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

The present application is a continuation-in-part of U.S. Ser. No. (09/824,583), filed Apr. 3, 2001 now abandoned.

The present invention is in the field of kinase proteins that are related to the serine/threonine 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 controlling 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 I: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 though 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 14:3679-86). 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, hyperosmolor 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 N K (1992) Annu. Rev. Cell. Biol. 8:463-93). Regulation of PTK activity may therefore be an important strategy in controlling some types of cancer.

Kinase proteins, particularly members of the serine/threonine 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 serine/threonine 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 serine/threonine 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 carcinoma and placenta.

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 carcinoma and placenta.

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, SNPs were identified at 33 positions.

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 serine/threonine 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 serine/threonine 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 serine/threonine kinase subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in lung carcinoma and placenta. 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 serine/threonine 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 serine/threonine 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 carcinoma and placenta. 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. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 13 by ePCR, and confirmed with radiation hybrid mapping.

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. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 13 by ePCR, and confirmed with radiation hybrid mapping. 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. Thirty-three SNPs were identified. 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% of 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 then 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 iselenoylation, 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 encompasses 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 bit 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 that kinase proteins of the present invention are expressed in lung carcinoma, as indicated by virtual northern blot analysis, and placenta, which is the cDNA library source from which the cDNA clone came from. A large percentage of pharmaceutical agents are being developed that modulate the activity of kinase proteins, particularly members of the serine/threonine 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 carcinoma and placenta. 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 serine/threonine 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 that kinase proteins of the present invention are expressed in lung carcinoma, as indicated by virtual northern blot analysis, and placenta, which is the cDNA library source from which the cDNA clone came from.

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 carcinoma and placenta. In an alternate embodiment, cell-based assays involved 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 that kinase proteins of the present invention are expressed in lung carcinoma, as indicated by virtual northern blot analysis, and placenta, which is the cDNA library source from which the cDNA clone came from.

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, including 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 carcinoma and placenta. 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 carcinoma and placenta. 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 agents. 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, shown 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 carcinoma and placenta. 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 peptides 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 comprises, 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 that kinase proteins of the present invention are expressed in lung carcinoma, as indicated by virtual northern blot analysis, and placenta, which is the cDNA library source from which the cDNA clone came from. 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 carcinoma and placenta. 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 carcinoma and placenta. 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 carcinoma and placenta. 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 nucleic 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 in 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 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB 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 those 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 additionally 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 be 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. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 13 by ePCR, and confirmed with radiation hybrid mapping.

FIG. 3 provides information on SNPs that have been found in a gene encoding the kinase proteins of the present invention. Thirty-three SNPs were identified. 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 45 C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to low stringent 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, SNPs were identified at 33 positions.

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 the 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 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. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 13 by ePCR, and confirmed with radiation hybrid mapping.

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 that kinase proteins of the present invention are expressed as lung carcinoma, as indicated by virtual northern blot analysis, and placenta, which is the cDNA library source from which the cDNA clone came from. 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 in a kinase gene has been mutated. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in lung carcinoma, as indicated by virtual northern blot analysis, and placenta, which is the cDNA library source from which the cDNA clone came from.

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 carcinoma and placenta. 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 assays 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 process 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 that kinase proteins of the present invention are expressed in lung carcinoma, as indicated by virtual northern blot analysis, and placenta, which is the cDNA library source from which the cDNA clone came from. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

Alternately, 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 carcinoma and placenta.

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 the 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. Thirty-three SNPs were identified. 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 indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 13 by ePCR, and confirmed with radiation hybrid mapping. 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., Landergran 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.

Alteratively, mutations in a kinase gene can be directly identified, for example, by alteration 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 cleave 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 cleave 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. 217:286-295 (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 technique 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 regiment for treatment. FIG. 3 provides information on SNPs that have been found in a gene encoding the kinase proteins of the present invention. Thirty-three SNPs were identified. 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 provides vectors for gene therapy in patients containing cells that are abnormal 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 that kinase proteins of the present invention are expressed in lung carcinoma, as indicated by virtual northern blot analysis, and placenta, which is the cDNA library source from which the cDNA clone came from. 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 WO95/11995 (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 cDNA, fixed to 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 WO95/251116 (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. Thirty-three SNPs were identified. The changes in the amino acid sequence that these SNPs cause can readily been 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 Immunoctyochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), 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 covalenty 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 hose 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 or 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 virsues, 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 or 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 gluthalione 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 11d (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 protcolytically 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. cerevisiase 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 (Kaufmann 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, or 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 tetracylcline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase of neomyin 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 involved 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 transgenic 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. No. 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 allows 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., Lasko 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 0 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 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.

4

1

2370

Human

1

cattggagac catggataag tacgatgtga ttaaggccat cgggcaaggt gccttcggga 60

aagcatactt agctaaaggg aaatcagata gcaagcactg tgtcataaaa gagatcaatt 120

ttgaaaagat gcccatacaa gaaaaagaag cttcaaagaa agaagtgatt cttctggaaa 180

agatgaaaca tcccaacatt gtagccttct tcaattcatt tcaagagaat ggcaggctgt 240

ttattgtaat ggaatattgt gatggagggg atctcatgaa aaggatcaat agacaacggg 300

gtgtgttatt tagtgaagat cagatcctcg gttggtttgt acagatttct ctaggactaa 360

aacatattca tgacaggaag atattacaca gggacataaa agctcagaac atttttctta 420

gcaagaacgg aatggtggca aagcttgggg actttggtat agcaagagtc ctgaataatt 480

ccatggaact tgctcgaact tgtattggaa caccttacta cctgtcccca gagatctgtc 540

agaataaacc ctacaacaat aaaacggata tttggtctct tggctgtgtc ttatatgagc 600

tctgcacact taaacatcct tttgagggta acaacttaca gcagctggtt ctgaagattt 660

gtcaagcaca ttttgcccca atatctccgg ggttttctcg tgagctccat tccttgatat 720

ctcagctctt tcaagtatct cctcgagacc gaccatccat aaattccatt ttgaaaaggc 780

cctttttaga gaatcttatt cccaaatatt tgactcctga ggtcattcag gaagaattca 840

gtcacatgct tatatgcaga gcaggagcgc cagcttctcg acatgctggg aaggtggtcc 900

agaagtgtaa aatacaaaaa gtgagattcc ggggaaagtg cccaccaaga tcaaggatat 960

ctgtgccaat taaaaggaat gctatattgc atagaaatga atggagacca ccagctggag 1020

cccagaaggc cagatctata aaaatgatag aaagacccaa aattgctgct gtctgtggac 1080

attatgatta ttattatgct caacttgata tgctgaggag gagagcccac aaaccaagtt 1140

atcaccctat tcctcaagaa aatactggag ttgaggatta cggtcaggaa acgaggcatg 1200

gtccatcccc aagtcaatgg cctgctgagt accttcagag aaaatttgaa gctcaacaat 1260

ataagttgaa agtggagaag caattgggtc ttcgtccatc ttctgccgag ccaaattaca 1320

accagagaca agagctaaga agtaatggag aagagcctag attccaggag ctgccattta 1380

ggaaaaacga aatgaaggaa caggaatatt ggaagcagtt agaggaaata cgccaacagt 1440

acctcaatga catgaaagaa attagaaaga agatggggag agaaccagag gacattgaaa 1500

aagacttgaa acaaatgagg cttcagaaca caaaggaaag taaaaatcca gaacagaaat 1560

ataaagctaa gaagggggta aaatttgaaa ttaatttaga caaatgtatt tctgatgaaa 1620

acatcctcca agaggaagag gcaatggata taccaaatga aactttgacc tttgaggatg 1680

gcatgaagtt taaggaatat gaatgtgtaa aggagcatgg agattataca gacaaagcat 1740

ttgaaaaact tcactgccca gaagcagcat ttacagaact gacttggctc agtttcctct 1800

tcctggaata ctctctgcct catttccttc tggaaaaatc tccattcagc aggcatctta 1860

ttgaggatct cctttgtgcc aacgactgct cactgaagga ctggagtgag aaggaaatgg 1920

agcttaggac ataaccctac cactacataa acaaactttg gagaatcagg agagagtaaa 1980

gccaaaggag gagagacagg tcatggggag gcacaggaat tggcagcatc aactggaaga 2040

gaaaggccag atgagggttt tccacgcaga ctgtagttgc tgtgggaaac aggaggcagt 2100

gggatggagg agcgcctcag actctgctgc agatgatggc agtggccgac atcacctcca 2160

cctgccccac ggggcctgac agtgagtctg tgcttagtgt cagtcgtcag gaagggaaga 2220

ccaaggaccc gtacagccca gtgctcatcc tgatgtgata gtctacttct cactatacac 2280

cctatagatc ttgtatcaga cactttcaaa tatgttgttt tgatatctca agaaaaaaaa 2340

aaaaaaaaca ctgtcatgcc gttacgagcg 2370

2

640

Human

2

Met Asp Lys Tyr Asp Val Ile Lys Ala Ile Gly Gln Gly Ala Phe Gly

1 5 10 15

Lys Ala Tyr Leu Ala Lys Gly Lys Ser Asp Ser Lys His Cys Val Ile

20 25 30

Lys Glu Ile Asn Phe Glu Lys Met Pro Ile Gln Glu Lys Glu Ala Ser

35 40 45

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

50 55 60

Ala Phe Phe Asn Ser Phe Gln Glu Asn Gly Arg Leu Phe Ile Val Met

65 70 75 80

Glu Tyr Cys Asp Gly Gly Asp Leu Met Lys Arg Ile Asn Arg Gln Arg

85 90 95

Gly Val Leu Phe Ser Glu Asp Gln Ile Leu Gly Trp Phe Val Gln Ile

100 105 110

Ser Leu Gly Leu Lys His Ile His Asp Arg Lys Ile Leu His Arg Asp

115 120 125

Ile Lys Ala Gln Asn Ile Phe Leu Ser Lys Asn Gly Met Val Ala Lys

130 135 140

Leu Gly Asp Phe Gly Ile Ala Arg Val Leu Asn Asn Ser Met Glu Leu

145 150 155 160

Ala Arg Thr Cys Ile Gly Thr Pro Tyr Tyr Leu Ser Pro Glu Ile Cys

165 170 175

Gln Asn Lys Pro Tyr Asn Asn Lys Thr Asp Ile Trp Ser Leu Gly Cys

180 185 190

Val Leu Tyr Glu Leu Cys Thr Leu Lys His Pro Phe Glu Gly Asn Asn

195 200 205

Leu Gln Gln Leu Val Leu Lys Ile Cys Gln Ala His Phe Ala Pro Ile

210 215 220

Ser Pro Gly Phe Ser Arg Glu Leu His Ser Leu Ile Ser Gln Leu Phe

225 230 235 240

Gln Val Ser Pro Arg Asp Arg Pro Ser Ile Asn Ser Ile Leu Lys Arg

245 250 255

Pro Phe Leu Glu Asn Leu Ile Pro Lys Tyr Leu Thr Pro Glu Val Ile

260 265 270

Gln Glu Glu Phe Ser His Met Leu Ile Cys Arg Ala Gly Ala Pro Ala

275 280 285

Ser Arg His Ala Gly Lys Val Val Gln Lys Cys Lys Ile Gln Lys Val

290 295 300

Arg Phe Arg Gly Lys Cys Pro Pro Arg Ser Arg Ile Ser Val Pro Ile

305 310 315 320

Lys Arg Asn Ala Ile Leu His Arg Asn Glu Trp Arg Pro Pro Ala Gly

325 330 335

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

340 345 350

Ala Val Cys Gly His Tyr Asp Tyr Tyr Tyr Ala Gln Leu Asp Met Leu

355 360 365

Arg Arg Arg Ala His Lys Pro Ser Tyr His Pro Ile Pro Gln Glu Asn

370 375 380

Thr Gly Val Glu Asp Tyr Gly Gln Glu Thr Arg His Gly Pro Ser Pro

385 390 395 400

Ser Gln Trp Pro Ala Glu Tyr Leu Gln Arg Lys Phe Glu Ala Gln Gln

405 410 415

Tyr Lys Leu Lys Val Glu Lys Gln Leu Gly Leu Arg Pro Ser Ser Ala

420 425 430

Glu Pro Asn Tyr Asn Gln Arg Gln Glu Leu Arg Ser Asn Gly Glu Glu

435 440 445

Pro Arg Phe Gln Glu Leu Pro Phe Arg Lys Asn Glu Met Lys Glu Gln

450 455 460

Glu Tyr Trp Lys Gln Leu Glu Glu Ile Arg Gln Gln Tyr Leu Asn Asp

465 470 475 480

Met Lys Glu Ile Arg Lys Lys Met Gly Arg Glu Pro Glu Asp Ile Glu

485 490 495

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

500 505 510

Pro Glu Gln Lys Tyr Lys Ala Lys Lys Gly Val Lys Phe Glu Ile Asn

515 520 525

Leu Asp Lys Cys Ile Ser Asp Glu Asn Ile Leu Gln Glu Glu Glu Ala

530 535 540

Met Asp Ile Pro Asn Glu Thr Leu Thr Phe Glu Asp Gly Met Lys Phe

545 550 555 560

Lys Glu Tyr Glu Cys Val Lys Glu His Gly Asp Tyr Thr Asp Lys Ala

565 570 575

Phe Glu Lys Leu His Cys Pro Glu Ala Ala Phe Thr Glu Leu Thr Trp

580 585 590

Leu Ser Phe Leu Phe Leu Glu Tyr Ser Leu Pro His Phe Leu Leu Glu

595 600 605

Lys Ser Pro Phe Ser Arg His Leu Ile Glu Asp Leu Leu Cys Ala Asn

610 615 620

Asp Cys Ser Leu Lys Asp Trp Ser Glu Lys Glu Met Glu Leu Arg Thr

625 630 635 640

3

cttggcaggc cgccgctgtg gcccaaagag taggaagccg ttccagtctc acgtccacct 60

tttggcaata tttgagacct tgtacaagaa acactcttcc tgtatcagtt tagctcattt 120

gtaaaactgg gagactactg ccttgacggg ttgtaaagaa aagagagaac gtttgcgaag 180

cgtctggtgc accttaagca agagcgggga gcgctactgt agactgcaaa gcaaaggaat 240

cccgacccaa ggcaacggga cggttgcggg gtgactctgc cgggtctcca aactccctgg 300

cgcctgaccc tgcctcgagg tggactggtc cccaggccat tccagacccg cgccccgccc 360

gcgtttcctt gcgcggctcc gccccggccg cagggaggcg cagcggcccc gggaacccgg 420

atccttccgg gacgcttcgt tggccccgcg gagccggcgg agcaggtacg cttgcagggg 480

ccgcccttag ttcttgcccg gagccgccac agggcttcgg gagctcggca gggtggggga 540

aagggatgga gtttcggcct ggggcggcgg gggcggccca gaaaaggcct agcgtcctgg 600

gctgtgtggg tgtagcgtcc agggcgcgtc ggtctctatg gcaacgctcc acacgcggag 660

gtcgggtacg ggtaagcgtc ttgccactca cccgcggccg cttccagggg cggccctagg 720

ggagaaggaa ttttcctaat ttgggggctt ccaccctttg gtgccacttg ggcgggaggg 780

tcgcgggccc tcagttcccg gcgagtcacc cccggcccca agtccgtatg cgtctctctc 840

agaacccgat cctccggtgt ctgcagcctc tcctggctgc ggagctggtt cccagccccc 900

tgcaacccag taccgacttc ccaccctgac gtaaaattat tcgaaaacaa gccccctgct 960

caccccatta acaacaacaa caaaactgta ttatgcccta actgtagcat aaagaggaaa 1020

tagaaggaaa gcaataagta agaaagtaca tatttcaatc tgaaaatgct tggcactact 1080

acccttggaa aatgtagaga agtagccagt agccgcgcct ggggagtcgc ctgaacgtga 1140

cggcagcaaa tgcagattgt tgggtctccg ggaccaggag cagcgtggcc agtgaagcgc 1200

gtggttttcc caaatggtga acaattcttg gtaaacctcc aaaccgaagt gcaatcaagc 1260

cttgatttac atgtagttgc attcctcgaa aaaaaaaaga agtgttcatt aaaactgcaa 1320

aaatacttag catttcgatg taaaatagag tttggttcta gccacaaacg gatttttcca 1380

cgcacaggaa tgtataggaa gactctccaa gattgtaggg ccgcggggta atcctttatt 1440

gtgcgggact gtctctcgaa tcgcagaatc ctaccatctc aggccccaac cacctgtaaa 1500

cctcatgcct ctgaatcttg gggaaacagc ttccccaccc ccatatattt ccagaattcc 1560

ccctaggggg cagtacgtcc ccactaagaa aggctgaact ataaaagtgc acaagcctaa 1620

ggacattcct gctttataaa ggtgcgaaac accggatata gtatctttca ttctcagaac 1680

aaacttgcaa aacaggtatt gttattccat tttagaaatt aggaaagtga ggttttgcca 1740

ggttaagtga cttacccgag aatacagggc aaaagtgtat caaagctgag ctatgacccg 1800

tgtctgacca agaaactctg tctcatttca gttatctgtg gccacaaaga aagttatttg 1860

tctctgtctt ggcaaggctg ggaggaaagt tttagctaag tgagttcttt tacactttag 1920

tcatcagttt tctgactttg ttagtcttta tgagacgtgt gtgataaatt tacattactc 1980

taattccagg aaactcagcc cattggagac catggataag tacgatgtga ttaaggccat 2040

cgggcaaggt gccttcggga aagcatactt agctaaaggg aaatcagata gcaagcactg 2100

tgtcataaaa gagatcaatt ttgaaaaggt aaagttaagt tcaaatttct gttaattttc 2160

agtgggatat tcagctggct tttaatccaa tataaaaagg aaatttttat tttttataat 2220

ttcgaatttt aagccataat tgatttttgt taattcaacc tcctaagtcc attgtccaaa 2280

cagcaaccaa tgatctcatt tttaaaaaga ggctggacgc actggctcac ccctgtaatc 2340

ccagcacttt ggatggccaa ggtgggagga ttgtgtgaag ccaggagttc gagatagcct 2400

gggcaacata gcaagaccct gtccctgcta aaaaaaaatt tttttaatga aaatagaaaa 2460

gaaataagat cacatccctg tggctcctat ggccctcctt agggtgccct gcaaggccct 2520

gtgagatgcc agcctcctct gttgccctga cttttctctg tggtgcactt cctctctcct 2580

tattcaggtc ctctacgagg ggttttctgc aaacatccta gctagagtag acccccagcc 2640

acaatcacac cttatcacct tatcacacca ccttggttcc tggtttcttt tttgttttct 2700

tttcttttct ttttttagac ggagtctcgc tctgtcacct aggctggagt gcagtggcat 2760

gatcttggct cactgcaatc tccacctccg ggattcaagc aattctccca cttcagcctc 2820

ctgaatagct gggactacag gtgcatgcca ccatgcctgg ataatttttt gtatttttag 2880

tagagatgga gtttcaccat gttgcccagg ctggtcttga actcctgagc tcaagtgatc 2940

tgcccgcctt ggcctcccaa agtgctggga ttacaggctt gagccactgc ttctggcctg 3000

gtttattttc ttactagcat gtataatgct ctgcaattac tttgctctct taattattca 3060

tttgtttatt gcttgtcttc ctcagtatgc agaacagttc ctgtcacata ataggtgcta 3120

aacacattta ttgagtgcac tgaatgaata gagaaaaact atatgtaatt gttggtctaa 3180

tgattttgga aaataaatat agttaattaa aaattaataa tttttgctaa atccaccttg 3240

gtcagtgttt atgtcaccct ctttagtgat atgttcattt cataatatat tgggacaaca 3300

atgtccattg tttgctagaa ttaattctaa ggcaagtctt gttggtcagc ttctagagga 3360

tttataaatg agagtagcat aaaaagttcc atacaaagtg tgtgcaaaat ggactaccca 3420

agttacacca tatgaatata cttaatgcca ttgaactgta cacttaaaaa tcgttaaaat 3480

gatataaatt ttatcttacc acaaaaaatt gcaagaaaac ctacccaaac ttaaagctca 3540

agagtagatg actggcttcc agggataatg atttatttcc caatataggt ctctttttgt 3600

gaatccatgg catattcata ataatgtcct cttattctag tggcccgcaa tagcttcctc 3660

ccatgacatt attctgctca ctctcttttg tttatctgac tgctctccct caggcttatc 3720

tctgtcttcg ccctgtgtat gtcctcaacc atgtgtcctt ttctgatttt ctttttctgt 3780

ccattgtcac ctaaactgcc ccacttcagt gtttaccaat aagtagatct ctcttaaatc 3840

tctgtctcta cccctggcat ctttcagtac cctagttctg catttcttct gccagctaga 3900

taacttcagg taatatctgt ggttttgttt tgaggtggag tctcgctctg tcgcccaggc 3960

tggagtgcag tggtgccatc tcggctcact gcaagctctg cctcccaggt tcatgccatt 4020

ctcctgcctc agcctcccga gtagctggga ctacaggcgc ccgccaccac gcctggctaa 4080

ttttttgtat ttttagtaga gacggggttt cactgtgtta gctaggatgg tctcaatctc 4140

ctgacttcgt gatccacccg cctcggcctc ccaaagtgct gggattacag gcatgagcca 4200

ccacacccat ccaacatcta tgttattaat ctattgctgt gtagcatatt accccaaact 4260

tagtggctta aagaataaac atttattgtc tcagagatcc tgtgcatcaa gaatttagga 4320

tgacgatcat tgagaccacc ttggaggctc ggtatcacaa ttgtacccaa aaacaagtat 4380

taatagtgat tcttccttgt tgtaagcaga cccacttcac ctcctatgtg ctgcgctgta 4440

ttaatgtcat cagtgtcctt atggttgcca gcctgaaaac cttgggatcg tttgtgagct 4500

tattccttct ccacattcaa ttatttggcg aatactgttg actcttcctc ttccttgaat 4560

ttgcttcagt ccttttgtcg aggccctggg tcacttggat ccttcaagtg gctccagccc 4620

aattttgata atgctccagc catgccccca aaccttcact gggacagagg ctgtaaagaa 4680

agagttgcct aggtttgact acataaaaat agaaaacgtt tgtatgtcaa aacaaacact 4740

ataaataaat tcaaagaaat cgagaaggtg ccaaaaatat ttgcaagtat tgacttaatg 4800

gtgttagcct tttattaaat caataaaaag ataaaatcca tatatgaagt catcgtacaa 4860

aaatttgaaa ctcagtagaa aactaagaaa ttaggagttt attcaaagaa aaaccccaca 4920

gataaacagt tagaaaacaa atgtccaaca gtaggtaatt tgttaagtaa tttataaaaa 4980

actaagtggc tattagcaat catgttgtag gtgaagcatt gacatgggaa aatttcaatg 5040

tttgcaatgt ttgagaaaat agtaagtgta aaataatata atctttggaa aaatatatat 5100

attctccata tatatgtata cctacaaata tgttcatata tgtacaaaga aagacacaaa 5160

ttgttattat tgaggtagaa agtggggttt gccttgtgca ttttttttga gacaggattt 5220

cattctgttg cccaggcagg agtgcagtga catcatcatg gctcactgta accttgaaca 5280

agccatcctc ctgcctcagc ctcctgagta gctaggactg caggcatgcg ccaccacacc 5340

cagctaattt tgaaattgtt tttagagaca ggatcttgct atgttgccca ggctggcctc 5400

aagtgatcct cccactttgg cctcccaaag tgctgggatt acaggtgtta gccactgtgc 5460

ctggtctgcc ttgtgctttt atattgtttc actcttcaga gaagttttga gaccctctct 5520

gatttgctcc aaaactacag ctcctatcac ataccctact ttttttcccc actccagcct 5580

ctgcatttgc ttctggggct acttcttcca aggtcgttgc ctgctgatct cccagcatca 5640

agatcccact tgttcaaggc tgagctctac catacctcca gaatcctccc actctaaaga 5700

atttatcctt ctctgtaaac ttgcataact tttattggaa cctctgttat agtactgact 5760

gctttctttc tggacatgct ttggctgttt attttgtgcc ttctcctcct tatttagctg 5820

taatatgttc tgtgaggacc gagtccatgt gtgttttgtg ctggtattcc acacagcacc 5880

taatgcttgg tgccaggaga tattcaataa cttcttattg gatagatgat tcactggaca 5940

gatgctttca ggccctcttg ctctactgtg aagctggtat atacttagga attataaaac 6000

cattttaatt ctatgtaaag agaaaatatt tgagaggtga atctctataa aaatgtacat 6060

taacattact gcatttcata gcatctctcc cattctttag tataatcaaa aattgactat 6120

atttttctaa tagagcacca atttttcatc actttactca tgaactactc ttgtcactat 6180

gccataaata agtagaatct tatattagac ctcattattc ttgttttccc atatctgttt 6240

atgttatcga atttacctat aacatctgtg tcacaatatt aacatttatt acttctttct 6300

tcctatctac tctcatgtag tttttcatta cttcttatct agagaaattt atatttcttc 6360

tctctaatgc ctccctactc cctacactag accccagaac taaattgctt gttttcttac 6420

aggtaccaaa aagctaatat ttctcttatc atcctaccat tatcaagcat gttcttttcc 6480

ttctgggctc aaataaaagt gttttatctt tcctcaattg tgaaaataaa aatgttcgtt 6540

gtagaaattt tgaaaagagc caaaggagaa aataagacca tttagaggaa aataaaaata 6600

gcatataacc tctttcttaa tcactatgaa cactttgctg aatttctctc tagactattt 6660

ttaatgtata agtatataag ttattagaat gattggtgtc atggtagata tactcttttt 6720

tttttttgag acggagtctc gctctgttgc ccaggctgga gtgcagtggc gtgatctctg 6780

ctcactgcaa tctctgcctc ccaggttcaa acaattctcc tgcctcagcc tcctgagtag 6840

ctgggactac aggcgcatgc caccatgcct ggctaatttt tgtattttta gtagaaacag 6900

gctttcacta tgttggccag gctggtctcg aactactgac ttcgtgatct gcctgccttg 6960

gcctcccaaa gtgctgggat tacaggtgtg agccactgcg cccagcccag gtatactctt 7020

ttgtaacagt tttttatatt agcaatatat tgtgaatatt tcctcatctc attcaatatt 7080

tttatataat aaaatgttgt catttaatga tattaaatgt gttcacacta atgataaagg 7140

gaccacctgc agggtgtcca ttatatgtca caccatcctg ggtgttttat tatgtatatc 7200

aactcaattt aatcttcaca accacttaaa aggtagctct cattactctc actgtacaag 7260

tgaaagagct gaggctaaag aggttaagca gttagctcca ggatgcacag taatcagcag 7320

atccatctaa gtctttctct gctctttcca tgatactaca ttgcctccct ttatttttaa 7380

tgactgcata gcattaaagt ggtagcaggt caaaaatacc ataatttagc tgggcatggt 7440

ggcaagtgcc tgtagtccca gctattctgg aggatgagtt gggaggatcc cttgacccca 7500

ggagttaaaa tccagcttag acaacatagc agaactctgt cttaaaaaaa aaaaaaaagc 7560

tagcaaaaca cccctgtaat ttatttaact ctttttctat tttcagataa ttacattgtt 7620

tggttggttt tttggctacg attcaataac atttaatatg taaagtatga ttcattttta 7680

ttaaacaaaa ctatgtatat atgcttgcct atatatgcat gaaataaaaa gctctaacta 7740

ttaacaacag ttatccctag ggaatatagt attaggttgg cgcaaaagta attgcatttt 7800

tgccattaag agtaaggtta ccacctatgg gctttcgtct gtgggctaga tgagaaagaa 7860

agagggaagt ttcactttta ccttattcac ttctatttga cttaaaacaa gcgtgcatta 7920

ttagagtaac ttaaaaacta gcaataaaac actgtaacaa agtcttttgt atgagaactc 7980

ttctgtaccc ttttattatc ttctttggat aaatttctag aagaattagt caaaaatagg 8040

aacatttccc tcatgcctgt aatcccagca ctttgggagg ctgaggcagc tagatcactt 8100

gaggccagga gttcgagagc agcctgggca acatggtgag accccatctc tactaaaaat 8160

acaaaaaatt agccgggtat gatggtgctt gcctgtggtc tcagctactc aggaggctga 8220

ggtgggagga tcacttgagc tcagtgggca gaggctgcag tgaaccaaga tcatgccact 8280

gcactccagc ctgggtgata gagcaagacc ttgtcttaaa aaaaaaaaaa aaaagatttc 8340

ttcagcagga tacagacccc ccacaaaaat gaacatttta aagattcata ttatatattg 8400

taaaactgcc ttcccagaaa tattttatca atttgtgtag ttttaccaga aataaatgag 8460

tgtccatttt gctgctttct ggccaatagt agttattgac attcttttca tctttgccag 8520

tttcatacat ggaatactat attacatttt gttttagctt ttattccttt tttttttttt 8580

ttgcaatgga gtcttactct gtcacccagg ctggagtgca gtggtgtgat tttggcttac 8640

tgcagcctcc atctcccagg ttcaagggat tctcctgcct cagcctcctg agtagctgag 8700

accacaggtg tgtgccacca cgcctggcta attttttgtg tttttagtag agacagggtt 8760

ttgctatgtt ggccaggctg gtcttgaact cctggcctca agtgatctgc ctgccttggc 8820

ttcccaaaga gctgggatta caggcatgag ctaccacacc cagccaaatt ttgctttagt 8880

ttttattcct ttgattactg catgagattg aatatttttt ctatcagcca tttttatttc 8940

tctttttttt tcgagttgac tattcttgta ctttgctatt tttctgttgg ggtgtttgcc 9000

tttttaaaaa ttatttgcca tcaattttta tattataaat atatttgtca tatatggtac 9060

aaatattgta tcttatcctt ttgtttgtct tttaattttg tttataatat tcttttaaat 9120

aaatagtagt taggaatttt ttaagttgct aaatgtatcc agctggtagg agtaatttag 9180

ctgtttttgt tttgaaactc ctatgtactg actatacaat ttaaattggg gcaggaaaca 9240

ctgaagctta gaggggttta aggaacttac tgaaggatcc ttcagctgag atgtagggaa 9300

gctagaattg agaatattaa tttttaagaa gttcttaagt ctaaatgaga atgagaaatc 9360

tggccaatgt tgaagacctc taatgggtgg aggccccgtg gacatcagaa aagcggggca 9420

gtcaggggct ggaagtcagg gtagaaatga caagtcagca aagcatcaag agtgaggaag 9480

aaaaagtaga aatgaggtgt ggccactggt actggcacca aaccccttgg caagtattgt 9540

ctataggtga aagtagaaca agaaaataca cccaaatact tctaaaatga agtcatgcaa 9600

gacaattttt atttgaaaat gaaaaatgta gtcatcttaa tacaaaattt tactgacctg 9660

atttctgtgg gatatgacac attttctttt tttagatttc atttgtttct tctcagcagt 9720

gattgctcct ggaatgttgc atttttataa agaattcctt cgctactgaa agatagatat 9780

taaaatatgg ctccatatgg ctagataatg aacacggtac caccagtcca acttttaata 9840

tagcaaaact tcaccagaaa tatttatttt cttgatgatg gttgtcaaca aaccattgat 9900

gagatgtagg gcactctgct aattctagaa atgttgtttc ctgccattga aagatcgttt 9960

tcaaagtgac attaaaagcc agtgaaatcc tagagaattt tagatggaaa tgagcagaaa 10020

gcatgttctt gaaaccaagt tagctttata gactactctg tctcttaatg taatttagat 10080

gcccatacaa gaaaaagaag cttcaaagaa agaagtgatt cttctggaaa agatgaaaca 10140

tcccaacatt gtagccttct tcaattcatt tcaaggtttg attttctaat attcgttaag 10200

tatttttata aagtataggc atgttgacat atgtaaaaag atttgttcct aaggactgtg 10260

tataaattaa tttttgtaaa tgggtcattt ccccatttac ttaaattgca gcttgagacg 10320

tcctcgttat ttcctctcta gtaagttttt gtagacggct ttcttatgtt ttcttgtttt 10380

tctgcctctc cttaattctc actctcccaa aaaattaatg actggcttat tagcttcttt 10440

gctgtagtaa caacccccaa atttaagtga cttacaataa gacacatcta tttctcactt 10500

acattacatg ctagctgtgg tgggctgggg tcttgagtct ggggcccagg ctgaaggagc 10560

agctcagata aggaaccagc tgttctcata agcaagagaa gaggggaaaa cacagagccc 10620

accacactat cgctctcaaa gccctgctag gatgtgtgtg tttgtgtgtg tgtgctgggg 10680

ggtactctgt ttacatgaga tcctgcatat cctcaggcaa cagatgggac tgtgtaatcc 10740

tcttacagag agccagcaaa cagccacgca ccatagccta gcacactgcc acggagaggg 10800

ggagaacttt agggaaggaa gtaccttcct ctgtacacct gaatacaatt ctgctgacaa 10860

ctttagggaa ggaagtccct tcctccatac atctgaatac aattctgcct ccacgcatca 10920

ctgtagtcca aaggtaaaaa ataaataata aatgaaggag cattggtcag acagcattca 10980

ttcactgaac tgatacttat tgagtgctta ctctatgcca ggcattgttc taggtgtcag 11040

gaatatagca gtgaacaaag cagatgaaaa tccctgtttt catgaaattt atattctagt 11100

gggaagagat agacaataaa caaatctaca gtatgtcagg tgtgtcttaa gttgtgacag 11160

ggctgtatgt gctgacagtt ttatgaaggg tcattcccca gcccagcccc cagcgcaggg 11220

ctgttttaag actgataatt agttcatgga gcagaagtgt taacctcaat atcttcaagc 11280

atcatcagtt gggtaaaagt cagtcaataa ataaatacag ccactgtgtc ttgagtatgt 11340

aaactgtgca gagcactgtg ttccttactg attaaaaccg ctacattcaa ggtacttctg 11400

tgtgtatggc ccttctttgg cttctgggta tttaaaaaga gctcttggga ctcttctgag 11460

gtcttcctgg gagcagaaca gtacacatgg tctggaattg ggttgcatgg aataactttc 11520

aaggaaagcc actgaataaa gtgccctgca ttcctgtcca ttggatactg ataatgctat 11580

aagatgatct ttctcttctt tattttgttt gagattattg tgactctctg gctaactcct 11640

acttatcctc aggccttttc tgaactcaca attcaaatta cagctccctt tggttctctt 11700

ccacagcagt tgtacttaca tatgtctatt tatataatta tgaatttgtt tcatatttgt 11760

cgccctttac atggtaaact taatgaattt tggggctcca tctgttttgc tcaccacttg 11820

atccttggca tgtagcacac aatggctgct caatacctat ttactgaatg agcaaatgga 11880

ctggaccact tttagagact ggagtatttc cttataccat gtgagattga tttttgagga 11940

cagtttacca ctggaagctt ttgcagaact aaggtcattt ttacagtata cataacctct 12000

gctgtgtttg ttgatactgt aagtttacat tttcttatga ctctttttaa gtagagcacc 12060

cctgtgttta ggaaagctag agctattgtg atgcctttga gtttgcttgg ctgattgctg 12120

ggacttgaac tactgagctt atctaaaagc ctcagaggcc ttgtagcctc tgtcttttag 12180

agagtgtagg taaaggcttg ttttccctca aatcgcttat ctctgatcat aagaaccatg 12240

gctctaatgt ttgtctatag aaaatagaat gttttggccg ggcgcagtgg ctcatgcctg 12300

taatcccagc accctgggag gccgaggcgg gcagatcacc tgaggtcagg agttcaagac 12360

cagcctggcc atggtgaaac cccgtctcta ctaaaaatac aaaaacgttt agccgggcat 12420

ggtggtgtgc acctgtaatc ccagctactt gggaggctga ggcaggagaa tcgcttgaac 12480

ctgggaggca gaggttgcag tgagctgaga ttgcgccact gcactccagc ctgggcaaga 12540

agagtgaaac tctgtctcaa aaaaaaaaaa agaaaatagg atgtttttat tggtttgaag 12600

caacataaga aaaataatga gaatgtagtg atattttcct aagacaaaat taattccatg 12660

tatattccat caataaacat tcactaagtg tctgttatat gccaggcatg ttctaggtct 12720

tggagatata tcagcaaaca aaataggcaa aaattcccat gctgttgtat ttgttttcta 12780

ttactacata acaaatgaac acaaatttag tggcttaaca acaacaccta tttattatct 12840

cttgatttct gtaggtcaga agcctgaggt tggcttagct ggattctctg cccggagtct 12900

cagctagttg aaatcaaggt gtcagctggg actgttatct gtggctcatg gtcctcttct 12960

aagcttattt aggttgttat agatttcatt tacttgcaat tgggttaatt ggatcatggc 13020

tcactgcagc cttgaactcc tggcctcaag tgatcctctc gccatggcct ccaaaagtgc 13080

tgtgagtact gtgcctggcc agaaagagct cttttacatt tatttaaaca cagagtttta 13140

ttttatatta ctctaatgca cacataaaaa agaaaatata agcaaacaaa gttggttaag 13200

gtattctaaa aattatttag gcagtgaaaa cattaagcct gccgggtcta cagcaagtga 13260

ttggaagatg ccaatgtctg taagaaacaa tcttgatttt tttttttttt ttttttgaga 13320

cagtcttact ctgttgccca ggctggagtg cagtggtgtg attacagctc actgcagcct 13380

tgaccttctg ggcttaaggg atcctcccac ctcagtctcc tgagtactgg gactacaggc 13440

atgtaccccc acacctggct aatttttgaa tattttttca ttatagagcc aggttttcgc 13500

catgttgccc aggctggtct caaacaccta agttcaagca atccacctgc cttagccttg 13560

gcctcccaaa gtgctgggat tacaggagag agctgctgcg ccaggccctt gattttttaa 13620

aagtgcattt tagaatgaat tataataatt gtttaataaa tgttggaatt tgacaaataa 13680

aaaggttatt tagtgcccct caattgtttt gaagtgtcag tgatccatga gctttacagc 13740

agatggaaaa tttgagagca taaatgattt ttccagacac ttccaataaa tataaaatta 13800

acagtggcta atgggggaaa atccttattt tacagtcaga taatgctaat tgacattaag 13860

tagtttcttt tttttttttt tttttttttt tttgacggag tctcactctg tcacccaggc 13920

tggagtgcag tggcacaatc ttggctcact gcacctccac ctcccgggtt caagcaattc 13980

tcctgcctca gcctcctgag tagctgggat tacaggcgcc cgccaccatg cctgnnnnnn 14040

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnntaataa tcagggattt 14100

agaaatagga tgaaatgaca agctaaagtc tttgagtctt agttcctcag ctgtaaagtg 14160

ggcatggtac catttcacag gaattgatac gaggattaga agagattagg gctgtgaagt 14220

gcctgacaca cagatagttc ttgaatcaaa tgagggtaaa ttgtagcact catctgtctc 14280

ataagcctaa actgcacatg tatctatata ttcatgacaa aaatcattcc aacacccagg 14340

ggctgtccaa aatacatctc tatctctctg tatacctatt ccctctccac cccccaaatc 14400

ctatacaaat tcctcttcct cggccgggca cggtggctca cgcttgtaat cccagaactt 14460

tgggaggcca aggcgggtgg atcacgaggt caggagatcg agaccacagt gaaaccctgt 14520

ctgtactaaa aatacaaaaa attagccggg catggtggcg ggcgcctgta gtcccagcta 14580

ctcggagagg ctgaggcagg agaatggcgt gaacccagga ggcggagctt gcagtgagcc 14640

gagattgcgc cactgcactc cagcccaggt gacagagcga gattccgtct caaaaaaaaa 14700

aaaaaaggta tctctgtcat gatgaatttt tagtcagttt ctttcaccaa aagccagtgt 14760

aaggttgagt gagttggagg ttggtgaaaa ggaagtgaga tgaaataatg tgtctcctct 14820

gtttcattat tctgattctt ccatggtttt gaaattgcca tcccttcatt gctgtggcag 14880

accttttact gactgagctt caatgacaag aaacatcaat tctcctaaaa gtaacacatt 14940

cctgaaaata accgatctct agacattaag gcatatgggg aagcatcttc tcagaaccct 15000

tcctagatct ccagtcttac ttgcatgcgc tttctcctca ccctccagcc tacagacttc 15060

ttcacagcac ttctcaccca atgctgaggg actccccaac tagacttcgc ctccttaaca 15120

gtagaatgga tggcttcttt gctttttatt cctacacagc attccttgct tttgcatgtc 15180

ctcaatagaa gtttgttaat aactgaatgg atcgtctctt aaagaagaga ggaggaaaaa 15240

ttgaaatatg tgaaagaaga tgcatggttt gtgaattaga agcaaccaag ggtagacact 15300

gccaggttac tgatatccac agtaaagttg gttagggtac tttaaagagt aggatagcaa 15360

aagatagata tttggcaaga gattttggca tttaatgggt actacagggg aaaatgttat 15420

caacaattgc ttataagact gattttggcg cttatgtttt gtgttccttc agggtttttt 15480

tgtttgtttg tttttaatga atccactcaa caaacattta agcccctttg atgtgctaac 15540

tactgtttag gtacaaaaga atgaagtgta gacaaacaag tgagtagaaa tccttctttt 15600

ctaacaagat cccagctgtt agttggttgg ctaatgaaga aagctggtta gagcagaaaa 15660

tcatctgttt tagtctattc cagcagctat aacaaaatac cataaactag gtggcttata 15720

aacagcagaa atttatttct ggcagttctg gaggctggga agtgcaagat caaggtgcag 15780

gcagattcag tgtctggtga gggttcactt tctggttcat agatggtccg cctcattgtg 15840

acttcacatg gtggaagggg agagggttct ctcttggaca gcaatccatt aatgtgggct 15900

ccaccctcat gacctagtca ccttccaaaa gccctatacc tcctaatacc atcactgtgg 15960

gggttaaaat ttcaacatat acatttgggg aagacaaaga catttggatc ctagcaatta 16020

tacagacata ttttaacata agaagacata atcatccttt gagtggaaat ggccaggaaa 16080

aaaaaaaaga aaaaaaattt aaggaaatga caagcatttg ttaaaggata atttcttttc 16140

tttaatacgg agcaagtgtt tgtggataat ctgtccacaa tccttttaga agttttctag 16200

ttatatttca ttcatttcat tcaacattta ggtcaatggt tatttattta tttattttta 16260

aactcactga gtcctccaaa atattcagca tagcttttgg aggaataatc acatctttca 16320

ttttctattc atatttcatc agtttatgta ataaagacaa gaataactca ctacagttca 16380

agaaaattca gaattatagt tggtagatta tgagtccact gactatagtt ctgaattttc 16440

tttcttatgt aagttatgtg tcttatttag aatttctagt ctcttttctt taatgtggag 16500

caaggatttg tggatagtct gtacataatc cttttagaag ttttccagtt atatttcatt 16560

catcccatca acatttaggt caatagctat tttttttaaa aactactcac ttttactgag 16620

tcctccaaaa tattcagcat agcttttgga aaataatcac ctttcatttt ctattcatat 16680

ttcatcagtt tatgttacaa agacaagaat aaatggcgta aacatatttg ggaaaaaaca 16740

aaatgatctt ggtaagattc agttcaattg gtaagagcag aggtacttgg acatactaga 16800

gagcctagta gtatttagtg gtaacgttga tggggcaata gcaatggaga gtgtcctgta 16860

atctagtgag tggtttaggc agaggtcagt tataagagtt tctattgtat tcaacaacat 16920

agataaataa gtgccatata aatatggctt tatgcccaaa ttcaagaagg ggctattaat 16980

tcttcctggg gtgttatgaa agagtcacag ctgctatttt tacagcagat ttctaacctc 17040

tcaaaggaat gtattaataa aaatagccaa catttcttaa actctcatta tgtacaggca 17100

ttattctaag catctgacat ggattaactc atttaatctt tccaacaatc caaagaagaa 17160

atttctataa tatttccatt ttacagatga ggtaatgagg cacggagaag ttaaagtgac 17220

ttgcccagag tcacagagct aagaaatata aatggtatag tggaattaga acccagtcca 17280

tcttgattcc acagccagaa catggctgat aaatatctga aatccttcca gcaccaaatg 17340

attccttttc ttacagagaa tggcaggctg tttattgtaa tggaatattg tgatggaggg 17400

gatctcatga aaaggatcaa tagacaacgg ggtgtgttat ttagtgaaga tcaggtaaaa 17460

acttctaatt tgctttttaa ttttatgtat gtgtggtggg gggagaatta aatttataat 17520

gtcataagta gtagactatg ttgatataaa gcatgtgttt tggtagacag attgaaacca 17580

taaaatagtg gatcaatatt cttggtagat tcagtccaga acaagtttgc aattgaactt 17640

aaactgaatt gttttttaga gagtgagatt ttcttgagaa aagataatct gtttggaaaa 17700

tctcatgtag gatgcctctg agataagtct tcatggttaa aaaaaaatct gaatgtgagt 17760

gttccttagc catttaacat gtaacatatt tatagcttca ctgttttctc ttttaccatt 17820

tggtgctctg ttttaaacta gatatcccac tttgctaggg aggatagaag cttgtcttgg 17880

caatgcctat ttagtttcat tggttattaa gagggggaga taaaagatga agaataatgg 17940

cctctcccac tttctctttg cacaaatgta tttctcttct atactccaag cctccctgga 18000

actctcttgg gagtggtact catgaaggag acaggtttgt atgtgtggag aggaatctga 18060

attagctcct ttatgattga tggtaaggcc attgcctcaa gaagcacaca ggaaaggccc 18120

accatctttc ctttggccat tgtttctttg ttctatttta gcatgtaaga gcatcatgcc 18180

catataattt cctcttaaag ttgaatattt tgaatatatg aaggattaaa aaatcaatat 18240

ctctaacttc tgtaagatta atcaagcatt ctttgtatgc tcatttatat tatatattaa 18300

attccatatt gatagaaact cttttttctt atctaggtat tattttgcca cattttataa 18360

aaatgcttct catgacaaaa ttttgagtta catttctttt tgttgggaat gaactaaaat 18420

tacaactgaa tattaatgtc tggaatatag ctttattcct attattttcc tctttctgta 18480

taatttggca gacaaagaac cagtgaaatt ttagaatagt ttaaataaat ctctgtaggc 18540

atagaacaca ttttcataaa gaggctcatg gtcaacaaag ataaaatcaa atcatgactt 18600

agaaataaaa ctaaacttca aaggtaaagt atttgttggt tttatattag atatactgat 18660

attttattac aattcctaac ctcacagatc ccccatttct tcctctttct ctccccaccc 18720

ttgtcaccct ccttccactg taaaggaaga accaatggct cccaggttat caggaaacag 18780

ggctgcttgt gtactattca cgatgcagtt agcaccccag ggttaagtag gaaaaaaaga 18840

aaaacatgaa cggcatgcct ctttcccttg cttctactta tctttttctg catgtggaat 18900

ttcccttgat tttaccagtg atatttggat tacttttctg tgcctccatt tttttagttg 18960

tagaatgaaa ataataatat gataaagtgt acctattaac ttcattccta taaatacaca 19020

tacactatat gtgtgtatgt gtgtgtgtgt gtgtgtataa tttctatctt tttgcatgtt 19080

accatgaaga catttcagtg actaccaggc tattcagtgg ctttgttttg tgttctctct 19140

atagatcctc ggttggtttg tacagatttc tctaggacta aaacatattc atgacaggaa 19200

gatattacac agggacataa aagctcaggt aacagctcag agagaagact aagacagaac 19260

tgatcttttc ttgaagtacc tcaaacaaca tgacattttc tccatttata gaacattttt 19320

cttagcaaga acggaatggt ggcaaagctt ggggactttg gtatagcaag agtcctgaat 19380

aagtaagtac tttgaaaata atttttcttt ctagtcaaaa tagcccaaat atgtattttt 19440

agatatcatg gattaagaag atattaaaat cttggttgtc taaataattt taggtagctt 19500

tatgtaaatg cattacatca gatggtactt tgagattaaa attctcaaga taaattgtgg 19560

tgtaatagaa tgatgttgct aatattctgt agtgtgattc cagtttgtca aatatggatg 19620

tgactgtaat atgcataaag ctagagagaa tttcgtgaaa taggcaggtt tacacttctt 19680

aatgaaaaaa gtcaaactct ataaaatatt tgaagagatt tattctgagc caaatacgag 19740

tgaccaaagg tccatgcctg tgacatagcc ctcaggagat cctaagaaca tgtacccaag 19800

gtggccggtc tacaacctgg ttttgtacat tttagggaga tgcaagacat caattagatg 19860

tacatgggtt tggtccagaa aagcaggaca actcaaagct gggaagaatg ggagggagct 19920

tccaggtcat aggtggatta aaaacttttc tgattggcaa ttgattgaaa gagtctatct 19980

gaagacctgg aattagtgga agggagtgtc tgggttaaga taaggggttg tggaaatgaa 20040

ggtttttatt atgcagatga aatctccaag tagcaggcct cagagagaat agattgtaaa 20100

tatttcctct tatcggattt aaaaaggtgc cagactctta gttaactttt tcctggatca 20160

ggaaaaagcc ttggaaaaag aagggaattt tcttcagaat gtagattttc cccacaagag 20220

atacctttgc aggactattt caagatatgg acaaagaaac atgatttggg gtaaaatatt 20280

ttgattcctt tcaggcctgc tatctgtcat gtgatgttat actagagtca ggctggactt 20340

tggtatctta ttgctacaag gagtctgctt tgtcagtctt aaggtctgtt ttaatgttaa 20400

tgctggtcaa ctgtgcctga attccaaagg ggaggaggag ttaatgaggc atatcagacc 20460

ctgcttccca tcatggcctg aactagtttt tcaggttaac tttggaatgt ccttggccaa 20520

agggagggtt tatgagttgg ttggggggct tagaatttta tttttggttt acacactttc 20580

tagcaaaata aatttgtgca cctgtttgga agacaatttg gtggcaatat gtaccaagag 20640

atttttaaat atcctgtttc tgggacttct tccaagggaa taatttgaaa tttggaataa 20700

cgtaaatgcc taaataattg ggaaatggtt aaatttaata aagcttggca tggccatggc 20760

catgtacctg aatatatcat aaacatttat ggttttgaag acttcttgat aactttgtta 20820

tactaagcaa agaaaatgga attctgaatt ttaaatacat tgtgatcacg gttatatgaa 20880

aaatatgtgt ggaaagaaga caggaaggaa atatatcaga attttaacaa tagttgtttt 20940

aggtgctaag attctgggta acttttttct cccttattca tttttgtatt ttccaagttt 21000

taaatcatga ggttgcaatt tgataatctc tacatctgag agatttttat aacatgacaa 21060

tttcatctct ttgtggagtc tttaagccat aaaaaatata ttttaatgtg taaatttttg 21120

ggaggtgaat tgtaagttta aaaatcagct gatttagtta ctttatcaac atacagtgtt 21180

ttgctttctt ctaacacatg tatgcatcaa atcttgtgtt atccattttc acattttttc 21240

ttgcatgtcc atgtcttaag acttttctta ctccaataaa aaatcatgct gatttattat 21300

ttaatataat ttactagttc catggaactt gctcgaactt gtattggaac accttactac 21360

ctgtccccag agatctgtca gaataaaccc tacaacaata aaacgtaagt tgctgactct 21420

tagtttgaaa gtgtcagtaa aatctgatgg atgacactga atgaagattc cagaaactaa 21480

aattcaaatc tcttctttct ttcttatggt acttttgtaa tttcatttgc ttcatgtgtg 21540

aaattgttct ggaccaaact gagggttggg ttgctatttc tcgcggtcca atacgagatg 21600

cagatgaact ggggaggaag agagttttta tttctgtaac cagtacaggg agaaggcctg 21660

gaaattatca ccagaccgac tcaaaattac aaagtttttc agagcttata caccttctaa 21720

gctatatgtc tatgtgtaag tgtgcattca tttaaagaca tactgattaa ctccttttaa 21780

tctataacta aggtctgagt cctgaagact ttcttctgga gcctcagtaa gcttacttaa 21840

tctaaatggg tctaggtcct ggggtgatta cccttatttt gtctcctgct aaatcatgga 21900

ggtttaggga gttcctgcag acctccaata aacttgtttg tggaggcctg gggagtttct 21960

tcagaccacc aataaaactt gtttaatctt aaaaggctcc ttgttaagaa ttccttcatt 22020

attttgtcat ggtttaaggc ccaggaaagg cctaggcaaa actcttggtg ggcttttgtt 22080

acattacagc ctttgtataa gggcactggc tttttttttt ttattttttg agatagagtc 22140

ttgctcttgt cacccaggct agagtgcaat ggcacgatct cggctcactg caacctccac 22200

ctcccaggtt caagcgattc tcctgcctca gcctcctgag tatctgggat tacaggtggc 22260

tgccaccatg cccagctaaa gttttgtgtt tttagtagag atggagtttc accatgttgt 22320

ccaggctggt ctcaaactcc tgacctcacg atctgcctgc ctcagcctcc caaagtgttg 22380

agattacagg tgtgagccac tgtgcctggc tgggcactgg ctttttaagc ttttaatatt 22440

taacttcacc actcagttag tatagaaaca gttgtgatgg aggcctgcat tggtaagacc 22500

tggcctgcca caaaatgggg atcccagtga ctatctctga gcagtgttac ctgaaggttt 22560

caaacttgtt tagaagaaag ccatttctct tcatttaaag atacaagtgg tataaaaaat 22620

aacatcgaaa attgcagtca ctgtgatgtc catttttgta ttatatgttc atatctttga 22680

agcactgttt agtctattgc aagaaagatt gaagaggatg aagtagaaga caatgtggtc 22740

tggtgaccgc tcactggatt aggagctagg aatcctagtc ttggctcagt tgctaacttg 22800

accaagtcag ttgacctctg tgggcttcag ttccctaact cataataatg agagtattga 22860

ctaggtaatc ttcaaggtgt cttccagctt taaaacccag ttagttttta tgtatgtgat 22920

atcagagtct ggttctcagc aataattttt tttttttttg agatggagtc tggctctgtc 22980

atccaggctg gagtgcagtg gtgtgatctc ggctcactgc aatctctgcc tcccaggttc 23040

aagcaattct cgtgcctcag tctcccaagt acctgggact gcaagcacgc cccaccatgc 23100

ccagctaata ttttgtattt ttagtagaga tggggtttca ccatgttggt cgggctggtc 23160

ttgaattctt gacctcaggt gatctgcccg cctcagcctc tcaaagtgct gggattgcag 23220

gtgtgagcca ccgcacctgg ccctcagcag taatgctaat gtatactgca agaaaaggtg 23280

aagaggagct tttgcttcct ataaggagaa ggaaaaaaat ttcatttttc aaagctggct 23340

gccattgaac aagttggcga taaggaagat tgagttccct ttggaagtta attgtccttt 23400

tgtttaggaa aaaatgccca agagatactt ggctattgga ctttgaagga gataaatgga 23460

aggcaaagct cagacaatag agatttacaa aaagaatagt aagaatttct ctgcataata 23520

aaataacagg gatttttttt ttttttgaga catcccctgg caccaaggag tttggcctca 23580

agttagttgt gcaggaattc aggtagggtg tgttggacgg aaagtaggct gttcagagca 23640

gggcatgcca cagacagcct tgggtcagct gcattgtttt gtttgcttgt actgcttttc 23700

aagaatttga atcaacattt aaatgctgct ggatatggtg gctcatgcct ataatcccag 23760

cactttggga ggctgagggg gatgattgct tgagtccagg ggttcaagac caacctggac 23820

aacattgtga gaccctgtca ctacatccaa aaaaaaatta aatattgaaa gactttaaaa 23880

tatgcatagt ttgtacctct gaaaattgga agatcttagc aataatcagg tgggtagccg 23940

ctggctccat tagaggactg gttcaccaca gtcctcaata tgcagagtgg tctcaggcct 24000

gcaactggcc ccacccaacc cccaggtggc tgcagtactg cctgagccct gggggcatat 24060

gaattctctg ccctggctgc agagggtcct ctgggaacag aagagaagtt tgggtctgtg 24120

gaagccctag taaagacaaa agtctgtgtg gtgtgaaatg gtcagtgagt ttctagaagg 24180

tctagaaagt tcatgtttgt ttcctgggtc aggtgcaggc ggctcacacc tgtaatccca 24240

gcactttggg aggccaagaa gggagtattg cttgagctca agagtttgag accagcctga 24300

gcaacatggt gaaaccttgt taatgaaaaa aaaaattatt aaaaaaaatc ccacaaattt 24360

gtttcccacc aatcttaccg tctattgtac ttactaccat cttttgtact caaactttta 24420

gtatgagtct atctctctct ccttctctct gacacacaca cacacacaca cacacacaca 24480

cacacactca tgcacaaagc attgctgcta gaggagccat ttacctcact cctcacttta 24540

atgattcctt cttgctttga ctccttgact tctgattaga cattttttga tcttttagat 24600

ttaattgtgc tttttgttct ataaaataac tcctcaaacc aatcacatat aaatatttat 24660

gaagtactaa atctgtaagg agcaaagctc atgatatata ttttaagtat atttttaaat 24720

gtttattgag aatcagatac tatgtttatc acataatata actttggttc tgtcaaaagc 24780

cttgagtagg atatatcttt caaaatcaac caaatattac cttttgagtc aaaacaaatc 24840

catgtttgag ttctgcctgc ctcctccaaa ttgctcaaca tttcatcata catacattgt 24900

ttttgagcag gaagctgaac taaatattaa gccaccaggt tgtagcaaag tttgtgtgcc 24960

tttctttgac tagaaatctg acaaactaca aatggttttc attttacctc ttatcttcta 25020

ataagaattg atgatatatc tgaaagcatt tgtaaaagct gatcaactta cataaaattg 25080

taaagcgaca caaatttaag gcactgtaag gataaaagct tttattaaga attatggata 25140

ttttcttggc atgtaaactc ttatcttctt tagggatatt tggtctcttg gctgtgtctt 25200

atatgagctc tgcacactta aacatcctgt aagtatgctc attgtcagac taatcttgaa 25260

ttattggaat tgtagaaaag aaattaactt ctgggagaaa aaggttaatg tttggtttta 25320

ttagattgtt aaaaattata tggataagct acttaaaata atgatagatg acatggaaag 25380

ctgtccaagc aatattataa agtaaaaagt ccaagttgga gaatagtatg tgtagcatat 25440

ttccattaaa aataaattgt gtgggcttgg cgtggtggct catgcctgta atcccagcac 25500

tttgggaggc tgaggcgggt ggatcacttg aggtcaggag ttggagacca acctggccaa 25560

catgatggtg acaccccgtc tctactaaaa atacaaaaat tagccaggca tggtggcatg 25620

tgcctgcagt cccagctagt tgggaggctg aggcacgaga attgctagaa cccaggaggc 25680

agaggctgca gtcagctgag attgcgccac tgcactccag cctgggtgac agcgagactc 25740

catctaaaaa aaataattaa ttaattaatt actgtatgaa tagatacgtt cagcaaaaga 25800

aaaatgtaca tgggcaaagt tcataggaaa ccaggcacaa gcttttaaga gtcttttccc 25860

agaggtcaca tgggatgtgc caaatcctcc agcattgtta cccacgtcac ctgtgaaatg 25920

tgatctataa gaaagctcat cggatatacc cagtgcccag gatttttact ggggactggt 25980

cacataggca ccctctacct ggcatatgcc aaacttccag actcctggaa agaaagcccg 26040

tgttcagcat aaaccatttt gttcacataa atagctgagg caaagatagc cactcttgac 26100

attcagggaa tggtgggaat tcttctgaaa tcttagttcc cagacaccag ccacgggcca 26160

acattgtaag caggcctttc tgaggagagc ttgctacatc aactcttttc tccacagctg 26220

tcatcattgt tattaattat tgtcaagggt tgcacagcca gtgtctgacc aaaatgtgta 26280

ctccattgtt tttttgagat ggagtcccgc tctgttgccc agactggagt gcggtggcac 26340

gatctcagct cactgcaacc tctgactcct gggtacaagc aattctcttg cctcagcctc 26400

ccgaggagct gggattacag gcacccacca ccacacccgg ctaatttttt tgtattttta 26460

gtagagtcag ggttttgcca tgttggccag gttggtcttg aactcctgac cttgggtgat 26520

ctgcccacct tggcctccca gagtgctggg attacaggcg tgagccacca tgcccggcca 26580

atgtgtacct ttattgctac accatggagt tgaatattat tatgtataaa taactattgg 26640

tttcatacaa tagaagattt ctggtctatg aagcatttta gaggaaatta aacgatgttt 26700

atgttaattt taaaaagcaa gagataaaat ttcatatcaa tatgacctca actttgtaaa 26760

ataaacatca tttttaaaag agatcagaag gagctatacc tctgagtggt aaaattatac 26820

atattttccc ctgtctttat aacttcctat accttccagt ttttttatta tgagtaaaca 26880

ttattttgat aataagacag aattaaaaca aaataaaaac ttgttttaaa taacatggca 26940

tcttgttgaa taactgcagt atctgctcat gaaagattag ttgatgaaaa caatttaagg 27000

tggaccacag tgcttctttt ttattttttg attgagacag ggtctcactc tgtcacccag 27060

gctggagtgc agtgacgcaa tcacggctta ctgcagcttt gaccgcctgg gcttagacaa 27120

tcctcttgcc tcagcctccc aagtagctgg gaccacaggc tcatgccacc aagcccagca 27180

aatgtttaaa aaccatgatt tggagagatg aggtctaact atgtttccca ggctggtctt 27240

gaactcctgg gctcaagtga tcctcctgcc ttggcctccc aaattgctgg gattacaggt 27300

gaccctagtg cttctaacta caatttaaaa acattgtttt gcttcttggt atatttgtta 27360

ctttaacact tttattattt gttactttag taacttttct ctgatttagt gtcatttctc 27420

cttgtccttt cagtttgagg gtaacaactt acagcagctg gttctgaaga tttgtcaagc 27480

acattttgcc ccaatatctc cggggttttc tcgtgagctc cattccttga tatctcagct 27540

ctttcaagta tctcctcgag accgaccatc cataaattcc attttgaaaa ggcccttttt 27600

agagaatctt attcccaaat atttgactcc tgaggtaagt tttgaggtga ctgtttggat 27660

tttggcagag attttgggtt gcaggtcctt gacacgtgtg ttcggtttta ggtcattcag 27720

gaagaattca gtcacatgct tatatgcaga gcaggagcgc cagcttctcg acatgctggg 27780

aaggtggtcc agagtaagtg tgactttggc atgcaatcaa aagtatttat tacacatgtc 27840

tcacacagag agtaatgcaa ggaaatttca ccaaacatat tgaaagtgga cattttaaaa 27900

aatacaagca gtataagcag gagaaaaatc atcttgtcaa atggcaacta gtgagtgtgc 27960

ctgaaagttg tatatctagc tcatgcatga cctgcagggt tccttctcgt tagtcaggaa 28020

acctccatga agcagaggac atgctaatag agatgcttga agaggttgag cccaaactta 28080

acttttgtgt agtgaaggga cagagtggga gaaggttgca gatagacatg gatgatgaga 28140

tgaaacttat ttttctaaaa gaggatagac tggcaattaa gaattctgtt gcaaaggacc 28200

attggagctg aagttaggat cttggggcct aattgataac agtaagaact gttactttgt 28260

ggttcccaaa gaaggcagga gatattttat ggtagtaata aatacagaaa actttttttt 28320

ttttccgaga cggagtctcg ctctgtcgcc caggctggag tgcaatggcg cgatctctgc 28380

tcactgcaaa ctccacctcc cgggttcatg ccattctcct gcctcagcct cccgagtagc 28440

tgggactaca gccgcccatc accactcccg gctaattttt tgtatttttt tagtagagac 28500

gaggtttcac tgtgttagct aggatggtct cgatctccgg acctcgtgat ccgcccgcct 28560

ctgcctccca aagtgctggg attacaggcg tgagccaccg cgccaggccg gagaaaacta 28620

ttttagtcct ggtgtcaaga atcagctaag ctgtgtgtca gagggagggg tacgttaaga 28680

aagagaaaat tactaattca tttgatgctg tgaaagtcaa agccccagaa tttagctgta 28740

actgaatgcc tggacttaca atatcaggag gagcagaaag cctctcaaag gaatccatga 28800

cagggaaatg ttatccattg agacagagat tctaaaatca aggaaagtta aagagaaagt 28860

gaatgagcct ctttgccatt taatttgact aacattgttg tataccagtc tagattgaga 28920

atgtttagaa aatagacaag tacagagtat gggactgtgt attgtccata tttctaatct 28980

aggtaagata ggagaacaag aacaattttt tttttattga gatggggtct cactgtgttg 29040

cccaggctgg tctcgaactc ctgagctcaa acaatcctcc taccttggcc tcccaaattg 29100

ctgggattac aggtgcgagc caccttactc agcccaagaa caaattttga tggagataaa 29160

gacaagcatt agaagatcta ctcatacctc agtcctggca ctttgggagg ccaaggaggg 29220

caggtcaccg gaggccagga gtttgatgcc agtctggcca acatggccaa accatgtctt 29280

tactaaaaat acaaaaatta gctggacctg gtggcccatg cctgtaatcc cagctccttg 29340

ggtggctgag gcacaagaat cgcatgaact cgggaggtga aggttgcagt gagctcagac 29400

cctgccactg caccgtagcc cgggtgacag agtgagactg tctcaacaaa aaaaaaagag 29460

agaagatcta ctcataaatt ccaaacaatg tggcatgaat ggagtggcct gataacccaa 29520

gctctaatga ccaaatttaa taacttttat tattacccca tacatattgt ttctgtaaat 29580

gttaatatta atttctattt ttctgaaaaa aagtgatgtt atatattact agaaatatgc 29640

aaagggactc tgaaaaaatg gtttttttca tttaaagaaa ttgcatatta atttttcatc 29700

agtactctca ctgtgtgtaa aatatctctg gctaaaaagt aaacttactg tgttatgaaa 29760

tgtagcttat gtttatactc ttacaagtat cagtattaat ggtgtacaat ttttaaaaaa 29820

ttgaagctgt tttattttgg ttaattaaga gtgtaaaata caaaaagtga gattccaggg 29880

aaagtgccca ccaagatcaa ggatatctgt gccaattaaa aggaatgcta tattgcatag 29940

aaatgaatgg agaccaccag ctggagccca gaaggccaga tctgtaagtc attctaaacc 30000

ctcctttgtg ttttttagct atggtatatg ctttttgttt gtttgtttgt ttgttttgag 30060

acggagtctc gctctgtcgc caggctggag tgcagtggcg cgatctcggc tcaccgcaaa 30120

ctccacctcc cgggttcaag caattcttct gccccagcct cctgagtagc tgggactaca 30180

gacgtgtgcc actatgccca gctaattttt gtatttttgg tagagatggg gtttcaccat 30240

attggccaga atggtctcca tctcttgacc tcgtgatcca cctgcctggg cctcccaaag 30300

tgctgggatt acaggtgtga gccatggcgc ccggccccgg ctaattttta tacttttagt 30360

agagacaggg tttcaccatg ttggtcagac tggtctcgaa ctcctgacct tgcgatcagc 30420

ctgcctcggc ctcccaaagt gctggtatta caagcataag ccactgcacc cagctgttat 30480

attctttttc tttaattttt taattaaaaa aaaaattttt gtgggtacat agtaagtgta 30540

tatatttatg gggtatatga gatgttttga tacaggcaag caatgtgaaa taagcacatc 30600

atggagaata gggtgtttgt cccctcaagt atttatcctt tgagttacaa acaacccagt 30660

tatactctgt aacttatttc aaaatgtaca attaagttac tattgaccat aggcagtcta 30720

ttgtgctatc aaatagtagg tcttattcat tcttttgttt ttttaaccca ttaagctatg 30780

gtatattctg acagacctat ctgcacatgt tcatgaggta caagcttatt gtttggagtc 30840

cacaaatttt gtacttaaaa tgaagtattc tgtactgagc attataatgg tattttgttg 30900

gacaacttct agtttttata ttttatgaaa caatgctgta tgctcttata agtatacttt 30960

aggcttaatt ttctttttat aactgaaatt cttctaattt ctaataaata agatttttct 31020

gtataggaaa agtgagtaac atagcaacag aaaacactct gcatttaata ttcttaattc 31080

taacatatta tgtataggat tgagaagttt ttatgatata ataattgata tttccctagt 31140

gattctttgt gtttaattat ttgaattcac ttcagcagag tgttgaatct tttaggtcat 31200

actagtgaaa tgcttctggt atgtaaatga taaaatggct actgtctttt aattaaagaa 31260

ttgtattttt aaagaaggct catggttaaa ttaagaacca tttggaagtg tatttactaa 31320

gtgtttactt gatatataga cattttagaa aatgtgttgg tatataaaca tttttttaaa 31380

aaccgattgt ttaagttatt gcccttcatt tgataaaggg ctttatttat ttatttattt 31440

atttatttat ttatttattt atttatttga aagagggtcc tgctgtgtca cccaggctag 31500

agggcagtgg catgtctcag ctcactgcag cctggatgta ttagtctgtt ctcatactac 31560

tataaagaac tgcttgagac tgggtagttg ataaagacaa gaggtttaat tggcttacag 31620

ttctgcaggc tgtacaggat gcattgctgg ggaggccgca ggaaacttat aatcatggca 31680

gaaggggaag caggctcatc ttaaatggcc agagcaggag aaagagagca aagggggagg 31740

tgctacacac ttgtaaacaa ccagatctct ggagaactta ctatcacaag aacagtaaga 31800

gggaaatctg tccccataat ctaatcacct tccaccaggc ccctcctcca acatcaggga 31860

ttacaattca acatgaaatt tgggcaggga cacaaatcca aaccatatca ttccaccttt 31920

ggcccctccc aattcccata tccttctcac attgcaaaat acaattatcc cttctcaaca 31980

gtcccccaag gcttaactca tttcagcatt aactcaaaag tccacaattc aaggtctctc 32040

tgagacaagt caagtccctt ccacctgtga ggctgtaaaa taaaaaacaa gttagttact 32100

tccaaaatac aatgagggta caggcattgg gtaaatacac ccatttcaaa agggagaaat 32160

cagccaaaac aaagggttta tagaccccat gcaaattcaa aacctagcag ggcagtcatt 32220

aaatcttaaa gctccaaatt cctttgaccc catgtctcac atccagggca tactggtgtg 32280

aggagtgggc tctcaaggcc ttgggcagct ctgctcctga ggctttgcag gctacagccc 32340

ctgcggctgc tctcacaggc tgctgttgag tgtctgcggc ttttccaggt gcgtggtgca 32400

agctgtcgtt caatctaccg tttttggagt caggagaatg gtggccctct tctcacagct 32460

ccactaagca gtgccccagt ggggactctg tgtggaggct ccaatgccac atttcccctc 32520

tgcactgccc tagtagaggg tccccctgaa acaggcttct gcctggacga ctaggctttt 32580

ccatacatct tctgagatct tggtggaggc tcccacgcct caactcttgc actctgtgca 32640

tctgcagact taacaccatg tggaagccac caagatttac ggcttgcacc ctctgaagca 32700

atggcctgag ctgtaccttg ggccgtttta accatggctg gagctggagc agccacaata 32760

caggacacca tgtcctgagg ctgcacagag cagtggggcc ctgggcttgg tcctcaaagc 32820

cattcttccc tcctaggcct ctgggcctgt gatgagaggg gctgcctcaa aggtctctga 32880

aatgccttca aggcatttcc cccattatct tggctaacaa catttgactc ctctttattt 32940

ttgaaaattt ctgcagctgg tttgaattgc tccccagaaa atgggttttt ctttctaggc 33000

tgcaaacttt cctaactttt acactctgct tctcttttaa gtataagctc tggttttaca 33060

tcatttattt gctcacaaat atgaccatag ggtgctagag cagccaggcc acatcttgaa 33120

tactttgttg cttagaaatt ttttctgtca gacgccttaa atcatcactc tcaaagttca 33180

aagttccaca gatcccctag ggtagtggca caatgcctcc aacctctttg ctaattcata 33240

acaaaagtgt cctttgctgc atttctcaat aagttcctca tctccatctg agacctcctt 33300

agcctggact ttattgacca tatcactatc agcattttgg tcactatgat tttaagaagt 33360

ctctagggca ttccaaactt tccatcatct tcctatcttc ttctgagccc tccacgctct 33420

tccaacctcc gcccattacc cagttccaaa gtcactttca cattttcagg tatctttata 33480

caatacccca ctcctggtat caatgtactg tgttagtcca ttctcatact gctataaaga 33540

acacctgaga ctgggtaatt tataaagaaa atacatttaa ttggctcaca gttctgcagg 33600

ctgtacagga agtatggctg gggaggcctc aggaaattta taatcatagc agaaggggag 33660

gcaggctcat cttacatgca ggaggaaaag agtgaagggg tagccgctac aaacttttga 33720

acaaccagat ctcatgaaaa ctcactcact atcacaagaa cagcaagggg ggaatctgcc 33780

ccaacgatcc atttaccagg cctcgtctcc caacattggg gattacagtg caacatgaga 33840

ttgggcagag acacaaatcc aaagcatatc actcgacctc ccaggctgag acacaaatcc 33900

aaagcatatc actcgacctc ccaggctcaa gtgatcctac cgtctcagcc tcctgaatag 33960

ctatactacc ggtatgcacc atgatgccca gctagttttt actttttgta gagtcagggt 34020

ctcactgtgt tgcccaggct gttcttgaat tcctgggctc tagtgatatg cccgcctcag 34080

cctcccaaag tgctgggatt ataggcgtga gccactgtgc ccagcctaag ggcttaattt 34140

tattaaagaa ataagaaaag tatgttgtga ttcagaggac tctttatcag acctgtagaa 34200

gggaaaacac atctaaaaga tttgaggatg aattaaatta cgaactgttg aacacgctga 34260

catttttcca gttccttgaa aaggtaaaat tgatttccac aggaactacc tctgatattc 34320

ctattactgt tgggatgtta gagaacattt taaagaaaat gtttattgcc tttcaatact 34380

tttctatatt ttttaccact tttcaacaag tcattagtag cattttcttc taggttgtat 34440

ataggtgaaa ttgtaaaaca aagaaaacta cttcttgttt taaaagattt taaaaatagg 34500

caggtgcagt ggctcacgcc tataatccaa cactttggga ggctgaggca ggaggatcat 34560

ttcagcccag gagttcgaga ccagcctggt caacacattg agaccccacc tctacaaaaa 34620

gtaaaattaa aaaaaaaatt ttttgttttt tactggacac agtagcatgt gcctgtagtc 34680

ccagttactt gggaggatga ggcaggagat ccctggatcc caggagtttg aagctgcgat 34740

gagctatgat cacaccacag tcctgcaggc tgggtgacag agtgagatcc tgtctcagaa 34800

tttaaaaaga aaagaaaata ttttaaaaat aaacatataa tttgtattta gattaatgaa 34860

ctaaatttta tacatttact taaatattta aatagaacta tatgaaagtg ccatttttct 34920

agattaatta tggtcaattc tgggcaattt ctttttttga gacggagtct cactctgtca 34980

cccaggctgg agtgcagtgg tacgatcttg gctcactgca agctccgcct cccgggtttg 35040

tgccattctc ccgagtagct gggactatag gcacccgcca tcacgcccag ctaattttgt 35100

ttttgtgttt ttagtagaga cggggtttca ccttgttagc catggtggtc tcgatctcct 35160

gacctcgtga tccgcctgcc tcagcctccc aaagttctgg gattacaggc atgagccact 35220

gcgcccagcc aattatgtgc aatttcatat ggtccaatct aacatatatg tgaaccatat 35280

agcagtaaaa acaacaaaga atataacatg ttacctcttt acatgaggac attttggttt 35340

taattgttct tgttattcat attcccaact attagttcct aggtctttcc agtagtttta 35400

tctttttttc tctttttatt attaactgta aactgtaaac tagacagagt tgccacgctt 35460

taggttaaat tgaccccact ttgctcttta gcaagaaggt cttgactggc ttttatatct 35520

taatttgatc tgtttcttgt cttctagctc agtggcttct actcagttgg aagataaact 35580

gtcatttctg gttctcctat tctcattctg ttctggttgg gaagggtggt gagggctggg 35640

atggtgatat gcccatcatg gctgttatat gacctttttt aatattttct ctggaagaat 35700

gattctgatt cagcatcttc tttcctttaa gtcatgatgc cattttgcat ttagtcaatt 35760

tatcagaaac taaaaatgtt gcaaatcccc atatgtgtga gtttcactat gctttttatt 35820

tccctgtaaa gtatggtaag gtataaatga gtttatgaaa aatagaaaac aataattctg 35880

agtttagttt tggatcttgg gttgcctggg catactcact agctaagtat ttttcacata 35940

ctagccatga agtatgcatg attcatatcc ataccttagc aaaattgtaa accactatac 36000

tatctagtac ttaggtcttt ttgtactcta ggatttgggg actcttaaga ttattctgga 36060

aaaaaaagta tagaagaaaa acagcaaaaa tacaccttca gtgccttatc ttagctatgg 36120

tcactgttat attgtcaagt attataaatt tgtattatgg tttttttttt tgagatggag 36180

tctcgctcac attgtgcagg ttggagtgca gtggcatgat ctcagctcac tgcaacctcc 36240

acctcctggg ttcaagtgat tctccttcct cagcctccca agtagctggg attacaggcg 36300

tgcgccacca tgcctggata atttttgtat ttttagtaca gacgaggttt tgccatgttg 36360

gccaggctgg tcttgaactc ctgacctcag gtgatccacc cgcctcagcc tcccaaagtg 36420

ctagggttac aggtgtgagc cactgcaccc agcctgtatt atggttttta aaaacatccc 36480

ctcttgtttt cttcagataa aaatgataga aagacccaaa attgctgctg tctgtggaca 36540

ttatgattat tattatgctc aacttgatat gctgaggagg agagcccaca aaccaagtta 36600

tcaccctatt cctcaagaaa atactggagt tgaggattac ggtcaggaaa cgaggcatgg 36660

tccatcccca agtcaatggt aatattgtgg tctagcttaa gctttggtta atctaaaaat 36720

atctttatat attaacattt attattctga aatccaaatt ctcctaacac aaataatcca 36780

agaagaactt tccaaatctt cattttaaac acatagttcc cttgaccttt ttcttttgtt 36840

tgcttttgta gacagtctca ctctgatgcc taggctagag tgcggtggcg caatctcagc 36900

tcactgcaac ctctgccttc tgagttcaag cgattctcgt gcctcggcct ctccagtagc 36960

tgggactaca ggcgtgcacc accatgccca actgattttt atatttttag taaagacagg 37020

gtttcaccat gttggccaac ctgatcttga actcctgacc tcaggtgatc tgcccgcctc 37080

agcctcccaa agtgctggga ttacaggcat gagccaccat gcctggccat gttagtccct 37140

tctttctatg tcagccctat acctgcttgt tagttggttc ttcaaattct caggtaccct 37200

ctcaccaggc agccactgac ctcatgtgat ccacctgcct tggcctccta aagtgctggg 37260

actacaggca agagccacta ttcccagcct ttctttcttt tttttttgtt agaaagattt 37320

tgtttttatt tccatcagaa tgtcatatat gttacacaaa tcaaatctgt tgacatctca 37380

agcttataac aattacgtgt tcttataaat tacgtgggaa ttacatgtac tgtgagaagt 37440

gttgtaatta tgatgtaatg tatattataa tttagcctac agaagtaaca aagtcttgta 37500

attaaataaa gcaataaatg tgttgataga ttattacaat tgataagtaa ttgataaatt 37560

atcttctttt tcctgtaacc cttcttcatc tcaagtctga tctagcttat tttcttattc 37620

atagagctgc ttaactgtag gcacagaccc atacccttgc tcttttaata ttctttcttc 37680

ctcctactaa attccactat atggcaggtg aaaaaatagt tgtgtatatt tcatttctca 37740

aagaggttac taatatgaat caataattga atcattaaaa tcaaatgatc atttgagaca 37800

ttttgagaaa taagatatat ttcattcggc atttatgttc tagggatttt caaaatatgg 37860

acatgttaga aagaaaatag tattcttaaa ttggtcttat ggtagatttt caaaaaattt 37920

actccataat agatttctgc agatctacaa tattttcaaa tttttttcac actgatgttg 37980

acattcctgt gttcagaata attgacacct aacagaggcc tgaagactta agtctaagag 38040

ttctatttta aaaatgtttt gtcatcaatt ttttttgttc agggttaaga atttgttact 38100

ttggcgactc tgattgttta ttgttgaaat tttggatgaa ttatgaaaaa cacaagatac 38160

tatgggatgg caatctcatt aatagtgaaa atgagataag caagaatgat aagagaataa 38220

tttcttcaga aacatataac gggaaaagca tatgttttat ctttaaagat attgggcact 38280

gttgtggttt ttatcgatct tcataacaca tttttaatta tctccacaat tatcaaaagt 38340

tatgtcttct gtccgttcaa attgtaatat ccatattgga ctcaattagt gagggacata 38400

gattttacag aagaactgga gcagccacaa aactcccttt ctcttttctc aaccactggg 38460

aaaatatatc ttccactctt tgtctagatt tgagagctgt caagctatca attattttga 38520

ccacatgtga ttttatatct cccaagctct cacatgaaac agtaggaagg gtccttctct 38580

ttcctggatg cccctttgat gcctggtaac ccctcctctt tgtatactcc ctcatcccca 38640

gctttctgtc tgctggaggt ccaattacag gccatgggat ggaggaaaag gatttttttt 38700

ttttttgaga cagagtctcg ctctgtcgcc caggctggag tgcagtgacg cgatcttggc 38760

tcactgcaag ctccgccccc tgggttcaca ccattctcct gcctcagtct ccagagtagc 38820

tgggactaca ggcgcctgcc acgacacctg gttaattttt ttgtattttt agaagagaca 38880

gggtttcact gtgtcagcca ggagggtctc aatctcctga ccttgtgatc cacccgcctc 38940

ggcctcccaa agtgctggca ttacaggcat gagccaccat gcccggccga ggaagaggaa 39000

tttgtatagg atttgggggg tggagaggga ataggtagac agagagatag agaatgtctt 39060

ttggacagcc cctgggtgtt ggaatcattt ttctcatgaa gatattgata catgtgccag 39120

ttaggcttat gagacagatg agtgctacaa tttaccctca tttgattcaa gaactatctg 39180

tgtgtcaggc acttcacagc cctaatctct tctaatcctc gtatcaattc ctgtgaaatg 39240

gtaccatgcc cactttacag ctgaggaact aagactcaaa gactttagct tgtcatttca 39300

tcctatttct aaatccctga ttattaactt gcctctttgt aaattgggga tgcttatcat 39360

gatgttcctt cctaaaggag ttatttctga aattacagtt ctgtctttgg agccttagaa 39420

gttactcgta ttccaaaaaa cttatggtct gaaatgcggt ttttatttag caaccaataa 39480

ttacagaaat gttttacagg aaattctgcc aaaaaaaaga tacataaaat gtgagtataa 39540

acttgaaaat tgtttgactg gaattgacta aaattgtgct ggaaaaatac cttaaacatt 39600

tggagagaca gctaaaccat tatttctttc ctcattaagc atttatgtgc ggagataaag 39660

ggatggatgg agggacacat tctgctctca gggagctcag tatgtggtgc aggaaacaga 39720

tatgcagcca ttcttttttt tcttttcttt tctttttttc tttttttttt gagatggagt 39780

ctcactctgt cactcaggct ggagtgcagt ggtgcgattt tggcttactg caacctctgc 39840

ctccctgctc agcctcccca gtagctggga ttacaggtgc ccaccaccac gccccactaa 39900

ttttttttag cagagacggg gtttcaccat gttggccagg ctgatctcga actcctgacc 39960

tcgtgatcca cccacctcgg cctcccaaag tgctggggtt acaggtgtga gccaccacat 40020

ctagccacta ttgttaaatc aggagcgaca acctgtacat tagacaccta cacaaagcgt 40080

gagaacttct gggtgtgggt ctgttttcct ccccacaaca tattatagag aatggaagga 40140

ctgaatcttg tcctgaagaa aaatcactgg ataagaatat ttttctgttt aatcctctcc 40200

tgtatcccca cttgttactc ttcatccttt tttccttttg attccaaaat tttcttttcc 40260

aatgtaaaga ttctgtaact gtgaactact tcttgaactt ggaacttcaa gccactggtg 40320

aattgtgaat ctcattacta aactgaaaat tactcgtcaa attggtgcct aagattcgtt 40380

caagtttcta cttaagctga acattcttat tttctaaggc ctgctgagta ccttcagaga 40440

aaatttgaag ctcaacaata taagttgaaa gtggagaagc aattggtaag taaaatacca 40500

aatatgggaa gcaattagga atttcctaat agtttttctg ttcacagatt ttcaagtcaa 40560

agttcattcc accagaaggt caagaatact ctctactagt ccccagtttt ttttgttttt 40620

gtttttgttg ttgttgttgt tgttgttttc tgagacagag tctcgctctg tcaccaggct 40680

ggagtgcagt ggtgtgatct tggctcactg caacctctgc ctcccaggtt caagcaattc 40740

tccttcccca gcctcctgag tagctgggat tacaggcgcc caccaccacg cccagctaac 40800

ttctgtattt ttagtagaga cagggtttca ccatgttgtc caggctggtc tcgaactcct 40860

gatctcgggt gatccaccca cctaggcctc ccaaagtgct ggggttacag acgtgagcca 40920

ctgcacctgg cccgagtccc cagtttttaa tagctaaata aaataatggg aacaggcttg 40980

aatcaccccc ttagcagtcc ggtttcttcc ttggctctat ctcttctgtg ggaccttgga 41040

cagttcattc agcctatctg agccttaatt tccttttcta taaatgacaa tttttagagt 41100

agatgagctt caaatttcct tgcagtgctg tagtgctttg gttctatttt gttaaagatt 41160

ctgctgcaca ttaaaaaaag tgacaagggg ccaggtgcgg tggctcatgc ctgtaatccc 41220

agcactttgg gaggccaagg tgggcggatc ataagatcag gagttcaaga tgagcctgac 41280

caacatggtg aaagcccgtc tctactaaaa atacaaaaat tagccaggca tgatggtgca 41340

cacctgtaat cccagctact tgggaggctg aggcaggaga attacttgaa cccaggagga 41400

ggaggttgca gtgagccgag atcgccctac tgcactctag cctgggcgac agaacgagac 41460

tctgtctcaa aaaaaacaaa aaaaacaaaa accaacaaca aaaaagtgat taggccagat 41520

attatggctc atgcctgtaa tcccagcact ttgggaggct gaggtgggtg gattgcttga 41580

gcccaggagt tcgagactag cctaggcaac ataatgagac cttatctcta ccaaaaaaaa 41640

caaaaattac ccaggtgttg tggtgtgtgc ctgtagtccc agctactgag ggggctgagg 41700

ccggaggatt gcttaagctt gggaggcaaa ggttacagtg agctaagatt gcgccactgt 41760

actccagcct gggtgacaga gtgagactct gtcttaaaaa aaaaaaaaaa aagaaaggct 41820

gggcttgatg gctcatgcct gtaatcccag cactttggga ggccaaggcg ggcagatcac 41880

gaggtcagga gattgagacc atcctggcta acacagtgaa accctgtctc tactgaaaat 41940

acaaaaaatt agccgggtgt ggtggcgggt gcctgtagtc ccagctactc ggaaggctga 42000

gacaggacaa ttgcttgagc ctaggagttg gaggctgcag tgagccaaga tcatgccgct 42060

gtactccagc ctgggtgaca gagtgagacg ctctcaaaca gaaaaaaata tatatttttt 42120

aatgctttat aattaagaaa attctactac ttaccacaaa aaaaactccc aaatactgag 42180

tttgcttagt gatataattc ttatttatag gaaaaagtca atgtcaaatc agaagatgtt 42240

tccgaaatca aagtatgcat tataaattat ttcattcaat aaatagggtc ttcgtccatc 42300

ttctgccgag ccaaattaca accagagaca agagctaaga agtaatggag aagagcctag 42360

attccaggag ctgccattta ggaaaaacga aatgaaggaa caggttaaaa actgtttaat 42420

tccagggcta cccttgtatt tctttgtatt actgtctttt gtactgtaat agggagttac 42480

ttctatttcc tacagtgccc ctgaatatgt caacaccatg ctgagtgtta taggggatac 42540

agagttaggt atttcacttt ctcagataat gcgatatggc taagttcata aagcttttca 42600

ccttgagatt catagagtaa ctgtccatca gtaacaggtt tgggatttgt attagtttcc 42660

tgggctgctg taacagagtt caacaaacta ggtggcttaa cacaatagaa atgtattgtc 42720

tcacaattct ggaagagtgg aaatagaaaa tcaaggtgtc agcaggacca agtgccctct 42780

gaaacctgta ggggaatcct tccttgcctc ttcctagctt ctggtgcgtc actggcaatc 42840

tttggcattt ctttacttgc agtcgcatca ctccattctc tgccttcatc acatgctgtt 42900

cttcctgtgt gtccctgtct tcacatggcc atcttcttgc aaggacatca gtcatattga 42960

actaagggcc cactggtatg atctcatctt aagtagtctc atctgcagcg accctgtttt 43020

caaatggggt cacattctaa ggcactgggg gttaggactt caacatatct ctttttgggg 43080

aggaacaaac ttcagtgcat aagagggtta tatataaaag tgggatttat aaagtaagtg 43140

tacatcatga acacatttgg gttatatata aaattgagct ctgtagctaa agccactgtc 43200

tcacagggag tgaagtactg cagccaaaac ataaggcaga ttatcatctt taggagcaac 43260

atatttttct aaccttattt tatattacac acttttgaaa ttgtaggctg cagaaagatt 43320

atttttgtta tggtgttcat aaacatttaa agtttctgga ttgggtttgc tttccaggaa 43380

tattggaagc agttagagga aatacgccaa cagtaccaca atgacatgaa agaaattaga 43440

aagaagatgg ggagagaacc agaggtaaat tcattcttct aggggaaaca ttgttctatc 43500

gatttagagc taactaaatt gagctggtat taaaagtaat gatttcctta tagaaaagat 43560

aaagttttat catagagata atcatgtaga cttctttttt aataggaaag ctgtcagacc 43620

tcattggagc ttcagtttat tatggtttat gagagactac acaagataat aaggatatct 43680

gagattctca ggaatggcta ttattaaaag tacttattga ttgtttcctt catgaatcac 43740

tcaatacata tttattgagt ggcaacttta gacaagagct ggattagatg cagaaagtcc 43800

aaaatgagtg taagttcatg cccagaaggt gggaaaaaaa caaacccaac acactagcat 43860

tttttcaact ctctgcaggg taaggttcta aaggctattt aaggcaaaat ctgagtgcag 43920

ttgaactgat tcttaaaaat ctcttaaagg cgccacattg gaaattcatc cttccatctc 43980

ccaagaaggt ctctagagtt ggcacagatc actgcttctt cagaagagct tcacatgaaa 44040

tagccagcct gtgtttggaa accatgttgt aagaaagaca catggctatt gaaacactag 44100

gaacacactc agtgccctgg aatgctctcc taggagaagc ttgcaggcac tgagacagct 44160

gtctcccatc ccacatgcac ttggccacac actcattgag tagagctacc atgctgctga 44220

aattgatctc tctctctctt tctcccaccg cagtgcatac agataaattc atataagtca 44280

aatgaatgta tggtgcaatt cagttgtgtt tgccaggcca tgaactagag ctttcacata 44340

ctgtattagt ctgctctcat actgctaata aagacatacc caagactggg taatttataa 44400

agaaaaagag gtgtaataga cgcacagttt cacatggctg gggaggcctc acaatcatgg 44460

cagaaggcaa aggaggagca aagtcatgcc ttacatgaag gcaggcaaga gagcttgtgt 44520

aggggaactc ctatttacaa aaccatcaga tcttgtgaga cttactcact accattaaaa 44580

tagtatggga gaaaccaccc cgatgattca gttatctcca cctggcccca cccttgacaa 44640

atgaggatta ttacaattca aggtgagatt tgggtgggga tacagagcca aaccatatca 44700

cgttcatact ttcttttatt tacccctgta cagagaagtt aagtagctca tccaaagtca 44760

cgtagctatt acaaggcaga cgaaatattt aaatatctga ctctaggctg ggcacggtag 44820

ctcatgcctg taatcccagc aatttgaaag gctgaggtgg gaggattgct tgagcctagg 44880

agtttgagac cagcctgggc aacataggga aaccctagct ctaaacacac acacacacac 44940

acacacacac acacacacac acacacacac acacacacac tctctctctc tctctctctc 45000

tctctcactc tctctctctc tctctctctc tttaaattag ccggacatgg tggtttgcac 45060

ctgtagtcct agctacttgg gaggctaaag cagaaggatt gcttgagcta ggagctaaag 45120

gctgcagtga gccatgattg tgccactgta ccccagcctg ggatacagag caagactcgg 45180

tctcaaaaaa ataaagtaaa ataaaatgaa aatctgactc taaaacccct actcatgttc 45240

atgcctgtaa tcctagcatt ttgggaggcc aaggcagaag gatcgcttga gcccaggagt 45300

ttgagaccgg cctgggcaac ataatgagac tccatatgta caaaaaattt aaaaaattag 45360

tgggtcatgg tggcaaatgc ttgtagtccc agctactcag gaggctgagt tgggaggctg 45420

aggttgaagc tgctgtgaac tgtgattttt ccactgcact ccagcctggg caacagaggg 45480

aggccctgtc ccaaaaaata aaaaaataca attataacca ctattctttc tggcatatgc 45540

agttctactt ataaatggtt ggaatgacga gacacatgta taaaacaatc atagagtaag 45600

ccctgtaggt gcacaggagc caagatgagc agaatgagca ggtagcgggt atttatggaa 45660

gaagtgggtg gggcctaaag agtggaattt ggagaggcag agttaaagga ggagagtggg 45720

cattctgaaa gaaccatgca aaggttggga cagaggactg tgtgtgctgg gaggggcagc 45780

ctggaggttg ttctctctgg agcagaggcc tggctcatgg gcagcctgga gccatcatca 45840

gccttatgtc taaggctgat ctgggatggg cagcctgagc cctgcaaaaa tggatagcag 45900

acattggtca ggcgcggtag ctcacgcctg taatccccag cactttggga ggctgaggtg 45960

ggcggatcac gaggtcggga gatcgagatc atcctggcta acacagtgaa accctgtctc 46020

tactaaaaag ccaaaaaatt agccaggcgt ggtggtgcgt gcctatagtc ccagctactt 46080

gggaggctaa ggcaggagaa tggcatgaac tcaggaggca gagcttgcag tgagccaaga 46140

tcgcaccact gcactccagc ctgggtgaca gagtgagact ccatctcaaa aaaaaaaaaa 46200

tggtggatat cagacatttc tacagaggct atggaggaag gatttgggaa atactgaagc 46260

tgtggcgggg aaaaggagct ataaaaaggg ttctgtccag ccatttcatc attgatcgcg 46320

catcagccaa gtagccttca gagctcactc agaaccaatc ttgttgactg tgtataattt 46380

gtgttaaagg agaactcaaa aataagtcat aaaacctatt tggtgaagaa gagtaacctg 46440

cctgtccatc aagatgcatc tgagggagaa gcacctgtgc aggtaatgat ggctatgatc 46500

agatgtgtgt gcttgcagtg tgtgtgctct accccaagtg gctcttaccc ttctctgtgc 46560

agcagaacca cgtagggaac tttttttttt tttctatgag acggagtctc actctgtcgc 46620

caggttggag tgcagtcgtg cgatcttggc tcactgcagt gtctgcctcc tgggttcaag 46680

tgatttcctg cttcaggctc ccgagtagct gggactacag gcatgcgcca ccatgcccag 46740

ctaatttttg tatttttagt agagacagga tttcaccatg ttggccagga tggtctttat 46800

ctcttgacct cgtgatccac cagcctcggc ctcctaaaat gctgggatta taggcatgag 46860

ccaccgcctt tttaccagaa cttttaaaaa ctcagatgcc tctctctgcc ccagatggtc 46920

tgggatgagg cccaggcatt ctgtctgcag aagcttgctg ggtgatttag taagcagcca 46980

agtttgagaa ctgctgctat ttagtataag aacgttccac tctctggagg gtctaagtca 47040

gtgtatcaga cacattggtc aggaaatctg agtcaagttc tcttccattt caaccttatg 47100

tttttggtgg aggtaagagc ctgggcagag ttgaaataac aaataaatct caagagagtt 47160

tttttccctt ctgagaaaag ataatgcaat tataatacaa gatgaatctg ttgatttcaa 47220

ccaattctga gaattattaa acctgtgaaa tgacctgata aacaatgctt ttatggttac 47280

ataaaataat tacataaaat gttttacttt ccaaggagtt atatttattt tgcgaataag 47340

aagcccaagt gtgcttttat ttttgcttag taagaagatt ctcaatgatt tggcccatac 47400

taagaattat tattatcttt tttttttaga tggaatttcg ctcttgttgc ccaggctgga 47460

gtgcaatggc acgatcttgg ctcaccgcaa cctccgcctc ccaggttcaa gcgattctcc 47520

tgcctcagcc tcctgagtag ctggaattat aggcgcctgc caccgcgccc agctaatttt 47580

tgtattttag tagagacagg gtctcaccat gttggccagg ctggtcttga actcctgacc 47640

tcgggtgatc cacctgcctc agcctcccaa agtgctggga ttataggcat gagccacccc 47700

gcctgagcga attattatta tctttataat tagagtaatt ctctgtgttt taaattatat 47760

ttattattag agcttggtcc agagtcaact agaaatggaa aatcctcaag gtattataaa 47820

cttgtcattt aaaggtgcca gtaggatcac agtcacattc cataaaaaca cggctcagat 47880

gttacagaca tgtttttctc tcacattttt taacctggtt agagtaaatc cagtgcctta 47940

aagtttttaa taagtcaggt aattaaaaat aaaccactgg aagcctcaaa aagtttgtat 48000

caggaattgg gtgaataaaa tcttgtatat tttatgcaag aggagtaact ttgaaagaaa 48060

acacaccaaa atgccaatgg tggtaattgg tggtatctgg attggtgtga gtaggaatga 48120

ttattgtctc tctacttttt agatttttta taagaaggtt acagaacttt tactacaaat 48180

atgtataata aagtatccgt tccttagttc tgtcagcact ctaatcaata tcttcaaaca 48240

aaaaagccat ctgaaagaca gaaatggtgg cacgagacta tagttccagc tatttaggag 48300

gccgaggatc ccttgagctc aggagtttga gaccagcctt ggtaatatag tgagacccca 48360

tctctaaaaa aaaagaaaag gcatctgata tttcctgaag gctcctccag agcaatccag 48420

cagcagatac ctttgcaaac ttttgtaaag gaaataatta tcacttaatt tgtctaattt 48480

ttggatttag gttttaatta tcttttttga agggaatatg cagctatata ataagacact 48540

ttaaaaaagt ctctacttgt agagttatct ttccaaaata ctgatttgaa cattatttct 48600

ctacacgaca atcaatggcg actgccattt ctcttagcat ggcatgctag acttttgtga 48660

gttgttccta acagaatgtt ccagcctcat tgctcacatt tcccccaaac atacccaaag 48720

ctctaaatgt ctcagattac cttttttttt tttaaatgac atatttttta tttctttaag 48780

tgattttttt cactgtggta aaatacatat aacatcgcct ttaccaccct aaccattttt 48840

tttttttttt ttaattgatc attcttgggt gtttctcgca gaggggtatt tggcagggtc 48900

ataggacaac agtggaggga aggtcagcag acaaacaagt gaacaaaggt ctctggtttt 48960

cctaggcaga ggaccctgcg gccttccgca gtgtttgtgt ccctgggtac ttgagattag 49020

ggagtggtga tgactcttaa cgagcatgct gccttcaagc atctgtttaa caaagcacat 49080

cttgcaccgc ccttaatcca tttaaccctg agtggacaca gcacatgttt cagagggcac 49140

agggttgggg gtaaggtcac agatcaannn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49200

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49260

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49320

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49380

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49440

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49500

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49560

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49620

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49680

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49740

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49800

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49860

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49920

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49980

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50040

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50100

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50160

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50220

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50280

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50340

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50400

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50460

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ntacgaaaac cagtcaggcg 50520

tggcggtgcg cccctgcaat cgcaggcact cggcaggctg aggcaggaga atcaggcagg 50580

gaagttgcag tgagccgaga tggcagcagt acagtccagc ttcggctcgg catcagaggg 50640

agaccgtgga aagaggggag agggagaggg agagggagag ggagagggat cagattactt 50700

tttaaagccc tacttattta aaaagacatc ttccttttaa cctccaggct tttgtaaaat 50760

gcttatttct ctactgaaat atcccttccc tctcttctct tcttgcagaa cacatctatc 50820

agacctcctg gtgaagtttc tagcacagct tttttctctt tctcccttag aattaataac 50880

tgcctcatct gtatttccac agcatttcca agtacttcat acaccagcct gtgtcagttt 50940

gaagcattat tagctatttg ccctgcaaac ttgggaaggg tttttctgcc ttgcagtagt 51000

atgaagtctg aaatcaggac tatgacttat ctatcttact tatatttgtg aagttggttg 51060

tctgatttgc ttggatagtc tggtcatctc aattgtacaa taagtgctcc attactttac 51120

tttccttaaa atacaacgat ctcagattcc aaccccaatc tactccagtg ggtgggacat 51180

tcaaccttag tgtgctgtca agctctccag ggtcatgtca tctgaaaggc cctcttggcc 51240

ctgtgaagac tgattaactg tgtagccatg gagtctggga tcttgaggca ggaactctag 51300

gctggtgtgc agtctcttgc tcactactcc aatgtactgc cacagattag gacttgagtc 51360

cgccatctct ttaaaaaaaa aaacagtttt attgagatat aattgatcat aataaaccac 51420

acatatttaa tgtatatact ttataaaatt tggcaggcac acccatgaaa cccatcacca 51480

caatcaatat agtgaacata tccatcacct gcaaaagttt gttgcgccct tttgtaaact 51540

cctctctctt attctcccta cctctcctcc catctcatcc ccatgcaatc acggatctgc 51600

tttctgtcgc tgtaggttgg tttgaatttt ctagatttgt ttggattaca taaatggagt 51660

cgtgctgtat gtgctctttt ttctggctac tttcattcac ataataatgt tgagatttat 51720

ctatgttgca catattaata gttcattatt attctttatt gctgagtata ttctattgta 51780

tgaatgtatc aaaatttatt gatccattca ctgtagatgg atatttgggt tgtctccagc 51840

ttttggctat tataaataaa gctgctagga acattcatat acaaatcttt tttttttttt 51900

ttgagacaag tttcgccctt gttgcccagg ctggagtgca atggcacaat ctcagttcac 51960

cacaacctct gcctgctgga ttcaagtgat tcttctgcct cagcctccca agtagctggg 52020

attacaggca tgcgccacca cgcctgacta attttgtatt tttaatagag acagggtttc 52080

accatgttgg tcaggctggt ctcgaactcc cgacctgagg tgatccaccc acctcagcct 52140

cccaaagtgc tgggattaca ggtgtgagcc actgtgcctg gctatcatgt acaaatcttt 52200

atgtggtcat gtgcttcttt tctttctttt gggtaaatac attggactgg gatggatgga 52260

tcatatagta ggtgtatatt taactttcag agaactacca aatggtgttc cagaatggtc 52320

gcactgtgtt acactcccct tgacattgta tgagtgtttc agttctctct gtgcagctct 52380

ctcctctttg ggtctttgtc tttcagactc tagcacctta atacccccca agccttgtct 52440

tatcaactca gggagttggc cacactcatc ttcggtttcc atccctgcac ctcttcagtt 52500

ccccatcccc gcaccatggc ttgcaaactc tctcaagaca ggaggctggg gcagttgcag 52560

ggcttgtctc attggttttc tgtttcttag ggattactgt ctttcattgc tggatgtcta 52620

atgtattaaa aaccatttat ctattatatg tttgatttgg ctctttggtt gtttcaggtg 52680

cggaattaaa tctggtttct gatactctgt cttggctgaa agcatacgtt ttcagtgccc 52740

actgctggag aggggtggag ggcactcaag agttccattt ggacattgag ttagagaagt 52800

tgtgagagtt tacatacctg ctctggagcc tttaccccac tgttccctct gcatggaaaa 52860

tgctctcccc agactggcat atgccaaggt ccaatatcat tccagggctt aaattgattg 52920

ccagataagc tttgcctgta ttactctcac tccctactca ttttctgtcc tgttatccta 52980

ttttgttccc ttgatagcac ttaacacttt ctgaaattat gtcattcgtt aactcattta 53040

ttacctatcc tactccagta aaatgtaatt ttcgtgtcag cagggacctt tctggtcatg 53100

tccactgtgc taacccattt tgagggtttc tggcccctgg ggagtgctca gtgtgaattt 53160

gtggagtgaa tattaagatg aagataatgc taagtaggca gttggatatg tgagtctgga 53220

gctcagagga gaggaaaagt gaagcctgaa gatacacatt taagagtctc tgcttaacag 53280

tggcatttaa atccatagga atgaatgaaa ccccttgtat tagggaatag aagagcagat 53340

ggcccaagat aggatgctaa gaaacctccg aatatggagt tcacatctca gttgtgcctt 53400

tgaaattctt gtcatccact tttagttttc ttctcttcct acttgaaatt gcctaccaat 53460

tttcagagcc ctctccttcc tttataccgt catgagttgc gcactttgct tattttcctg 53520

attaagatca taagcctctt aagggaaaga tcctgtagtc aaaattacat tcttgaattg 53580

aattgggttg gactggagtg gactggagtg ataagtattg tcacattata gaattccacc 53640

cactgaagtg caagtgttaa atgtattaat atttcaagtt aatggatact ctgcccaagt 53700

ttttagttaa ttattattaa ctttccatta taaaagcttg tttttgttat taaatcaatc 53760

atcagattta acgcagaaat caactcatgt aaacatacag tgagagaatt gtatttttct 53820

ctaaattttc aggacattga aaaagacttg aaacaaatga ggcttcagaa cacaaaggaa 53880

agtaaaaatc cagaacagaa atataaagct aaggtaagaa atacttttgt ctttgggttc 53940

catattaaat agctggctgg ggagccacct tgtgatctcg gttgcctgca tgattttccc 54000

cctagtattt tatagaattg ctctattttg tgatatgaga ccaatggttt taagaatcta 54060

taatgtcaaa caaaattgac ctagggagtt gtaattttaa ggcttttact gaattgctaa 54120

actttttttt tttttttgct ttctcctaga agggggtaaa atttgaaatt aatttagaca 54180

aatgtatttc tgatgaaaac atcctccaag aggaagaggt atgccattaa gtctaaattt 54240

ccattagtag gtatcagaaa atgcatatat cttaatagca tgtttcatga aattatttca 54300

caggctgtag ggataatttt tttcaacttt tattttagat tcaggtggta catgtgcagg 54360

tttgttacct ggatatgttg tgtgatgttg aggtttggga tatgaatgat cccgtcaccc 54420

aggtattgag cataataccc agtagttagt ttttcaagcc ttgcttccct cctttcttac 54480

ccccactgta gtagctccca gtatctattg ttgctatctt tatgtccatg agtacccaat 54540

gtttagctcc cacttataag tgagaacatg cagaatttgg ttttctatcc ctatgtaatt 54600

ggttttctat ccctatgtaa tttgcttagg atagtagcct ccagctgcat ccatgttgca 54660

tggacatgat ttcattcttt tttatggctg catagtatcc catggtgtat atgtaccaca 54720

ttttctttat ccagaccacc actgatgggc acctaggttg attccatgac tttgctattg 54780

tgaatagtgc tgggatgaac atgtgagtat atgtgtcttt ttggtagaat ggtttgtttt 54840

cttttggata tatacccagt aatgggattg ctgggttgaa cagtagttct aagttctttg 54900

agaaatatcc aaactgcttt ctacagtggt tgaactaatt tacattacat ttccgccaac 54960

actacataag cattcccttt tctctgcagc ctcgccaata tttgtttttt gactttttag 55020

taatagccat tctgactcgt gtgagatggt gtctcattgt ggttttgatt tgtagttctc 55080

tgataattag tgatgatgag tattctttta tatatttgtt ggctgcttgt atgtcttctt 55140

ttgagaagtg tctctttcta tcttttgtcc actttaaaat ttgggttgtt ttttcttgtt 55200

cagttaagtt ccttatagag tctggatatt agacctttgt tggatgcata gtttgcaaat 55260

attttcttct attctgtagg ttgtctattt actctgttga tagtttcttt tgctgtgcag 55320

aagctcctta gtttaattag gttccacttg tcaattttgt ttttgttgca attgcttttg 55380

aggacttaat cacaaattct ttcccaaggc ccatgttcat aatggtgttt cctaggtttt 55440

cttttaggat tcttatagtt taaggtctta cttttaaatt gttaagtcat ctttagctga 55500

tttttgtata cagtgaaagg taggggtcca gtttcattct tctgcatgta gctaaccagc 55560

tatcccagca ccacttattg gataggaagt cctttcccca ttgcttattt ttgtcgattt 55620

tgtcaaagat tatatggctg tagatgagtg gctttatttc tgggttctct attctgttcc 55680

ttggtttatg tgtttgtttt tgaaccagta ccatacagtt ttgattactg tagtcttatg 55740

gtatagtttg aagttgggta atgtgacgac tctggcgttg ttctttttgc ttagaattac 55800

tttggctatt tgggctcttt tttgtttaca tatgaatttt agaatagttt tttttttctc 55860

caatcctgtg aaaagttaca ttggtagttt gacaggaata gtgttgaatc tatagattac 55920

tttgggcagt atggccattt taatgatatt gattattcca atccatgcat gtggcatgtt 55980

tttccatttg tttatgtcat gtatgatttc tttctgtgtt gtgtagctct tcttgtagag 56040

atctttcacc tccttggtta gatgtactcc taggtatttt attttatttt ttggtggcta 56100

ttgtaaatgg gattacgttc ttgatttggc tctctgcttg aatgttattg gtgtatagga 56160

atcctattga ttattgtact tcgatattgt atcctgaaac tttgctgaag ttgttcatca 56220

gttccaggaa cctttgggtc gagtctttgg gttttcaacc tatagtatca taagcgtgaa 56280

gagatggttt gacttcttct tttcttattt ggatgcctag aattttagaa aatatttcta 56340

gaaaaatgtt tggtgctcaa ggccagggaa cggtggctca cagctgtaat cccagcactt 56400

tgggaggctg agacgggcag atcatgagat caggagattg agaccatcct ggctaacatg 56460

gtgaaacccc atctctacta aaaatacaaa aaattagctg ggtgtggtgt cacccacctg 56520

tagtctcagc tacttaggag gctgaggcag gagaatcact tgaacccagg aggcagaggt 56580

tgcagtgagc tgagatcgct gtactgcact cgagcctggg caacagagtg agacactgtc 56640

tcaaaaaaaa aaaaagggaa agaaaaatgt ttggtgttca aatgagtcct ccaaatactt 56700

tttattctcc cattttattt tattggtgtt atttctttag ataaattatt acattttaat 56760

ttacttttct ttaaataaaa gagctatttt actcataata ttaattttta tcatagccaa 56820

attaaaatag aagacctgat acattgtcaa caactaatat actgacctaa aaaattgaac 56880

aggtaccctg aaaccaggca catttatttt aggtcttaat tagttattga taactttaag 56940

taaatctcat ttatgcattt gggctctcct tgccacagca aggagtaaat acagtaaatc 57000

caatacagta aatccaaatt tcattttatt agttgatttc aaaatctttt tttatcctgg 57060

ttttatcaga cctataacaa atgtcaaaat taattggttt atttttccat tttacctttt 57120

ctgaattcac cttttaagtc aatataagta tgaataatta tacctgatgc tcagttttta 57180

tttaatgttc tttattagct taaaacattt tcatgttagc atttcttatt tttatgagca 57240

tttgctacat aaagacttca ttagagtggt gatagttagc attcacctct gttcaaccat 57300

aaattcctaa atgccccaga ggtgagacat cagagtggag cagatctggg gacctgcttc 57360

tgagtgggaa cttgagaagt ggtactctca cagagcttct gtgaagtgag gtgctgacgt 57420

tgccctgctg aaatgaaaga atggagtcca aaaagtttta actgccactc tttcttattc 57480

tttgctttga tctgcgtgaa acagaagtgt tcattttggt attgactaca aaatactagg 57540

agcagattta gggagctggt taagaatgtt gtacacttaa aacagggcat gaatgagaaa 57600

agcttgagag cactgtagaa tggagctgaa gtggaatact attgaagtca gaaagtctag 57660

ataaaattaa gttgccttat gaccagtgct tgacactgtt aacatggaga agaaatgaaa 57720

acatttctgt ttttatctaa catagctcta gttttaaaac tctatggatt tatttgttta 57780

gtaaacattt gttgaatatt tactatatac cttgctaatt aattttacta ggaacacgaa 57840

aatatggttt ttctttcttt caaaatatgg ctaatttatc atgaaacact gtggaattga 57900

tttaggcaat ggatatacca aatgaaactt tgacctttga ggatggcatg aagtttaagg 57960

aatatgaatg tgtaaaggag catggagatt atacagacaa agcatttgaa aaacttcact 58020

gcccagaagc aggtatgtgt ttcttgaaag ttgtaaatga gaaggaactg ttttattagc 58080

aacccatttt gaactctgtc cccatgcatc tgcctcggct ccactgttac ttgacccctt 58140

tctgccctct ctaagcaagg cagaaacaca cttattattc tcctgccacc catgcagtgg 58200

ccacactccc tgagatccag ccctcctctc ctgctccata cccactccct cttgcagctt 58260

tggcttctcc caggagctcc agacttacca gtctttctca ttgtcttctg ggaagctcca 58320

tggacaagtg ttgccagtat ctgaaactca gctgtgtaaa gtcaagctct tctgtgctct 58380

tcccagtgac cctttatttt ggttagtgtc acagatgcaa ctggctgggg ccagtgttgt 58440

gggcagtaaa agaatttatc aacacaattg taagtaaaga aaggcagatt tattaaagta 58500

cagagatacg ttgcaagagt gcaatgggca gcacagcaga gaagaggctg tctgctaaga 58560

ggcaggggct agagggaagt tttatagggt catattggag gagctacatg ctgataaggt 58620

gtgcagataa ggttttgctg cttgggctac atgtggaagg aatgaggtat ttgggaacag 58680

gatgtgacag cagcttgtct gtgatgagtc atctctcaga acagttgttc ccccatcccc 58740

acccccaacc tgggacccct ccctttttgt tgtttactta tcttatgaga acttcacagt 58800

cagtgctgtc accagggtgc acccttagca tagtgtctat tctgagatgt ctctggagtc 58860

ttcctcttcc ttcattctct ctgttactgg tttagggctc tgtcatctct cagtagtgtg 58920

gtgtaggctt cagagacaga tgggaattga atctcagctg ttgctgccac cttctggtta 58980

tgtgaccttt ctttcacaag ttattccaac actgaatctc agtttcacct taggaacagg 59040

ggataatagt agtaggaata accacacagg gtaattgtga ggaccaaagt gagttttgat 59100

gtataaacga cctggcacat actaggtgcc taaattaagt gctgtctttt cattttccct 59160

tttccttccc cttgctgtat tgccttattt gcttatgtga ccttctttct ctagtatttc 59220

cccttcattc tctaaatggt tactgtatta gtccattttc ttgctgctga taaagacata 59280

cctgagactg agcaatttac aaaagaaaga ggtttaattg gacttacagt tccacatggc 59340

tggggaagcc tcacaatcat ggtggaaggc aaggaaaagc aagtcacatt ttacatggat 59400

ggcagcaggc aaagagaaag aacttgtgca gaggaactcc tctttttaaa accatcttat 59460

ctcgtgagac tcattcacca tcacgagaac agcatgggaa agatccgccc ccatgattca 59520

accacctccc tctgggtcac cccacaacac acaggaattc aagatgagat ttgggtggga 59580

cacagccaaa cgatatcagt tactaaagtt atcttggcat attattactc tgctcagatt 59640

ttttttttgg ataatacctg cagaataagg tccattccac atattatcac atttaacact 59700

acatggccta attctgctgt gacccacttt tctcatccca gcatggcctc tttccttcca 59760

tggaaaatgg gatccataca gcctgctgga atgcccattt tctcctacag ctggaatgcc 59820

cattttctcc tacagcattt acagaactga cttggctcag tttcctcttc ctggaatact 59880

ctctgcctca tttccttctg gaaaaatctc cattcagcag gcatcttatt gaggatctcc 59940

tttgtgccaa agactgctca ctggtaggga gctcaaagat gaatgaaatc tgggccctgt 60000

tctcaatatc acagaagtgt tatgagcaaa aaagtcacaa aacatgtttt ctgagcctga 60060

aatgttaatc actgtttgaa gtgcgagctg ggtggagagt cagggaggtc cgcactcctc 60120

cagggcttca catgccatca tttttgtgat tgagaaggat catgctggct gcagagcaaa 60180

ggatggcatg gagggcaaga ctgaaggcag gagaagagtc caagtgcatg agccagagtg 60240

gtgcagggag aatagatact gagtgtggga actgaggaag agaaggggct caaggatatt 60300

cccagttttc taattcaaat gcatgaagct ttcatcaacc aaaaatacat cacatggagg 60360

gtaatggggt cgggagagac aaggtagtga tctaaatttg gaacatgttg agatttaggt 60420

ctatagagca tcagttgcag attctatata agactgaagg cctggggcat atcagggata 60480

aagatatagc ttggtggccc ttagcatatc cgtggttttt aactttggtg atggtcaaaa 60540

tacctatgca gaaggactgg agtgagaagg aaatggagct taggacataa ccctaccact 60600

atataaacaa actttggaga atcaggagag agtaaagcca aaggaggaga gacaggtcat 60660

ggaggaggca caggaattgg cagcatcaac tggaagagaa aggccagatg aggtgagtgg 60720

gatttggccc ttcaggagcc gttaatggcc tcagggaaag cagtcaactg tgtaaggggt 60780

aaattcaatg gttatctttg catcagtttg ctgggaaaag cagagggggt tggctgtttt 60840

ttagatgaaa gaaaaaaaaa ccttcatcag tagtatactg aaaattgtct ctcattttaa 60900

tctgtattcc tgtaattatt atttaggctg aaggattttt ccgtatgttt gttgaccatt 60960

catatttctc cttttttttt cttttttctt tttttgtttt ttttttgaga gggagtctcc 61020

ctctgtcgcc caggctggag tgcagtggca caatcttggc tcactgtaac ctccgcctcc 61080

tgggttcaag cgattctcct gcctcagcct ccctgagtag ctggcatagg tgcgcgccac 61140

cacgcctagc tgatttttaa aatattttta gtagagatga ggtttcacca tgtttgccag 61200

gctggtattt gaactcttga tctcaggtga tctgcccacc ttggcctccc aaagtgctgg 61260

gattacaggc atgagccacc acgcctggcc aacccttcat atttctgtta tgaattatgt 61320

actcatgccc ttcatccttt tttctactga aaatggcatg tttgtttttt tctttataag 61380

actgatttaa atcaaacctt tgcctgtaat atgtattgca aatgttttcc tcagttggtt 61440

gtcagatctc atttatagta ataacagcaa atatatatga gtgtgtgtgt gtgtgtgtgt 61500

gtgtgtgtgt gtgtatttgt gtattcatcc acttaggaat aaattttatg agaattgtgc 61560

ggcatataga aagaaaactg taaaacctta ctgaggtatt tacagaccac ttgaataaat 61620

ggagagaaat aacggtgcta tatattggaa atattttttc caaataaata ttgcagtatc 61680

gttgtctgag gtattaccca gaactctttg tctcacgacc aaaagaataa ggagggtgga 61740

cagtaagggt gagtttggac cgaaaattta ataaacaaaa gaggaaagct cttcactgtg 61800

gagaggggac ccaagagggt tgccatttca cagctgagta caaaggcttt tatgaggaac 61860

ctgatagggc tgggggtttc atttgcataa ggcatgaatt tctggcagct ccaccctgtt 61920

ctcctagtat gctgactggc taggggttgt ttttggaaaa ggcaccactc agaaaatgac 61980

atgatggttg accaggcatg gtagttcatg cctgtaatcc cagcactttg ggaggctgag 62040

gtgggcagat ctctcaaggc caggagttcg agactagcct ggccaatatg gctaaagccc 62100

atctctacta aaaatacaaa aattagccag gtgtggtggt gcacacctgt aatctcagct 62160

acttgggagg ctgagccaca agaatcactt ggacctggga ggtgaaggtt gcagtgagac 62220

gagattgtgc caccacactc cagtcacact ccagctgggt gacagagcaa gcaagactcc 62280

atctcaaaaa aaaaaaaaaa aaaaatgacg tggtgtaaag accagttgga gccttggccc 62340

acaaccagct gagtgttgga gtgatggttc acagaggctt ggctcacagt ccaaagtatg 62400

ccccaaaaag gaaaggaatg tgctcactgg ggcccaccat gtacatgccc acaaaaggag 62460

aaggaactat ttgctagagg cccactgatt gcacaaagaa caaaggcatt tctgtgttgg 62520

actttgctcc cttatctgtg cagctgtggg catgttttag gcaagcttcc tgtgctagtt 62580

cccttatctg tgtctgcagc ttgatttttc agactgttct tttgtttgaa agaattctga 62640

ggacctgccc taactgcctg cctaactgat tctttctttc tcctccctca atatgtggat 62700

ttatggctat ttcaatcaaa accacagtag gatttttttt ttaatggtat agggagatct 62760

tggcaggttg gagaatcctg gagcttctta agtggccaaa aattttgaaa aagaagaaca 62820

gtgaagtggt acttacattt ccaaatgtca aaatatatta cagaaattat agtcattcac 62880

acaatatgat agtagcaccc aaatagttaa aacagtgaga agagaaagtt agaaacagat 62940

cctagtatgt atcataattc agcacaaatg aaaagtaaca tcacaagtca gcgtgaaaag 63000

aaaggattat tcagataaat gctgctgggc caattggtta acagtttggg gaagattgtg 63060

aaatcagacc ctatataata tgatacaaca aaataaattt tttaaaaaag agttatatgt 63120

aaaaagttat acattagaaa atgaaataaa agaacatagg tcattttttt ttttttttga 63180

gacagcgtct cactctgtca ccaaggctgg agtgcaaagg cgtgatctcg gctcactgca 63240

aactccgcct tctgggttca agcgattctc ctgcctcagc ctcccgagta gctgggacta 63300

caggcacccg ctaccacgcc cagctaattt ttatattttt gatagagacg gggtttcacc 63360

atgttggcca ggatggtttc gatctcttga ccttgtgatc cgcccgcctc ggcctcccaa 63420

agtgctgaga ttacaggcgt gagccactgc acccggccga gttaattttt tttgaacagg 63480

gaagagctat ctgttcaaaa tacatagaaa aaaaaaccac agaataaatt agtaataatt 63540

caactttaac aacaaaaagc tgtaataaag caaatcatac taacccct 63588

4

616

Mouse

4

Met Glu Lys Tyr Val Arg Leu Gln Lys Ile Gly Glu Gly Ser Phe Gly

1 5 10 15

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

20 25 30

Lys Glu Ile Asn Ile Ser Arg Met Ser Asp Lys Glu Arg Gln Glu Ser

35 40 45

Arg Arg Glu Val Ala Val Leu Ala Asn Met Lys His Pro Asn Ile Val

50 55 60

Gln Tyr Lys Glu Ser Phe Glu Glu Asn Gly Ser Leu Tyr Ile Val Met

65 70 75 80

Asp Tyr Cys Glu Gly Gly Asp Leu Phe Lys Arg Ile Asn Ala Gln Lys

85 90 95

Gly Ala Leu Phe Gln Glu Asp Gln Ile Leu Asp Trp Phe Val Gln Ile

100 105 110

Cys Leu Ala Leu Lys His Val His Asp Arg Lys Ile Leu His Arg Asp

115 120 125

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

130 135 140

Gly Asp Phe Gly Ile Ala Arg Val Leu Asn Ser Thr Val Glu Leu Ala

145 150 155 160

Arg Thr Cys Ile Gly Thr Pro Tyr Tyr Leu Ser Pro Glu Ile Cys Glu

165 170 175

Asn Lys Pro Tyr Asn Asn Lys Ser Asp Ile Trp Ala Leu Gly Cys Val

180 185 190

Leu Tyr Glu Leu Cys Thr Leu Lys His Ala Phe Glu Ala Gly Asn Met

195 200 205

Lys Asn Leu Val Leu Lys Ile Ile Ser Gly Ser Phe Pro Pro Val Ser

210 215 220

Pro His Tyr Ser Tyr Asp Leu Arg Ser Leu Leu Ser Gln Leu Phe Lys

225 230 235 240

Arg Asn Pro Arg Asp Arg Pro Ser Val Asn Ser Ile Leu Glu Lys Gly

245 250 255

Phe Ile Ala Lys Arg Ile Glu Lys Phe Leu Ser Pro Gln Leu Ile Ala

260 265 270

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

275 280 285

Pro Gly Lys Arg Pro Ala Ser Gly Gln Gly Val Ser Ser Phe Val Pro

290 295 300

Ala Gln Lys Ile Thr Lys Pro Ala Ala Lys Tyr Gly Val Pro Leu Thr

305 310 315 320

Tyr Lys Lys Tyr Gly Asp Lys Lys Leu Leu Glu Lys Lys Pro Pro Pro

325 330 335

Lys His Lys Gln Ala His Gln Ile Pro Val Lys Lys Met Asn Ser Gly

340 345 350

Glu Glu Arg Lys Lys Met Ser Glu Glu Ala Ala Lys Lys Arg Arg Leu

355 360 365

Glu Phe Ile Glu Lys Glu Lys Lys Gln Lys Asp Gln Ile Arg Phe Leu

370 375 380

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

385 390 395 400

Asn Arg Ala Arg Glu Gln Gly Trp Arg Asn Val Leu Arg Ala Gly Gly

405 410 415

Ser Gly Glu Val Lys Ala Ser Phe Phe Gly Ile Gly Gly Ala Val Ser

420 425 430

Pro Ser Pro Cys Ser Pro Arg Gly Gln Tyr Glu His Tyr His Ala Ile

435 440 445

Phe Asp Gln Met Gln Arg Leu Arg Ala Glu Asp Asn Glu Ala Arg Trp

450 455 460

Lys Gly Gly Ile Tyr Gly Arg Trp Leu Pro Glu Arg Gln Lys Gly His

465 470 475 480

Leu Ala Val Glu Arg Ala Asn Gln Val Glu Glu Phe Leu Gln Arg Lys

485 490 495

Arg Glu Ala Met Gln Asn Lys Ala Arg Ala Glu Gly His Val Val Tyr

500 505 510

Leu Ala Arg Leu Arg Gln Ile Arg Leu Gln Asn Phe Asn Glu Arg Gln

515 520 525

Gln Ile Lys Ala Lys Leu Arg Gly Glu Asn Lys Glu Ala Asp Gly Thr

530 535 540

Lys Gly Gln Glu Ala Thr Glu Glu Thr Asp Met Arg Leu Lys Lys Met

545 550 555 560

Glu Ser Leu Lys Ala Gln Thr Asn Ala Arg Ala Ala Val Leu Lys Glu

565 570 575

Gln Leu Glu Arg Lys Arg Lys Glu Ala Tyr Glu Arg Glu Lys Lys Val

580 585 590

Trp Glu Glu His Leu Val Ala Arg Val Lys Ser Ser Asp Val Pro Leu

595 600 605

Pro Leu Glu Leu Leu Glu Thr Gly

610 615