Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof

The present invention is in the field of kinase proteins that are related to the SNF-like kinase subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

Protein Kinases

Kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Uncontrolled signaling has been implicated in a variety of disease conditions including inflammation, cancer, arteriosclerosis, and psoriasis. Reversible protein phosphorylation is the main strategy for con trolling activities of eukaryotic cells. It is estimated that more than 1000 of the 10,000 proteins active in a typical mammalian cell are phosphorylated. The high energy phosphate, which drives activation, is generally transferred from adenosine triphosphate molecules (ATP) to a particular protein by protein kinases and removed from that protein by protein phosphatases. Phosphorylation occurs in response to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc), cell cycle checkpoints, and environmental or nutritional stresses and is roughly analogous to turning on a molecular switch. When the switch goes on, the appropriate protein kinase activates a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor.

The kinases comprise the largest, known protein group, a superfamily of enzymes with widely varied functions and specificities. They are usually named after their substrate, their regulatory molecules, or some aspect of a mutant phenotype. With regard to substrates, the protein kinases may be roughly divided into two groups; those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate serine or threonine residues (serine/threonine kinases, STK). A few protein kinases have dual specificity and phosphorylate threonine and tyrosine residues. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The N-terminal domain, which contains subdomains I-IV, generally folds into a two-lobed structure, which binds and orients the ATP (or GTP) donor molecule. The larger C terminal lobe, which contains subdomains VI A-XI, binds the protein substrate and carries out the transfer of the gamma phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue. Subdomain V spans the two lobes.

The kinases may be categorized into families by the different amino acid sequences (generally between 5 and 100 residues) located on either side of, or inserted into loops of, the kinase domain. These added amino acid sequences allow the regulation of each kinase as it recognizes and interacts with its target protein. The primary structure of the kinase domains is conserved and can be further subdivided into 11 subdomains. Each of the 11 subdomains contains specific residues and motifs or patterns of amino acids that are characteristic of that subdomain and are highly conserved (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Books , Vol 1:7-20 Academic Press, San Diego, Calif.).

The second messenger dependent protein kinases primarily mediate the effects of second messengers such as cyclic AMP (cAMP), cyclic GMP, inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cyclic-ADPribose, arachidonic acid, diacylglycerol and calcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) are important members of the STK family. Cyclic-AMP is an intracellular mediator of hormone action in all prokaryotic and animal cells that have been studied. Such 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 cyclic-AMP 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, N.Y., pp. 416-431, 1887).

Calcium-calmodulin (CaM) dependent protein kinases are also members of STK family. Calmodulin is a calcium receptor that mediates many calcium regulated processes by binding to target proteins in response to the binding of calcium. The principle target protein in these processes is CaM dependent protein kinases. CaM-kinases are involved in regulation of smooth muscle contraction (MLC kinase), glycogen breakdown (phosphorylase kinase), and neurotransmission (CaM kinase I and CaM kinase II). CaM kinase I phosphorylates a variety of substrates including the neurotransmitter related proteins synapsin I and II, the gene transcription regulator, CREB, and the cystic fibrosis conductance regulator protein, CFTR (Haribabu, B. et al. (1995) EMBO Journal 14:367-86). CaM II kinase also phosphorylates synapsin at different sites, and controls the synthesis of catecholamines in the brain through phosphorylation and activation of tyrosine hydroxylase. Many of the CaM kinases are activated by phosphorylation in addition to binding to CaM. The kinase may autophosphorylate itself, or be phosphorylated by another kinase as part of a “kinase cascade”.

Another ligand-activated protein kinase is 5′-AMP-activated protein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem . 15:8675-81). 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.

The mitogen-activated protein kinases (MAP) are also members of the STK family. MAP kinases also regulate intracellular signaling pathways. They mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Several subgroups have been identified, and each manifests different substrate specificities and responds to distinct extracellular stimuli (Egan, S. E. and Weinberg, R. A. (1993) Nature 365:781-783). MAP kinase signaling pathways are present in mammalian cells as well as in yeast. The extracellular stimuli that activate mammalian pathways include epidermal growth factor (EGF), ultraviolet light, hyperosmolar medium, heat shock, endotoxic lipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL- 1).

PRK (proliferation-related kinase) is a serum/cytokine inducible STK that is involved in regulation of the cell cycle and cell proliferation in human megakaroytic cells (Li, B. et al. (1996) J. Biol. Chem . 271:19402-8). PRK is related to the polo (derived from humans polo gene) family of STKs implicated in cell division. PRK is downregulated in lung tumor tissue and may be a proto-oncogene whose deregulated expression in normal tissue leads to oncogenic transformation. Altered MAP kinase expression is implicated in a variety of disease conditions including cancer, inflammation, immune disorders, and disorders affecting growth and development.

The cyclin-dependent protein kinases (CDKs) are another group of STKs that control the progression of cells through the cell cycle. Cyclins are small regulatory proteins that act by binding to and activating CDKs that then trigger various phases of the cell cycle by phosphorylating and activating selected proteins involved in the mitotic process. CDKs are unique in that they require multiple inputs to become activated. In addition to the binding of cyclin, CDK activation requires the phosphorylation of a specific threonine residue and the dephosphorylation of a specific tyrosine residue.

Protein tyrosine kinases, PTKs, specifically phosphorylate tyrosine residues on their target proteins and may be divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembrane protein-tyrosine kinases are receptors for most growth factors. Binding of growth factor to the receptor activates the transfer of a phosphate group from ATP to selected tyrosine side chains of the receptor and other specific proteins. Growth factors (GF) associated 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.

Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors. Such receptors that function through non-receptor PTKs include those for cytokines, hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes.

Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells where their activation was no longer subject to normal cellular controls. In fact, about one third of the known oncogenes encode PTKs, and it is well known that cellular transformation (oncogenesis) is often accompanied by increased tyrosine phosphorylation activity (Carbonneau H and Tonks NK (1992) Annu. Rev. Cell. Biol . 8:463-93). Regulation of PTK activity may therefore be an important strategy in controlling some types of cancer.

SNF Kinases

The kinase proteins of this invention are homologous to the SNF kinases, involved in signal transduction and development. SNF kinases derepress glucose-repressible genes in yeast and in higher plants. SNF-like kinases are also found in mammals. For example, the hunk1 kinase is expressed in murine mammary gland and may play a role in tissue development and carcenogenesis. Also, msk (myocardial SNF1-like kinase) is expressed in developing heart. Experimental evidence indicates that msk regulates HEK kinase, an ephrin receptor type kinase. For more information on SNF-like kinases, see Bielke W, et al., Gene Feb. 25, 1994;139(2):235-9; Le Guen L, et al., Gene Oct. 21, 1992;120(2):249-54; Gardner H P, et al., Genomics Jan. 1, 2000;63(1):46-59; Ruiz J C, et al., Mech Dev 1994 Dec;48(3):153-64; and Yoshida E N, et al., Genome 1999 Dec;42(6):1077-87.

Kinase proteins, particularly members of the SNF-like kinase subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of kinase proteins. The present invention advances the state of the art by providing previously unidentified human kinase proteins that have homology to members of the SNF-like kinase subfamily.

The present invention is based in part on the identification of amino acid sequences of human kinase peptides and proteins that are related to the SNF-like kinase subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate kinase activity in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers.

FIG. 1 provides the nucleotide sequence of a cDNA molecule or transcript sequence that encodes the kinase protein of the present invention. (SEQ ID NO:1) In addition, structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers.

FIG. 2 provides the predicted amino acid sequence of the kinase of the present invention. (SEQ ID NO:2) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding the kinase protein of the present invention. (SEQ ID NO:3) In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. As illustrated in FIG. 3, known SNP variations include C1681A, T6888-, A7939G, G9790A, G11689A, A12145G, A12437-, A12437G, G12437-, C22982T, C23239T, A23859G, G24862A, G25097A, C25751T, T25934-, G26179A, A26277G, -26354T, T26356-, T26359G, G29484A, A29885T, C30601T, C30920T, C31057A, C31057G, G31097A, T31204C, T32660A, T32875-, C33477G, C34059T, -36681A, C36821T, A38079G, and A38432T.

General Description

The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a kinase protein or part of a kinase protein and are related to the SNF-like kinase subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human kinase peptides and proteins that are related to the SNF-like kinase subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these kinase peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the kinase of the present invention.

In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known kinase proteins of the SNF-like kinase subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known SNF-like kinase family or subfamily of kinase proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the kinase family of proteins and are related to the SNF-like kinase subfamily (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3 ). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the kinase peptides of the present invention, kinase peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprise the amino acid sequences of the kinase peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the kinase peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

The isolated kinase peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. For example, a nucleic acid molecule encoding the kinase peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the kinase peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

The kinase peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a kinase peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the kinase peptide. “Operatively linked” indicates that the kinase peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the kinase peptide.

In some uses, the fusion protein does not affect the activity of the kinase peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant kinase peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology , 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A kinase peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the kinase peptide.

As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the kinase peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. ( Computational Molecular Biology , Lesk, A. M., ed., Oxford University Press, New York, 1988 ; Biocomputing: Informatics and Genome Projects , Smith, D. W., ed., Academic Press, New York, 1993 ; Computer Analysis of Sequence Data , Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994 ; Sequence Analysis in Molecular Biology , von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer , Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ( J. Mol. Biol . (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res . 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. ( J. Mol. Biol . 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. ( Nucleic Acids Res . 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the kinase peptides of the present invention as well as being encoded by the same genetic locus as the kinase peptide provided herein. The gene provided by the present invention is located on a genome component that has been mapped to human chromosome 5 (as indicated in FIG. 3 ), which is supported by multiple lines of evidence, such as STS and BAC map data.

Allelic variants of a kinase peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the kinase peptide as well as being encoded by the same genetic locus as the kinase peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. The gene provided by the present invention is located on a genome component that has been mapped to human chromosome 5 (as indicated in FIG. 3 ), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been found in a gene encoding the kinase proteins of the present invention. The following variations were seen: C1681A, T6888-, A7939G, G9790A, G11689A, A12145G, A12437-, A 12437G, G12437-, C22982T, C23239T, A23859G, G24862A, G25097A, C25751T, T25934-, G26179A, A26277G, -26354T, T26356-, T26359G, G29484A, A29885T, C30601T, C30920T, C31057A, C31057G, G31097A, T31204C, T32660A, T32875-, C33477G, C34059T, -36681A, C36821T, A38079G, and A38432T. The changes in the amino acid sequence that these SNPs cause can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a base.

Paralogs of a kinase peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the kinase peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

Orthologs of a kinase peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the kinase peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

Non-naturally occurring variants of the kinase peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the kinase peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a kinase peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant kinase peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to phosphorylate substrate, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2 . The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as kinase activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol . 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

The present invention further provides fragments of the kinase peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2 . The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a kinase peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the kinase peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the kinase peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2 .

Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in kinase peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2 ).

Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties , 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins , B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. ( Ann. N.Y. Acad. Sci . 663:48-62 (1992)).

Accordingly, the kinase peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature kinase peptide is fused with another compound, such as a compound to increase the half-life of the kinase peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature kinase peptide, such as a leader or secretory sequence or a sequence for purification of the mature kinase peptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a kinase-effector protein interaction or kinase-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, kinases isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the kinase. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. Specifically, a virtual northern blot shows expression in lung, infant brain, germ cell tissue, germ cell tumor tissue, and primary cancers. In addition, PCR-based tissue screening panel indicates expression in liver, kidney, bone marrow, thyroid, brain, fetal brain, and placenta. A large percentage of pharmaceutical agents are being developed that modulate the activity of kinase proteins, particularly members of the SNF-like kinase subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1 . Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. Such uses can readily be determined using the information provided herein, that which is known in the art, and routine experimentation.

The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to kinases that are related to members of the SNF-like kinase subfamily. Such assays involve any of the known kinase functions or activities or properties useful for diagnosis and treatment of kinase-related conditions that are specific for the subfamily of kinases that the one of the present invention belongs to, particularly in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. Specifically, a virtual northern blot shows expression in lung, infant brain, germ cell tissue, germ cell tumor tissue, and primary cancers. In addition, PCR-based tissue screening panel indicates expression in liver, kidney, bone marrow, thyroid, brain, fetal brain, and placenta.

The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems. Cell-based systems can be native, i.e., cells that normally express the kinase, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the kinase protein.

The polypeptides can be used to identify compounds that modulate kinase activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the kinase. Both the kinases of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the kinase. These compounds can be further screened against a functional kinase to determine the effect of the compound on the kinase activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the kinase to a desired degree.

Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the kinase protein and a molecule that normally interacts with the kinase protein, e.g. a substrate or a component of the signal pathway that the kinase protein normally interacts (for example, another kinase). Such assays typically include the steps of combining the kinase protein with a candidate compound under conditions that allow the kinase protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the kinase protein and the target, such as any of the associated effects of signal transduction such as protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′) 2 , Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

One candidate compound is a soluble fragment of the receptor that competes for substrate binding. Other candidate compounds include mutant kinases or appropriate fragments containing mutations that affect kinase function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention.

The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) kinase activity. The assays typically involve an assay of events in the signal transduction pathway that indicate kinase activity. Thus, the phosphorylation of a substrate, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the kinase protein dependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the kinase can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2 . Specifically, a biological function of a cell or tissues that expresses the kinase can be assayed. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. Specifically, a virtual northern blot shows expression in lung, infant brain, germ cell tissue, germ cell tumor tissue, and primary cancers. In addition, PCR-based tissue screening panel indicates expression in liver, kidney, bone marrow, thyroid, brain, fetal brain, and placenta.

Binding and/or activating compounds can also be screened by using chimeric kinase proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a substrate-binding region can be used that interacts with a different substrate then that which is recognized by the native kinase. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the kinase is derived.

The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the kinase (e.g. binding partners and/or ligands). Thus, a compound is exposed to a kinase polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble kinase polypeptide is also added to the mixture. If the test compound interacts with the soluble kinase polypeptide, it decreases the amount of complex formed or activity from the kinase target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the kinase. Thus, the soluble polypeptide that competes with the target kinase region is designed to contain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable to immobilize either the kinase protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., 35 S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of kinase-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a kinase-binding protein and a candidate compound are incubated in the kinase protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the kinase protein target molecule, or which are reactive with kinase protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

Agents that modulate one of the kinases of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

Modulators of kinase protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the kinase pathway, by treating cells or tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. These methods of treatment include the steps of administering a modulator of kinase activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

In yet another aspect of the invention, the kinase proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem . 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the kinase and are involved in kinase activity. Such kinase-binding proteins are also likely to be involved in the propagation of signals by the kinase proteins or kinase targets as, for example, downstream elements of a kinase-mediated signaling pathway. Alternatively, such kinase-binding proteins are likely to be kinase inhibitors.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a kinase protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a kinase-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the kinase protein.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a kinase-modulating agent, an antisense kinase nucleic acid molecule, a kinase-specific antibody, or a kinase-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

The kinase proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. The method involves contacting a biological sample with a compound capable of interacting with the kinase protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered kinase activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. ( Clin. Exp. Pharmacol. Physiol . 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the kinase protein in which one or more of the kinase functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other substrate-binding regions that are more or less active in substrate binding, and kinase activation. Accordingly, substrate dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. Accordingly, methods for treatment include the use of the kinase protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′) 2 , and Fv fragments.

Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments of the kinase proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or kinase/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2 ).

Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. Specifically, a virtual northern blot shows expression in lung, infant brain, germ cell tissue, germ cell tumor tissue, and primary cancers. In addition, PCR-based tissue screening panel indicates expression in liver, kidney, bone marrow, thyroid, brain, fetal brain, and placenta. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the kinase peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nuleic acid arrays and similar methods have been developed for antibody arrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid molecules that encode a kinase peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the kinase peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5KB, 4KB, 3KB, 2KB, or 1KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1 ), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the kinase peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the kinase proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3 .

A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. The gene provided by the present invention is located on a genome component that has been mapped to human chromosome 5 (as indicated in FIG. 3 ), which is supported by multiple lines of evidence, such as STS and BAC map data.

FIG. 3 provides information on SNPs that have been found in a gene encoding the kinase proteins of the present invention. The following variations were seen: C1681A, T6888-, A7939G, G9790A, G11689A, A12145G, A12437-, A12437G, G12437-, C22982T, C23239T, A23859G, G24862A, G25097A, C25751 T, T25934-, G26179A, A26277G, -26354T, T26356-, T26359G, G29484A, A29885T, C30601T, C30920T, C31057A, C31057G, G31097A, T31204C, T32660A, T32875-, C33477G, C34059T, -36681A, C36821T, A38079G, and A38432T. The changes in the amino acid sequence that these SNPs cause can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a base.

As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology , John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to low stringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2 . As illustrated in FIG. 3, known SNP variations include C1681A, T6888-, A7939G, G9790A, G11689A, A12145G, A12437-, A12437G, G12437-, C22982T, C23239T, A23859G, G24862A, G25097A, C25751T, T25934-, G26179A, A26277G, -26354T, T26356-, T26359G, G29484A, A29885T, C30601T, C30920T, C31057A, C31057G, G31097A, T31204C, T32660A, T32875-, C33477G, C34059T, -36681A, C36821T, A38079G, and A38432T.

The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene provided by the present invention is located on a genome component that has been mapped to human chromosome 5 (as indicated in FIG. 3 ), which is supported by multiple lines of evidence, such as STS and BAC map data.

The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. Specifically, a virtual northern blot shows expression in lung, infant brain, germ cell tissue, germ cell tumor tissue, and primary cancers. In addition, PCR-based tissue screening panel indicates expression in liver, kidney, bone marrow, thyroid, brain, fetal brain, and placenta. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in kinase protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a kinase protein, such as by measuring a level of a kinase-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a kinase gene has been mutated. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. Specifically, a virtual northern blot shows expression in lung, infant brain, germ cell tissue, germ cell tumor tissue, and primary cancers. In addition, PCR-based tissue screening panel indicates expression in liver, kidney, bone marrow, thyroid, brain, fetal brain, and placenta.

Nucleic acid expression assays are useful for drug screening to identify compounds that modulate kinase nucleic acid expression.

The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the kinase gene, particularly biological and pathological processes that are mediated by the kinase in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta , bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. The method typically includes assaying the ability of the compound to modulate the expression of the kinase nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired kinase nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the kinase nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

The assay for kinase nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the kinase protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of kinase gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of kinase mRNA in the presence of the candidate compound is compared to the level of expression of kinase mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate kinase nucleic acid expression in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. Specifically, a virtual northern blot shows expression in lung, infant brain, germ cell tissue, germ cell tumor tissue, and primary cancers. In addition, PCR-based tissue screening panel indicates expression in liver, kidney, bone marrow, thyroid, brain, fetal brain, and placenta. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

Alternatively, a modulator for kinase nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the kinase nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers.

The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the kinase gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in kinase nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in kinase genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the kinase gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the kinase gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a kinase protein.

Individuals carrying mutations in the kinase gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been found in a gene encoding the kinase proteins of the present invention. The following variations were seen: C1681A, T6888-, A7939G, G9790A, G11689A, A12145G, A12437-, A12437G, G12437-, C22982T, C23239T, A23859G, G24862A, G25097A, C25751T, T25934-, G26179A, A26277G, -26354T, T26356-, T26359G, G29484A, A29885T, C30601T, C30920T, C31057A, C31057G, G31097A, T31204C, T32660A, T32875-, C33477G, C34059T, -36681A, C36821T, A38079G, and A38432T. The changes in the amino acid sequence that these SNPs cause can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a base. The gene provided by the present invention is located on a genome component that has been mapped to human chromosome 5 (as indicated in FIG. 3 ), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

Alternatively, mutations in a kinase gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant kinase gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr . 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol . 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol . 21 7:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res . 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl . 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the kinase gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been found in a gene encoding the kinase proteins of the present invention. The following variations were seen: C1681A, T6888-, A7939G, G9790A, G11689A, A12145G, A12437-, A12437G, G12437-, C22982T, C23239T, A23859G, G24862A, G25097A, C25751T, T25934-, G26179A, A26277G, -26354T, T26356-, T26359G, G29484A, A29885T, C30601T, C30920T, C31057A, C31057G, G31097A, T31204C, T32660A, T32875-, C33477G, C34059T, -36681A, C36821T, A38079G, and A38432T. The changes in the amino acid sequence that these SNPs cause can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a base.

Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs to control kinase gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of kinase protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into kinase protein.

Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of kinase nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired kinase nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the kinase protein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in kinase gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired kinase protein to treat the individual.

The invention also encompasses kits for detecting the presence of a kinase nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates expression in lung, liver, kidney, thyroid, brain, infant brain, fetal brain, placenta, bone marrow, germ cell tissue, germ cell tumor tissue, and primary cancers. Specifically, a virtual northern blot shows expression in lung, infant brain, germ cell tissue, germ cell tumor tissue, and primary cancers. In addition, PCR-based tissue screening panel indicates expression in liver, kidney, bone marrow, thyroid, brain, fetal brain, and placenta. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting kinase nucleic acid in a biological sample; means for determining the amount of kinase nucleic acid in the sample; and means for comparing the amount of kinase nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect kinase protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996 ; Nat. Biotech . 14: 1675-1680) and Schena, M. et al. (1996 ; Proc. Natl. Acad. Sci . 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

Using such arrays, the present invention provides methods to identify the expression of the kinase proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the kinase gene of the present invention. FIG. 3 provides information on SNPs that have been found in a gene encoding the kinase proteins of the present invention. The following variations were seen: C1681A, T6888-, A7939G, G9790A, G11689A, A12145G, A12437-, A12437G, G12437-, C22982T, C23239T, A23859G, G24862A, G25097A, C25751T, T25934-, G26179A, A26277G, -26354T, T26356-, T26359G, G29484A, A29885T, C30601T, C30920T, C31057A, C31057G, G31097A, T31204C, T32660A, T32875-, C33477G, C34059T, -36681A, C36821T, A38079G, and A38432T. The changes in the amino acid sequence that these SNPs cause can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a base.

Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques , Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al, Techniques in Immunocytochemistry , Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology , Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified kinase gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

Vectors/host cells

The invention also provides vectors containing the nucleic acid molecules described herein. The “term vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli , the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual . 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual . 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli , Streptomyces, and Salmonella typhimurium . Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11 (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli . (Wada et al, Nucleic Acids Res . 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J . 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell Biol . 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J . 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual . 2nd, ed., Cold Spring Harbor Laboratory , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. ( Molecular Cloning: A Laboratory Manual . 2nd, ed., Cold Spring Harbor Laboratory , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as kinases, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

Where the peptide is not secreted into the medium, which is typically the case with kinases, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a kinase protein or peptide that can be further purified to produce desired amounts of kinase protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involving the kinase protein or kinase protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native kinase protein is useful for assaying compounds that stimulate or inhibit kinase protein function.

Host cells are also useful for identifying kinase protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant kinase protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native kinase protein.

Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a kinase protein and identifying and evaluating modulators of kinase protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the kinase protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the kinase protein to particular cells.

Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo , (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G o phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect substrate binding, kinase protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo kinase protein function, including substrate interaction, the effect of specific mutant kinase proteins on kinase protein function and substrate interaction, and the effect of chimeric kinase proteins. It is also possible to assess the effect of null mutations, that is, mutations that substantially or completely eliminate one or more kinase protein functions.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

6

1

1544

Human

1

ctaccacgtt cactgccttc ctctcactaa agccgagagg gaggctgctc agctctcagg 60

aaaactcttt tgaaccctgg gcacctgctg tcctcagttg gcatctccca ccctctgagc 120

ctcttctgtt cctgcacaac ctgcctcttc gctgagatgg agacgtgagc ccccgtggac 180

gatgactgca gtgtatatga atggaggtgg cctggtgaac ccccactatg cccggtggga 240

tcggcgcgac agtgtagaaa gtggctgtca gaccgagagt agcaaggagg gtgaggaggg 300

acagccccgc cagctgacgc ccttcgagaa actgacacag gacatgtccc aggatgagaa 360

ggtggtgagg gagatcacgc tggggaaacg gataggcttc taccgaattc gaggggaaat 420

cggaagtgga aacttctccc aagtgaagct tgggattcac tccctaacca aagaaaaggt 480

ggccattaag atcctggaca agaccaagtt agaccagaaa acccagaggc tactatcccg 540

agaaatctcc agcatggaaa agctgcacca tcccaacatc atccgccttt acgaagtggt 600

ggagacccta tccaagctgc acttggtgat ggagtatgca gggggtgggg agctcttcgg 660

aaaaattagc actgagggga agctctctga accagaaagc aagctcatct tctcccagat 720

tgtgtctgcc gtgaagcaca tgcatgaaaa ccaaattatt catagagatc tgaaagcaga 780

aaatgtattc tataccagta atacttgtgt gaaggtgggc gattttggat tcagcacagt 840

aagcaaaaaa ggtgaaatgc tgaacacttt ctgtgggtct cctccctacg ctgcgcctga 900

actcttccgg gacgagcact acatcggcat ttacgtggat atctgggcct tgggggtgct 960

tttgtacttc atggtgactg gcaccatgcc atttcgggca gaaaccgtgg ccaaactaaa 1020

aaagagcatc ctcgagggca catacagtgt accgccgcac gtgtcagagc cctgccaccg 1080

actcatccga ggagtccttc agcagatccc cacggagagg tacggaatcg actgcatcat 1140

gaatgatgaa tggatgcaag gggtgccata ccctacacct ttggaacctt tccaactgga 1200

tcccaaacat ttgtcggaaa ccagcactct caaggaagaa gaaaatgagg tcaaaagcac 1260

tttagaacat ttgggcatta cagaagagca tattcgaaat aaccaaggga gagatgctcg 1320

cagctcaatc acaggggtct atagaattat tttacataga gtccaaagga agaaggcttt 1380

ggaaagtgtc ccagtcatga tgctaccaga ccctaaagaa agagacctca aaaaagggtc 1440

ccgtgtctac agagggataa gacacacatc caaattttgc tcgattttat aaattgcact 1500

agactgcttg taactaacca agatgattgt tgctgcttct aaat 1544

2

436

Human

2

Met Thr Ala Val Tyr Met Asn Gly Gly Gly Leu Val Asn Pro His Tyr

1 5 10 15

Ala Arg Trp Asp Arg Arg Asp Ser Val Glu Ser Gly Cys Gln Thr Glu

20 25 30

Ser Ser Lys Glu Gly Glu Glu Gly Gln Pro Arg Gln Leu Thr Pro Phe

35 40 45

Glu Lys Leu Thr Gln Asp Met Ser Gln Asp Glu Lys Val Val Arg Glu

50 55 60

Ile Thr Leu Gly Lys Arg Ile Gly Phe Tyr Arg Ile Arg Gly Glu Ile

65 70 75 80

Gly Ser Gly Asn Phe Ser Gln Val Lys Leu Gly Ile His Ser Leu Thr

85 90 95

Lys Glu Lys Val Ala Ile Lys Ile Leu Asp Lys Thr Lys Leu Asp Gln

100 105 110

Lys Thr Gln Arg Leu Leu Ser Arg Glu Ile Ser Ser Met Glu Lys Leu

115 120 125

His His Pro Asn Ile Ile Arg Leu Tyr Glu Val Val Glu Thr Leu Ser

130 135 140

Lys Leu His Leu Val Met Glu Tyr Ala Gly Gly Gly Glu Leu Phe Gly

145 150 155 160

Lys Ile Ser Thr Glu Gly Lys Leu Ser Glu Pro Glu Ser Lys Leu Ile

165 170 175

Phe Ser Gln Ile Val Ser Ala Val Lys His Met His Glu Asn Gln Ile

180 185 190

Ile His Arg Asp Leu Lys Ala Glu Asn Val Phe Tyr Thr Ser Asn Thr

195 200 205

Cys Val Lys Val Gly Asp Phe Gly Phe Ser Thr Val Ser Lys Lys Gly

210 215 220

Glu Met Leu Asn Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala Pro Glu

225 230 235 240

Leu Phe Arg Asp Glu His Tyr Ile Gly Ile Tyr Val Asp Ile Trp Ala

245 250 255

Leu Gly Val Leu Leu Tyr Phe Met Val Thr Gly Thr Met Pro Phe Arg

260 265 270

Ala Glu Thr Val Ala Lys Leu Lys Lys Ser Ile Leu Glu Gly Thr Tyr

275 280 285

Ser Val Pro Pro His Val Ser Glu Pro Cys His Arg Leu Ile Arg Gly

290 295 300

Val Leu Gln Gln Ile Pro Thr Glu Arg Tyr Gly Ile Asp Cys Ile Met

305 310 315 320

Asn Asp Glu Trp Met Gln Gly Val Pro Tyr Pro Thr Pro Leu Glu Pro

325 330 335

Phe Gln Leu Asp Pro Lys His Leu Ser Glu Thr Ser Thr Leu Lys Glu

340 345 350

Glu Glu Asn Glu Val Lys Ser Thr Leu Glu His Leu Gly Ile Thr Glu

355 360 365

Glu His Ile Arg Asn Asn Gln Gly Arg Asp Ala Arg Ser Ser Ile Thr

370 375 380

Gly Val Tyr Arg Ile Ile Leu His Arg Val Gln Arg Lys Lys Ala Leu

385 390 395 400

Glu Ser Val Pro Val Met Met Leu Pro Asp Pro Lys Glu Arg Asp Leu

405 410 415

Lys Lys Gly Ser Arg Val Tyr Arg Gly Ile Arg His Thr Ser Lys Phe

420 425 430

Cys Ser Ile Leu

435

3

aaagcacaag ttcctgccca cctggtgctt tttttttttt tcagatggcg tttcactctt 60

gtcgcccacg ctggagtgca atggcatgat ctcagctcac tgcaccctct gcctcctggg 120

ttcaaacaat tctcctgcct cagcctccca agtagctggg attacaggtg tgcgccacca 180

tgcttggcta atttttgtat ttttagtaga gatggagttt caccatgttg gccaggctgg 240

tctcaaactc ctaacctcag gtgatccacc cacctcagcc tcccaaagtg ttgggattac 300

aggcttgagc cactgtgccc ggcgccctcc tagtgctgat gtagttgtgg cccaagtaca 360

ttagtgacaa ggacagcaca gtaatgagga cacagagcca gtaagttctt cagagagtct 420

gttagaacac aaagtgatca aagtgatcaa aagtactgca ttctatggaa aatctatatt 480

tttctttttc cttaggttaa gctaaagttt cttgcagaat tactacttca ttctagtctg 540

tcataagctg catggtgttt ttctgccagg aatctgggga gatctagtgc cacttgacct 600

tcacagagat tcttaacctg atgggtttca gaatgtcatc ttaaaattgg atgcaaattg 660

tatgggtgag ggtttttttt tttttcctcc tggggataaa gtcataggtt ttgttagcat 720

ctcaaaggag tctgtgagcc caggatgatt aagaatcagt gattttggct gagtacagtg 780

gttcacacct gtaattccag cactttggga ggtagaggtg ggtaaagaaa aggaagaaga 840

atcagtgact tagattttct tcactctaat taaggacctt tacctttttt gtttgtttgc 900

tttgctttgt ttttttgaga cagggtccgg cattgtcacc caggctggag tacagtggct 960

tgatctcagc tcactgcaac ctccacttcc tgggctcaag ccatcctccc acctcagtct 1020

gtaggtgtag ctgagactac aggtgcacac cctgctaagg ggtacaccac cacaccctgc 1080

taggaggtac aacaccacac cctgctaagg ggaactttat acttaaaaat gtttttaaaa 1140

aacatcctaa ttggtctcta acctagagat gtgcttagtt actgatacct ggtccctttt 1200

aagcctatgt tcgtcatcct tcttccagat tcctttctct ctatccagaa ataggagcag 1260

tactgggcat caacctcctc tttacttccc ttatttatgt ttatttggca accccatact 1320

taaccttaag tataagctta ggaagcctta tttatactta attgtaataa tgtgatatta 1380

ttgagcagag ttggaaatcc atacttccaa ggcagtctct catgttttca gtcaaatttt 1440

tggtttggaa tgctcaagtc caaaaatcaa gtttaacaca tatttactta gtaaacactt 1500

tctatatgta agaacttata ctccattttg aaggagagag gaggtgagca ggacatacct 1560

acaagtcaaa accagttccc accttcactg agatgggaca ttttggaaaa tgcacgaagt 1620

gctgtgggaa ttcagagagg aagagagtat ttcctgctgg agagtgcaga aaaaaaacat 1680

cccgatgaca tcgtaggtgg gcagcatttc ttgtcactgg caatgctgta aaccctcctt 1740

ttccacatct tcaaaataag gtgctgggcc tagaaactga cacctgctca gaaaaattca 1800

aaaagtgaat gtaggaggaa aaatcagcct ttgtcataaa atgacatagg aagaaagagt 1860

ggaagcaact agtataggac aagttgtgca agaacatgaa gtagtaaagg agataaaata 1920

ttattatttg aataaatatg agaaattttg aaaaccatta gtaatccgga atctttcatc 1980

tttgctatca tattattttt aaaatttggt tttaaagcaa cacaaggcaa tgtgcatatt 2040

ttcttaaact ttaagaaata gaaaaattgt tttgctagaa atatgtacgg accttagggg 2100

cttttctaaa atgtaggttg agaggtgtgt gtatatgctc ttttcttctt actagatcac 2160

atgtaatcaa gcatctacta ctgatatgag tcataaattt gtggggtact tttagatcaa 2220

tgcttacaaa gcagttgcac attcattaca aatcctgtac agtacattag cataagctaa 2280

taaatttctt actctctatt cttaggttga accagccaaa ttttcgagac agctcacggc 2340

ttagaggaag gttcatctaa ataaaggccg gctaaagtga cattgcaggg attaaatcct 2400

tctttggctg cctgtgtgac cagaaggctt atttgcaagt ttcttctttc ctggggtcca 2460

gattattagg tctccagcgc cctgcagctt gacagaaaga gaagcatgaa atgaaggtca 2520

gagatgagat cccgcagcag ggacgtgggg gcctcccagg ggcatttacg caccagagtg 2580

caagattctc tggccatcaa gggaaatagc aaacagaagc ctttgtcctg gggcacagcc 2640

acctaccaca aagcatcaga ctccacgtct ggccagaaag ttcctggagt cccatcaggc 2700

cagtgggtat gtaacatgtg cctaattgta cagctagagc ctgcaagttc aacgtgaggg 2760

aaggtgggaa atgtcttgag tgaggcgagc agctcctggc tgggctgggc agactcagct 2820

accacgttca ctgccttcct ctcactaaag ccgagaggga ggctgctcag ctctcaggaa 2880

aactcttttg aaccctgggc acctgctgtc ctcagttggc atctcccacc ctctgagcct 2940

cttctgctcc tgcacaacct gcctcttcgc tgagatggag acgtgagccc ccgtggacga 3000

tgactgcagt gtatatgaat ggaggtggcc tggtgaaccc ccattatgcc cggtgggatc 3060

ggcgcgacag tgtagaaagt ggctgtcaga ccgagagtag caaggagggt gaggagggac 3120

agccccgcca gctgacgccc ttcgagaaac tgacacagga catgtcccag gatgagaagg 3180

tggtgaggga gatcacgctg gggaaacgga taggcttcta ccgaattcga ggggaaatcg 3240

gaagtggaaa cttctcccaa gtgaagcttg ggattcactc cataaccaaa ggtaggatcc 3300

gacttcccaa gggtcatccc tggcagtatt gggacctagt gtaggaaagg ggttaggtgg 3360

ccagggccaa ggaagcaagt aaagtgacct cagcagagcc cctgcaaggc ccacatcctg 3420

tgccagccgc cttctgtggt cttctcagtt aattttcaca gtaaccatgt gaggtcaata 3480

tttttttcca ttttgcagat aaagaaactg agatccaaag aaggcaaatg tgtcttcagt 3540

tcnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3600

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3660

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3720

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3780

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3840

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3900

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3960

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4020

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4080

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4140

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4200

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4260

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4320

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4380

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4440

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4500

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4560

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4620

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4680

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4740

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4800

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4860

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4920

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4980

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5040

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5100

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5160

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5220

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5280

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5340

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5400

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5460

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5520

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5580

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5640

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5700

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5760

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5820

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5880

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5940

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6000

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6060

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6120

nnnnnttgac acataaaata aaccatcaga gttggtaact gattggatgt agaactgaag 6180

atggaaggat ggacgaaggc aatgaccttg ctttagttga ctatgtgaac caaaacaggt 6240

gatgccttta ataggaatag gagaaagagc aaatttggga gggaaaaagt tgagtgaaaa 6300

cactgacatc cagaaggagc tgtctactgg gcatgctcca gtcatggaca agggcatgag 6360

gtcagaccag gtatttgcat tgagaagatt gtagaaatgg atgcagtcac tgaaggagac 6420

tgtggagaag ggtgatgagc aaccttttta cacttagtaa ctagttctac atcctagttg 6480

acattttatg tatggattag tttttttttt tttttttttt tgagatggag tctcgctctg 6540

ttgcccacgc tggagtgtag tggtgtgatc ttggctcact gcaagctctg cctcctgggt 6600

tcatgccgtt ctcctgcctc aacctcccga ggagctggga ctacagacac ccaccaccac 6660

gcctggctaa ttttttgtat ttttagtaga gacggggttt caccatgtta gccaggatgg 6720

tctcgatctc ctgacctcgt gatctgcccg cctcggcctc ccaaagtgct gggattacag 6780

gcatgagcca ccgcgcctgg ccggattagt tttaaatatg ctaatggaat cattcactgt 6840

tgctccatga acactatgat ttttcaatgt cattaaatat agatttttaa aatatattta 6900

tgtgtatata ttatctatat atgtgtattt attatctata ctctcatctt ttaaagagga 6960

catattgttt cacatatagc tgtggcataa attctttaac ttagccacta ttggttaaca 7020

tttggatcat ttctaaagtt ttgctcttaa ttataatgct acagagatta ttcttttcca 7080

tacttctttg catatacgta tgatttctct cagcatacat tcctagaagt tagctgtctt 7140

ggtcagaggg catgcattga aaaattttaa cacacagcac caaaagagca gcttttgaat 7200

ccttactcat agagtacatc tatctgtaac tcaaatctca cagggactaa tgttgctttt 7260

caatagtcta gagccagcat gaaagacaaa agatgaaaca catagagcga tgaaaggcca 7320

aaaatcgaga cagaagatgt gatttggaag gttacaagag aagttggata aagccaaaca 7380

aggaaaggaa tttagaaaaa agtgtccaat gctgcagaag ggtcacagag ctacagtctg 7440

agcaaaggct actgcataag gccaactttg agaagtttca tacaagtgtg ggttcagatg 7500

ttagattcca atagaaagga agcaggatgg gacagtagga aaagcgtgga cttggggacc 7560

agacaaattt atctgaaata ctcattcagt aacttactag gcttctgact ttaggcatat 7620

taatctcttt ggtcctcagt tgcctcagct gtaaaataga gataatgatt tttagaattg 7680

ttggggaaat tggaaataat atatgtgaag tacatgacat atagtaacca ctcaaaatgg 7740

tagctgccat tgctataact ttacttttta attaatctta tttcaaaaag gtatttaatg 7800

ttattacaac aataaaaacc agtatcactt actatgaata gaaggtgatg gtaaaaataa 7860

atacaatgca aacaaattaa aaagcattaa aggaaaatta caagtaacaa agtaattttt 7920

aaaacatgct aacaatcaat ctcagacttt ctctttgcct taattaggag aattaaagga 7980

gagctgaaat gggaatcatg tttttacgct gtgattcagt gctatttaag gctgtttcca 8040

cgccctaaaa tcatttcaac ctcatgagtc ttaaaggttc cactttggga agcactgaca 8100

tcatctagtc cttccatgtt ttacaaacga ggagatggag gctcagaaga ggctgtccca 8160

aggtttagaa cctgtttaca ggaattcaca tggaattgag tactcacatg tattgagtac 8220

aggggaattc tatggaccca cagcagggaa gtagggccgg gtcttaagat gatattcttt 8280

tgatcttcac tttctctggg gctgtgtgct ttctcatttc tacttttctt gatgaatttg 8340

ttccatcttt atacctcttt ctgcaaaaag gttttccaca cttaattatc agaaggtata 8400

tcatggccaa aaatggttgt tgccagtccc acatctacat aaactctcaa tgttattgac 8460

cgcctccatc ccatgtgttt cattctgttt cagttcaaat tttaaaaaga aagaatatac 8520

ttaattacta gccagctgaa aatgaactac ctttgagtca agggcatgcc cgtgtcagtg 8580

agctgtaacc ctttgagtca cgtgggccac tactgtctca gcagagccaa aggtccaaag 8640

aagtctctgg gaagggagaa aattgacaga catatccacc atagagacat tagctagcta 8700

agccgagaag tttctacaag ccgggagcaa gacagtgccc aattctgtag acaggatatt 8760

atgtgaaagg acaaaaaaaa aaggtggaat aataattagt gtttatatac tgacttactt 8820

ctcgggttcc aaagacttct tgtctgttgt attacaaaat taacttcaaa cttgtcccca 8880

tgagtttcag agagcatggc attttcccac tgtgtgggtg tgggtgggtg ccttgtgtgg 8940

cctcactgac atgccctcct tatacctaac ctgctacaaa gtagctctat tgtcaaagaa 9000

acagtctcac ccgctggttc tgggacagta tgtgcaaaaa gggacaggaa aataaatagc 9060

agtttgtaca aatatggccc ccacaccaaa ccaacttgcc cagaattgtc atccggtttt 9120

ctaggaatat aaggacagtt tgaataagat aatagaggag attcccagga aagcagagag 9180

tttcagtgcc aggagcagct cccaggacag tagtctaaaa aattgaaaaa aaaaaaaata 9240

aagaaataca aagcaccaag tgctctggat ctctacaaag gaggctccaa tatgggtccc 9300

tgctggctgg ggtttatttt tatagctcac agtttggaat tcccttgggc tctccaagct 9360

gatagtgaag gttgacagtc atcatgagcc cccttttagg ctgattagaa atttctcata 9420

agagatgata cacttagctt ctgaatattt tatgtcccat ctgttgtcac aaacaaaaga 9480

agtggcagcg aatgtgggtg tttttttctg ttgctgccct tctgacttct tccttctgct 9540

actctagtct catgtttcta ggatcctttc ttcctcccac tttatttatc tatttatcta 9600

ttaattcatt ctttccttcc ttcctttttt ttttttttat cttttgagga agagttttgc 9660

ttcattgccc aggctggagt gcagtgatgc gatcaaagct cactgctgcc ttggactcct 9720

gggctcaagt gattcaccca cctcagcctc ccaaagcact gggcattaca ggtgtgagac 9780

accatgactg gccttcattc ctttatatat atatataata taatatataa tgtaatatat 9840

aatataatat ataatataat atatgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtaa 9900

gcacatgcct gataaggaac ttacagcagt ttaacagagg cagacaggaa aaaattgatg 9960

agtgctgtcc agagtaaggc agcaagaatg tgcttacctg tgccccgtgg atccaggaag 10020

ggtctattga gcaggaaaag cttgcaatgg gtcttgaggt ctgaggagca atttcacagg 10080

agaaggagga aagagaactc cagaaagaga gataggatat aaagtacatg gaagcatgaa 10140

ccagtgtggg gagtatttgg agatttaaaa gtaattcagc aaagctagag catgaggtgc 10200

acccacagag tcatgggaaa gagataaggt ggatggggct tgatcatgga aaatcttctt 10260

gcatatctgg attttgtcat ttaggtattg gaaagctact gaagagtttt tttttttttt 10320

gagacggagt cttgccctgt cacccagcct ggagtacaat ggtgtgatct ctgttcattg 10380

aaacctctgc ctcctgggtt caagtgattc tcctgcctca gtctcctgag tagctgggat 10440

tacaggcaca cgccaccatg ctcggcaatt tttttttgta tctttagtag agatggggtt 10500

tcaccatctt ggccaggctg gtctcgaatt cctgacctcg tgatccgccc gcctcagcct 10560

cccaaagtgc tgggattaca ggcatgagcc actgcgctca gtcgtttagg agctttaagc 10620

aagccattaa tgcaagtgat taatatgatc ttatttcagt gcggaatgtg aattgaagtt 10680

ggaaagtaag gagagtcaag gaagtcagaa aaaaatgtta gtcaaatggt ccagatggat 10740

gatgttgttc atctcaacta aggagaagtg gcagtgagaa tggagagaag agcatgtatt 10800

tgaaaaatgt cttaggatat tgaaaaacct tgtgatcaat ggtatgggtg agagcaggac 10860

gggacatggg agaggaagaa ttgacgatga ctcccagttt ccagttttaa tgccttacca 10920

gtaaataatt acaggagaag tggatttgag agtaaatata tggaagcact gacttggagg 10980

tatatggaat tcatgaaatg aatgggtcta gcagctttct tggagcccaa gagaaagtat 11040

gatacataaa gatttgaggc ttaacaaact ctgggatgta gtgacatctt gggagtccta 11100

gttacaagct ccttggtgtc agatactata tcacattgat ccttaaagtt cctccatgtg 11160

tgttataaat aataaacact cgacaaatat ttggtgagtt aagttagtat aacttctcat 11220

gaaaatccag gagtggtgga ggaagaaata ttcgagggag caatgcatag cagcaccttc 11280

tggtaggtgc tgtatgctgt gatctccctg gaggacaggg ctagagcacg aactcagtct 11340

gtagcagagc ttcataaaga tagcccaatg gttttgtcgt tttgcttttt ctagagatga 11400

agtctcaact atggttatcc aggccgttca agaacttctg ggctcaagca atcctcctgc 11460

ctcagtttcc taagtagcat gggggacagc tatgcaccac tgcacctgac tgctcactgg 11520

ttttaaaaat tgattccccc ctcttgaata cattcattaa aatagcaatt caaaattgta 11580

ttttcttact ttaaaaatgg ttagttagtg atttagggaa ttcaccctaa aaataaatca 11640

tatattggaa atactacaac caaaaaaatg ttcattgtcg tgttatttat agtgtcgaga 11700

actagtaata acttatggga tccaatgata taggaatggt caggtaaagc atatacactg 11760

ggcagaagat tccatagcca tgagaaatca tgattatgaa gactctgtgg taacatggaa 11820

gatgggcagg acatgagtgt aagtgaaaag ccaggtgcaa atggagcgca cactattatg 11880

ataatactta ctccaggttc ttcagtgaga ttattcactc agcaaatact tgttgagcac 11940

ctactatgca atagccacta ttctagagac tggtgataca gcagtgaaca aaatagacag 12000

caatctctgt ccttatggaa cttacactct agtgagaggg aagccagaca aaaataaatg 12060

tgtaaaatat tcattgtatt atgtagcgat aagtgataag aaagaaaaga gggccgggcg 12120

cggtcggtca cgcctgtaat cccagcactt tgggaggcca aggtgggtgg attatgagat 12180

caagagatgg aggccatcct ggccgacatg gtgaaaccac aattctacta aaaatacaaa 12240

aattagctgg gcatggtggt gtgcaccttt agtctcagct actcaggaga ctgaggcagg 12300

aaaatcgctt gaacccagga gggggaggtg gcagtgagcc aagatcgtgc cactgtactc 12360

cagcctggtg acagagccag actctgtctc aaaaaaaaaa aagaaagaaa gaaagaaaga 12420

aagaaagaaa gaaagagcag gaggggagat agaaagcatt gaggatgaca gtttttagat 12480

aagttggcca ggacagcctt actgaggagg taataattaa actgaaagaa atgagggaaa 12540

tggctataca tgggggaact ccatcaagca gaaggcgtag caggcgtaag agtgtccaga 12600

tgggagatgc ctgcagtact cttgaaacag ctgaggggaa agggcagaga gtaacaggga 12660

acagattgtg gaggaccaag tttgtcaaag taagaagttt ggcttttcct gggattttac 12720

aggagccatc agaaggaact ggggatttca caaagggaac cagggatttc ctgggatttc 12780

acaggagcca tcagagggaa ctgggggagt gacatgatct ggcttannnn nnnnnnnnnn 12840

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnncttg ggcaacatag caagaccctg 12900

tctttaaaac caaaaccaaa accaaaaaaa ccacacacat ataacctttt tatgtttaac 12960

gcatatcaat accaaaaaag tttaaaaatc atgagtattc attttgatga attttccaaa 13020

ctgaaaagac ccatggaagc accaccaaca tcaagacaca gaacacttct ggtactcaga 13080

gcgctgccct ccacctgctc gtcatcctcc attctattct ctacatcctg aagtgtaacc 13140

actattctga cttctaatac catagattgc ttttgactgt tttaaacttt acataaatgg 13200

catcatccaa tgtgtattct tttgtcttac ttcaattgtc caattttgag tttgtgacta 13260

ccatccatac tgttttatgt agttcagatg tttcatgctc attgccatat atacagtaat 13320

tcattgtatg aatgtagtac cacaatttat ttatccattc aactgttgct gatgtgacta 13380

gtttctagtt tccagtttgg ggctattatg aataacattg atgtgaacat tctagtgcaa 13440

gcgtttttgg taaacttttt tttttttttt gaggcagggt tttgctttgt cacccaggct 13500

ggaatgtggt ggcacaatca cagctcactg caaccttgac tttctgtatt tgagtgatcc 13560

tccaacctca gcctctggag tagctggaac cacaggggca tgccatcatg cctggctaat 13620

tttttaaatg ttttgtagat atggggtcct gctatgttgc ccagactggt ctcaaactcc 13680

agggctcgag taatcctctt gccttggcct cccagagtgt tggtattaca gatgtgagcc 13740

actgagtctg gctcgtatgc atttttattg gggacatttt tacttataaa ttttttttca 13800

atgacttttg gggtacaagt ggtttttggt tacaatgatg aactgtatac tggtgaaatc 13860

ttagatttta gtgcacctat cacctgagta gtgtacattg tacccaatac gtagttttta 13920

atccctcacc ccctcccacc ctaccccttc taagtctcca aagcccatgc cttttcatac 13980

ccatagctaa actctcattt ctaagtaaga acatacagta tttgattttc cattcctgag 14040

ttacttcact tggaacaatg gcctccagct ccatccaagt tgctgcaaaa tacattattt 14100

tattcttttt catagctgag tagtattcta tggtatatac accacgtttt ctttacccac 14160

tcattggttg gttgacactt aggttggttc cagatctttg caattgtgaa ttgtgctgcc 14220

ataaacatgc atgcaggtgt tttttgatat aaggacttct tttcctttgg ggagataccc 14280

agtaggggat tgttggatca aatggtagat ctactctcag atttagattt taatttactt 14340

ttagatttta gatttagaaa tctccattct gttttccata gaggctgtac taatttacat 14400

tcccaccagc agtgtataaa tgttaccttt tcaccacatt catgccaaca tctactgttt 14460

tttgactttt taataatgac cattctggct ggggtaaagc ggtatctcac tgtggtttta 14520

atttggattt ttgtgatgat tagtgatgta gagtgttttt ccatatgttt gttggccatt 14580

tgtatgtctt cttttgagaa atatttattc atatcatttg cccacttttt aatgagatct 14640

ttttttgttt tttgtttgct aatttgtttg agttccctgt aaattctgga tattagttct 14700

ttgtcagata catagtttgc aaatattttc tcccattctg tggattgcct gtttgctttg 14760

atgattattt tttctgttgt gcagaagctt tcagtttaat gaggtcccat ttatttattt 14820

ttgtttttgt tgcatttact tttggggtct tagtcacaaa ttcttatcta agtcaatgtc 14880

cagtagagtt tttcctgttt tcttctataa tttttgtggt ttcaggtctt agatttaagt 14940

ctttaatcat attgagttga tttttgtata aggtgagtgg gatccagttt tattctccta 15000

catggggcta tccagttttc ccagcaacat ttactaaata gggtgtcctt tccctaattt 15060

atgtttttgt atgctttgtt gaagatcagt tggttgttan nnnnnnnnnn nnnnnnnnnn 15120

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15180

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15240

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15300

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15360

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15420

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15480

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15540

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15600

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15660

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15720

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15780

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15840

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15900

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15960

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16020

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16080

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16140

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16200

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16260

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16320

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16380

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16440

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16500

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16560

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16620

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16680

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16740

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16800

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16860

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16920

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16980

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17040

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17100

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17160

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17220

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17280

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17340

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17400

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17460

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17520

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17580

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17640

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17700

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17760

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17820

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17880

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17940

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18000

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18060

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18120

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18180

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18240

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18300

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18360

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18420

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18480

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18540

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18600

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18660

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18720

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18780

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18840

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18900

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18960

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19020

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19080

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19140

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19200

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19260

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19320

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19380

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19440

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19500

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19560

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19620

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19680

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19740

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19800

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19860

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19920

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19980

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20040

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20100

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20160

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20220

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20280

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20340

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20400

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20460

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20520

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20580

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20640

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20700

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20760

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20820

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20880

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20940

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21000

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21060

nnnnnnnnnn nnnnnnnnnn nctgtggggg gctccaccca gtttgagctt cccagctgct 21120

ttgtttacct actcaagcct cagcaatggc ggatgtccct ccaccagcct cattgccacc 21180

ttgcagttca atctcagact gctgtgctag cagtgagtga ggctccatgg gcgtgggacc 21240

ctctgagcca ggtgcgggat atgatctcct ggtgtgccat ttgctaggac tgttggaaga 21300

gcacagtatt agggtgggag tgtcccgatt ttccaggtac cttctgtcat cacttccctt 21360

ggctaagaaa ggggattccc tgaccccttg cacttcctgg gtgaggcgat gccctgccct 21420

agttcagctc acacttcgtg ggctgcaccc actgtccaac aagccccagt gcgatgaacg 21480

cggtacctca gttggaaatg cagaaatcac ctgtcttctg cgttgctcat gctgggagct 21540

gtagactgga gctgttccta tttggccatc tttgaacctc ccccctgact tgcgtatgtt 21600

aaatcttccc tctatctctg ggacaaagcc catttgatca tgatgtatta tgtttttgat 21660

ttgctattgg attctgtttg ctggtatttt gttgaggact tttgcatcta tgttcatcag 21720

gaatattgat ctgtagtttt cttttttgtt atgtcatttc ctgggtttga cacagggtga 21780

tactggcttc atagaatgag tttgggagga ttttctcttt ctcaatcttt ttgaataatc 21840

tcaatagtat tgcttccaat tcttctttga ctgtctggta gaattcagct atgaatccat 21900

ctggccctag gcttttttgt tgctggcaat ttcaatcagt aaattttact tagtcagtct 21960

cactgcttgt tatctggtct gctcaggatt tctatttctt cctgatttaa tctgggatgg 22020

ttgtaaattt ccaggaattt atccatttct tctatgtttt ttagtttgtg tgaatagaga 22080

tgttcatagt aatctcaact gatcttttgt atttctgttt cattggttgt agtgtctcta 22140

ttttcatttc taattgaact tattcaaaac ttctctcctc ttagttaatc tagttaatgg 22200

tctatcaatt ttgtttatct tttcaaagaa ccaacttttt gttttactaa ccttgtgtaa 22260

tatttttgtt tcaatttcat ttatttctgc tctggtcttt gttgtttctt ttcttctcta 22320

gctttgggtt cctttgttcc tgtttcttta gctccttgag gtgcgatgtt agggtgtcaa 22380

gttgtgatct ttcagacttt ttgatgtagg catttagtgc tataaacttt cctcttagca 22440

ccacttttgc tgtatctcag aagttttgac aactgtgtca tattatcatt catttcaagt 22500

aatttttaaa tgttcatctt tatttcattg ttaacccaaa atttatacag gagcagattt 22560

tttaatttcc atgtatttgt atagttttga gaattccttt tggagttggt ttctagtttt 22620

ttcctactgt ggtctgagaa gatgcttgat atgattttga tttttaaaaa ttattgatac 22680

ttgttttgta gccaatcgta tggcctatgt tggaatatat tccatgtgct gatgagaaga 22740

aagtatattc tatagttttt gggtaggatg ttctgtaaat atctgttagg tctacttgtt 22800

ttagagtatt gtttaagtca ttgtttcttt gttgactttc tgcctcgatg atctgtctag 22860

tgctgtcagt ggggtgttga agtatcccac tattattggg ttgctcatta tctctttcct 22920

taggtctagc agtaattgtt ttatgaattt tggagctcca tagttaggta cacatatatt 22980

ttggattata atattctctt gtcagattaa tccttttatc attatataat tgtaaccacc 23040

taacaggtta tttctgccca ctgcacaagc aaaatcaatt catggcattg tggtgaagaa 23100

agtgtttaat tgatgcaagg ccagtcatgc catgtggtca aactaggatg gagatattac 23160

tcaaatcaat ctcatcaaag gcttgttggt taggggtttt tcaaaggcag ttttggggaa 23220

gggattgggc tgaccagacc aggtgcttgc tgctgattgt ttgggttgga gatgaaatca 23280

tatggagttg aagctgtcct cttgtgctga gtcacttctg tggggccaca ggagtggttg 23340

gtgggtccat atgggtccaa gtgaagccat cggtgtcaga catgcaaaaa acctgaaaag 23400

atatctcaaa aggctaatct taggttctac aatagtgatg ttatctgcat gagtaattgg 23460

gggagttgca tatctgtgac ctctggaata atggctgtca atcatttata tccacacctt 23520

agcagaattc aggctcttct cctcctccta agtctggtgg tttctcatta gctttacaaa 23580

ggtgactgag ttttgggaaa gattattatc atttaaacta caaactaaat gtctcccaaa 23640

gctagccaag actaagccca ggaataatta aggcagcttg aaggctaaag gcaagaggga 23700

ggttggctag atcagatgtc ccccactgcc ataattgtct cagtgatata atttttgcaa 23760

aggtggtttt ataatgacct tctttgtctt tttctttttt ttaactgttg ttgctttaaa 23820

gtctgtttta tttaatataa gaatagccac tcctacccag gcacggtggc tcacacctgt 23880

aaatcccagc actttgggag gctgaggcag gtatatcacc tgaggtcagg agttcaagac 23940

cagccagaca gagatggtga aactccatct ctactaaatt tacaaaaaca ttagccaggc 24000

atggtggggg gcacctgtaa tcccagctac tcaggaggct gaggcaggag aattgcttgt 24060

acccgggagg cagaggttgc agtgagccaa gattgcgcct tgctctccag cctgggcaac 24120

aaagtgagac ttcatctcaa aaaaaaaaaa aaaaaaaaaa aagaatagct actcctgcta 24180

gcctttggtt tccatataca cagaacatct tttcccaccc ctttaccttg tgcttgtgtg 24240

aatccttatg cattagatga gtctcttgaa gacagcagat atttggtttg tgatatttta 24300

tccattctgc caatctgtat gttttcagtg gagcatttat gccatttatg tttaatatta 24360

acattgagat gtaaggtacg attctcctca tcatgttgat tattacctgg atccttttgt 24420

gtgtgtgtgt gtgtgttatt gtttttattt ttttatttta atttttttga gacagagtct 24480

tgctctgtca cccaggctgg agtgcagtgg cgagatctct gctcactgca agctccgcct 24540

cctgggttca tgccattctc ctgcctcagc ctcccgagta gctgggacta cagacacccg 24600

ccactacgcc tggctaattt tttgtatttt tagtagagac ggggtttcac catgttagcc 24660

aggatggtct cgatctcctg acctcgtgat ccacccccct tggcctccca aagtgctggg 24720

attacaggtg tgagccaccg tgcccggcca tgtgtgtgtg ttattgtttt ataggccctg 24780

tgagttttat gctttcaaga ggttttattc tgatgcatgt caacttttgg tttcaagatt 24840

tagaagtcct tttagaattt tgtgtatggc tggtctggta gttacaaatt ccttcatcat 24900

ttgcttgtct ggaaaagact ttatttctct ttcatttatg aaacttagtt ttgctggaca 24960

caaaattctt gactgacact tattctgttt aaggaggcta aagattggac cccaatccct 25020

tctggcttgt aaggtttctg ctgaggggaa gatttttatc aatgacataa atgtgtgatg 25080

atggaaacaa tccagtggga agagaaaatt gatgcaggag agaagaaaga aagctttttt 25140

ggagggctgc tctttagtat atgagagtag atggaatcta ttcaacatgt aaaggagtct 25200

tagctaggcc ttagatagga gtaggaattg tttatccaaa gtaaaatgaa ggaagagaga 25260

ttccagtagg ttggaagaca tggtgggaag gacttgagga aattctcctc tattgcttat 25320

tttactcaat aaagtaggaa gcaaggttac cagctgtgaa tgaggagtgg aagaaggcat 25380

tagagacttg atgagagaaa agaagccatg aaagagttat acaaaagtgt atgaggttta 25440

ttggactagg gaaagtatgt ctgccaggcg ctttaaaggg ccaactgaag ttagtgacca 25500

gaaattcaaa gtgagacaag ccattatgtc caacagcatg ggtgcaggca gagagtagtg 25560

ggaagctgga tttaaccaga gtcatggttt agcaaagtga actcaacaac ctgggagaga 25620

gacaggggaa cttagggagt gcccgaaggc atgataacat taatagagat cctcctccag 25680

tgaatgtcac cagcctccca gttgctcaac tgagaaacct tgaccttttc ctcaacccct 25740

aacacccaaa caccaagtgg tattgatttc acttcctaaa tatctcctcc tttcctcatt 25800

ggattaagct actgtaattt gtcttcatgt ttatatcatt tcccccatag cagccagagg 25860

tgatgtttta gaaattacaa gcctgatcat gtaactacct acttacaagc ttccagcagc 25920

ttcttttttt ttttcaagtg gcagttttaa gtttgtgggt ttttaattta tatatgtgaa 25980

ataccttttt ataggcaaaa ataataatag catgcttctt actgccctta atgtgaaatg 26040

actcacaaga ccttggtgtg gccaactctc cagcctcttt tttggcttat atttttcctt 26100

cttccccata tgccagccct actttcttct ttcaattctt caaagtcagt catctctgag 26160

ggtgcatata cccccagaga atgcccaaga tgattcactg ggatgaggaa aaaaaatact 26220

agaacttcct tttatattta tttgtgatta tatattttta atttctgtga ttatatgtat 26280

tgaaaatgtg taaatcagta gtacatggat acactttata aatacatata catatattag 26340

agtagtgctc aaatatattt tttattatgc acatgcagtt gtaagaatat gtacagagac 26400

cacatgtacc atttacccag tttcacacaa acttgcatct tgcaaaacta tagtcataac 26460

cttgatatta atattgatct aattcactga tcttattaag attttcccag ttcttgtatt 26520

catgcatgtg tatgtatttt aactctatgg aatgttatca cataaatagg ttgttatatt 26580

ccccaccaca gtaaagatac aggacaagtc cattatcaca agggcccttc aagtggctct 26640

tctgtagcca cctcctaacc ccctataccc tgtccctgac tcctgccagc cactaatccg 26700

ttctccattt ctataatttt gtcattccaa gaaaattata tacaagaaat catacagtag 26760

taacctttta ggattggcat ttttcactca ccatacttct cgggcaattc atctaaactg 26820

ttgtatcaat agttggttca tttttattgc tgagtggtat tccacgtatg taggtaccac 26880

aatttgttta accattcacc agctgaggac atgtagattc tttgcagtta ggagctatta 26940

tcaataaagc aactaatgat gttgaatatt aaaaaaatgc tttattgaca aaggaagcca 27000

atcaaaaaag cttgtagacc ataaccctaa tgtttcaggt ctttcctacc tcaagatctt 27060

ctgtcctacc catccctact tttagaacct ctccctactt ttctctagct aactcttgga 27120

ctccagagaa agtgttaaaa gtcttcccta acccccagac caggagtaca tccccctgtt 27180

ttatgctcct aatgctcctg gcacttttcc ccctaagccc actgtacctg atattttcta 27240

tctgcccctc cagatctact ctccaccctt cttcattctg ctctcagccc aggactacat 27300

caaatctcct tagtcctctg ggttccagtt gactttgatc tctagggagc ccttgcaggg 27360

gaaggaagga gaaggagggc ctgattttat tcccccagcc cctcacggta aggctgactc 27420

aggcaacttt tgtgtctaga aggaaagcct cagcttctct ctacacaaaa ctatcttcct 27480

aggttttaat atctgctccc ttccctttgt ccccttggtc ctgtagacgt attttgcttg 27540

gtctaatcct aggttttaaa atttgaatta gttgttgata cttaataatt ggaagatttt 27600

gcataaagtt tcagatgtct ggcttctctt gaaaaaaaat cagaaggttt gacaacactg 27660

agcctccatt ccaaatagta gccattgtgt ggccttgagt agagccaccc cttccactgg 27720

ggcagctact cttaaggatt caccatcccc actaactccc tgttccatac aataaccttg 27780

ctttgctatt accaccctgg cactaggagt cctggtttaa agaatgtgtt gcccgctagg 27840

tgggacctgt gtcagacttc tcatcagatt tggagaaagg gcatgctggc ctctcccatc 27900

tgtctacttc tctctgtctc cccttgcacc acatagagca ccctttctct ggggtttcta 27960

tcatcccctc ctagactccc acttgctccc aggcttgctg ctcctctctg cacttctcac 28020

atcttgccag agtgcttttt tttttttctt ttttttgagg tggagtctca ctctgtcacc 28080

taggctggag tgcagtggtg tgatcttggc tcactgcaac ctctgccccc tgggttcaag 28140

caattctcct gtctcagcct cccaagtagc cagaattaca ggcggctgcc accatgcctg 28200

gctaattttt tgtatttata gtagagacgg ggtttcactt tcaccatctt gaccaggctg 28260

gtcttgaact cttgaccttg tgatccaccc gcctcggcct cccaaagtgc tgggattaca 28320

ggtgtgaggc atcacacccg gcccagagtg ctctttttaa atgggactca gattacaacc 28380

ctttttctta aaaccttgct atgttttctc attgccctat agttaaagcc tgcatttcta 28440

aagcagtccc ctggcttatc tgaccttttc ctactaccag gctgatctgg ggccacccct 28500

cctcattctg cttttccaga acagtgggtt cctcaagcac acaccttatg tcttgcctgg 28560

gcagcacact tgccaagctc ctaggccaag tatgaggcct ctctcctcac ttcctggtca 28620

caatttaagt gcccttaccc agatagtgcc tttccattct ctgtctgtat ctcttcttaa 28680

taggtgctat gcttgtaaga agggattttt tttttagtct ttctctcatt ggactgtgag 28740

aatagtgttt atctttttca ctgcctggtg tatagtaggt gctcattgaa catttattga 28800

aagagcagat ttagagatct agctcttcat ggaatagcag tttctaccac ctagcgacaa 28860

tgtggaatac cagggcttct aaagtgttta gtatgatcat acatttatat tatgaggcct 28920

gcctgtgggg tgattgcaca tgaggtgagg ccattgaggc acctagggca gaggatttaa 28980

ggaagctttc acccttacaa tgcctctcct gccacactct agacctggct ctgggaggga 29040

agcctttgct aagaaccatg aaaggaaagg agaaaaagaa aactagggtg gtgctaaagg 29100

gacatttgtg atggcagcct gacacctacc acctgattct tgaacactca ctgtggatag 29160

aatggtgtat gatggccaca gggcaggagg gggcagcaga gtgatgatac cttaactgac 29220

acctgagtca gtaatgggag aactgaggag aagcagtggg ttcttctaca ggaaatcgaa 29280

gggtggaatt cattaatcta gcccttaaaa caacagctta cccaacatgc tacacaaaac 29340

caggtttctg tttcagataa ataggtgaag ggactcttaa tcctaaagac tgaaaagtag 29400

aaaagaaagg ggaagaagag tgcctcaagg attgccattg gaggttcttt tgctggggct 29460

gattgccagc tgagattatt caagccccag agcaaacatt ctgctcctgc tcccttagag 29520

ctgccctccc accgctcagt attgcctcct gcgaggggcg ggctggctgc cgcagacacc 29580

agtgaaccct ttttccattc cagaagtccc agtggaccta ctttaatata ccaataacac 29640

tcctatttta aactagctgt atccattttc gttttaatag tcccagtgct aaagtttttc 29700

aaagcagtta ttttgtaagt aggtcaaaca ggtactttgg gatcctgttc tgtctgtttg 29760

cttgccaggt aacctctttg ttatctaatt caaagtctgg tacagtttga accaaaacaa 29820

aaaaggaatg atgtttcact ttggagtcaa gattcattca ttttctaaca ttaatcattt 29880

tcgtaataca gtaagtctat attcatgata aaaaatagaa aatatgaata agcaaaacta 29940

aattgaaagg aaaaccatct gtgatctgcc aattagaaaa tctctattct aaacattttg 30000

gtaaatatgc taccagattt ttatctatgc aaatgtgtat ctgtattttt cctcacttgt 30060

atagtggaca tcttttcata ttaataaata agttagcatc attacttttg atagcttcat 30120

ggtgtgaaat tataaaatta gacctaccac actctatttt aactatctct cttgctgggt 30180

acttaagcta ttcccaatat cacagaagcc tttttacaca tgcatccttg tacatgcatc 30240

agattcttta agattgccaa aggtgaaatg gttgggtccc agggtgcaac tattttttag 30300

catttcaata cataatacca aagtgcaatc cagaaccatt gtaccaatta atgctcccgc 30360

cagcagcaca ttgcagtgtt gatttcctcc tggcctcacc agcacgctgt caacactggg 30420

tactggtgtg tcgtgtacat gacattacat gccaggcact gagagtgaaa taaaacagtt 30480

catgttggga atggggcaag agagggagat aaacaaggga ggtgacatct acagaccggt 30540

tagctgagat tacagtacat tggggctcca agtgcacaga atctgaacat catgttcatc 30600

ctaggacaag tgttgcagag gcttcctgaa gaggttcatt ctgaggctta ccttgaagga 30660

tcatcaggaa ttaatgatga gtgtattaag ccattctcgc atttctataa agaaacgcct 30720

gaaattgagt aatttttttt tttttttttt tttctgagac ggagtcttgc tttcttacct 30780

aggctggaga gcagtggtgc gatctcggct cactgcaacc tctgcctccc tgggttcaag 30840

tgattctcct gcctcagcca cccgagtagc acaggtgcct gccaccgggt ccatctaatt 30900

tttgtatttt tagtagagac agggtttcac catgttggcc aggctggtct caaactccca 30960

acctcgtgat cctcccgcct tggcctcccg aagtgctggg attacaggcg tgagctgccg 31020

cgcccagcca aaattgagta atttataaga aaaaaaggtt ttattagctc atgattctgc 31080

aggctgtaca ggaagcgtgg tggcatctgc ttctgtagag gcctcaggaa gctccaatca 31140

tggtggaagg tggaaggcac atcacactgt gaaagcagga gcaagagttg ggggaacatg 31200

ccataagatc ccatcacttt taaagaacca gatctcatgt gaactcagag cgagagctca 31260

cttatcacta aggagatggc ccaagccatt catgagacat cagcctccat gacccaaaca 31320

cgtcccacca gactccacct ccaacattgg ggattacatt tcaatgagat ttgggtgggg 31380

acaaatgtcc aaactatatt gatgagtgaa agcaagagta aagaacaggg aaggaatgat 31440

ggagagaatg aaccaggtag agaaaatctt ctaggcatga atgttcagcc ccttttcagg 31500

gagcaacaaa aagttctgta ttggttgcaa tgaagggtgc atggggcatt ggggatggtg 31560

gacaggaatg atggacagtg aggctgatga gatgagctgg gtctcatcaa gaagttctct 31620

ccgggcttag caaaggagct ctagctttat ctggaaactt tccttgggag gcaggacttg 31680

gcatcaaaac ctgtgtgtgg aagactcaca ccgatccaag ggaagggcac tgatgctggg 31740

cttaagcatt gctttgtcat tccctgatga gctctctcag cttgggaaca gccactctct 31800

gtctagtaaa ggaaaggagc tgatccaggt cctctccagt tctaactatg ctcctgtgaa 31860

ttctggcaca atttttactt tttttccttg cagaaaaggt ggccattaag atcctggaca 31920

agaccaagtt agaccagaaa acccagaggc tactatcccg agaaatctcc agcatggaaa 31980

agctgcacca tcccaacatc atccgccttt acgaagtggt ggagacccta tccaagctgc 32040

acttggtgat ggagtatgca gggggtgggg agctcttcgg aaaaattagc actgagggga 32100

agctctctga accagaaagc aagctcatct tctcccagat tgtgtctgcc gtgaagcaca 32160

tggtgagcag gggtgacgag tgagaacctt gctcccattg cactgacact gggagcacag 32220

ggctttaggt tactaaccct caagtgtccc agagggcttt tgtcctacaa agcagacagt 32280

agtcccttct gagagtcaga agtctggctg ggatcatgct ccttgcctgt gaagcaacat 32340

ccagggatgt ccagttacac gtcaacctcc tagcttctgc agacattggc aggaatcact 32400

gggagcagct gatgaatatc acccactccc tggccatacc catttctcca acctcatctt 32460

ccactatctg gatagctggc tcctcactgc ttcttggttt tgctttccta cttctgtcta 32520

ggatgccctc atatgacatc tcacattcta aatcctatcc atacttcatg cccagcttgg 32580

ttctctctct ctctctctcc cccacccccc ctctccgtgt gtgtgtgtgt gtgtgtgtgt 32640

gtgtgtgtgt gtgtgagaga gagagagaga gagagagaga tgttcttcaa tctcacatca 32700

gttatactga tattttgcct tcagtaagat cccctagctc ttattgcctc tgtgcccagt 32760

agattaacac ttggttgtgt ctttcttttt tcccttcctt cctgcctgcc ttccatcctt 32820

ccttccttcc ttttcttcct cccttccttt cttttccttc cttccttcct tcctttcttt 32880

ctgtttcttt tttttttttt tagacagagt cttcctctgt cacccaggat ggagtgcagt 32940

ggcatgatct tggctcactg caacctccgc ctcccgggtt caagcgattc tcctgcctca 33000

gcctcccaag tagctgggat tacaggcatg tgccaccatg cctagctgat tttgtgtttt 33060

taatagagat gaggtttcac catgtttgtc aggcttgtct tgaactcctg acctcaagcg 33120

atccacctgc ctcggtctcc caaagtagtg ggattacagg cgtgagccac cgtgcctggc 33180

cgcttgtgtc attcttattt tagttctttg gttacttctt tagataaatc tggactccta 33240

atgcacactc tgtagttctc ccaagaactg gctgttttgc tgcctcctta attttggccg 33300

tattgcgggc catacaggag gaacctgact tgcaggcctg agtatcactt ttactaccaa 33360

aagttttagt cagtcaccat ctttactcag atcccagttg aacggatctg tcagaaaaga 33420

gacacttggc tagggtctat attagttgag aggctatttc caattcactt ctcagcctgc 33480

atggttgtag gagcaagaac tgggatcagt gagccacagt gaacctcatt acatgccagg 33540

cactgaaaaa gtcagataaa acagtccatg ctagcaatgg ggcaaaagag ggagaatttg 33600

caagggaatc tgtggcctag agggcccaaa atagcagtag ggaaggagag gaggaagaaa 33660

aagaagtgag ccagggagaa gagcttgact gtagtggtgg tggatgtttc agacctgtga 33720

tatatgacca atcatcattt aagtggcttt gggctgtggt gagctttccc agggccagag 33780

aataagccag acccatacca agtaatagaa cagatgggca aagagtcttc cacattctac 33840

ttggaaaagt aacttggtgg gagaacagaa gagaatccag caatagaagg tacagatgtt 33900

agtcttcagc agctctactc cctgccattt ctccaacgct ctgcaaacag gaagtgtgtt 33960

tcatagccat agatccacat cttattgagt ttgtttatat gccaagacca tgctgagcac 34020

tgttctcatc atacgtagtt agcgtcatat atagttctca ccacagccta tggttatcag 34080

tatcccactt tacagagaag gaaactgaca ttcacagtac ccagctggta tagttgaacc 34140

tagaattgac cagactgtcc cctccaaagc ctggattctt attccaggag ggccatcagg 34200

aaagctgaca aacacaaagc cattttccaa gagcccttgg gactgaacag gtcaagggtc 34260

ctctgaagat tgtctggatt caggatgcaa gggtgggagt ggagcatgtg cccacaatcc 34320

acagtgtgtt ctgtggctag atccttgcca aatgcaacca cctcccttcg gctgaattct 34380

gtaaggataa aagagtccac cccaaaaagc atggccggaa agtcagggga gggctccaag 34440

ccttcctggt caaacgatcc atcagccaga aaaacccatg tatgacctca acaactaagg 34500

attactgttt cattgtattt cagaatgtgt agtttcataa gatctgggtc tgatttctgg 34560

tgttagtttc tgagtccaca tgtgtggaac agactccaac ccttaccaca tagaatagga 34620

actttggtct atttggggag gtgtaggcat tacattgggc taaaggttat gacagggttt 34680

gctatcatga cttaaggtga tcctcactga atttgttatt ccaccatcat tatctcactg 34740

tttcagagca actgatacat tttttatttt gacattttac tgtaaaaatg acttttctct 34800

atgtcttctt ccacagcatg aaaaccaaat tattcataga gatctgaaag cagaaaatgt 34860

attctatacc agtaatactt gtgtgaaggt gggcgatttt ggattcagca cagtaagcaa 34920

aaaaggtgaa atgctgaaca ctttctgtgg gtctcctccc tacgctgcgc ctgaactctt 34980

ccgggacgag cactacatcg gcatttacgt ggatatctgg gccttggggg tgcttttgta 35040

cttcatggtg actggcacca tgccatttcg ggcagaaacc gtggccaaac taaaaaagag 35100

catcctcgag ggcacataca gtgtaccgcc gcacgtgtca gagccctgcc accgactcat 35160

ccgaggagtc cttcagcaga tccccacgga gaggtacgga atcgactgca tcatgaatga 35220

tgaatggatg caaggggtgc cataccctac acctttggaa cctttccaac tggatcccaa 35280

acatttgtcg gaaaccagca ctctcaagga agaagaaaat gaggtcaaaa gcactttaga 35340

acatttgggc attacagaag agcatattcg aaataaccaa gggagagatg ctcgcagctc 35400

aatcacaggg gtctatagaa ttattttaca tagagtccaa aggaagaagg ctttggaaag 35460

tgtcccagtc atgatgctac cagaccctaa agaaagagac ctcaaaaaag ggtcccgtgt 35520

ctacagaggg ataagacaca catccaaatt ttgctcgatt ttataaattg cactagactg 35580

cttgtaacta accaagatga ttgttgctgc ttctaaattt ttttcaagga caacttgagt 35640

ggagacattt ttgtaatttt taaataaact taaatttgag atatgcattt tttttctcca 35700

aaaagtctat tagctcagat tctggcttga tttgggatct tgttttatta tcaaatttca 35760

gcattcattc atttaatcaa gaaatattta tccagtgcct cctgtgtgtc aggcactatt 35820

taaggtgcta gggaaacagc aagaaataaa taggcaaggt ccctcctctc atgtgcagga 35880

cgttctagcc caggacaaag gtactaacaa ctacattttt taaaaaagag aaagatcagt 35940

gatagatgct atgcagagga ttcaaaaagg gtgatctgat atcagaggct tgctactttg 36000

gattagtcat taaggaaggc cactttgagt acatataagt ttaagatctg aatgagaagc 36060

aggagtgact tttacaaaat gccagagcat tccaggcagc taggggtttc agactcagtt 36120

ccccccaaaa cagagcctaa gacaaaggct tccatataaa gtagtttatt tgggaactga 36180

tcccagagca caggagtgaa ggacagagga aacagggaaa gctaatacac agacacatta 36240

gcaagttagc tagtgctaca gtttctggtg cttggttttg caggatcctc cagaggagcc 36300

tgatgaaaca catctcaggt gtctgccctg ggtatgaaag gggaaagcat ttaaccataa 36360

gctcttgctc accgctgatc aagggtggcc ctttgtgtca tctgccctgc actcccagat 36420

tgggctgtgt gaatgccaag tgggttccct aagcttccct tgccttcatg tcagagaagt 36480

cttggagcag gagggaagag gtacaccacc agtacgatac ctcctttctt tgcctgcttg 36540

tccactggca tggggagccc aaagtgacca tgtgacactt gtagattcca attcactgga 36600

gcccttatca agtcccctga tgaaagcatc cctgatcccc tgggaaccaa accttttaat 36660

ctagcctaga ttgtagaaaa aaaaagaata gaaattaatc aagtgagtca ccaattgcgt 36720

gatggtgaca ggagtcaatc tacttccacc tagtggtttc cagacctgtg ttttaatgat 36780

agggacatgt atcagtcatt attaaagcat gttactgaat cctggaggac agcaccccaa 36840

ccctgcaggc tgctgtctca tctctggtgt cttagctgag tcattccatt gttctactca 36900

tgttggctgc ttctgatgtt gaggtatatg gtaagatgag gggatcccgt tgtcatgtgc 36960

ccattgcacc acctccttta tcaaaaaagt ggtccttgag gcaatgtttg agatatcatg 37020

acagtgagtc agttattctc tatgtcttag tgtgcttgta tggaaagaca cattgcaggc 37080

agaaaaggca aacccatacc ctgagatatc aattccaata aggatgaatc actcctgggc 37140

ccatctgaat tcatgactcc atggatctga taatgcccag ctcatacgtt aacagctctg 37200

tttgcctagt cctcagtatc tcataatcag gttctcagcc tccactgggg cccagtagca 37260

catcgggagt ctgagtagtt ctgtcgcagg aagcatggcg ttactacagg atcccacaga 37320

gctgtgctgt tatctcttat tggggctttc cagaggcccc acactacaca gcctgagtag 37380

cagtttgctc agaagccaaa attgctgcaa tctttctttt atgggatcca cttgaaactg 37440

gcagccgtgt taaatgggtg ggaggaatac accagataca atatatgtgc ttccaaagtc 37500

cagagggacc tactaaatcc tatgcctcta tcttagtgtt aagaggtggg aggtgcaaga 37560

aattgttctt tactatagaa gaaatatcta ggcatgcctc aactcattgg tcttctaaac 37620

tcttcatcaa tgtggcatgc cccagattct tcatagagtc tataatccac accctggcac 37680

atgtaagcct tactagggca ttggtaatac ttgccacttc ctgatagcca atccccctag 37740

catgctctca caatatagtg ggccagcatg atgtcctgta gaatgtcaag atgatcaagg 37800

tccttttgga ttatattgtg atatagagaa ggagatttaa gattgttttg gggcaataga 37860

gtaaatgaga atcgctctcc atcccagaga aatgtgaact gcttttgatc ctcctttctg 37920

atggaaattt agaagaatga atttgccaaa tcaatagctg catatcaaat gccagaggct 37980

gccagtaaag ataccacatc caatatggca gctacaactg agactagcag tagtccacca 38040

tcattctcca ttatccatct gctctttttc agaagtgtaa tggtaactta aatgagatta 38100

tgatggagcc cattacactt gcatcccaaa attttttgat ggtgacactg atgtctgcca 38160

ttcctcatgt ggattgctca tgatttatta acatggccag aaagaggttt cctctttctc 38220

ccataatggt tgttacttca caggtctaag acctccacag aggtctaagt atattgatcc 38280

caaatatgta cttagaggtt gggagaataa ttccaaggtg ggaaccatta tgtgatagac 38340

tttggccagg actcccttta tcatatggtt tccataaccc ctcatctacg tacaaagcca 38400

taatggtatc cggttctgaa aaattctggg tatgtacatc atatatactt gtgaaagcaa 38460

ggcctttcct caagaccagt cctccttttc attgtgcact ggatctgtaa attgacttag 38520

ctctggaagc tgatgaagag accgtgattt tcccattgtg acag 38564

4

290

Caenorhabditis elegans

4

Gln Val Ala Leu Gly Arg Arg Ile Gly Phe Tyr Arg Leu Gly Lys Glu

1 5 10 15

Leu Gly Ala Gly Asn Phe Ser Lys Val Lys Leu Gly Val His Gln Leu

20 25 30

Thr Lys Glu Lys Val Ala Val Lys Ile Met Asp Lys Ala Lys Met Asp

35 40 45

Ala Lys Ala Gln Lys Leu Leu Ser Arg Glu Ile Gln Ala Met Glu Glu

50 55 60

Met Asn His Pro Asn Ile Val Lys Leu Phe Glu Val Val Glu Thr Leu

65 70 75 80

Thr Arg Val His Leu Val Ile Glu Tyr Ala Ser Gly Gly Glu Leu Tyr

85 90 95

Thr Tyr Val His Glu Arg Gly Lys Leu Thr Glu Gly Asp Ala Lys Pro

100 105 110

Leu Phe Ala Gln Ile Val Ser Ala Val Ser His Met His Ser Arg Asn

115 120 125

Ile Val His Arg Asp Ile Lys Ala Glu Asn Val Met Phe Ser Ser Pro

130 135 140

Asn Thr Val Lys Leu Val Asp Phe Gly Phe Ser Cys Leu Val Asp Arg

145 150 155 160

Glu Gln Met Leu Arg Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala Pro

165 170 175

Glu Leu Phe Gln Asp Thr Ser Tyr Ala Gly Glu Leu Val Asp Val Trp

180 185 190

Ala Leu Gly Val Leu Leu Phe Phe Met Leu Ile Gly Val Thr Pro Phe

195 200 205

Lys Ala Glu Thr Val Pro Asp Met Lys Val Leu Ile Thr Ala Gly Lys

210 215 220

Tyr Gln Ile Pro Asp Tyr Val Ser Leu Leu Ala Thr Glu Leu Ile Lys

225 230 235 240

Ser Met Leu Lys Thr Asp Thr Gly Gln Arg Ala Asp Ile Asp Ser Val

245 250 255

Lys Lys His Phe Trp Met Arg Asp Cys Arg Phe Thr Lys Ser Tyr Leu

260 265 270

Ser Ile Lys Ala Thr Ala Lys Ile Asp Asn Glu Glu Glu Lys Lys Ala

275 280 285

Ile Asp

290

5

268

Drosophila melanogaster

5

Gln Val Thr Ile Gly Arg Arg Ile Gly Leu Tyr Arg Phe Cys Gly Asp

1 5 10 15

Ile Gly Arg Gly Asn Phe Ser Lys Val Lys Leu Ala Val His Gln Leu

20 25 30

Thr Arg Asp Lys Val Ala Ile Lys Val Val Asp Leu Asp Arg Ala Gly

35 40 45

Leu Asp Ala Lys Ala Leu Arg Met Leu Ser Ser Glu Ile Ala Thr Leu

50 55 60

Glu Cys Val His His Pro Asn Ile Leu Arg Leu Phe Glu Val Val Glu

65 70 75 80

Thr Leu Gly Arg Val Tyr Leu Val Thr Glu Trp Ile Arg Gly Gly Glu

85 90 95

Leu Tyr Asn His Ile Thr Gln Gly Gly Pro Leu Arg Glu Ile His Ala

100 105 110

Ala Pro Leu Leu Lys Gln Leu Leu Leu Ala Val Lys His Met His Ser

115 120 125

Leu Gly Tyr Val His Arg Asp Ile Lys Ala Glu Asn Val Leu Leu Leu

130 135 140

Ser Glu Asp Arg Leu Lys Leu Ala Asp Phe Gly Phe Ser Thr Gln Leu

145 150 155 160

Ile Asn Gly Thr Gly Ala Asn Gln Lys Leu Asp Thr Phe Cys Gly Ser

165 170 175

Pro Pro Tyr Ala Ala Pro Glu Leu Phe Ser Asp Asp His Tyr Ile Gly

180 185 190

Ala Pro Val Asp Val Trp Ala Leu Gly Ile Leu Leu Tyr Phe Met Val

195 200 205

Val Gly Asn Met Pro Phe Arg Ala Pro Thr Ile Pro Gly Leu Lys Ala

210 215 220

Ala Ile Leu Lys Gly Asp Tyr Leu Leu Pro Gly Gln Leu Ser Leu Pro

225 230 235 240

Cys Ile Arg Leu Ile Gln Arg Ile Leu Ile His Ile Pro Ala Gln Arg

245 250 255

Pro Thr Ile Asp Asp Met Leu Asn Ser Gln Phe Val

260 265

6

74

Human

6

Pro Thr Asn Thr Glu Asp Leu Ser Gln Leu Gly Ala Leu Glu Phe Glu

1 5 10 15

Ala Arg Gln Ile Leu Ala Glu Leu Gly Leu Thr Ser Glu Met Leu Ile

20 25 30

Asn Ala Arg Gln Ser Gly Pro Arg Ser Asp Ile Ile Gly Ala Tyr Arg

35 40 45

Ile Val Val Asn Arg Leu Gln Lys Gln Ser Trp Leu Ala Arg Lys His

50 55 60

Val Glu Met Ala Leu His Ser Glu Pro Lys

65 70