Genomics). Array elements that exhibit at least about a two-fold change in expression, a signal-to-background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentiaUy expressed.
• XII Complementary Polynucleotides Sequences complementary to the KPP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring KPP. Although use of oUgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with smaUer or with larger sequence fragments. Appropriate oUgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of KPP. To inhibit transcription, a complementary oUgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oUgonucleotide 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 Umited to, the trp-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 mammaUan cell Unes with recombinant Autographica cahfornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
• AcMNPV Autographica cahfornica 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.
• KPP is synthesized as a fusion protein with, e.g., glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
• GST glutathione S- transferase
• a peptide epitope tag such as FLAG or 6-His
• FLAG an 8-amino acid peptide
• 6-His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). Purified KPP obtained by these methods can be used directly in the assays shown in Examples XVD, XVm, XIX, XX, and XXI, where appUcable. XIV. Functional Assays
• KPP function is assessed by expressing the sequences encoding KPP at physiologicaUy elevated levels in mammaUan ceU culture systems.
• cDNA is subcloned into a mammaUan expression vector containing a strong promoter that drives high levels of cDNA expression.
• Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU Une, for example, an endotheUal or hematopoietic cell Une, using either Uposome 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 ceUs and is a reUable predictor of cDNA expression from the recombinant vector.
• Marker proteins of choice include, e.g., Green Fluorescent Protein
• FCM Flow cytometry
• KPP 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 cells and bind to conserved regions of human immunoglobulin G (IgG).
• Transfected ceUs are efficiently separated from nontransfected 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 skiU in the art. Expression of mRNA encoding KPP and other genes of interest can be analyzed by northern analysis or microanay techniques. XV. Production of KPP Specific Antibodies
• PAGE polyacrylamide gel electrophoresis
• the KPP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oUgopeptide is synthesized and used to raise antibodies by means known to those of skiU in the art.
• LASERGENE software DNASTAR
• Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophiUc regions are well described in the art (Ausubel et al, supra, ch. 11).
• oUgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (AppUed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-malei ⁇ dobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the oUgopeptide-KLH complex in complete Freund's adjuvant.
• 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 immunoaffinity 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 disrapt 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.
• KPP KPP, or biologicaUy active fragments thereof, are labeled with 125 I Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539).
• Candidate molecules previously arrayed 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.
• molecules interacting with KPP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Natore 340:245-246), or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (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 all interactions between the proteins encoded by two large Ubraries 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. 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.
• ATP adenosine triphosphate
• the binding sample then undergoes a centrifugal ultrafiltration process with a membrane which wiU retain the product-avidin complex and aUow passage of free gamma 32 P-ATP.
• the reservoir of the centrifuged unit containing the 32 P-peptide product as retentate is then counted in a scintillation counter.
• This procedure aUows the assay of any type of protein kinase sample, depending on the peptide substrate and kinase reaction buffer selected.
• This assay is provided in kit form (ASUA, Affinity Ultrafiltration Separation Assay, Transbio Corporation, Baltimore MD, U.S. Patent No. 5,869,275).
• Suggested substrates and their respective enzymes include but are not Umited to: Histone HI (Sigma) and p34 cdc2 kinase, Annexin I, Angiotensin (Sigma) and EGF receptor kinase, Annexin D and src kinase, ERKl & 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 neutraUzed 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, may be 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% ⁇ -mercaptoethanol 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. CeU. 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 Ught absorbance at 410 nm resulting from the hydrolysis of PNPP is measured using a spectrophotometer. The increase in Ught absorbance is proportional to the activity of KPP in the assay.
• KPP activity is determined 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 -i ⁇ miunoaffinity 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 seram 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 seram albumin, and 0.05% Triton X-100
• KPP activity is measured for each weU and the abiUty 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.
• XXI Identification of KPP Substrates
• a 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.
• DNA fragments containing the mutation are exchanged with the conesponding wild-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 COS1 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. Forty- eight hours after transfection, cells 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 from lysates 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 visuaUzed by PAGE or further purified to identify substrate molecules (Flint, AJ. et al. (1997) Proc. Natl. Acad. Sci. USA 94:1680-1685).

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