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Definitions

• This invention relates to nucleic acid and amino acid sequences of human kinases and to the use of these sequences in the diagnosis, treatment, and prevention of cancer, immune disorders, disorders affecting growth and development, cardiovascular diseases, and lipid disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of human kinases.
• 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. There are two classes of protein kinases.
• 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 LX comprise the highly conserved catalytic core.
• PTKs and STKs also contain distinct sequence motifs in subdomains VI and VET 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. If a protein analyzed includes the two protein kinase signatures, the probability of that protein being a protein kinase is close to 100% (PROSITE: PDOCOOl 00, November 1995). Protein Tyrosine Kinases
• Protein tyrosine kinases may be classified as either transmembrane, receptor PTKs or nontransmembrane, nonreceptor PTK proteins.
• Transmembrane tyrosine kinases function as receptors for most growth factors. Growth factors bind to the receptor tyrosine kinase (RTK), which causes the receptor to phosphorylate itself (autophosphorylation) and specific intracellular second messenger proteins.
• Growth factors (GF) that associate with receptor PTKs include epidermal GF, platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascular endothelial GF, and macrophage colony stimulating factor.
• Nontransmembrane, nonreceptor PTKs lack transmembrane regions and, instead, form signaling complexes with the cytosolic domains of plasma membrane receptors.
• Receptors that function through non-receptor PTKs include those for cytokines and hormones (growth hormone and prolactin), and antigen-specific receptors on T and B lymphocytes.
• 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.
• Substrates for tyrosine kinases can be identified using anti-phosphotyrosine antibodies to screen tyrosine-phosphorylated cDNA expression libraries.
• Fish so named for tyrosine- phosphorylated in Src-transfromed fibroblast, is a tyrosine kinase substrate which has been identified by such a technique.
• Fish has five SH3 domains and a phox homology (PX) domain. Fish is suggested to be involved in signalling by tyrosine kinases and have a role in the actin cytoskeleton (Lock,P. et al (1998) EMBO J. 17:4346-4357).
• SHP-2 an SH2-domain-containing phosphotyrosine phosphatase
• RTKs receptor tyrosine kinases
• cytokine receptors Phosphotyrosine phosphatases are critical positive and negative regulators in the intraellular signalling pathways that result in growth-factor-specific cell responses such as mitosis, migration, differentiation, transformation, survival or death.
• Signal-regulatory proteins (SIRPs) comprise a new gene family of at least 15 members, consisting of two subtypes distinguished by the presence or absence of a cytoplasmic SHP-2-binding domain.
• the SIRP-alpha subfamily members have a cytoplasmic SHP2- binding domain and includes SIRP-alpha- 1, a transmembrane protein, a substrate of activated RTKs and which binds to SH2 domains.
• SIRPs have a high degree of homology with immune antigen recognition molecules.
• the SIRP-beta subfamily lacks the cytoplasmic tail.
• the SIRP-beta-1 gene encodes a polypeptide of 398 amino acids.
• SERP family members are generally involved in regulation of signals which define differnet physiological and pathological process (Kharitonenkov,A. et al (1997) Nature 386:181-186). Two possible areas of regulation include determination of brain diversity and genetic individuality (Sano,S et al (1999) Biochem. J. 344 Pt 3 -.661-615) and recognition of self which fails in diseases such as hemolytic anemia (01denborg,P.-A et al (2000) Science 288:2051-2054). Protein Serine
• STKs Protein serine/threonine kinases
• a subclass of STKs are known as ERKs (extracellular signal regulated kinases) or MAPs (mitogen-activated protein kinases) and are activated after cell stimulation by a variety of hormones and growth factors.
• Cell stimulation induces a signaling cascade leading to phosphorylation of MEK (MAP/ERK kinase) which, in turn, activates ERK via serine and threonine phosphorylation.
• MEK MAP/ERK kinase
• a varied number of proteins represent the downstream effectors for the active ERK and implicate it in the control of cell proliferation and differentiation, as well as regulation of the cytoskeleton.
• ERK Activation of ERK is normally transient, and cells possess dual specificity phosphatases that are responsible for its down- regulation. Also, numerous studies have shown that elevated ERK activity is associated with some cancers.
• Other STKs include the second messenger dependent protein kinases such as the cyclic- AMP dependent protein kinases (PKA), calciun -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 calciun -calmodulin dependent protein kinases
• MAP mitogen-activated protein kinases
• 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.
• the mammalian circadian mutation tau was found to be a semidominant autosomal allele of CKI-epsilon that markedly shortens period length of circadian rhythms in Syrian hamsters.
• the tau locus is encoded by casein kinase I-epsilon, which is also a homolog of the Drosophila circadian gene double-time.
• CKI-epsilon is able to interact with mammalian PERIOD proteins, while the mutant enzyme is deficient in its ability to phosphorylate PERIOD.
• CKI- epsilon plays a major role in delaying the negative feedback signal within the transcription-translation- based autoregulatory loop that composes the core of the circadian mechanism. Therefore the CKI- epsilon enzyme is an ideal target for pharmaceutical compounds influencing circadian rhythms, jet-lag and sleep, in addition to other physiologic and metabolic processes under circadian regulation (Lowrey, P.L. et al. (2000) Science 288:483-491).
• HIPKs Homeodomain-interacting protein kinases
• HIPKs are serine/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 HTPK2 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 murine homology to Caenorhabditis elegans UNC51 a serine/threonine kinase, has been determined to be required to signal the program of gene expression leading to axon formation from granule cells of the cerebellar cortex (Tomoda, T. et al (1999) Neuron 24:833-346.
• the human homolog of UNC-51, ULK1, for UNC-51 (C. elegans)-like kinase 1, is composed of 1050 amino acids, has a calculated MV of 112.6 kDa and a pi of 8.80.
• ULK1 has 41 % overall sequence similarity to UNC-51 and is highly convserved among vertebrates including mammals, birds, reptiles, amphibians, and fish.
• Kuroyanagi et al have shown ULK1 to be ubiquitously expressed in adult tissues, including skeletal muscle, heart, pancreas, brain, placenta, liver, kidney, and lung while UNC-51 has been specifically located in the nervous system of C. elegans.
• Fish and RH mapping confirmed the localization of ULK1 to human chromosome 12q24.3.
• CaM kinases are involved in regulation of smooth muscle contraction, glycogen breakdown (phosphorylase kinase), and neurotransmission (CaM kinase I and CaM kinase H). 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 ⁇ 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 postnatalfy.
• 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, 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).
• TNF tumor necrosis factor
• IL-1 interleukin-1
• Altered 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.
• MAPKKK6 is one of numerous MAP3Ks identified. Isolated from skeletal muscle, MAP3K6 is 1 ,280 amino acids in length with 11 kinase subdomains and is detected in several tissues. The highest expression has been found in heart and skeletal muscle. MAP3K6 has 45% amino acid sequence identity with MAP3K5, while their catalytic domains share 82% identity. MAP3K6 interaction with MAP3K5 in vivo was confirmed by coimmunoprecipitation. Recombinant MAP3K6 has been shown to weakly activate the JNK but not the p38 kinase or ERK pathways (Wang,X.S. et al. supra)
• 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.
• 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 similar deficiency in a checkpoint kinase, such as Chkl, may also contribute to cancer by failure to arrest cells with damaged DNA at other checkpoints such as G2/M.
• Proliferation-related 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. 5'-AMP-activated protein kinase
• a ligand-activated STK protein kinase is 5 -AMP-activated protein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 271:8675-8681).
• AMPK 5 -AMP-activated protein kinase
• Mammalian AMPK is a regulator of fatty acid and sterol synthesis through phosphorylation of the enzymes acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase and mediates responses of these pathways to cellular stresses such as heat shock and depletion of glucose and ATP.
• AMPK is a heterotrimeric complex comprised of a catalytic alpha subunit and two non-catalytic beta and gamma subunits that are believed to regulate the activity of the alpha subunit.
• Subunits of AMPK have a much wider distribution in non-lipogenic tissues such as brain, heart, spleen, and lung than expected. This distribution suggests that its role may extend beyond regulation of lipid metabolism alone.
• Apoptosis is a highly regulated signaling pathway leading to cell death that plays a crucial role in tissue development and homeostasis. Deregulation of this process is associated with the pathogenesis of a number of diseases including autoimmune disease, neurodegenerative disorders, and cancer. Various STKs play key roles in this process.
• ZIP kinase is an STK containing a C-terminal leucine zipper domain in addition to its N-terminal protein kinase domain.
• 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 NEH3T3 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 is then cleaved into inositol triphosphate (rP 3 ) and diacylglycerol. These two products act as mediators for separate signaling pathways.
• Cellular responses that are mediated by these pathways are glycogen breakdown in the liver in response to vasopressin, smooth muscle contraction in response to acetylcholine, and thrombin-induced platelet aggregation.
• PI 3-kinase 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 (J S)-l , the catalytic subunit is activated and converts PI (4,5) bisphosphate (PIP 2 ) to PI (3,4,5) P 3 (PIP 3 ). PIP 3 then activates a number of other proteins, including PKA, protein kinase B (PKB), protein kinase C (PKC), glycogen synthase kinase (GSK)-3, and p70 ribosomal s6 kinase. PI3K also interacts directly with the cytoskeletal organizing proteins, Rac, rho, and cdc42 (Shepherd, P.R.
• SPP sphingosine-1 -phosphate
• SPP levels are regulated by 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 AdK is found in almost all cell types and is especially abundant in cells having high rates of ATP synthesis and utilization such as skeletal muscle. In these cells AdK is physically associated with mitochondria and myoiibrils, the subcellular structures that are involved in energy production and utilization, respectively. Recent studies have demonstrated a major function for AdK in transferring high energy phosphoryls from metabolic processes generating ATP to cellular components consuming ATP (Zeleznikar, R.J. et al. (1995) J. Biol. Chem. 270:7311-7319). Thus AdK may have a pivotal role in maintaining energy production in cells, particularly those having a high rate of growth or metabolism such as cancer cells, and may provide a target for suppression of its activity to treat certain cancers.
• 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 ras and promote oncogenesis.
• Increasing GuK activity to increase levels of GDP and reduce the GTP:GDP ratio may provide a therapeutic strategy to reverse oncogenesis.
• GuK is an important enzyme in the phosphorylation and activation of certain antiviral drugs useful in the treatment of he ⁇ es 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 drags and possibly for reducing the necessary dosages of the drugs. Pyrimidine Kinases
• the pyrimidine kinases are deoxycytidine kinase and thymidine kinase 1 and 2.
• Deoxycytidine kinase is located in the nucleus, and 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.
• pyrimidine kinases like purine kinases, in phosphorylation is critical to the activation of several chemotherapeutically important nucleoside analogues (ArnerE.S. and S. Eriksson (1995) Pharmacol. Ther. 67:155-186).
• the invention features purified polypeptides, human kinases, referred to collectively as “PKfN” and individually as “PKIN-1,” “PKIN-2,” “PKIN-3,” “PKJ -4,” “PKIN-5,” “PKIN-6,” “PKIN-7,” “PKTN-8,” “PKTN-9,” “PKIN-10,” “PKTN-11,” “PKIN-12” “PKIN-13,” “PKIN-14,” “PKTN-15,” “PKIN-16,” “PKTN-17,” “PKTN-18,” “PKF -19,” and “PKTN-20.”
• the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1
• the invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ JD NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l- 20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20.
• the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-20.
• the polynucleotide is selected from the group consisting of SEQ ID NO:21-40.
• the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20
• the invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprismg an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:1-20.
• 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.
• the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ JD NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ JD NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.
• the invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ JD NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:21-40, 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 comprises at least 60 contiguous nucleotides.
• the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ JD NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:21-40, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
• the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof.
• the probe comprises at least 60 contiguous nucleotides.
• the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:21-40, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
• the method comprises a) amplifying said 5 target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
• the invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected
• the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.
• the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional PKIN, comprising administering to a patient in need of such treatment the composition.
• the invention also provides a method for screening a compound for effectiveness as an
• the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
• the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
• the invention provides a method of
• composition treating a disease or condition associated with decreased expression of functional PKIN, comprising administering to a patient in need of such treatment the composition.
• the invention 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 JD NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:1-20.
• the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
• the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient.
• the invention provides a method of treating a disease or condition associated with overexpression of functional PKIN, comprising administering to a patient in need of such treatment the composition.
• the invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ JD NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ JD NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-20.
• the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
• the invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ JD NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ JD NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.
• the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
• the invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ JD NO:21-40, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
• the invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ED NO:21-40, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO :21-40, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv
• Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ JD NO :21-40, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO :21-40, 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 comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
• Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probability score for the match between each polypeptide and its GenBank homolog is also shown.
• Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
• Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
• Table 5 shows the representative cDNA library for polynucleotides of the invention.
• Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
• Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
• a reference to “a host cell” includes a plurality of such host cells
• a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
• all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
• any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the ptupose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. None herein is to be constraed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. DEFINITIONS
• PKIN refers to the amino acid sequences of substantially purified PKIN obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
• agonist refers to a molecule which intensifies or mimics the biological activity of
• PKIN may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of PKIN either by directly interacting with PKIN or by acting on components of the biological pathway in which PKIN participates.
• allelic variant is an alternative form of the gene encoding PKLN. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
• altered nucleic acid sequences encoding PKIN include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as PKIN or a polypeptide with at least one functional characteristic of PKIN. Included within this definition are polymo ⁇ hisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding PKIN, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding PKIN.
• the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent PKIN.
• Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of PKIN is retained.
• 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
• amino acid and amino acid sequence refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
• Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
• PCR polymerase chain reaction
• Antagonist refers to a molecule which inhibits or attenuates the biological activity of PKIN.
• Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of PKIN either by directly interacting with PKIN or by acting on components of the biological pathway in which PKTN 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 PKIN polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
• the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
• an animal e.g., a mouse, a rat, or a rabbit
• RNA e.g., a mouse, a rat, or a rabbit
• antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
• an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
• antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a specific nucleic acid sequence.
• Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine.
• Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
• the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
• 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 PKIN, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
• Complementary describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
• composition comprising a given polynucleotide sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
• the composition may comprise a dry formulation or an aqueous solution.
• Compositions comprising polynucleotide sequences encoding PKIN or fragments of PKIN may be employed as hybridization probes.
• the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
• 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 (GCG, Madison WI) 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.
• 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, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
• Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a stractural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
• a “fragment” is a unique portion of PKIN or the polynucleotide encoding PKIN which is identical in sequence to but shorter in length than the parent sequence.
• a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
• a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues.
• a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
• 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.
• these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
• a fragment of SEQ ID NO:21-40 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:21-40, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
• a fragment of SEQ JD NO:21-40 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:21 -40 from related polynucleotide sequences.
• the precise length of a fragment of SEQ ID NO:21-40 and the region of SEQ ID NO:21-40 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended pu ⁇ ose for the fragment.
• a fragment of SEQ JD NO:1-20 is encoded by a fragment of SEQ ID NO:21-40.
• a fragment of SEQ JD NO: 1-20 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO: 1-20.
• a fragment of SEQ ED NO:1-20 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-20.
• the precise length of a fragment of SEQ JD NO: 1 -20 and the region of SEQ ID NO: 1 -20 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended pu ⁇ ose for the fragment.
• a “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
• a “full length” polynucleotide sequence encodes a "full length” polypeptide sequence.
• Homology refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
• Percent identity and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as inco ⁇ orated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WT).
• CLUSTAL V is described in Higgins, D.G. and P.M. Sha ⁇ (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS 8:189-191.
• the "weighted” residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
• NCBI National Center for Biotechnology information
• BLAST Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410
• NCBI National System for Mobile Science
• BLAST 2 Sequences a tool that is used for direct pairwise comparison of two nucleotide sequences.
• BLAST 2 Sequences can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
• the "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below).
• BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
• Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ED number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
• percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
• Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge andjiydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
• 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: Matrix: BLOSUM62
• Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ JD number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• "Human artificial chromosomes" are linear microchromosomes which may contain
• humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
• Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
• Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
• 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.
• 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 will be readily apparent to those of ordinary skill in the art.
• Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
• hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
• a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which ' cells or their nucleic acids have been fixed).
• insertion and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
• 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 oligopeptide fragment of PKEN which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
• the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of PKEN which is useful in any of the antibody production methods disclosed herein or known in the art.
• the term “microarray” refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
• element and “array element” refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
• modulate refers to a change in the activity of PKEN. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of PKIN.
• nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
• PNA peptide nucleic acid
• “Operably liriked” 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 oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
• Post-translational modification of an PKIN may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of PKIN.
• Probe refers to nucleic acid sequences encoding PKIN, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences.
• Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
• Primmers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme.
• Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
• PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that pu ⁇ ose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
• Oligonucleotides for use as primers are selected using software known in the art for such prupose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have inco ⁇ orated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome- wide scope.
• the Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.)
• the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
• this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
• the oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
• a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
• the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
• a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
• such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia viras, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
• a “regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
• Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
• RNA equivalent in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
• sample is used in its broadest sense.
• a sample suspected of containing PKIN, nucleic acids encoding PKIN, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
• binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
• substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
• substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
• Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers,' magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
• the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
• a “transcript image” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
• Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
• transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
• a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
• the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant viras.
• the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
• the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
• the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
• a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 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 "allelic” (as defined above), “splice,” “species,” or “polymo ⁇ bic” variant.
• a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
• the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
• Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other.
• a polymo ⁇ hic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
• Polymo ⁇ bic variants also may encompass "single nucleotide polymo ⁇ hisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
• a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identify 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 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
• the invention is based on the discovery of new human human kinases (PKIN), the polynucleotides encoding PKIN, and the use of these compositions for the diagnosis, treatment, or prevention of cancer, immune disorders, disorders affecting growth and development, cardiovascular diseases, and lipid disorders.
• PKIN human human kinases
• Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Encyte project identification number (Incyte Project ED). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ED NO:) and an Incyte polypeptide sequence number (Encyte Polypeptide ED) as shown.
• Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ED NO:) and an Encyte polynucleotide consensus sequence number (Incyte Polynucleotide ED) as shown.
• Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database.
• Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ED NO:) and the corresponding Encyte polypeptide sequence number (Encyte Polypeptide ED) for polypeptides of the invention.
• Column 3 shows the GenBank identification number (Genbank JD NO:) of the nearest GenBank homolog.
• Column 4 shows the probability score for the match between each polypeptide and its GenBank homolog.
• Column 5 shows the annotation of the GenBank homolog along with relevant citations where applicable, all of which are expressly inco ⁇ orated by reference herein.
• Table 3 shows various stractural features of the polypeptides of the invention.
• Columns 1 and 2 show the polypeptide sequence identification number (SEQ ED NO:) and the corresponding Encyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention.
• Column 3 shows the number of amino acid residues in each polypeptide.
• Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTEFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WE).
• Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
• Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
• SEQ ID NO:2 is 97% identical to mouse tousled-like kinase (GenBank ED g2853031) as determined by the Basic E >cal Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
• SEQ ED NO:2 also contains an eukaryotic protein kinase domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
• HMM hidden Markov model
• SEQ D NO:2 is a tousled-like kinase.
• SEQ JD NO: 10 is 63% identical to human serine/threonine protein kinase (GenBank ID g36615) as determined by the Basic E ical Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 7.7e-122, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
• SEQ ED NO: 10 also contains an eukaryotic protein kinase domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein domains.
• HMM hidden Markov model
• SEQ JD NO: 10 is a serine/threonine kinase.
• SEQ ED NO:16 is 53% identical to human receptor protein-tyrosine kinase (GenBank ED g551608) as determined by the Basic Local Alignment Search Tool (BLAST).
• SEQ ED NO:16 also contains an eukaryotic protein kinase domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and PROFELESCAN analyses provide further corroborative evidence that SEQ ED NO: 16 is a receptor tyrosine kinase.
• SEQ ED NO: 19 is 93% identical to rat Calcium/calmodulin-dependent protein kinase isoform EV (GenBank ED gl 836161) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 6.0e-257, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ED NO: 19 also contains an eukaryotic protem kinase domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein domains. (See Table 3.) Data from BLIMPS, MOTEFS, and
• SEQ ED NO: 19 is a protein kinase.
• SEQ ID NO:l, SEQ ED NO:3-9, SEQ ED NO:l l-15, SEQ ED NO:17-18, and SEQ ED NO:20 were analyzed and annotated in a similar manner.
• the algorithms and parameters for the analysis of SEQ ID NO: 1-20 are described in Table 7. As shown in Table 4, the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
• Columns 1 and 2 list the polynucleotide sequence identification number (Polynucleotide SEQ ED NO:) and the corresponding Incyte polynucleotide consensus sequence number (Encyte Polynucleotide ED) for each polynucleotide of the invention.
• Column 3 shows the length of each polynucleotide sequence in basepairs.
• Column 4 lists fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ED NO:21-40 or that distinguish between SEQ ED NO:21-40 and related polynucleotide sequences.
• Column 5 shows identification numbers corresponding to cDNA sequences, coding sequences (exons) predicted from genomic DNA, and/or sequence assemblages comprised of both cDNA and genomic DNA. These sequences were used to assemble the full length polynucleotide sequences of the invention.
• Columns 6 and 7 of Table 4 show the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences in column 5 relative to their respective full length sequences.
• the identification numbers in Column 5 of Table 4 may refer specifically, for example, to Encyte cDNAs along with their corresponding cDNA libraries.
• 2564295H1 is the identification number of an Incyte cDNA sequence
• ADRETUT01 is the cDNA library from which it is derived.
• Incyte cDNAs for which cDNA libraries are not indicated were derived from pooled cDNA libraries (e.g., 71191190V1).
• the identification numbers in column 5 may refer to GenBank cDNAs or ESTs (e.g., gl 164223) which contributed to the assembly of the full length polynucleotide sequences.
• the identification numbers in column 5 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (ie., those sequences including the designation "ENST”).
• the identification numbers in column 5 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (/. e. , those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records ⁇ le., those sequences including the designation "NP”).
• the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
• ⁇ L K XKXJ ⁇ 1 J ⁇ 2 pYYYYY_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 applied, and YYYYY s the number of the prediction generated by the algorithm, and N 1 3 coordinate , if present, represent specific exons that may have been manually edited during analysis (See Example V).
• the identification numbers in column 5 may refer to assemblages of exons brought together by an "exon-stretcbing" algorithm.
• FLXXXXXXX_gAAAAA_gBBBBB_l_N is the identification number of a "stretched" sequence, with XXXXX being the Encyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB being the GenB ank identification number or NCBI RefSeq identification number of the nearest GenB ank protein homolog, and N referring to specific exons (See Example V).
• a RefSeq sequence was used as a protein homolog for the "exon-stretching" algorithm, a
• RefSeq identifier (denoted by " ⁇ M,” “ ⁇ P,” or “NT”) may be used in place of the GenBank identifier (le., gBBBBB).
• a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
• the following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
• Incyte cDNA coverage redundant with the sequence coverage shown in column 5 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Encyte cDNA identification numbers are not shown.
• Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences.
• the representative cDNA library is the Encyte cDNA library which is most frequently represented by the Encyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
• the tissues and vectors which were used to constract the cDNA libraries shown in Table 5 are described in Table 6.
• the invention also encompasses PKEN valiants.
• a preferred PKEN variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the PKEN amino acid sequence, and which contains at least one functional or structural characteristic of PKEN.
• the invention also encompasses polynucleotides which encode PKIN.
• the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO:21-40, which encodes PKEN.
• the polynucleotide sequences of SEQ ED NO:21 -40, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
• the invention also encompasses a variant of a polynucleotide sequence encoding PKEN.
• a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identify to the polynucleotide sequence encoding PKEN.
• a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO:21- 40 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 ED NO:21-40. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or stractural characteristic of PKIN.
• nucleotide sequences which encode PKEN and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring PKIN under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding PKIN or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
• RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
• the invention also encompasses production of DNA sequences which encode PKEN and
• PKEN derivatives, or fragments thereof, entirely by synthetic chemistry After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding PKEN or any fragment thereof. Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ED NO:21-40 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507- 511.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions.”
• Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention.
• the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
• sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology. John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853.)
• the nucleic acid sequences encoding PKIN may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
• various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
• restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.)
• inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
• the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
• a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic.
• primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
• commercially available software such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
• libraries that have been size-selected to include larger cDNAs.
• random-primed libraries which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
• Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
• Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
• capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
• Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
• Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
• polynucleotide sequences or fragments thereof which encode PKEN may be cloned in recombinant DNA molecules that direct expression of PKIN, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express PKEN.
• nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter PKEN-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
• DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
• oligonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
• the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDENG (Maxygen En ⁇ , Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of PKIN, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
• MOLECULARBREEDENG Maxygen En ⁇ , Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et
• DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
• genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
• sequences encoding PKIN may be synthesized, in whole or in part, using chemical methods well known in the art.
• chemical methods See, e.g., Carathers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.
• PKIN itself or a fragment thereof may be synthesized using chemical methods.
• peptide synthesis can be performed using various solution-phase or solid-phase techniques.
• PKEN amino acid sequence of PKEN, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
• the peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.)
• the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
• the nucleotide sequences encoding PKEN or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
• these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding PKIN. Such elements may vary in their strength and specificity.
• Specific initiation signals may also be used to achieve more efficient translation of sequences encoding PKEN. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
• exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
• Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. CeU Differ.
• a variety of expression vector/host systems may be utiUzed to contain and express sequences encoding PKEN. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculoviras); plant cell systems transformed with viral expression vectors (e.g., cauUflower mosaic viras, CaMV, or tobacco mosaic viras, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems.
• 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., baculoviras); plant cell systems transformed
• Expression vectors derived from retrovirases, adenoviruses, or he ⁇ es or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or ceU population.
• the invention is not Umited by the host ceU employed.
• a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding PK N. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding PKIN can be achieved using a multifunctional E.
• coli vector such as PBLUESCREPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding PKEN into the vector's multiple cloning site disrupts the Z ⁇ cZ gene, aUowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
• these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem.
• vectors which direct high level expression of PKIN may be used.
• vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
• Yeast expression systems may be used for production of PKEN.
• 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 intraceUular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
• Plant systems may also be used for expression of PKEN. Transcription of sequences encoding PKEN 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. 3:17-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Corazzi, G. et al. (1984) EMBO J. 3:1671-1680; Bro ' glie, R. et al.
• En mammaUan ceUs a number of viral-based expression systems may be utilized.
• sequences encoding PKEN may be ligated 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 PK N in host ceUs.
• transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host ceUs.
• SV40 or EBV- based vectors may also be used for high-level protein expression.
• HACs Human artificial chromosomes
• HACs may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid.
• HACs of about 6 kb to 10 Mb are constructed and deMvered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic proposes.
• liposomes, polycationic amino polymers, or vesicles for therapeutic proposes.
• sequences encoding PKEN can be transformed into ceU lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. FoUowing the introduction of the vector, ceUs may be aUowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
• the ptupose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfuUy express the introduced sequences.
• Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the cell type.
• Any number of selection systems may be used to recover transformed cell lines. These include, but are not Umited to, the he ⁇ es simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
• dhfr confers resistance to methotrexate
• neo confers resistance to the aminoglycosides neomycin and G-418
• als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
• Additional selectable genes have been described, e.g., trpB and hisD, which alter ceUuIar requirements for metabohtes.
• 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. (See, e.g., Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131.)
• marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
• sequence encoding PKEN is inserted within a marker gene sequence
• transformed ceUs containing sequences encoding PKEN can be identified by the absence of marker gene function.
• a marker gene can be placed in tandem with a sequence encoding PKEN 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 nucleic acid sequence encoding PKEN and that express PKIN 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 ampMfication, 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 PKEN using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
• ELISAs enzyme-linked immunosorbent assays
• RIAs radioimmunoassays
• FACS fluorescence activated cell sorting
• a two-site, monoclonal-based immunoassay utiUzing monoclonal antibodies reactive to two non-interfering epitopes on PKIN is preferred, but a competitive binding assay may be employed.
• a competitive binding assay may be employed.
• Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding PKIN include oUgolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
• sequences encoding PK N, 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 Pharmacia Biotech, Promega (Madison WI), and US Biochemical.
• Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.
• Host ceUs transformed with nucleotide sequences encoding PKEN 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 PKIN may be designed to contain signal sequences which direct secretion of PKEN through a prokaryotic or eukaryotic ceU membrane.
• a host ceU strain may be chosen for its abihty to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
• modifications of the polypeptide include, but are not limited 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 correct modification and processing of the foreign protein.
• ATCC American Type Culture CoUection
• Manassas VA American Type Culture CoUection
• natural, modified, or recombinant nucleic acid sequences encoding PKEN may be Ugated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
• a chimeric PKIN protein containing a heterologous moiety that can be recognized by a commerciaUy available antibody may facilitate the screening of peptide Ubraries for inhibitors of PKIN 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), calmoduUn 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, calmoduUn, 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 PKEN encoding sequence and the heterologous protein sequence, so that PKEN may be cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10).
• a variety of commerciaUy available kits may also be used to faciUtate expression and purification of fusion proteins.
• synthesis of radiolabeled PKEN 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-metbionine.
• PKEN of the present invention or fragments thereof may be used to screen for compounds that specificaUy bind to PKEN. At least one and up to a pluraUty of test compounds may be screened for specific binding to PKEN. Examples of test compounds include antibodies, oUgonucleotides, proteins (e.g., receptors), or smaU molecules.
• the compound thus identified is closely related to the natural Ugand of PKEN, e.g., a Ugand or fragment thereof, a natural substrate, a stractural or functional mimetic, or a natural binding partner.
• PKEN e.g., a Ugand or fragment thereof, a natural substrate, a stractural or functional mimetic, or a natural binding partner.
• the compound can be closely related to the natural receptor to which PKEN binds, or to at least a fragment of the receptor, e.g., the Ugand binding site.
• the compound can be rationaUy designed using known techniques.
• screening for these compounds involves producing appropriate cells which express PKEN, either as a secreted protein or on the ceU membrane.
• Preferred ceUs include ceUs from mammals, yeast, Drosophila. or E. coU. CeUs expressing PKIN or cell membrane fractions which contain PKIN are then contacted with a test compound and binding, stimulation, or inhibition of activity of either PKIN or the compound is analyzed.
• An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label
• the assay may comprise the steps of combining at least one test compound with PKEN, either in solution or affixed to a soUd support, and detecting the binding of PKIN to the compound.
• the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
• the assay may be carried out using ceU-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support.
• PKIN of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of PKEN.
• Such compounds may include agonists, antagonists, or partial or inverse agonists.
• an assay is performed under conditions permissive for PKEN activity, wherein PK N is combined with at least one test compound, and the activity of PKEN in the presence of a test compound is compared with the activity of PKEN in the absence of the test compound. A change in the activity of PKEN in the presence of the test compound is indicative of a ' compound that modulates the activity of PKEN.
• a test compound is combined with an in vitro or ceU-free system comprising PKEN under conditions suitable for PKEN activity, and the assay is performed.
• a test compound which modulates the activity of PKEN 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. En another embodiment, polynucleotides encoding PKEN or their mammaUan homologs may be
• mouse ES ceUs such as the mouse 129/SvJ ceU Une, are derived from the early mouse embryo and grown in culture.
• the ES ceUs are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R.
• the vector integrates into the corresponding region of the host genome by homologous recombination.
• homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, JD. (1996) CUn. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res.
• Transformed ES ceUs are identified and microinjected into mouse ceU blastocysts such as those from the C57BL/6 mouse strain.
• the blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
• Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
• Polynucleotides encoding PKEN may also be manipulated in vitro in ES ceUs derived from human blastocysts.
• Human ES cells have the potential to differentiate into at least eight separate ceU Uneages including endoderm, mesoderm, and ectodermal ceU types. These cell Uneages differentiate into, for example, neural ceUs, hematopoietic Uneages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
• Polynucleotides encoding PKIN can also be used to create "knockin” humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
• knockin technology a region of a polynucleotide encoding PKEN is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome.
• Transformed ceUs are injected into blastulae, and the blastulae are implanted as described above.
• Transgenic progeny or inbred Unes are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
• a mammal inbred to overexpress PKEN e.g., by secreting PKIN in its milk, may also serve as a convenient source of that protein (Jaime, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
• THERAPEUTICS e.g., by secreting PKIN in its milk.
• PKEN Chemical and stractural similarity, e.g., in the context of sequences and motifs, exists between regions of PKEN and human kinases.
• the expression of PKEN is closely associated with bladder cancer, prostatic, ovarian, brain, colon, ileum, penis, skin, adrenal tumor, digestive, and cancerous tissues. Therefore, PKEN appears to play a role in cancer, immune disorders, disorders affecting growth and development, cardiovascular diseases, and Upid disorders.
• PKEN appears to play a role in cancer, immune disorders, disorders affecting growth and development, cardiovascular diseases, and Upid disorders.
• PKEN 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 PKEN.
• disorders include, but are not Umited to, a cancer, such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gaU bladder, ganglia, gastrointestinal tract, heart, kidney, Uver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, saUvary glands, skin, spleen, testis, tbyrnus, thyroid, and uterus, leukemias such as multiple myeloma and lymphomas such as Hodgkin's disease; an immune disorder, such as acquired immunodeficiency syndrome (AIDS),
• AIDS acquired immuno
• a vector capable of expressing PKEN 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 PKEN including, but not Umited to, those described above.
• a composition comprising a substantiaUy purified PKEN in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PKIN including, but not Hmited to, those provided above.
• an agonist which modulates the activity of PKEN may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PKEN including, but not Umited to, those Usted above.
• an antagonist of PK N may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of PKEN.
• disorders include, but are not Umited to, those cancer, immune disorders, disorders affecting growth and development, cardiovascular diseases, and Upid disorders described above.
• an antibody which specifically binds PKEN may be used directly as an antagonist or indirectly as a targeting or deUvery mechanism for bringing a pharmaceutical agent to cells or tissues which express PKEN.
• a vector expressing the complement of the polynucleotide encoding PKIN may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of PKEN including, but not Umited to, those described above.
• any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary 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 PKEN may be produced using methods which are generaUy known in the art.
• purified PKEN may be used to produce antibodies or to screen Ubraries of pharmaceutical agents to identify those which specificaUy bind PK N.
• Antibodies to PKEN may also be generated using methods that are well 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
• various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with PKEN or with any fragment or oUgopeptide thereof which has immunogenic properties.
• various adjuvants may be used to increase immunological response.
• 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 baciUi Calmette-Guerin
• Corynebacterium parvum are especially preferable.
• the oUgopeptides, peptides, or fragments used to induce antibodies to PKIN have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these oUgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of PKIN amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
• Monoclonal antibodies to PKEN may be prepared using any technique which provides for the production of antibody molecules by continuous cell Unes in culture. These include, but are not Umited to, the hybridoma technique, the human B-cell hybridoma technique, and the EB V-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al (1985) J.
• 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.
• techniques developed for the production of "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.
• techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce PKEN-specific single chain antibodies.
• Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobuUn Ubraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
• Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobuUn Ubraries or panels of highly specific binding reagents as disclosed in the Uterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
• Antibody fragments which contain specific binding sites for PKEN may also be generated.
• such fragments include, but are not Umited to, F(ab 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. (See, e.g., Huse, WD. 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 PKIN and its specific antibody.
• a two-site, monoclonal-based immunoassay utiUzing monoclonal antibodies reactive to two non-interfering PKEN epitopes is generaUy used, but a competitive binding assay may also be employed (Pound, supra).
• K. association constant
• High-affinity antibody preparations with K ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the PKEN- antibody complex must withstand rigorous manipulations.
• 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 PKIN-antibody complexes.
• Procedures for evaluating antibody specificity, titer, and avidity, and guideUnes for antibody quaUty and usage in various appUcations, are generaUy available.
• the polynucleotides encoding PKEN 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 PKIN.
• 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 PKIN.
• 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(3):469-475; and Scanlon, K.J. et al.
• Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated viras vectors.
• viral vectors such as retrovirus and adeno-associated viras 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(l):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997) Nucleic Acids Res. 25(14):2730-2736.
• polynucleotides encoding PKIN may be used for somatic or germUne gene therapy.
• Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCJD)-Xl disease characterized by X- Unked 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.
• SCJD severe combined immunodeficiency
• ADA adenosine deaminase
• hepatitis B or C viras HBV, HCV
• fungal parasites such as Candida albicans and Paracoccidioides brasihensis
• protozoan parasites such as Plasmodium falciparam and Trypanosoma cruzi.
• the expression of PKIN from an appropriate population of transduced ceUs may alleviate the clinical manifestations caused by the genetic deficiency.
• PKIN are treated by constructing mammaUan expression vectors encoding PKEN and introducing these vectors by mechanical means into PKEN-deficient cells.
• Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (u) ballistic gold particle deUvery, (hi) 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 PKIN include, but are not Umited to, the PCDNA 3.1, EPFTAG, PRCCMV2, PREP, PVAX vectors (invitrogen, Carlsbad CA), PCMV-SCP PT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF,
• PKEN may be expressed using (i) a constitutively active promoter, (e.g., from cytomegaloviras (CMV), Rous sarcoma viras (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycUne-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al.
• CMV cytomegaloviras
• RSV Rous sarcoma viras
• TK thymidine kinase
• ⁇ -actin genes e.g., the tetracycUne-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:
• CommerciaUy available Uposome transformation kits allow one with ordinary skill 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.
• a retrovirus vector consisting of (i) the polynucleotide encoding PKEN under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (n) appropriate RNA packaging signals, and (Mi) a Rev-responsive element (RRE) along with additional retrovirus cis-ac ng 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 ubUshed data (Riviere, I. et al. (1995) Proc.
• the vector is propagated in an appropriate vector producing ceU Une (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 AD. MiUer (1988) J. Virol 62:3802-3806; Dull, T. et al. (1998) J. Virol.
• VPCL ceU Une
• U.S. Patent Number 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging cell Unes and is hereby inco ⁇ orated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4 + T-ceUs), and the return of transduced ceUs to a patient are procedures well known to persons skilled in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J.
• an adenoviras-based gene therapy deUvery system is used to deUver polynucleotides encoding PKEN to ceUs which have one or more genetic abnormaUties with respect to the expression of PKEN.
• adenoviras-based vectors The construction and packaging of adenoviras-based vectors are weU known to those with ordinary skiU in the art.
• RepUcation defective adenoviras 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).
• PotentiaUy useful adenoviral vectors are described in U.S. Patent Number 5,707,618 to Armentano ("Adenoviras vectors for gene therapy"), hereby inco ⁇ orated by reference.
• Addenoviras vectors for gene therapy For 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, both in
• a he ⁇ es-based, gene therapy deUvery system is used to deUver polynucleotides encoding PKEN to target ceUs which have one or more genetic abnormaUties with respect to the expression of PKEN.
• the use of he ⁇ es simplex viras (HSV)-based vectors may be especiaUy valuable for introducing PKEN to ceUs of the central nervous system, for which HSV has a tropism.
• HSV simplex viras
• the construction and packaging of he ⁇ es-based vectors are weU known to those with ordinary skill in the art.
• HSV he ⁇ es simplex viras
• Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for pmposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73 :519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby inco ⁇ orated by reference.
• an alphaviras (positive, single-stranded RNA viras) vector is used to deUver polynucleotides encoding PKEN to target cells.
• SFV SemUki Forest Viras
• SFV SemUki Forest Viras
• This subgenomic RNA repUcates to higher levels than the fuU length genomic RNA, resulting in the ove ⁇ roduction 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 PKEN into the alphaviras genome in place of the capsid-coding region results in the production of a large number of PKEN- coding RNAs and the synthesis of high levels of PKEN in vector transduced ceUs.
• alphaviras 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 viras (SEN) indicates that the lytic repUcation of alphaviruses 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 alphaviruses wiU aU ow the introduction of PKEN 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.
• the methods of manipulating infectious cDNA clones of alphaviruses, performing alphaviras cDNA and RNA transfections, and performing alphaviras infections, are well known to those with ordinary skill in the art.
• OUgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
• inhibition can be achieved using triple heUx base-pairing methodology.
• Triple heUx pairing is useful because it causes inhibition of the ability of the double heUx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
• Recent therapeutic advances using triplex DNA have been described in the Uterature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Emmunologic Approaches, Futura PubUshing, Mt. Kisco NY, pp.
• a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of
• RNA The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage.
• engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of sequences encoding PKIN.
• Specific ribozyme cleavage sites 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.
• RNA sequences of between 15 and 20 ribonucleotides 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 and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemicaUy synthesizing oUgonucleotides such as soUd phase phosphoramidite chemical synthesis.
• RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding PKEN. Such DNA sequences may be inco ⁇ orated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
• these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into ceU Unes, ceUs, or tissues.
• RNA molecules may be modified to increase intraceUular stabiUty and half-Ufe. 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 Unkages within the backbone of the molecule.
• An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding PKEN.
• 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 specifically inhibits expression of the polynucleotide encoding PKIN may be therapeutically useful, and in the treatment of disorders associated with decreased PKEN expression or activity, a compound which specificaUy promotes expression of the polynucleotide encoding PKEN may be therapeuticaUy useful.
• At least one, and up to a pluraUty, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
• a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commerciaUy-available or proprietary Ubrary of naturaUy-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or stractural properties of the target polynucleotide; and selection from a Ubrary of chemical compounds created combinatoriaUy or randomly.
• a sample comprising a polynucleotide encoding PKEN 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 PKEN 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 PKEN.
• 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.
• a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomvces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human ceU Une such as HeLa ceU (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
• 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 (Braice, 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 ceUs 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. (See, e.g., Goldman, CK. 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 pharmaceutically acceptable excipient.
• Excipients may include, for example, sugars, starches, ceUuloses, 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 PKEN, antibodies to PKEN, and mimetics, agonists, antagonists, or inhibitors of PKEN.
• compositions utilized in this invention may be administered by any number of routes including, but not Umited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal intranasal, enteral, topical, subUngual, or rectal means.
• compositions for pulmonary administration may be prepared in Uquid or dry powder form. These compositions are generally aerosoUzed immediately prior to inhalation by the patient.
• aerosol deUvery of fast- acting formulations is weU-known in the art.
• macromolecules e.g. larger peptides and proteins
• Pulmonary deUvery has the advantage of administration without needle injection, and obviates the need for potentiaUy toxic penetration enhancers.
• compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
• the determination of an effective dose is weU within the capabiUty of those skiUed in the art.
• SpeciaUzed forms of compositions may be prepared for direct intraceUular deUvery of macromolecules comprising PKIN or fragments thereof.
• Uposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular deUvery of the macromolecule.
• PKEN or a fragment thereof may be joined to a short cationic N- terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al (1999) Science 285:1569-1572).
• 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 PKEN or fragments thereof, antibodies of PKEN, and agonists, antagonists or inhibitors of PKEN, 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 preferred.
• the data obtained from ceU culture assays and animal studies are used to formulate a range of dosage for human use.
• the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with Uttle or no toxicity.
• the dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
• the exact dosage 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 generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, deUvery of polynucleotides or polypeptides will be specific to particular ceUs, conditions, locations, etc. DIAGNOSTICS
• antibodies which specificaUy bind PKIN may be used for the diagnosis of disorders characterized by expression of PKEN, or in assays to monitor patients being treated with PKEN or agonists, antagonists, or inhibitors of PKEN.
• Antibodies useful for diagnostic pmposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for PKEN include methods which utiUze the antibody and a label to detect PKIN in human body fluids or in extracts of cells or tissues.
• the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
• a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
• PKEN PKEN-specific kinase
• ELISAs ELISAs
• REAs REAs
• FACS fluorescence-activated cell sorting
• PKEN expression normal or standard values for PKEN expression are estabUshed by combining body fluids or ceU extracts taken from normal mammaUan subjects, for example, human subjects, with antibodies to PKEN under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of PKEN 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.
• the polynucleotides encoding PKEN may be used for diagnostic purposes.
• the polynucleotides which may be used include oUgonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
• the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of PKEN may be correlated with disease.
• the diagnostic assay may be used to determine absence, presence, and excess expression of PKEN, and to monitor regulation of PKEN levels during therapeutic intervention.
• hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding PKEN or closely related molecules may be used to identify nucleic acid sequences which encode PKIN.
• the specificify 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 PKEN, aUeUc 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 PKEN encoding sequences.
• the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:21-40 or from genomic sequences including promoters, enhancers, and introns of the PKEN gene.
• Means for producing specific hybridization probes for DNAs encoding PKEN include the cloning of polynucleotide sequences encoding PKEN or PKEN derivatives into vectors for the production of mRNA probes.
• Such vectors are known in the art, are commerciaUy available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
• Hybridization probes may be labeled by a variety of reporter groups, for example, by radionucUdes such as 32 P or 35 S, or by enzymatic labels, such as alkaUne phosphatase coupled to the probe via avidin/biotin coupUng systems, and the Uke.
• Polynucleotide sequences encoding PKEN may be used for the diagnosis of disorders associated with expression of PKEN.
• a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, gangUa, gastrointestinal tract, heart, kidney, Uver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, saUvary glands, skin, spleen, testis, thymus, thyroid, and uterus, leukemias such as multiple myeloma and lymphomas such as Hodgkin's disease; an immune disorder, such as acquired immunodeficiency syndrome (AEDS), Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondyUtis, amyloidosis, anemia, asthma,
• AEDS acquired immunodeficiency syndrome
• the polynucleotide sequences encoding PKEN may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-Uke assays; and in microarrays utiUzing fluids or tissues from patients to detect altered PKEN expression. Such quaUtative or quantitative methods are well known in the art. En a particular aspect, the nucleotide sequences encoding PKEN may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding PKEN 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 nucleotide sequences encoding PKIN in the sample indicates the presence of the associated disorder.
• assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in cUnical trials, or to monitor the treatment of an individual patient.
• a normal or standard profile for expression is estabUshed. This may be accompUshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding PKEN, under conditions suitable for hybridization or ampUfication.
• 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
• 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 cUnical symptoms.
• a more definitive diagnosis of this type may aUow 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 PKIN may involve the use of PCR. These oUgomers may be chemically synthesized, generated enzymatically, or produced in vitro.
• OUgomers wiU preferably contain a fragment of a polynucleotide encoding PKEN, or a fragment of a polynucleotide complementary to the polynucleotide encoding PKEN, 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 the polynucleotide sequences encoding PKEN may be used to detect single nucleotide polymo ⁇ hisms (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 polymo ⁇ hism (SSCP) and fluorescent SSCP (fSSCP) methods.
• SSCP single-stranded conformation polymo ⁇ hism
• fSSCP fluorescent SSCP
• En SSCP, oUgonucleotide primers derived from the polynucleotide sequences encoding PKEN 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 Uke.
• 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 ampUmers in high-throughput equipment such as DNA sequencing machines.
• AdditionaUy sequence database analysis methods, termed in siUco SNP (isSNP), are capable of identifying polymo ⁇ hisms 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 MASS ARRAY system (Sequenom, Inc., San Diego CA).
• Methods which may also be used to quantify the expression of PKIN include radiolabeUng or biotinylating nucleotides, coampUfication of a control nucleic acid, and inte ⁇ olating results from standard curves.
• radiolabeUng or biotinylating nucleotides See, e.g., Melby, P.C et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.
• the speed of quantitation of multiple samples may be accelerated by running 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 polynucleotide sequences described herein may be used as elements on a microarray.
• the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
• the microarray may also be used to identify genetic variants, mutations, and polymo ⁇ hisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
• this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient.
• therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
• PK N, fragments of PKEN, or antibodies specific for PKEN may be used as elements on a microarray.
• the microarray may be used to monitor or measure protein-protein interactions, drag-target interactions, and gene expression profiles, ' as described above.
• a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or ceU type.
• a transcript image represents the global pattern of gene expression by a particular tissue or ceU type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al, "Comparative Gene Transcript Analysis," U.S.
• 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 microarray.
• 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 Une.
• 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 well as toxicological testing of industrial and naturally-occurring environmental compounds.
• AU compounds induce characteristic gene expression patterns, frequently termed molecular finge ⁇ rints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol Lett. 112-113:467-471, expressly inco ⁇ orated by reference herein).
• Ef a test compound has a signature similar to that of a compound with known toxicity, it is hkely to share those toxic properties.
• These finge ⁇ rints or signatures are most useful and refined when they contain expression information from a large number of genes and gene famiUes.
• IdeaUy a genome- wide measurement of expression provides the highest quaUty signature. Even genes whose expression is not altered by any tested compounds are important as weU, as the levels of expression of these genes are used to normaUze the rest of the expression data. The normaUzation procedure is useful for comparison of expression data after treatment with different compounds.
• the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
• proteome refers to the global pattern of protem 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 cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU type.
• the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
• the proteins are visuaUzed in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
• the optical density of each protein spot is generally proportional to the level of the protein in the sample.
• the optical densities of equivalently positioned protein spots from different samples 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 the present invention. En some cases, further sequence data may be obtained for definitive protein identification.
• a proteomic profile may also be generated using antibodies specific for PKEN to quantify the levels of PKIN expression.
• the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103- 111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
• Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in paraUel with toxicant signatures at the transcript level.
• There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. 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 corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
• Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
• the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
• Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g.,
• nucleic acid sequences encoding PKEN may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentiaUy 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.
• HACs human artificial chromosomes
• YACs yeast artificial chromosomes
• BACs bacterial artificial chromosomes
• PI constractions or single chromosome cDNA Ubraries.
• nucleic acid sequences of the invention may be used to develop genetic Unkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymo ⁇ hism (RFLP); (See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357.)
• RFLP restriction fragment length polymo ⁇ hism
• Fluorescent in situ hybridization may be correlated with other physical and genetic map data.
• FISH Fluorescent in situ hybridization
• Examples of genetic map data can be found in various scientific journals or at the OnUne MendeUan Inheritance in Man (OMEM) World Wide Web site. Correlation between the location of the gene encoding PKIN on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
• In situ hybridization of chromosomal preparations and physical mapping techniques such as Unkage analysis using estabUshed chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammaUan species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely locaUzed by genetic Unkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
• Unkage analysis using estabUshed chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammaUan species, such as mouse, may reveal associated markers even if the exact
• PKEN 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.
• PKEN 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 PKEN 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.
• En this method large numbers of different smaU test compounds are synthesized on a soUd substrate. The test compounds are reacted with PKEN, or fragments thereof, and washed. Bound PKEN is then detected by methods well known in the art. Purified PKIN can also be coated directly onto plates for use in the aforementioned drag screening techniques. Alternatively, non-neutraUzing antibodies can be used to capture the peptide and immobiUze it on a soUd support.
• nucleotide sequences which encode PKEN may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not Umited to, such properties as the triplet genetic code and specific base pair interactions.
• Incyte cDNAs were derived from cDNA Ubraries described in the LEFESEQ GOLD database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
• RNA was treated with DNase.
• 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 (QEAGEN).
• RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
• RNA was provided with RNA and constructed the corresponding cDNA Ubraries. Otherwise, cDNA was synthesized and cDNA Ubraries were constructed with the UNEZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using 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.
• cDNA was size-selected (300- 1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
• cDNAs were Ugated into compatible restriction enzyme sites of the polyUnker of a suitable plasmid, e.g., PBLUESCREPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Envitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), orpENCY (Encyte Genomics, Palo Alto CA), or derivatives thereof.
• PBLUESCREPT plasmid e.g., PBLUESCREPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Envitrogen, Carlsbad CA), PBK-CM
• Recombinant plasmids were transformed into competent E. coU ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies. II. Isolation of cDNA Clones
• 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 QEAWELL 8 Plasmid, QEAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QEAGEN. FoUowing precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophiUzation, at 4°C
• plasmid DNA was ampUfied from host ceU lysates using direct Unk PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycUng 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). III. Sequencing and Analysis Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows.
• Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (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 Pharmacia Biotech 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 (Molecular Dynamics); 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 (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VEEI.
• the polynucleotide sequences derived from Incyte cDNAs were vaUdated by removing vector, Unker, 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, and hidden Markov model (HMM)-based protein family databases such as PFAM.
• PubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases
• BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein family databases such as PFAM.
• HMM is a probabiUstic approach which analyzes consensus primary structures of gene famiUes. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.
• the queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
• the Incyte cDNA sequences were assembled to produce fuU length polynucleotide sequences.
• GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences see Examples EV and V) were used to extend Encyte cDNA assemblages to full length.
• FuU length polypeptide sequences were translated to derive the corresponding full length polypeptide sequences.
• a polypeptide of the invention 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 GenB ank protein databases (genpept), SwissProt, BLOCKS, PRJNTS, DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-based protein family databases such as PFAM.
• FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence aUgnments are generated using default parameters specified by the CLUSTAL algorithm as inco ⁇ orated 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 inco ⁇ orated 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- pu ⁇ ose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge, C and S. Kariin (1997) J. Mol. Biol 268:78-94, and Burge, C and S. KarUn (1998) Curr. Opin. Struct. Biol. 8:346-354).
• the program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
• Genscan is a FASTA database of polynucleotide and polypeptide sequences.
• the maximum range of sequence for Genscan to analyze at once was set to 30 kb.
• the encoded polypeptides were analyzed by querying against PFAM models for human kinases.
• Potential human kinases were also identified by homology to Encyte cDNA sequences that had been annotated as human kinases. These selected Genscan- predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubUc databases.
• Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons.
• BLAST analysis was also used to find any Incyte cDNA or pubUc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.
• Encyte cDNA coverage was available, this information was used to correct 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. Alternatively, full 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 EV. Partial cDNAs assembled as described in Example EH 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 full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
• Partial DNA sequences were extended to full length with an algorithm based on BLAST analysis.
• the nearest GenBank protein homolog was then compared by BLAST analysis to either Encyte 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 determine whether it contained a complete gene. VI. Chromosomal Mapping of PKIN Encoding Polynucleotides
• sequences which were used to assemble SEQ ED NO:21-40 were compared with sequences from the Encyte LEFESEQ database and pubUc domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ ID NO:21-40 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from pubUc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
• pubUc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a
• 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.
• Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular ceU type or tissue have been bound.
• a membrane on which RNAs from a particular ceU type or tissue have been bound See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.
• Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LEFESEQ (Encyte Genomics). This analysis is much faster than multiple membrane-based hybridizations.
• the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
• the basis of the search is the product score, which is defined as:
• 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 follows: 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.
• polynucleotide sequences encoding PKEN are analyzed with respect to the tissue sources from which they were derived. For example, some fuU length sequences are assembled, at least in part, with overlapping Encyte cDNA sequences (see Example EEE). 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 stractures; 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 categoiy is counted and divided by the total number of Ubraries across aU categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding PKEN.
• cDNA sequences and cDNA Ubrary/tissue information are found in the LEFESEQ GOLD database (Encyte Genomics, Palo Alto CA). VIII.
• 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 hai ⁇ in stractures and primer-primer dimerizations was avoided.
• the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the foUowing parameters for primerpair PCI A and PCE B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C En the alternative, the parameters for primer pair T7 and SK+ were as foUows: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20
• the concentration of DNA in each weU was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PECOGREEN; 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), allowing 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.
• 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.
• Hybridization probes derived from SEQ ED NO:21-40 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeUng of oUgonucleotides, consisting of about 20 base pairs, is specificaUy described, essentially 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 Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), 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 Pharmacia Biotech). 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 Rl, Pst I, Xba I, or Pvu D (DuPont NEN).
• the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40°C To remove nonspecific signals, blots are sequentiaUy washed at room temperature under conditions of up to, for example, 0.1 x saUne sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuaUzed using autoradiography or an alternative imaging means and compared. X. Microarrays
• the Unkage or synthesis of array elements upon a microarray can be achieved utiUzing photoUthography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof.
• the substrate in each of the aforementioned technologies should be uniform and soUd with a non-porous surface (Schena (1999), supra).
• 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 arrange and Unk elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
• a typical array may be produced using available methods and machines weU known to those of ordinary skiU in the art and may contain any appropriate number of elements.- (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
• Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or 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 array elements are hybridized with polynucleotides in a biological sample.
• the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
• a fluorescence scanner is used to detect hybridization at each array element.
• laser desorbtion and mass spectrometry may be used for detection of hybridization.
• the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed.
• microarray preparation and usage is described in 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 Pharmacia Biotech).
• the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBREGHT kits (Incyte).
• Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeUng) 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 SPEN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
• Sequences of the present invention are used to generate array elements.
• Each array element is ampUfied from bacterial cells containing vectors with cloned cDNA inserts.
• PCR ampUfication uses primers complementary to the vector sequences flanking the cDNA insert.
• Array 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 array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech). Purified array elements are immobiUzed on polymer-coated glass sUdes.
• Glass microscope sUdes are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distiUed water washes between and after treatments.
• Glass sUdes are etched in 4% hydrofluoric acid (VWR Scientific Products Co ⁇ oration (VWR), West Chester PA), washed extensively in distiUed 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 US Patent No. 5,807,522, inco ⁇ orated herein by reference.
• 1 ⁇ l of the array element DNA is loaded into the open capiUary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per sUde.
• Microarrays are UV-crossUnked using a STRATALENKER UV-crossUnker (Stratagene). Microarrays are washed at room temperature once in 0.2% SDS and three times in distiUed water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saUne (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes in 0.2% SDS and distilled water as before.
• PBS phosphate buffered saUne
• 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 arrays are transferred to a wate ⁇ roof chamber having a cavity just sUghtly larger than a microscope sUde.
• the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
• the chamber containing the arrays is incubated for about 6.5 hours at 60° C
• the arrays are washed for 10 min at 45° C in a first wash buffer (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 Unes at 488 run 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 array used in the present example is scanned with a resolution of 20 micrometers.
• a mixed gas multiUne 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) corresponding to the two fluorophores.
• Appropriate filters positioned between the array 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 array 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 correlated with a weight ratio of hybridizing species of 1 : 100,000.
• the caUbration is done by labeUng 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 EBM-compatible PC computer The digitized data are displayed as an image where the signal intensify is mapped using a Unear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
• the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore 's emission spectrum.
• a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
• the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
• the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
• Sequences complementary to the PKEN-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring PK N.
• 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 PKEN.
• a complementary oUgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
• a complementary oUgonucleotide is designed to prevent ribosomal binding to the PKEN-encoding transcript.
• PKIN PKIN-specific antigen-binding protein
• 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 PKEN upon induction with isopropyl beta-D-thiogalactopyranoside (EPTG).
• PKEN in eukaryotic ceUs is achieved by infecting insect or mammaUan ceU Unes with recombinant Autograpbica cahfornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculoviras.
• AcMNPV Autograpbica cahfornica nuclear polyhedrosis virus
• the nonessential polyhedrin gene of baculoviras is replaced with cDNA encoding PKIN by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
• baculoviras Recombinant baculoviras is used to infect Spodoptera fragiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculoviras.
• Spodoptera fragiperda Sf9 insect ceUs
• human hepatocytes in some cases. Infection of the latter requires additional genetic modifications to baculoviras.
• PKEN 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 ceU 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 (1995, supra, ch. 10 and 16). Purified PKEN obtained by these methods can be used directly in the assays shown in Examples XVI, XVfl, XVDJ, and XEX where appUcable. XIII. Functional Assays
• PKEN function is assessed by expressing the sequences encoding PKEN at physiologicaUy elevated levels in mammaUan cell 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 (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU Une, for example, an endotheUal or hematopoietic ceU 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 cells 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 (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
• Flow cytometry (FCM) an automated, laser optics- based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the ceUs and other ceUular properties.
• FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward Ught scatter and 90 degree side Ught scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
• PKIN The influence of PKIN on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding PKIN and either CD64 or CD64-GFP.
• CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobuUn G (IgG).
• Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
• mRNA can be purified from the ceUs using methods well known by those of skiU in the art. Expression of mRNA encoding PKIN and other genes of interest can be analyzed by northern analysis or microarray techniques.
• PKEN substantially purified using polyacrylamide gel electrophoresis PAGE; see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
• PAGE polyacrylamide gel electrophoresis
• the PKIN 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. (See, e.g., Ausubel, 1995, 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-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
• MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
• Rabbits are immunized with the oUgopeptide-KLH complex in complete Freund's adjuvant.
• Resulting antisera are tested for antipeptide and anti-PKEN activity by, for example, binding the peptide or PKEN to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
• Naturally occurring or recombinant PKEN is substantiaUy purified by immunoaffinity chromatography using antibodies specific for PKEN.
• An immunoaffinity column is constructed by covalently coupUng anti-PKEN antibody to an activated chromatographic resin, such as
• PKEN e.g., high ionic strength buffers in the presence of detergent.
• the column is eluted under conditions that disrapt antibody/PKIN binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and PKEN is collected.
• disrapt antibody/PKIN binding e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion
• PKIN or biologicaUy active fragments thereof, are labeled with 1 5 I Bolton-Hunter reagent.
• Bolton-Hunter reagent See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
• Candidate molecules previously arrayed in the weUs of a multi-weU plate are incubated with the labeled PKEN, washed, and any weUs with labeled PKEN complex are assayed. Data obtained using different concentrations of PKIN are used to calculate values for the number, affinity, and association of PKIN with the candidate molecules.
• molecules interacting with PKEN are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
• PKIN may also be used in the PATHCALLENG process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine aU 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 PKEN in the presence of [ ⁇ - 32 P] ATP.
• PKEN is incubated with the protein substrate, 32 P-ATP, and an appropriate kinase buffer.
• the 32 P inco ⁇ orated into the substrate is separated from free 32 P-ATP by electrophoresis and the inco ⁇ orated 32 P is counted using a radioisotope counter.
• the amount of inco ⁇ orated 32 P is proportional to the activity of PKEN.
• a determination of the specific amino acid residue phosphorylated is made by phosphoamino acid analysis of the hydrolyzed protein.
• protein kinase activity is measured by quantifying the transfer of gamma phosphate from adenosine triphosphate (ATP) to a serine, threonine or tyrosine residue in a protein substrate.
• ATP adenosine triphosphate
• the reaction occurs between a protein kinase sample with a biotinylated peptide substrate and gamma 32 P-ATP. FoUowing the reaction, free avidin in solution is added for binding to the biotinylated 32 P-peptide product.
• the binding sample then undergoes a centrifugal ultrafiltration process with a membrane which will 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 scintiUation counter.
• This procedure allows assay of any type of protein kinase sample, depending on the peptide substrate and kinase reaction buffer selected.
• This assay is provided in kit form (ASUA, Affinity Ultrafiltration Separation Assay, Transbio Co ⁇ oration, 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, ERK1 & ERK2 substrates and MEK, and myeUn basic protein and ERK (Pearson, JD. et al. (1991) Methods Enzymol. 200:62-81).
• protein kinase activity of PKEN is demonstrated in an assay containing PKIN, 50 ⁇ l of kinase buffer, l ⁇ g substrate, such as myeUn basic protein (MBP) or synthetic peptide substrates, 1 mM DTT, 10 ⁇ g ATP, and 0.5 ⁇ Ci [ ⁇ - 32 P]ATP.
• the reaction is incubated at 30°C for 30 minutes and stopped by pipetting onto P81 paper.
• the uninco ⁇ orated [ ⁇ - 32 P]ATP is removed by washing and the inco ⁇ orated radioactivity is measured using a scintiUation 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 followed by autoradiography.
• the amount of inco ⁇ orated 32 P is proportional to the activity of PKEN.
• adenylate kinase or guanylate kinase activity may be measured by the inco ⁇ oration of 32 P from [ ⁇ - 32 P]ATP into ADP or GDP using a gamma radioisotope counter.
• the enzyme in a kinase buffer, is incubated together with the appropriate nucleotide mono-phosphate substrate (AMP or GMP) and 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 enzyme activity.
• PKEN scintiUation proximity assays
• useful substrates include recombinant proteins tagged with glutathione transferase, or synthetic peptide substrates tagged with biotin.
• Inhibitors of PKEN activity such as smaU organic molecules, proteins or peptides, may be identified by such assays.
• Agonists or antagonists of PKEN activation or inhibition may be tested using assays described in section XVEE. Agonists cause an increase in PKEN activity and antagonists cause a decrease in PKEN activity.
• Binding of PKEN to a FLAG-CD44 cyt fusion protein can be determined by incubating PKIN to anti-PKIN-conjugated immunoaffinity beads foUowed by incubating portions of the beads (having 10-20 ng of protein) with 0.5 ml of a binding buffer (20 mM Tris-HCL (pH 7.4), 150 mM NaCl, 0.1 % bovine serum albumin, and 0.05% Triton X-100) in the presence of 125 l-labeled FLAG-CD44cyt fusion protein (5,000 cpm/ng protein ) at 4 °C for 5 hours.
• a binding buffer (20 mM Tris-HCL (pH 7.4), 150 mM NaCl, 0.1 % bovine serum albumin, and 0.05% Triton X-100
• ABI FACTURA A program that removes vector sequences and Applied Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences.
• ABI/PARACELFDF A Fast Data Finder useful in comparing and Applied Biosystems, Foster City, CA; Mismatch ⁇ 50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA.
• ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA.
• fastx score 100 or greater
• HMM hidden Markov model
• Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8:175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194.
• TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.
• HMM hidden Markov model

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