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

Abstract

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

Description

FIELD OF THE INVENTION

The present invention is in the field of kinase proteins that are related to the MEK kinase alpha 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.

BACKGROUND OF THE INVENTION

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 thought to account for the effects of cyclic-AMP in most of these cells. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison's Principles of Internal Medicine , McGraw-Hill, New York, N.Y., pp. 416-431, 1887).

Calcium-calmodulin (CaM) dependent protein kinases are also members of STK family. Calmodulin is a calcium receptor that mediates many calcium regulated processes by binding to target proteins in response to the binding of calcium. The principle target protein in these processes is CaM dependent protein kinases. CaM-kinases are involved in regulation of smooth muscle contraction (MLC kinase), glycogen breakdown (phosphorylase kinase), and neurotransmission (CaM kinase I and CaM kinase II). CaM kinase I phosphorylates a variety of substrates including the neurotransmitter related proteins synapsin I and II, the gene transcription regulator, CREB, and the cystic fibrosis conductance regulator protein, CFTR (Haribabu, B. et al. (1995) EMBO Journal 14:3679-86). 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, hyperosmolar medium, heat shock, endotoxic lipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).

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

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

Protein tyrosine kinases, PTKs, specifically phosphorylate tyrosine residues on their target proteins and may be divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembrane protein-tyrosine kinases are receptors for most growth factors. Binding of growth factor to the receptor activates the transfer of a phosphate group from ATP to selected tyrosine side chains of the receptor and other specific proteins. Growth factors (GF) associated with receptor PTKs include; epidermal GF, platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascular endothelial GF, and macrophage colony stimulating factor.

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

Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells where their activation was no longer subject to normal cellular controls. In fact, about one third of the known oncogenes encode PTKs, and it is well known that cellular transformation (oncogenesis) is often accompanied by increased tyrosine phosphorylation activity (Carbonneau H and Tonks 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.

MEKKα, probably encodes a MEK kinase, since it has very high homology in the kinase domain to known MEKKs, the first kinase in MAP kinase cascades. MEKKα plays a key role in a new regulatory pathway by which cell-type differentiation, morphogenesis, spatial patterning, and developmental timing are controlled. The components of three MAP kinase pathways required for chemotaxis, activation of adenylyl cyclase, and prespore cell differentiation have been identified in Dictyostelium. These pathways seem to be independent pathways and are unrelated to the pathway containing MEKKα. MEKKα protein contains an F-box and a WD40 repeats. The F-box has a domain known to control ubiquitin-mediated degradation of proteins. WD40 repeats are important for targeting MEKKα to the cell cortex or possibly the plasma membrane. Cells deficient in MEKKα, develop precociously and exhibit abnormal cell-type patterning with an increase in one of the prestalk compartments (pstO), a concomitant reduction in the prespore domain, and a loss of the sharp compartment boundaries, resulting in overlapping prestalk and prespore domains. Overexpression of MEKKα, or MEKKα lacking the WD40 repeats results in very delayed development and a severe loss of compartment boundaries. MEKKα activity is differentially regulated temporally and in a cell-type-specific fashion via developmentally regulated ubiquitination/deubiquitination, wherein MAP kinase cascade components can be controlled. Cells lacking the ubiquitin hydrolase have phenotypes similar to those of MEKKα, null (MEKKα-) cells, which indicates a direct genetic and biochemical interaction between MEKKα, the UBC, and the UBP. UBC and UBP differentially control MEKKα ubiquitination/deubiquitination and degradation through the F-box/WD40 repeats in a cell-type-specific and temporally regulated manner. (Chung et al., Genes Dev Nov. 15, 1998;12(22):3564-78).

Kinase proteins, particularly members of the MEK kinase alpha 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 MEK kinase alpha subfamily.

SUMMARY OF THE INVENTION

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 MEK kinase alpha 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 the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte).

DESCRIPTION OF THE FIGURE SHEETS

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 the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte).

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, including insertion/deletion variants (“indels”), were identified at 35 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

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 MEK kinase alpha 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 MEK kinase alpha 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 MEK kinase alpha subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). 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 MEK kinase alpha 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 MEK kinase alpha 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 the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). 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 2 by ePCR.

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 2 by ePCR. 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 identified in a gene encoding the kinase protein of the present invention. 35 SNP variants were found, including 6 indels (indicated by a “−”) and 3 SNPs in exons.

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

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

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

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

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

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

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

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

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

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

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

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

Protein/Peptide Uses

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

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

Substantial chemical and structural homology exists between the MEK kinase alpha protein described herein and MEKK alpha in Dictyostelium (see FIG. 1 ). As discussed in the background, Dictyostelium MEKK alpha is known in the art to be involved in cell signaling, cell differentiation. Accordingly, the MEK kinase alpha protein, and the encoding gene, provided by the present invention is useful for treating, preventing, and/or diagnosing diseases or other disorders associated with regulatory pathway, such as cancer.

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 the multiple sclerosis lesions by a virtual northern blot analysis. In addition, PCR-based tissue screening panel indicates expression in the mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). A large percentage of pharmaceutical agents are being developed that modulate the activity of kinase proteins, particularly members of the MEK kinase alpha 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 the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). 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 MEK kinase alpha 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 the multiple sclerosis lesions by a virtual northern blot analysis. In addition, PCR-based tissue screening panel indicates expression in the mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte).

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 the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). In an alternate embodiment, cell-based assays involve recombinant host cells expressing the kinase protein.

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

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

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

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

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

Any of the biological or biochemical functions mediated by the kinase can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2 . Specifically, a biological function of a cell or tissues that expresses the kinase can be assayed. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the multiple sclerosis lesions by a virtual northern blot analysis. In addition, PCR-based tissue screening panel indicates expression in the mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte).

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

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

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

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

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

Modulators of kinase protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the kinase pathway, by treating cells or tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). 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 the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). The method involves contacting a biological sample with a compound capable of interacting with the kinase protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

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

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

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

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

The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). Accordingly, methods for treatment include the use of the kinase protein or fragments.

Antibodies

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

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

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

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

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

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

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

Antibody Uses

The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the multiple sclerosis lesions by a virtual northern blot analysis. In addition, PCR-based tissue screening panel indicates expression in the mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). 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 the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). 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 the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). 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 the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

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

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

Nucleic Acid Molecules

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

As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 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 these nucleic acid molecules can be readily made/isolated is provided below.

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

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

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

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (antisense 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 2 by ePCR.

FIG. 3 provides information on SNPs that have been identified in a gene encoding the kinase protein of the present invention. 35 SNP variants were found, including 6 indels (indicated by a “−”) and 3 SNPs in exons.

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

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2 . As illustrated in FIG. 3 , SNPs, including insertion/deletion variants (“indels”), were identified at 35 different nucleotide 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′ non-coding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

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

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

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

The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. As indicated by the data presented in FIG. 3 , the map position was determined to be on chromosome 2 by ePCR.

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 in the multiple sclerosis lesions by a virtual northern blot analysis. In addition, PCR-based tissue screening panel indicates expression in the mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in kinase protein expression relative to normal results.

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

Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a kinase protein, such as by measuring a level of a kinase-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a kinase gene has been mutated. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the multiple sclerosis lesions by a virtual northern blot analysis. In addition, PCR-based tissue screening panel indicates expression in the mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte).

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 the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). The method typically includes assaying the ability of the compound to modulate the expression of the kinase nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired kinase nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the kinase nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

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

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

The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate kinase nucleic acid expression in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the multiple sclerosis lesions by a virtual northern blot analysis. In addition, PCR-based tissue screening panel indicates expression in the mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

Alternatively, a modulator for kinase nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the kinase nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in the multiple sclerosis lesions and mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte).

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

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

Individuals carrying mutations in the kinase gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been identified in a gene encoding the kinase protein of the present invention. 35 SNP variants were found, including 6 indels (indicated by a “−”) and 3 SNPs in exons. As indicated by the data presented in FIG. 3 , the map position was determined to be on chromosome 2 by ePCR. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241: 1077-1080 (1988); and Nakazawa et al., PNAS 91 :360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

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

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

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

Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 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 techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the kinase gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been identified in a gene encoding the kinase protein of the present invention. 35 SNP variants were found, including 6 indels (indicated by a “−”) and 3 SNPs in exons.

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

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

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

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

The invention also encompasses kits for detecting the presence of a kinase nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the multiple sclerosis lesions by a virtual northern blot analysis. In addition, PCR-based tissue screening panel indicates expression in the mixed tissue (brain, heart, kidney, lung, spleen, testis, leukocyte). 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 cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

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

In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application 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 identified in a gene encoding the kinase protein of the present invention. 35 SNP variants were found, including 6 indels (indicated by a “−”) and 3 SNPs in exons.

Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques , Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry , Academic Press, Orlando, Fla. Vol. 1 (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 covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

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

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

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

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

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

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

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

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

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

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

As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 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 proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli . (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kujan 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., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

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

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

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

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

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

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

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

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

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

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

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

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

Uses of Vectors and Host Cells

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

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

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

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

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

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

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

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

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

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

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

#             SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 7
<210> SEQ ID NO 1
<211> LENGTH: 4307
<212> TYPE: DNA
<213> ORGANISM: Human
<400> SEQUENCE: 1
tttccttgga tttccagttt tccacccagc tctgaagaca ctgttggtac tt
#aaaaatat     60
ttaactaaga ctgtgtcatt ttgcaggttg ttggatttct tctggaaaag tg
#agtagata    120
tcaccctttg caattacagc aatcgaaccg caattcatgt agctaattgc aa
#tatccaaa    180
gacaactctt ggcagtcaat agaatccagg ctccccaaat gcaacttcta ca
#aagttcat    240
ggcaaggtga tcttgagcaa gttcaacatt tactgagatc ctaaactttg tg
#attttagt    300
ggaaaatcag caatacatta tgtgtcacaa atagagagtt caaagaaaca gc
#agcttttg    360
gacattttaa tgagttctat gccaaaacca gaaagacatg ctgagtcatt gc
#ttgacatt    420
tgtcatgata caaactcttc tccaactgat ttgatgacag ttaccaaaaa tc
#aaaacatc    480
atcttgcaaa gcatcagcag aagtgaggag ttcgaccaag atggtgactg ca
#gtcattcc    540
acactggtta atgaagaaga agatcccagt ggtggtagac aggactggca ac
#ccaggaca    600
gaaggtgttg agatcactgt aacttttcca agagatgtca gtcctcccca ag
#aaatgagc    660
caagaagact taaaagaaaa gaatctgata aactcatcgc ttcaagaatg gg
#cacaagca    720
catgcagttt ctcatccaaa tgaaatagaa acggtggagc tcaggaaaaa ga
#agctgacc    780
atgcggccct tagttttgca aaaagaggaa agttccaggg agctctgcaa tg
#tgaacttg    840
ggctttttgc taccaagatc ttgtttagaa ctgaacattt ccaagtctgt aa
#ccagagaa    900
gatgctcctc attttctgaa ggagcagcaa agaaaatctg aagagttttc ga
#cctctcat    960
atgaagtaca gtggccgaag catcaagttc cttctgccac cactgtcact ct
#tgcccacg   1020
cgatctggtg tccttactat cccccaaaat cacaagtttc caaaagaaaa ag
#aaagaaac   1080
attccaagtc tcacatcttt tgtgcctaag ctctcagtgt ctgttcgtca at
#ctgatgag   1140
ctcagcccat caaacgagcc tccgggagcc ctagttaagt cgttgatgga tc
#cgactctc   1200
aggtcttctg atggcttcat ttggtcaaga aacatgtgct cttttcctaa ga
#ctaaccat   1260
cacaggcaat gcctggagaa ggaggaaaac tggaaatcca aggaaataga ag
#aatgtaac   1320
aaaattgaaa tcactcactt tgaaaaaggg cagtctttgg tgtcttttga ga
#atttgaag   1380
gaaggcaata ttcctgcagt tagggaagag gatattgact gccatggtag ta
#aaacgcga   1440
aaacctgaag aagagaactc tcaatatctt tcatcaagaa agaatgagag tt
#cagtagcc   1500
aaaaactatg aacaagatcc agaaatagta tgtaccattc caagcaagtt cc
#aagaaacc   1560
cagcattcag aaataactcc aagccaggat gaagagatga gaaataataa ag
#ctgcttca   1620
aaaagagttt cattacataa aaatgaagca atggaaccaa acaatatttt ag
#aagagtgt   1680
actgtactta aaagcttatc cagtgtagtc tttgatgacc ccattgataa ac
#tcccagaa   1740
ggttgtagca gcatggagac aaacataaaa atatcaatag cagaaagagc ca
#aaccagaa   1800
atgagtagga tggtgcctct tatccacatc accttccctg tggatggaag cc
#ccaaggaa   1860
ccagtgatag ccaaaccaag cctccaaaca agaaagggaa ccattcataa ca
#accatagt   1920
gtcaacatac ctgtacacca agaaaatgac aagcataaga tgaattccca ta
#ggagcaga   1980
cgtatcacca ataaatgtcg atcttcacac agtgagagga agagcaatat ca
#gaacaaga   2040
ctttctcaga aaaaaacaca tatgaaatgc ccaaagactt catttggcat ta
#aacaagag   2100
cacaaagtct taatttctaa agaaaagagt tccaaggctg tacatagcaa cc
#tacatgac   2160
attgaaaatg gtgatggtat ttcagaacca gactggcaga taaagtcttc ag
#gaaatgag   2220
tttctatctt ccaaagatga aattcatccc atgaacttgg ctcagacacc tg
#agcagtcc   2280
atgaaacaga atgaattccc tcctgtctca gatttatcca ttgttgaaga ag
#tttctatg   2340
gaagagtcta ctggtgatag agacatttct aacaatcaaa tactcaccac aa
#gcctcaga   2400
gatctgcaag aacttgaaga gctacatcac cagatcccat ttatcccttc ag
#aagacagc   2460
tgggcagtgc ccagtgagaa gaattctaac aagtatgtac agcaagaaaa gc
#agaataca   2520
gcatctctta gtaaagtaaa tgccagccga attttaacta atgatctaga gt
#ttgatagt   2580
gtttcagatc actctaaaac acttacaaat ttctctttcc aagcaaaaca ag
#aaagtgca   2640
tcttcccaga catatcaata ttgggtacat tatttggatc atgatagttt ag
#caaataag   2700
tcaatcacat atcaaatgtt tggaaaaacc ttaagtggca caaattcaat tt
#cccaagaa   2760
attatggact ctgtaaataa tgaagaattg acagatgaac tattaggttg tc
#tagctgca   2820
gaattattag ctcttgatga gaaagataac aactcttgcc aaaaaatggc aa
#atgaaaca   2880
gatcctgaaa acctaaatct tgtcctcaga tggagaggaa gtaccccaaa ag
#aaatgggc   2940
agagagacaa caaaagtcaa aatacagagg catagtagtg ggctcaggat at
#atgacagg   3000
gaggagaaat ttctcatctc aaatgaaaag aagatatttt ctgaaaatag tt
#taaagtct   3060
gaagaaccta tcctatggac caagggtgag attcttggaa agggagccta cg
#gcacagta   3120
tactgtggtc tcactagtca aggacagcta atagctgtaa aacaggtggc tt
#tggatacc   3180
tctaataaat tagctgctga aaaggaatac cggaaactac aggaagaagt ag
#atttgctc   3240
aaagcactga aacatgtcaa cattgtggcc tatttgggga catgcttgca ag
#agaacact   3300
gtgagcattt tcatggagtt tgttcctggt ggctcaatct ctagtattat aa
#accgtttt   3360
gggccattgc ctgagatggt gttctgtaaa tatacgaaac aaatacttca ag
#gtgttgct   3420
tatctccatg agaactgtgt ggtacatcgc gatatcaaag gaaataatgt ta
#tgctcatg   3480
ccaactggaa taataaagct gattgacttt ggctgtgcca ggcgtttggc ct
#gggcaggt   3540
ttaaatggca cccacagtga catgcttaag tccatgcatg ggactccata tt
#ggatggcc   3600
ccagaagtca tcaatgagtc tggctatgga cggaaatcag atatctggag ca
#ttggttgt   3660
actgtgtttg agatggctac agggaagcct ccactggctt ccatggacag ga
#tggccgcc   3720
atgttttaca tcggagcaca ccgagggctg atgcctcctt taccagacca ct
#tctcagaa   3780
aatgcagcag actttgtgcg catgtgcctg accagggacc agcatgagcg ac
#cttctgct   3840
ctccagctcc tgaagcactc cttcttggag agaagtcact gaatatacat ca
#agactttc   3900
ttcccagttc cactgcagat gctcccttgc ttaattgtgg ggaatgatgg ct
#aagggatc   3960
tttgtttccc cactgaaaat tcagtctaac ccagtttaag cagatcctat gg
#agtcatta   4020
actgaaagtt gcagttacat attagcctcc tcaagtgtca gacattatta ct
#catagtat   4080
cagaaaacat gttcttaata acaacaaaaa actatttcag tgtttacagt tt
#tgattgtc   4140
caggaactac attctctagt gttttatatg acatttcttt ttatttttgg cc
#tgtcctgt   4200
caattttaat gttgttagtt taaaataaat tgtaaaaaca ccttaaaaaa aa
#aaaaaaaa   4260
aaaaaaaaaa aaaacatgtc ggccgcctcg gcccagtcga ctctaga
#              4307
<210> SEQ ID NO 2
<211> LENGTH: 1167
<212> TYPE: PRT
<213> ORGANISM: Human
<400> SEQUENCE: 2
Met Pro Lys Pro Glu Arg His Ala Glu Ser Le
#u Leu Asp Ile Cys His
1               5
#                10
#                15
Asp Thr Asn Ser Ser Pro Thr Asp Leu Met Th
#r Val Thr Lys Asn Gln
20
#            25
#            30
Asn Ile Ile Leu Gln Ser Ile Ser Arg Ser Gl
#u Glu Phe Asp Gln Asp
35
#        40
#        45
Gly Asp Cys Ser His Ser Thr Leu Val Asn Gl
#u Glu Glu Asp Pro Ser
50
#    55
#    60
Gly Gly Arg Gln Asp Trp Gln Pro Arg Thr Gl
#u Gly Val Glu Ile Thr
65
#70
#75
#80
Val Thr Phe Pro Arg Asp Val Ser Pro Pro Gl
#n Glu Met Ser Gln Glu
85
#                90
#                95
Asp Leu Lys Glu Lys Asn Leu Ile Asn Ser Se
#r Leu Gln Glu Trp Ala
100
#           105
#           110
Gln Ala His Ala Val Ser His Pro Asn Glu Il
#e Glu Thr Val Glu Leu
115
#       120
#       125
Arg Lys Lys Lys Leu Thr Met Arg Pro Leu Va
#l Leu Gln Lys Glu Glu
130
#   135
#   140
Ser Ser Arg Glu Leu Cys Asn Val Asn Leu Gl
#y Phe Leu Leu Pro Arg
145                 1
#50                 1
#55                 1
#60
Ser Cys Leu Glu Leu Asn Ile Ser Lys Ser Va
#l Thr Arg Glu Asp Ala
165
#               170
#               175
Pro His Phe Leu Lys Glu Gln Gln Arg Lys Se
#r Glu Glu Phe Ser Thr
180
#           185
#           190
Ser His Met Lys Tyr Ser Gly Arg Ser Ile Ly
#s Phe Leu Leu Pro Pro
195
#       200
#       205
Leu Ser Leu Leu Pro Thr Arg Ser Gly Val Le
#u Thr Ile Pro Gln Asn
210
#   215
#   220
His Lys Phe Pro Lys Glu Lys Glu Arg Asn Il
#e Pro Ser Leu Thr Ser
225                 2
#30                 2
#35                 2
#40
Phe Val Pro Lys Leu Ser Val Ser Val Arg Gl
#n Ser Asp Glu Leu Ser
245
#               250
#               255
Pro Ser Asn Glu Pro Pro Gly Ala Leu Val Ly
#s Ser Leu Met Asp Pro
260
#           265
#           270
Thr Leu Arg Ser Ser Asp Gly Phe Ile Trp Se
#r Arg Asn Met Cys Ser
275
#       280
#       285
Phe Pro Lys Thr Asn His His Arg Gln Cys Le
#u Glu Lys Glu Glu Asn
290
#   295
#   300
Trp Lys Ser Lys Glu Ile Glu Glu Cys Asn Ly
#s Ile Glu Ile Thr His
305                 3
#10                 3
#15                 3
#20
Phe Glu Lys Gly Gln Ser Leu Val Ser Phe Gl
#u Asn Leu Lys Glu Gly
325
#               330
#               335
Asn Ile Pro Ala Val Arg Glu Glu Asp Ile As
#p Cys His Gly Ser Lys
340
#           345
#           350
Thr Arg Lys Pro Glu Glu Glu Asn Ser Gln Ty
#r Leu Ser Ser Arg Lys
355
#       360
#       365
Asn Glu Ser Ser Val Ala Lys Asn Tyr Glu Gl
#n Asp Pro Glu Ile Val
370
#   375
#   380
Cys Thr Ile Pro Ser Lys Phe Gln Glu Thr Gl
#n His Ser Glu Ile Thr
385                 3
#90                 3
#95                 4
#00
Pro Ser Gln Asp Glu Glu Met Arg Asn Asn Ly
#s Ala Ala Ser Lys Arg
405
#               410
#               415
Val Ser Leu His Lys Asn Glu Ala Met Glu Pr
#o Asn Asn Ile Leu Glu
420
#           425
#           430
Glu Cys Thr Val Leu Lys Ser Leu Ser Ser Va
#l Val Phe Asp Asp Pro
435
#       440
#       445
Ile Asp Lys Leu Pro Glu Gly Cys Ser Ser Me
#t Glu Thr Asn Ile Lys
450
#   455
#   460
Ile Ser Ile Ala Glu Arg Ala Lys Pro Glu Me
#t Ser Arg Met Val Pro
465                 4
#70                 4
#75                 4
#80
Leu Ile His Ile Thr Phe Pro Val Asp Gly Se
#r Pro Lys Glu Pro Val
485
#               490
#               495
Ile Ala Lys Pro Ser Leu Gln Thr Arg Lys Gl
#y Thr Ile His Asn Asn
500
#           505
#           510
His Ser Val Asn Ile Pro Val His Gln Glu As
#n Asp Lys His Lys Met
515
#       520
#       525
Asn Ser His Arg Ser Arg Arg Ile Thr Asn Ly
#s Cys Arg Ser Ser His
530
#   535
#   540
Ser Glu Arg Lys Ser Asn Ile Arg Thr Arg Le
#u Ser Gln Lys Lys Thr
545                 5
#50                 5
#55                 5
#60
His Met Lys Cys Pro Lys Thr Ser Phe Gly Il
#e Lys Gln Glu His Lys
565
#               570
#               575
Val Leu Ile Ser Lys Glu Lys Ser Ser Lys Al
#a Val His Ser Asn Leu
580
#           585
#           590
His Asp Ile Glu Asn Gly Asp Gly Ile Ser Gl
#u Pro Asp Trp Gln Ile
595
#       600
#       605
Lys Ser Ser Gly Asn Glu Phe Leu Ser Ser Ly
#s Asp Glu Ile His Pro
610
#   615
#   620
Met Asn Leu Ala Gln Thr Pro Glu Gln Ser Me
#t Lys Gln Asn Glu Phe
625                 6
#30                 6
#35                 6
#40
Pro Pro Val Ser Asp Leu Ser Ile Val Glu Gl
#u Val Ser Met Glu Glu
645
#               650
#               655
Ser Thr Gly Asp Arg Asp Ile Ser Asn Asn Gl
#n Ile Leu Thr Thr Ser
660
#           665
#           670
Leu Arg Asp Leu Gln Glu Leu Glu Glu Leu Hi
#s His Gln Ile Pro Phe
675
#       680
#       685
Ile Pro Ser Glu Asp Ser Trp Ala Val Pro Se
#r Glu Lys Asn Ser Asn
690
#   695
#   700
Lys Tyr Val Gln Gln Glu Lys Gln Asn Thr Al
#a Ser Leu Ser Lys Val
705                 7
#10                 7
#15                 7
#20
Asn Ala Ser Arg Ile Leu Thr Asn Asp Leu Gl
#u Phe Asp Ser Val Ser
725
#               730
#               735
Asp His Ser Lys Thr Leu Thr Asn Phe Ser Ph
#e Gln Ala Lys Gln Glu
740
#           745
#           750
Ser Ala Ser Ser Gln Thr Tyr Gln Tyr Trp Va
#l His Tyr Leu Asp His
755
#       760
#       765
Asp Ser Leu Ala Asn Lys Ser Ile Thr Tyr Gl
#n Met Phe Gly Lys Thr
770
#   775
#   780
Leu Ser Gly Thr Asn Ser Ile Ser Gln Glu Il
#e Met Asp Ser Val Asn
785                 7
#90                 7
#95                 8
#00
Asn Glu Glu Leu Thr Asp Glu Leu Leu Gly Cy
#s Leu Ala Ala Glu Leu
805
#               810
#               815
Leu Ala Leu Asp Glu Lys Asp Asn Asn Ser Cy
#s Gln Lys Met Ala Asn
820
#           825
#           830
Glu Thr Asp Pro Glu Asn Leu Asn Leu Val Le
#u Arg Trp Arg Gly Ser
835
#       840
#       845
Thr Pro Lys Glu Met Gly Arg Glu Thr Thr Ly
#s Val Lys Ile Gln Arg
850
#   855
#   860
His Ser Ser Gly Leu Arg Ile Tyr Asp Arg Gl
#u Glu Lys Phe Leu Ile
865                 8
#70                 8
#75                 8
#80
Ser Asn Glu Lys Lys Ile Phe Ser Glu Asn Se
#r Leu Lys Ser Glu Glu
885
#               890
#               895
Pro Ile Leu Trp Thr Lys Gly Glu Ile Leu Gl
#y Lys Gly Ala Tyr Gly
900
#           905
#           910
Thr Val Tyr Cys Gly Leu Thr Ser Gln Gly Gl
#n Leu Ile Ala Val Lys
915
#       920
#       925
Gln Val Ala Leu Asp Thr Ser Asn Lys Leu Al
#a Ala Glu Lys Glu Tyr
930
#   935
#   940
Arg Lys Leu Gln Glu Glu Val Asp Leu Leu Ly
#s Ala Leu Lys His Val
945                 9
#50                 9
#55                 9
#60
Asn Ile Val Ala Tyr Leu Gly Thr Cys Leu Gl
#n Glu Asn Thr Val Ser
965
#               970
#               975
Ile Phe Met Glu Phe Val Pro Gly Gly Ser Il
#e Ser Ser Ile Ile Asn
980
#           985
#           990
Arg Phe Gly Pro Leu Pro Glu Met Val Phe Cy
#s Lys Tyr Thr Lys Gln
995
#       1000
#      1005
Ile Leu Gln Gly Val Ala Tyr Leu His Glu As
#n Cys Val Val His Arg
1010
#   1015
#  1020
Asp Ile Lys Gly Asn Asn Val Met Leu Met Pr
#o Thr Gly Ile Ile Lys
1025                1030
#                1035
#               1040
Leu Ile Asp Phe Gly Cys Ala Arg Arg Leu Al
#a Trp Ala Gly Leu Asn
1045
#               1050
#              1055
Gly Thr His Ser Asp Met Leu Lys Ser Met Hi
#s Gly Thr Pro Tyr Trp
1060
#           1065
#          1070
Met Ala Pro Glu Val Ile Asn Glu Ser Gly Ty
#r Gly Arg Lys Ser Asp
1075
#       1080
#      1085
Ile Trp Ser Ile Gly Cys Thr Val Phe Glu Me
#t Ala Thr Gly Lys Pro
1090
#   1095
#  1100
Pro Leu Ala Ser Met Asp Arg Met Ala Ala Me
#t Phe Tyr Ile Gly Ala
1105                1110
#                1115
#               1120
His Arg Gly Leu Met Pro Pro Leu Pro Asp Hi
#s Phe Ser Glu Asn Ala
1125
#               1130
#              1135
Ala Asp Phe Val Arg Met Cys Leu Thr Arg As
#p Gln His Glu Arg Pro
1140
#           1145
#          1150
Ser Ala Leu Gln Leu Leu Lys His Ser Phe Le
#u Glu Arg Ser His
1155
#       1160
#      1165
<210> SEQ ID NO 3
<211> LENGTH: 64467
<212> TYPE: DNA
<213> ORGANISM: Human
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(64467)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 3
gctggctgtg agagatgtgg acctgtttga gagtcttgac atgttaacag tg
#tacaaacc     60
tgtggaagtt ctgtcccagc tcctaaggca tcatgcgtga atatgagcag tt
#agtcagcc    120
cagctgaagg gtgtcaattc aattgttatt tacagaaatc acatgtaaac cg
#agacacaa    180
agcttctttt ttaccctttc cctccctccc tcccatcctt ttctttcttt ct
#tttctttc    240
tttctttttt ctttctttct ctctctcttt ctttctttct ctctttcttt ct
#ttctttct    300
ttatttctct gtctctttct tttccctctc cttccttcct tccttccttt ct
#ctctctct    360
ctctttcttt cttcctttcc tcttttttat acaggatctt gctctgttgc ct
#aggctgga    420
gtgcagtgat gcaatcatag ctcactgtga cctcaaactc ctgggctcca tg
#gatcctcc    480
tgcctcagcc tctcgagtag ctggaactac aggcacatac cactatgccc gg
#ctaatttt    540
taattttttg tggagatgga gtcccactat attgcccatg ctggtctcaa ac
#ccctgccc    600
tcaagctgct ctcccatctt ggcctcccaa gctgtggaga ttacaggctg tt
#ttctacta    660
tatatgccaa atgcacatgc atcatcataa aagtgacttc acaattgcaa ag
#tgatgtgc    720
agtttctaaa atttgctacc tattattctt atgatatctg gctctttgtt tc
#atttcttg    780
aaatgattac tgttctggta gttactggga atgtcaaata atttcttgag ta
#tccagctc    840
tctaccccca agatattact aattatttca gaaaacactg tcaatgtctg aa
#aagcaatt    900
tataatagtg ttttcaagtt atcttaaaat tactatatgt caaatgctct tt
#taggaggg    960
aggagataaa caatgcactt tttttttaaa taagagggtt aataagcaat ct
#cttatgtt   1020
acaattgcag tttcctaaag ctgttactta gttatcttgt catcaaataa ga
#acagatgg   1080
cctgagctct ttctcagtac ttcatatgaa ttttgttttg aaaaaaaaag ga
#ggagggag   1140
cttcaagaac aaaattatag tcaagaatac aagatattgt aaaaggatca gt
#tagtataa   1200
tggaatgaaa agggaatttt gaagctactt cagcctagtg ttgagaaata gt
#ttggccaa   1260
ttgataaaag tggagattcc tgggacgtca tcccagagat gttcagtagg tc
#tggattgg   1320
ggtccagaat actggaaatc aaggtatgcc acttggagac accctaatct ag
#gcagatga   1380
ggagaggccc caatgagttt atctttactt gtttttatgc acccttaaat aa
#ttataaaa   1440
atttttgtcc aaagttggga attctctgca aatatgataa gtggcttgct ta
#aagccata   1500
tatcaaggta gtggcaaccc caattctcag tcctatgcta tttcttttga at
#tacaatct   1560
ttgatgaaga aaagtccata agagaatatt actgtggctc atgacacatt ac
#cctgtccc   1620
atagcaacga agagattcaa attcaaatgt tttaggacag agaccatgat ca
#acttgctc   1680
cttgtcctag aataggataa gtaaagcaag tttcatcatt gtttccctca ct
#gtaatcta   1740
ttaatgggat tctcatcatt taactttgga tttctctgag ctgatatcta at
#gcaagggt   1800
tcagtacaac atagagagga taagaagaga cttgtgctgt cataatagag ag
#gataagaa   1860
gagacttgtt ctgttgtaaa tggtcctaag atcagccagt tgggcttacc aa
#ccacaaag   1920
ccaggtaaag aggaatgaaa aggccatgtg ggggctgggc gcggtggctc ac
#gcctgtaa   1980
tcccagcact ttgggaggcc gaggcaggca gatcacgagg tcaggagttc ga
#gaccatcc   2040
tggctaacac ggtgaaaccc cgtctctact aaaaatacaa aaaaattagc cg
#ggcatggt   2100
ggcgggcccc tgtagtccca gctactctgg aggctgaggc aggagaatgg cg
#tgaacccg   2160
ggaggcagag cttgcagtga gccgagatcg cgccactgca ctccagcctg gg
#tgacagag   2220
caagactccg cctcaaaaaa aaaaaaaaaa aaaaaaaaag gaaaagaaaa gg
#ccatgtgg   2280
agaggcacac tttggttttt atgacaagat tgctccactc atccaagaga cc
#atgaaata   2340
aaagtatcag cttaatttta aagagaagat tctatgccat tccaccattt tg
#aatcataa   2400
aagagctagc tgttagcatt agaaaaaaga aatatcaaaa aagtcagcag tt
#agcttaat   2460
tattgaaaag aaaaaaatca agtgagctat ttggaatgat aagacaatca tt
#tatcaaaa   2520
tgttttaatc cttatgactc attgaaaaaa atttaaaaat ataaaaaaaa ac
#aacaaaag   2580
atgtttttat ctttacttga ttttatgtac ccttaaagaa ttataaaaat tt
#ttgtccaa   2640
agttgggaat tctctgcaaa cctcagaatg tttttagaat ggggatggga at
#aaagatac   2700
acagcaaatt ccttttattt aaaatcttgt aaaattgtca tcctctattc ac
#acattttg   2760
aaatcattat tattatcccc aaactacata agattacttt ttatttattt ga
#tgtaaatg   2820
tttccctctc atattagttt tcttttttca acagattcaa ctaaataaac tt
#taatgttg   2880
attctgttct tcctagagat cctaaacttt gtgattttag tggaaaatca gc
#aatacatt   2940
atgtgtcaca aatagagagt tcaaagaaac agcagctttt ggacatttta at
#gagttcta   3000
tgccaaaacc aggtaaatac tttcactcca catgcataat ttcccaaccc aa
#aaattcct   3060
gttagatctc tttctttttc tacatctgtt tgatggatcc atttaaagaa at
#caagtcca   3120
cggctattta tggagcagtg actctgtaca aggcccacga ctaggtgctc tg
#ggggatgc   3180
aaagaagtct aacgcaaggt ttaaactttt gaaaatatac agcatggtca gg
#ggcacatc   3240
acataagtca gagagagctt gctgtaaact tgaaagggga agagggactt at
#agtggttc   3300
ttgaaggctg gataacagtg ggaaggtttg atataggtag gaaaagagtc ca
#aacaaaga   3360
caaagaaaca gccacagcaa gaagtataat gaaaagtgtg ccactgagca gc
#gtgtgact   3420
ttgtgaaagc tgcctgactt tattgtttga ttcgctttct gtttgaagct tc
#gggggcag   3480
aggacaaagc tatacctaag aaggtttcat gaaagaggtg agacttgatc tg
#acctttga   3540
aaaaaggatg caatttgatt ttgtggagca gaggcccctt gctgggagtg ag
#catagctt   3600
atcccagggg caaacaagaa actagaactg aaagttcatg tcagggaaaa ga
#gaaacaga   3660
aggtcagata cataaagaaa ctgggcccat ggaggggaga gccttagatg tc
#aggctgaa   3720
ggacatcact tttttttttc aataaaacag acactaaaga attttaagcc ag
#agaatgat   3780
gaaggccatg ttttaggaat attaacctgt tcctatcgtg ttggctacat ct
#gagggaaa   3840
aggcagggat ctctattaag aaattataga agtgcccata tgtatggtgg ta
#agaactag   3900
ggaatgtgtc cttgggtggg gtgtgagagt gagcctaaga gatgctggga gt
#ggtgggtc   3960
taggagacat tgtgaaagaa caattcacag aactgcgaga tgtgatgttg ac
#agcggaga   4020
cacagagaca accgctgaga aacttgagtc aaagatgact aaattttaag gc
#ctggaaag   4080
tgcaggagat ggaaatacaa ccaacaaaat gggagcacat tggaactttc aa
#gtagcaag   4140
tttctgtagg actgggcttg tgggaaagga ccggttgaaa ggttagtttg gg
#agttctct   4200
acagagagga gattgtgagg acatgatggt gggtgaggtc attgagggac tg
#atgagagt   4260
gagaaaattg cagagggctg agccgaggag gtgcacccgc agatagagag ga
#gcggtggg   4320
gacatcagga ctcaggaagt gagaggagga ggagaatcag aagagagttg cg
#ggaagaaa   4380
ggagcaggaa acaatgttaa attggaaaag agattacaaa gcagatgtgg ta
#aggatgtg   4440
agacgtttca atggcaggat gtaggcagaa gatagattgc agagagtaaa ga
#aggaaaat   4500
atgatgaaga aatggaaagt ctgggtatag atcacttgtt caattttgtt tc
#cactacaa   4560
gataaatgga ggagccactg aaagaggggg agtttttgtt gaaagaagcc aa
#tgcttatt   4620
agaagaagcc aggatagcag gaggggatac atatgagagc aatgtcccta gg
#gtacaaac   4680
tgggagtctg ctgtttggtg ttaggaactc tgtccattta atgtggcttt aa
#tcactaga   4740
taggaagtgt gttcagagga gctgagtgtc tttgtcctgg gcaactatag ag
#caaatgtg   4800
atttccagct tatcattagg gtttcactta gcaactttgc ctaccacaaa cc
#attaatcc   4860
caaacatttg aagtgataac tgttgatcgc tattaattta acttcatgat ca
#ctcccttc   4920
tacaaactaa agaagaaagt ttgagcgatc taaatttttt aaattatagg at
#ggtctgta   4980
aggccctgtg ttgctttgat ttcagttgtt agccaaattg tgcagaaatt at
#cctcaatt   5040
cccaagaaat aacttcaggg gcttcagggc agtgcacaga ttcagagaaa ga
#aaatacag   5100
tatcgattga gccagcaata agtcttcagt accctgaaaa atacatggta gt
#ttttcagg   5160
gtttagttgg aagaggccaa gaagcatctc ctaatcttcc accagtagaa gt
#ctgtaatg   5220
atgggtcatc ctcaggaaac atggaagaca gatgtccttc ctctgcgcag ct
#ctggagaa   5280
gaggattccc taaccttgaa ctgctgatgg ctttaatggt taaaaagttc tt
#actcatgt   5340
cccagcaccc tacagagggt tttgcaatga cgacgtagac attaagtatg aa
#gtgactag   5400
atttaagctg aactaaaatc tgactcttgt taagttttaa tttctcatac ag
#cttaaaat   5460
ttggtgggtg ctcagatcag ataggatgat cgattcatcc taactctcta aa
#aaatattt   5520
cacttgctca aaatctcaaa ctacctgttt gatttttttg tccttatgta at
#agcagtta   5580
ccatcaaagc cttaaaaaaa aatagtaagc catccactcc gtggactctt gt
#cttcacat   5640
ctcttcttgt gaaaattagt gcttgaagct tcatcaggat cccagaccac ta
#tttcagga   5700
aaatctttga caaaatggag ctgattttag aacatagagc tagatcttct tt
#tgaaattg   5760
ctggagatga atcttatcaa aacatactat tatgtttctt ttgatagaaa ga
#catgctga   5820
gtcattgctt gacatttgtc atgatacaaa ctcttctcca actgatttga tg
#acagttac   5880
caaaaatcaa aacatcatct tgcaaagcat cagcagaagt gaggtaagag cc
#tcccttta   5940
aagaaacaac ggacagccta ctccatctac tactttattt gtgttgcttg aa
#tacttcat   6000
aacactcata tattacaatt ttatttttaa gtgtaatcat aaaaaagcat at
#ttggtaag   6060
acactcttct gaaagtttaa tctcagagca gtaattagct agtaaactct ga
#gactcatg   6120
cataagatgt gtgtgtacac gtgtgtgtgt gtgtgtgtgt gtgtatgtgt gt
#gtgtctta   6180
gtcagttctg gctgctataa caaagtacca tagattgggt agcttataaa ca
#gaaattta   6240
tttcttacag tcctggaagt ctgagatcag ggtgccagca ggtttgagtc tg
#gtgagggc   6300
tgtcttctgg actgcagatt gccaacctct catatgctca cttgatggac ag
#agagctag   6360
ctagtgctct ggggtccctt ttataagagg cactaatccc atcatgagga ct
#ctactttc   6420
ataatctacc tcccaaaggc cctacctcct acttgccatc acattggtag tt
#aagatttc   6480
aacatataaa ttttggtggg acacaaatat tcagttcttt actctgggtg ag
#cgtgcctg   6540
tgtgtgtgtg tctatgtgtc tccagtacca cagaatattg tttcagctga at
#ccatacta   6600
aataatcaaa tgtaccttcc tttttatgta cattaatatt gaaaaggaag tc
#taggctag   6660
ccgtggtggt ccacaccttg tattagtcca tttcacactg ctatagatac ta
#cctgagac   6720
tgggtaattt ataaacaaaa gaggtttaat tgactcagag ttccacatgg ct
#ggggaggc   6780
cccaggaaac ttacaatcat ggtgggaggc aaaggggaag caggcacatc tt
#cacaaggt   6840
ggtaggagag acagagagag tgcaggggaa actgccactt ttaaaaccat ca
#gatcttgt   6900
gagaactccc ccactatcac aagaacagta tgggggaaac cgcccccatg at
#ccaatcac   6960
cttctacaaa gtccctccct tgacatgtgg ggattacaat tcaagattag at
#ttgctggg   7020
gaacacagag ccaaatcata tcacacctgt aattccagca gtttgtgagg ct
#gaagatct   7080
gttgaggcca ggagttctgg actggcatgg gtaacaaaaa gagacctcat ct
#ctactaaa   7140
aataaaaaaa attagctggt catgatggca cacgcctgta gtcgcagcta ct
#tgggaggc   7200
tgaggtggaa gaatcacttg agcccaggag tttcaggcct cagtgagcta tg
#attgcacc   7260
agtgaactct agcctgggtg acagagcaag accctgtctc aattttttaa aa
#aagaaaga   7320
gacaggcacg gtggctcacg ccagtaatcc cagcactttg ggaggccaag gc
#aggtggat   7380
cgcctgaggt caggagttca agaccaacct ggccaacacg gtgaaagccc at
#ctctacta   7440
aaaatacaaa aaattagcca ggcttggtgg tgggcacctg taatcccagc ta
#ctcaggag   7500
gctgaggcag gaggatcgct tgaaccaggg aggcagaggt tgcagtgagc ca
#agattgtg   7560
ccattgcact ccagcctggg caataagagc gaaactccat ttcaaaaaaa aa
#aggaaaag   7620
aaaaggagat cattaatctg atcatatcaa acccatcaca gggtaccaaa aa
#ggaggtgc   7680
ctcctcgtgg ccctggttat cattctgtct atgatgaatg actttacaaa aa
#gtccccta   7740
tagtacagta acagtattag taacaagcat tgcagcccat agaaaaccgt gg
#aatgagac   7800
ccaagatgta caacaaactg gcaacagtga ttgcctacag agagagaact gg
#agatgcaa   7860
tttgcactgt ttactcattt gtaccttttg aatgtttata aaaattaaca ta
#tcccaaat   7920
aaagatccta ctactctata ttttattggt taaaaaaaaa agtccaaaaa at
#tttttatt   7980
tattttgaga ttgggtctca ttctgttgcc caggccgaag tgccctggca ta
#aacatggc   8040
tcactggagc ctcaatctcc caggctcaag caatcctcct acctcagcct cc
#tgactagc   8100
tgggactgca ggcacatgcc accacaccca gctaatttaa aaaatttttt ga
#actcctag   8160
cctcaagcaa tcctcctgcc tcggcctcct aaagtagtgg gattacaggc at
#gagccacc   8220
attgccattt tctaattgga ttatttgctt tctaactgat aggtttagag aa
#gcctttat   8280
atattctagg tatatgcttc ataaaatatt ttctcctagt caagaaaata at
#ttgacttt   8340
ttttcatcct tttaatgttt tattaaaaag aagttttaaa ttttgataaa aa
#acaacatc   8400
catttttttc tttatggatc atgatttttg tgactaggaa ttgttcaccg aa
#gcccagga   8460
cacaatttta tcctatgctg tcttctaaaa gatttatagt ttcacatttt ac
#atttagag   8520
tcataatcca attagagctt ttttttttct ttttttttga gatggagtct ca
#cttctgtc   8580
acccaggctg gagtgcagtg gcacgatctc tgctcactgc aacctctgcc tc
#ccaagcaa   8640
ttttcccgtc tctgcctcct gagtagctgg gattaaaggt gcccaccacc ac
#gcctggct   8700
aatttttgta tttttagtag agatggggtt tcaccatgtt ggccaggcta gt
#ctcgcatt   8760
cctgagctca ggtgatctgc ctgccttggt ttcccaaagt gttggggtta ta
#agtgtgag   8820
ccgccacgcc cagcggaatt tgagttaatt tttacaaagt acaaggttta gg
#tcgaggta   8880
cgtatttttg cctgttgttc ctctatcatt tgttgaaaag accatacttc ct
#ccactgat   8940
ttactttcac atctttgtaa aaaaagaaag aaagaaaaag aaaaagaaaa aa
#gatctggt   9000
ccaggtgcag tggcttatgc ctgtactccc agcactttgg gaagccaaga ca
#gtaggatc   9060
actttgtggg ggcaagagtt tgaaaccagc ttgaacaaca tagcaagagc tg
#tctctaca   9120
agaactttta aaaattagct gggcatggtg gtgtatacct gtagtaccta gc
#tatacagg   9180
aggctgaggc aggataattg cttgagccca ggaaattgag gcctcagtga gc
#caagacca   9240
tgccactatg ctccagcctg gccaacaaga ggcccaatcc cttaaaaaaa ta
#tatatgtt   9300
gagcttcttt cttcaattaa actactatca attctttttt tttttttttt tt
#tttttgct   9360
gttgttgcca aggctgctgg agtggaatgg ctcgatctcg gctcaccaca ac
#ctccgctg   9420
cccgggttca agtgattgtc ctaactcagc ctctggagta gctgggatta ca
#ggcatggg   9480
ccaccatgcc cggctaattt tgtattttta gtagagacgg ggtttctcta tg
#ttggtcag   9540
gctggtcttg aactctcgac ctgaggtgat ctgcctgcct cggcctccca ga
#gtgctggg   9600
attacaggca tgagccaccg tacccggcct aaactactat caattctaag at
#gtgtactt   9660
tgcattttaa cctctttgaa gtcagacatc ttaaaattgt cactgtcaaa tt
#ggtaccgt   9720
tttgtcattt ttagtggtac ataaaacaac agtgtagctt ttaatcaagg ac
#atcttaga   9780
tttagtgaaa catggtagga tacattgcta aacccaagtc acaatataaa at
#gtcagaaa   9840
gtggatagag aagtgagaaa tgattttgca gcatggagaa tggtaaaacc ta
#atttccag   9900
agaaaggata ttaatgagaa tcaagatgat gtactgcaaa gaaccatgga aa
#agcccagg   9960
aattagaggc accaggtact gcagacgttg ggagttagca tgaggttgaa aa
#acaggagg  10020
gtttggttga aaatgtatat aaggagcaga gagatcccca acattctact tc
#cactctat  10080
gtaactacat cactactcct tccccaccct cacagaaggc aggaagattt gg
#tggaggat  10140
tatttgagct ggaggaattc tggacttagt aacaacatac aaagtgaaag at
#gggaatca  10200
ggtctcaacc tgcaggctta agtctgaata ttgacagaga gattgcatcc at
#cctccttc  10260
cccacctagc tcccatatgg ccagcagccc gtttatacta ctaagccaaa ag
#actggaag  10320
attcttttct ggagatttaa taaccccaga aaataaacct accgatactg ac
#atttttaa  10380
gttccctgaa acacaagcat ttcaccagat taacccagcg aagcccacca ac
#aggtaaat  10440
agcaatatac atagagaact tctagtcata tttttagagt catattttat ct
#tccttaat  10500
atgaagagcc aagatagcca agggttatca ggtatttgag gaaagcctcc aa
#tatgaaaa  10560
gtagcatcaa aacaacaagg aatgcagatg acatcaggag cacaaagaaa tg
#aaggggaa  10620
gaaatagttt taaagggagg agagaaaaat aaagaaaaaa atgttatcag aa
#ccaaatga  10680
tatgagtttt caagtttaaa gcacccatca ctgcaagacc catcattgca gg
#acagtgac  10740
taagtacatt accttaaagt attatgaact tttaaagcac tgatgctaca ag
#agaatcct  10800
aaaagttttc aaagaaagag agagagataa tataaaggat aggaaactgg aa
#tggcacca  10860
gatgtcttaa aaataccatt gtaagctaca aatatatgga gctacaaata ta
#tggagcaa  10920
taaaagacct ctacactgaa agtagtaaaa tattgctgaa aattttaaga ag
#acttaaat  10980
aaatagaatg atgtaacatg ttagtggatt ggaaaattta cttttataaa ga
#tgtcaatt  11040
ctgccaaatt cgtttgtaga ttcaacacag tcccaatcaa aacctagcag gt
#ttgtgtgt  11100
gtgtgtaaat taacaagctg attctaaatt catatagaaa ggcaaaaggc ca
#agaatact  11160
gagggcaata ttgaagaaga acaaagtagg aagatttaca ctactagata tc
#acatccta  11220
ttataaagct aaatcaatta aggcagtgtg atattgctag aaatatagat aa
#atccatta  11280
cctgatttat gacaaagttc atgctgcagt gaaatagggg aaagaatttt ca
#atacatgg  11340
ttctgggttg catggatagt catatacaaa acaatatgca tgttgacccc ta
#cctcacac  11400
catatacaaa atcaattcca cattgattgg aacagatcac tgcagcctag ca
#ttcctgag  11460
cccaagcaaa actcctgctt cagtctcctg agtagctggg actgcaggca ca
#tgccacca  11520
ttcccggata atttttttca atttgttttt ggtagagatg gggtcttgct tt
#gttgccca  11580
gggtgttctt gaactcctgg cttcaaacaa tgtccctgcc tcatcctccc aa
#agtgctgg  11640
aattatagat gtgagccatt ttgcctgacc acactaaccc ttttgaaaga aa
#atgtaaga  11700
aaatctttgt gaccttggag ctggcaacaa atattttttt tttttttgag at
#ggaggctt  11760
gcgctgttgc caggctagag tgctgtggtg caatctcggc tcactgcaac ct
#ccaactcc  11820
ctggttcaag ggattctcct gcctccgcct cccgagttgc tgggattata ag
#catgcacc  11880
accatgcccg gctaattttt gtatttttag tagagatggg gtttcactat gt
#tggccagg  11940
atggtcttga tctcctgacc tcgtgatcca cccacctcgg cctcctaaag tg
#ctggaatt  12000
acaggcatga gccactgtgc ccggccaaca atttttacaa cagaacacac at
#cacaaaaa  12060
atgcttgcca taaaagaaaa gttaattaaa tgggctatat taaatgaagc at
#ttcttttt  12120
atctaaagac atcattaaga taataataag caactcataa ggtgagaaaa ga
#tacttaaa  12180
atgtatgtat ctgacaaagg acctgcattc agaaaaaatt taaaaactcc ca
#caaattag  12240
gaacagatag gctaatagaa agtgggcaaa aacttgatca gacacttagc aa
#aaaaaaga  12300
tgtctaaatg gccaacaaaa tatattaaaa gatgctcagc ttttagtcat ta
#gataaatg  12360
taattttaaa caacaatgtg ataacactgc acatccacag aatgattaca at
#tttacaag  12420
ttgggaaata tcaagtgttg acaaggatgt agggcaacaa gaactttcat gc
#actgctga  12480
tgggagaatg aactgttaga ataatttaga aagctgtctt ttggtgtctg tt
#aaagagaa  12540
atatatgcat actccataat ccagcaattc tgctcctaaa tacataccta ac
#agaaatgc  12600
atcatatgtt taccataagc tacatattat aatgatcata gcagcactat ta
#taatagcc  12660
cccaaatgga aaatacccaa gtgcctatca agaatagaaa ggatacataa at
#tgtggtat  12720
attcacatag tgtaaaacta cacataaatg agaatgagag tgaatgatct aa
#aattacat  12780
gcaaaaatac agatgaatct cacaaataca ctgttgagca aaagaaacca ga
#cataaaaa  12840
attaaatcct gtatgggtct atttatataa aaacaaaagg aggaataaca aa
#gctaatct  12900
atggtgttag aattcagaat agcacttgca tgagagtgtt ctttggggat at
#tggtagtg  12960
ttcttttatt tgatctgggt cctggataca caaatgtatt gggtttatta aa
#attaatct  13020
atacacatat ggtaagtgaa cttttctgaa tgtatgctat actaaaatca aa
#agtaatgg  13080
aaaaggggtg gagtagggaa tgtcttcaaa tatctgacac acacaaaaaa ga
#atatggtt  13140
ttcagccagg catggctgtg gatacctgta cctgtggtcc cagctactca gg
#aggctgag  13200
atgggaggat aacttgagcc caggagtttg agacgagcct ggacaacatg cc
#tttttttt  13260
ttcttttctt cttttttgga gacagggtct cactctgtca cctaggctgg gg
#tgcagtgg  13320
ccagtggcat gatcacagct tactgcaacc tccgccttcc aggcttcagc aa
#acctccca  13380
cctcagcctc ctaagtagct gggattacag gcatgctcca ccaggctccg ct
#aatttttg  13440
catttttttg tagagatggg gtttcaccat gtcacccagg ctgatctcga ac
#tcctggcc  13500
tgaagtgatc tgcccacctc agattcccaa agtgctggga ttacagatgt ga
#accactgg  13560
cccgaatgaa tgtggttttc aaactaggat tctatgccca aacaaactct ca
#gttaagca  13620
ttagagttga ataaagacat ttttcagaca cagaaatctc aaacatatta ct
#tctgatat  13680
accttttaag gaagctacta agtgctctat taaattgaaa aagtaaataa ag
#aaagagga  13740
aaaaatagga tctgtgactc agaggatcaa gagagaggag ggaatcattg gt
#ataatgaa  13800
gaaggcaggt cccaggactt cagctaatta gcaactctag aaaacaaaga gc
#tcagatga  13860
ttgggggatt ggggtggggg cagggagcag gacagaggag ggaaacagaa ca
#gatattgt  13920
tgtctgataa atttcaccaa gtggcaagac cattgtaggt tgggaagatt ta
#ggctttaa  13980
ataaaaggac ataagaaagt aaataaaata aaccaactag aaattaaaaa ac
#caagggat  14040
gaggggaagg aaggatgaat aggcagagca aagaagattt ttagggcact ga
#aactactc  14100
tgtatgattc tataatggtg gatacaggtc attatacatt tgttcaaacc ca
#tagaatgt  14160
acaacaccag gagtgaacct taatgttaac tacagacaac tgtaacaaat gt
#accactct  14220
ggagggcgat gttaataatg ggtgaggctg tgcatgtatg ggagcagggg gt
#atatggga  14280
aatccctata ccttgttctt cttcttcttc ttcttttttt tttttttggg ac
#ggactctt  14340
actctgtcgc ccaggctgga gcgcgatctt ggctcactgc aaccttcacc tc
#ctgggttc  14400
aagtgattct tctgcctcag cttcctgagt aactggggtt acaggcatgc ac
#caccatgt  14460
ctggctaatt tttgtatttt tagtagagac agggtttcac catgttgacc ag
#gctggtct  14520
caaacccttg accttaggag atccatccac cttggcctcc caaagtgtta gg
#attacagg  14580
cgagagccac tgtgcccggc ctataccttc ctcttaattt ctctgtgaac tt
#aaaatgtc  14640
cctaaaaata aagtctattc aaacaaacat acaaacaaac aaacaaacaa ac
#aagggttt  14700
gggggtttgt tctggaaaat aaaacagtta tacaagaaag aaagcataat ca
#tactatat  14760
tacaattgta ctactacata gtacaatatc ctcataatca aaattagcca tt
#gactattg  14820
atttaacagc aaagaaggta aatgtattgg gaggatggag gcagggcata ag
#aacattaa  14880
attattaact gccataataa gtcaatagat gatgcctcac tttgatgaat ca
#agagacag  14940
catgataact atgcagaaat acggaagaaa ataccaaaag aaacagctaa aa
#gtttggaa  15000
gtggttgcct ctgaggaaaa cggtgactgt ttttctcggt ataagtcttt ta
#ccattatt  15060
tgattttttt tacatgtgca gtttaatttt gataaaaatt aagtgaaaat ta
#aaaataaa  15120
cggttaaatc aagacttctc tgggacatgg gatgggatga gctaccatgg aa
#acattcct  15180
ttttaaatcc tatttgaata ttttagcttt gcgcatttat aaattttcta ag
#tagtttag  15240
tctgcttcct accaaagtgg aatttagtac cctggttccc aacaagggag tg
#attcccag  15300
cgcccacctc ccacccctcc caccctaggg ggtcatttga caatgtttgc ag
#acatttct  15360
ggtatcatca ctaggggaga atgcaactgg catcttgtgg gtacaagcca gg
#gacgctcc  15420
taaacatcct atcagacaca cgacagcccc cacagccaag aattatctgg tc
#ttgaatgt  15480
caacagtgca gagactgaga aatttgctac atgttgtcac aatattgaag gt
#tgcactgt  15540
gtttggttac taatattata tagtaatcaa aataaaatac ctagagacaa at
#ctttaagg  15600
tgagtgtcat gcataagata ttgataaaca aaaacatact ttttattttt at
#ggtctatt  15660
taagcaattt tctttttaaa aggactaact atatcacttc atattaatac at
#tgaaataa  15720
atgtttaaaa acatttttgt agagatgggg tctcactatg ttgcccaggc tg
#gtctcaaa  15780
ctcctggcct cagccaggtg tggtggcatg cacctgtaga accaactact tg
#ggaggctg  15840
aggcaacagg atcattcaag cccaggagtt caaagttaca gtgagctatg at
#cacaccac  15900
tgcactccag ccaggatgac agagggagag tctgtttcta aaaaacaaac aa
#acaaacaa  15960
acaaacaaac aacatcaaac tcttagtctc aagagattct cccacttctg tc
#tcctaaag  16020
tgcaggaatt acaggtgtga gccaccgtgc ctgatcagta cattttttga gg
#caacttta  16080
agactttttt tttttttttt ttgagacaga gtctcgctct gtcgcccagg ct
#ggagtaca  16140
gtggcgcgat ctcggctcac tgcaagctcc gcctcccggg ttcacgccat tc
#tcctgcct  16200
cagcttcccg agtagctggg actgcagggg cccgccacta cgcctggcta at
#tttttgta  16260
tttttagtag agacggggtt tctccgtgtt agccaggatg gtctcgatct cc
#tgacctcg  16320
tgatccaccc gccttggcct ccaaaagtgc tgggataaca ggcgtgagcc ac
#cgcgcctg  16380
gcaaaacttt ttttaaaaac ctttcattag gtgttttttc ttattgtagc cg
#aaataaag  16440
tttaaactcc tttttgaggg agaaatggac tttttcagta ttatatttgc ct
#ttccttcc  16500
ctagtggttt aactggggtt taaatccctt tcactctttt ctttaaatga aa
#gctttgtt  16560
ttctttttgg ttgtctgaaa taggttttta tagtttacaa atataagcag ct
#gccttgca  16620
tgtaggacag ctccagagag gctcgttata gactcgccca gtcatctttt tt
#cacctgag  16680
gagaatcttc tttcaaaatt ttatcatagg ctggatatgg tggctcatgt ct
#gtgatctc  16740
ggcacttggg gaggctgaag tgggaagatc ccttgagtcc aggcattcga ga
#cacccctg  16800
ggcaacataa taagactttg tctctacaaa aaaattaaaa aattagctgg tt
#atgggggc  16860
gtgcctctgt agttccagtt acttcctgga ggctgaggtg ggagaaccac tt
#gaacacag  16920
gagtttgagg ctgcagtgaa ctataattgt gctgctgcat tccagcctcg gc
#gacagagt  16980
gagctcccat gtctctaaaa tataaaaata aaaaaacttt aatcacgtct ga
#tttccatc  17040
gtgcctttac attctgtatg tttggtatgc tgttgtctgc aggctagaat gc
#gatgctct  17100
atttcttatc catctatcag ctcccgtggt gttgtcaatg gtttatgaaa tc
#catctatg  17160
tttgggactt gctattctga tgttttctct cttttactca ctcctagatg ac
#actatttc  17220
aattctcctc cttgtggcac ccaagcacat cttaaagtca ttgctggtta ga
#tttataaa  17280
ataagttaga aaattctgag ctgtttctgt ttgagtcttc acttccgtca tc
#accttcaa  17340
agtagatctt actccctaca tcctttttga ttgtgatact tatggttttt ca
#gtttgttc  17400
cagggtttaa atttttgtca ggtacttata gggatcacac atcttttatt at
#tatttttt  17460
ctatgcaaaa cttatcaatt aggtttgagt atcctttccc tttattttgc tc
#attaattc  17520
tttttttttt tctggttctt gttgaaattc attgtttcaa acttttcatg ct
#aacaagat  17580
cactgagtgg tcacaacctc tggacccaga tttcacagtc tgggtgtaaa tt
#ctggctct  17640
gccactggct agctgtgtga cctcgtgtaa gctacttaac ttttctgggc ct
#caggtaca  17700
aaatgaagat aatagatcct aactttagag ttgtgaggat taaattagtt aa
#cccattta  17760
tgcttagtgt tccattattg gaacggtgag cttgtggggg ttatttatat cc
#cactgctc  17820
aaggtcattg ccaaggtctg atttttcaca caaaaaaatt tgcaacctcc ga
#gataaatg  17880
ggttaatatg tgtaacgcat atagaacagt gtctggtact atatatgtaa at
#gctagtca  17940
tcattatgga ttttgtaggt gggtatgacc acactgccgg cttccaactt tt
#cctacagg  18000
accaactgac aaatgaactg agtagctgag attgaccaca gcccagtaat ca
#acatggaa  18060
acttgatgtg agaacctgct gtatgactaa cacttccaaa tgaaggctgc tg
#ttttctca  18120
aagctcagca taaaaatttc actgaatcac tgtaattaaa tgaaatggta ga
#aatgtgtt  18180
ttgaggtctc ttagagtgtt ctagactaag gatctacaca aaaactatat at
#aatactaa  18240
aaaagaaaaa ttcaaatgac ccataagcat ctaaaactat ctccaacttt tc
#tattaatc  18300
aaagtatgaa cattcaaaca ataataagaa gaccaattcc atctattaca tt
#aaatataa  18360
taaaatgaaa agtcggccag acgtggtggc aggtgcctgt aatcccagct ct
#ttgggagg  18420
cagagggagg tggattactt gagatcagga gatcgagacc agcctggtaa ac
#atattgaa  18480
acgccatctc tactaaaaat acaaaaaaaa aaaaaaaaaa ttagccaggc at
#ggtggtgg  18540
gcacctgtaa ccccagctac ttgggaggct aaggcaggag aatcgcttga ac
#ctggaagg  18600
tggagtttgc agtgagccga gattgcacca cagcactcca gcctgggcaa ca
#gagcaaga  18660
ctctgtctca aaaaataaaa aataaaatga aaaggggcag ggcatggtgg ta
#catactta  18720
tagttccagc tactcaggag gctgaggtgg gaggatcacc tgagcccagg ag
#ttcaaggc  18780
tgcagtgagc catgatagtg ccactgcatt ccagcctgtg tgccagagtg ag
#acactgcc  18840
tcaaaataat aacaataatc atataaacac ctgtgaaaag aagggaaaac aa
#taataatt  18900
aattaattaa ttaaatgaaa aggaatgatg ataagggaga agatatgata ta
#gcacattc  18960
atgcactgct ggtggattat aaatagggat aaactttact tttaaggcaa tg
#tgtgtaca  19020
aaaaaacaac tttttcatag tatttgggtc aataattcca tatctaggga tc
#tactctaa  19080
agaaataatg caaaattggg gtattagttg caaatattta ataatactat gt
#gtaagagt  19140
gaagaatttt aaattaccta ctaagcatca tgggagttac attgtaagac ta
#acggggct  19200
tattaaagaa gtactattgg ctgagcgtgg tggctcacgc ctgtaatccc ag
#cactttgg  19260
gaggccgagg caggcagatc acgaggtcag aagtttgaga ccagcctggc ca
#gcatggtg  19320
aaacctcgtc tctactaaaa atacaaaaat aaggcgggca tggtggcggg tg
#cctgtagt  19380
ctcagctact cgagaggttg agacaggaga atctcttgaa cccgggaggt gg
#aggttgca  19440
gtgagctgag gtcgcaccac tgcactccag cctgggcgac agagcaagac tc
#catctcaa  19500
aaaaaaaaaa gaagtactgt tatgaccctc tgatatttgt tgaaggaaag aa
#attttaaa  19560
ttccattaaa attaaaatga cacttactta gtaaattgct tatgaattta ca
#cttaagtg  19620
aaaaagccag atacaaaaat tatgtgatgc aactatattt ttaaatactt aa
#gagaaaca  19680
caaagaaaat atgattcctg tgttaacagt gtttgctctt tggttgtcag gt
#tataggtg  19740
attttttaaa ttttgcttta aaaaatactt ttttgtattt ttgtatgttc ta
#ggaagaat  19800
aaatccccct ttgtgatttg ataggaagga ggaggaattt gccagataat gg
#tagaattt  19860
ttgaaataca gagaaggtta agcagtgaaa ttgacaacag cctaggagct ga
#gtgaaccc  19920
atccgccatt gacaaccagg atagtctgag gtagggcacc caacttttgc ca
#ggagatag  19980
aaaacgcttt agaaagtatt aataagggta gtggggagta gggaggaagg gg
#gatggtta  20040
atgggtacaa aaaaaaatag ctagaaagaa tgaataatca acccaatgag ag
#aacaaaaa  20100
gaaaaaaaaa ggaaaagaaa gagtaagaac tagtactgat agcacaacag gg
#tgactata  20160
gtaataattt aattgtacgt ttaaaaataa ctaaaagtat aattgaatca tt
#tgtaacac  20220
aaaggataaa tgcttgatgt gatgaatact ccatttaccc tgatgtcatt at
#tatgcatt  20280
gcatgcctat atcaaaatat ctcctgtatc ccataaatat atatacctat gt
#acccataa  20340
aaattaaaaa acaatgttta agtataaact gctgaataaa agtaaggtat ga
#caactaag  20400
ttattatgat tgaataccta aaatattttt aatgactgta taaatggagg gt
#tttacttc  20460
tggttttttt ttttgagaca gggtctcact cagttgccca ggctggagtg ca
#gtggtgca  20520
atcatggctc actgcagcct caacctccta tggctcaaat gatcctcgca cc
#tcagcctc  20580
ctgagtagtt gggactacag gcacgtgcca ccatgcctgg ctaatttttg ta
#ttttttgt  20640
agagatgggg cttcaccatg ttgtccaggc tggcctcaag caatccaccc at
#ctcggcct  20700
cccaaagtgc taggattata ggtgtgactc accatgcctg gccaggtttt ac
#ttttattt  20760
cctttttctt ttcttcttct tttctttttt ctctctctct ctttctctct ct
#cttttctt  20820
ttctttcttt tgacagggtc tcactctgtc acccaggctg gagtgcagtg gc
#gtgaccct  20880
agctcaccat agccttgacc tcccgggttc aagccatcct cctgcctcag cc
#tgccaagt  20940
agctgggaca acaggggtgt gccatcacgt ccagctaatt tttgtatttt ca
#gtagagac  21000
agggttttgc catgttgccc aggctggtct cgaactcctg agctcaagtg at
#ccacccgc  21060
ctcagcctcc caaagtgctg ggattacagg agtgaaccac catgcctggc ca
#acttttat  21120
tatttgctac gacaattaaa atgaacaagg agagaaaagc aagaaatttc ct
#agctctct  21180
tgggaattaa taaatgagct atcagagaat ttttgtgact cgccacttct ct
#gacatttc  21240
agatgacagg cttgagcact tagggcaaag acttattgtc catcagtccc ct
#taaatagg  21300
tagtccacct agatcataga aaccagacag atagttgtaa cattcgggtt gt
#gatgggat  21360
gttttaacta ttacttggat ctatcatggt tctagaaatt taaaggcaca aa
#atcatcag  21420
ctataacttc gaatgaagga aactatcaaa aacaatgaat tctactaaga aa
#acatttct  21480
tctttaaaat gtttggagta ctttttgtaa atcaagttgg ttttcaacta ta
#atgattat  21540
tttctagaga gtgaaaagga agtttaagag gttatgcacc atgatttaga tc
#agaacgcc  21600
gtcataggga agactttaat cagctttgct gcctcctttc agtctagggt ta
#tatctgta  21660
gcttccacag gggcagggat ttccattctt gccatatgta aatgatgccc ca
#gggaggca  21720
ttatggaaaa gatcatgctc ctttggggtt gttcactgtg actgtggcca aa
#ggattctt  21780
tccagttacc tacccagatg gaatttgggg cagcttagca gcctgggcac tg
#agatgata  21840
aagtataaaa tactgagttt ctatgtgtcg atgtgatttc agctttgctc ct
#catttttg  21900
attatgcaat taatcacaac catgactgtc tgagcctagt gctccaaggg ca
#gatacttt  21960
cttattattt tagtcctaaa tactttatcc aatttaaaag gaatccatgg tg
#taaatctt  22020
tagcccagaa aaatcaacat tcactctgcc aacaaactgg tacatcgaat aa
#ctaataac  22080
tgagttttga attttatgat attgcaggag ttcgaccaag atggtgactg ca
#gtcattcc  22140
acactggtta atgaagaaga agatcccagt ggtggtagac aggactggca ac
#ccaggaca  22200
gaaggtactg ggctttactc cttgatgtgt ttacaaagat aacattatca ta
#tgggcttc  22260
tctccaattt cagaagggct tattgtagaa gtttgaacaa catacactgg ag
#cctatcag  22320
agggtagagg gagtggagga gggagaggat caggaaaaat aattaatggg ta
#ctaggctt  22380
aatacctggg tgataaaata atttgtacaa caaaccccat gatacatgtt ta
#cctatgga  22440
acaaacctgc acttgtaccc ctgagcttaa ctcttgcaat aaaagttaaa aa
#aattataa  22500
taaataagtt tgaaaacaca gaaaaagccc aaaggagaag aaaaaccact ca
#caatacta  22560
ccttttggtc catatctgaa tcagtgggtc taggcagctt gactggccag aa
#taggcaaa  22620
tgctctctgg ctcttttatt ccacctcact ccagctcagc cgacccattc cc
#tgtccatt  22680
tctttttgtc tgataacatc ctttccccaa tttcttcctc tcagaatctt cc
#agcggctt  22740
cagtgatcgg ttcccttccg gaaccacacg tgtctccatg agccgttgtc cc
#tgagggga  22800
aggtggggga gtgtacgaga cctgaaagtc cccaagtctc ggtcttttat tt
#acaaggcc  22860
ataagtctgg aatcttccag aacaccaccc atttcaaaca tgttatcctg tc
#acaccgta  22920
agtgcccttg cacttaacag accacaaggt atttgcagat tctcgcctca ga
#gcatagtt  22980
gccacggcta tcccatttgt ctgtcatcta ttcatccata accttcttaa ag
#taaatgtt  23040
tatttgaact gctgcaattt ctcccgggca atcttctggc ttctatttct ag
#cactccag  23100
ggaagccgcc ctctttgatg cccgtgtttc tcatcccttc gcacctctca ga
#aggctgca  23160
gctctcccga gtagcgtctc ctccgggagg tggtgcgatg ctgccctctc ct
#gggcagcc  23220
gcctgccttt ctcacgccca ctgggaatct tccctcccca ggctgagggc cg
#agagtaat  23280
ttagtaacca ttaaaattat gaaaaccatt aagcctgaaa gagctaacag aa
#agaaaata  23340
aaccccgaaa cccttcagaa cggtccttgc agtcctcctt cgactttcat ag
#acttcaaa  23400
gccaagctct tagaagccta atggtgtccc aagcaccttc caggaggtta aa
#tatttcat  23460
ttattctgct ccatatggag ataactcacc atttgggatg ttagtcattc tt
#ttaaactt  23520
gatttgcaat attttcagtt ttcatatggg agccataata cttatgaggc at
#ctccacta  23580
agttatttca gttttaagct tttaacaact tgagttacac atttggaaga ag
#caattctc  23640
ttcctgataa aattgcatct cacagttgat agagacttca gttgagctag ct
#actctttc  23700
taatcagaaa ttctgaaata aaagtgtttt agatattatt gtccattata tt
#cattttaa  23760
atatcggttt aaatctcttt aaatggaccg ggcactgtgg ctcacgcctg ta
#atcccagc  23820
actttgggag gccaaggtgg gcggatcacc tgaggtcagg agttcaagac ca
#gcctggac  23880
aacatggtga aaccccgtct ctactaaaaa tacaaaatta gccgggtgtg gt
#ggtgcgca  23940
cctgtaatcc cagctactcg ggaggctgag gcagaagaat cgtttgaacc cg
#ggaggcgg  24000
aggttgcagt gagctgagat tgtaccattg cactccaacc tgggtgacag ag
#tgaggctc  24060
cgtctcaaaa caaacaaaca aacaaacaaa caaacactat tttctcagaa ca
#taacagac  24120
acaaatctta tagactagaa attgagccta caaatttact gttttcatga gt
#gaacaaga  24180
gagcctattc cctaaaacta atgggcttaa aaatatttta attcagtata aa
#ttcatcag  24240
gatttgtagt tgcaggtata caagaaccta ctcttggttg ggttaaaaag ga
#agggaatt  24300
ttgaaagata ttaggaagtt catataccat tgaaaaacca gaggagagga ac
#tttcttag  24360
tccactcatg ctgccaaaac aaaataccat agactggggg gcttaaatag ca
#gacattta  24420
ttttctcaca gttcttaaga atgggaagtc caagatcaag attctagcag gg
#atgggttt  24480
ctggtgaggg ctctcttcct ggttgcagat ggctggtctg tccccacgtg gt
#ctttcctc  24540
tgtgcacaca gaggcagagc acaagtgagt gagctctctt cttagaagga ca
#caaatcca  24600
gctggatcgg ggccccaccc ttgacacctc atgtaacctt cgtttcttcc tt
#agaggccc  24660
catctccaaa taagccacac ttgggggtta gggcttcaac atattaatgt gc
#ggtggggg  24720
acacaaacat tcagaacatc cagtccataa caggaaggcc ccaggttgga tt
#gtcaggaa  24780
ggattcccat aactgcattt caaaactggc tgctactgac cctcaaatca tg
#ccacgtct  24840
gccataacca gagagccgct cccactatca atgtaagaac cccctccctc tg
#ctggtacc  24900
cacatcagca cacagcatgc ctgcacctta tcttttttca tgtaactcac at
#gcatcagt  24960
ctctgaagta agctttctga atctagcagc gcaggaagcc ggaaatacag ct
#gttttttt  25020
tttttaaagt ctgtgttgag cttcacaatt taggaaatca tcaaaatgtg aa
#gatggcat  25080
caaaatattt tgaacctcca tgctcgcaat ccagacagat atgcacatcc at
#tgaaatag  25140
aacaaggacc tcattgatat atgctcctat tatgtaccca cggaaattta ac
#aaataaaa  25200
taaaataaaa taaaataaaa taaggagacc aaacaggaaa gtaaggcttt tc
#tggagaaa  25260
ataatttttc tttattgaaa tcagttaagc tgggcctgat tttaagtttt tg
#ttttaata  25320
atggttttga cactaacaac aacaaattaa tgatcatttt tctgactggt ta
#tgaatgtc  25380
attttcacct cttctataaa gaaaatatat tcgtggctat gttgaaatgt tg
#tcttttaa  25440
tttctctcta tggtaatatt ttctgatagc gttaatttac cctcattatg tg
#aaaaatgc  25500
acttgctaag agcaagtgtt ttgtctttac ctgtgacaat gcatcctctt cc
#ctggccta  25560
ctgggtagct tgagaggcct tatccacagc aacgtcagca actcacagta tt
#caagaggc  25620
agaacaaaga gaacatctgt atgtttctag tggatttcag aatcaatatt ct
#gtaatctt  25680
ttttccaatt taggaccaac aattaggacg gtggccatta gctcttaaca at
#atcttaaa  25740
aggcaggtat ttcttacatg tgcttgttat atctttgttt cttggtttga aa
#aagaagtc  25800
agctgatgaa cagactttga agcacattac atttgtttga aaacattctg gg
#tttattaa  25860
ttcttgacaa ctgcaaaagt acagttgttc ttaaatatgg ttcatgtgaa ta
#cactcagt  25920
tttctaactt ccacagcaaa gaactaaata catttagctt ttgtaccaga ac
#atcctttt  25980
cactgacagt ttagttttta ggaatgtatg ctgtatgttt ttctcactct aa
#catgtcag  26040
ctaggtgttt gcactcaagg ataactacaa aaatattatg aaagacatcc at
#cttccttt  26100
caaataggag aacgaccttg agcatgtcat gcaaactcat tgctatcagt tt
#cttcgtct  26160
ctaaaacgaa aggcttgggt taggtgacct ctaagttcct tccagctcaa ta
#attccaag  26220
tctctcattt tttgctacat agtctggtga tagcctcttt gaaaacttaa aa
#aacaatgg  26280
actattccag ggaaacttca tttttagaca agtgttccat gcaattgtat ag
#tattagaa  26340
aacatgcaat caagttgtct cctttgagaa acattaagaa aaccaaagct ag
#ctacattt  26400
ttatggtagc acaaaacata atattggata acaatgatag taaacactat ta
#tcatttgc  26460
ctgattgtaa acaaaacttt tcattttgga attttttact gtgttttttt tt
#ttaatgca  26520
cttgtttcat taaatggcac aggtataaaa attgaacaac aaaaatgctt tc
#actatggt  26580
agttcctatg tattacacaa atatatccaa agtcctttaa aataataaaa at
#ctactaat  26640
ttagataatg atgatagcta ttaagcaact ttcccaaggt cacccaggta gt
#ggcagaaa  26700
agggatgtct gattcacacc ttaaccttat cctccctgcg atactccttc cc
#cagccttt  26760
aattagtgga gctcatacag ccattgctcc tccaggcaca agcagattga gt
#gaataaat  26820
ggctctgaca gataaatgga tagaaatgaa taccggggca agcattgcgt cc
#tcccggaa  26880
ggacacgcct ctctgctccc acatcaccac ttgcttctat cacagtgctt at
#ctcactgc  26940
attctttatt ttcttatcag ctctactagg gcctcagctg catcttgttt at
#ttccctgt  27000
tttcagcact aagtgctggg cttggcatat ccttaataaa agttcgttaa at
#ggaaaaaa  27060
ggaatgaatg aacacacctt aaagaacagg caatgttaga atagttcaca ct
#agtttttt  27120
acataatttt gcttaacatc ttatattgtg agcaagcgcg tattctataa gt
#tggaactt  27180
tctgtcttaa gggttattct ggaaattagt tcatgaaatg agacaggaga tg
#accaaaat  27240
tacaaataca agcaaacatc tttggtgtta cataaattat ctcattgaat gc
#tcacaata  27300
gttctgggag ataggtgtcg ttacacttta tagaaggtgg ttctgttttc tc
#catcctga  27360
ggacaacata gtttgttata aaactttatt ttacatctgt aaaatattat ta
#tatggttt  27420
tttgctcttt aagcaagcat ttatttagga tctatcatat cctgaacagg aa
#agatacaa  27480
agatggctaa acctcagctc ctgattaatg tccattttgt aatcattaag aa
#gagattag  27540
ccaaacagaa ataaagtacg tctcccgctt tccgctggaa ttcattactt tc
#ttccttct  27600
actactgtgg tatgtttcta caggtgttga gatcactgta acttttccaa ga
#gatgtcag  27660
tcctccccaa gaaatgagcc aagaagactt aaaagaaaag aagtaaggaa ta
#ttctttga  27720
agtatcagat ttgaaatgaa gtatgaagca atgatagtca tatggcaacc ta
#cattatta  27780
gtaattgaat ccataataat gcttaaaagt agaggtcaca ataaatagta tg
#tggcagag  27840
gccagatcat aaacactttg gctgtgtggg ccatatggtc tctgttgtga tg
#attcaact  27900
ctgcaactgt catttgaatg cagccataga caatatgtaa acaaatgagt gt
#ggctgtga  27960
tccaattaaa gctacagaaa aaggctgaag gctggatttg gtccccgggc tg
#tagtttgc  28020
tgaccctact gcagggcaga gctacttaga atgtggttct gtggtcctgt ta
#ctagtcca  28080
tgatgaggtc aggacaggct gcgagggtga ccattaaaaa agttgcaaag ca
#atttggca  28140
aataacttac gttcattgat cgggtagtga aacaaattga ggcttatttt tt
#gcatgtct  28200
tttatttttc ttccattttt atggcaattc atttatattt tacaaaagta tc
#agttcaca  28260
atgactggaa atttaaatgc tggttcttca tcaaaaatag tttgagaaac ac
#tgggctag  28320
tcagtacctg tggagataaa agggtacatc ccccaggcct gcccttggtc tc
#tgatttct  28380
ctgtgcaagg aaatggtgat tgggaaaacg agagtgagct gaaacctaat cc
#atccaagc  28440
gatggtacag agggctaagg aggccaggag gagtcagcag gtggagatgt ct
#taccctct  28500
caggattcag cctttccttt cagcagcacc atttggaagt agttcttcaa ac
#tttctagt  28560
gatgtcaggc tgaactgagg actggagatc cagaagatgt ggcaactgag tt
#ttaaatca  28620
aacatttccc tcccccttta gtctgataaa ctcatcgctt caagaatggg ca
#caagcaca  28680
tgcagtttct catccaaatg aaatagaaac ggtggagctc aggaaaaaga ag
#ctgaccat  28740
gcggccctta gttttgcaaa aagaggaaag ttccagggag ctctgcaatg tg
#aacttggg  28800
ctttttgcta ccaagatctt gtttagaact gaacatttcc aagtctgtaa cc
#agagaaga  28860
tgctcctcat tttctgaagg agcagcaaag aaaatctgaa ggtaagttga ac
#attgaatt  28920
ccacagtgag tccttttggt caacaaatat ttatgtattg tgccaggcac tg
#ttctaaat  28980
gctagagata aagcagtgaa cataccagaa aataacccca gcccttgtgt aa
#tttacatt  29040
cctgggggtc atgggggtgc gagacagaca ataaactagt aaacaagtaa aa
#tgaaggat  29100
ggcataatgt tccagcagaa agagcactat atgtgcaaat gtttatatat tt
#gttcaagg  29160
aatagaaggc aaatattttg agaatatctt attgtttaga ttatttcatt gc
#ttatttct  29220
ttaaacattt cagacattat tttgtaaact atacttgata atttcagtat ca
#atatccta  29280
ctgatttgca ctatggttga cttttttctt gagtgtttga aaatcttcat tg
#tgagctca  29340
tatttggttg atgtttatct gtgggaatct tgggggccca cgctgtggat gc
#ttttggcc  29400
agaagtattg aatttacttc taccaggtcc cagggtcatt atggacttga gt
#ctacgtta  29460
gccttattcc tggatcccca agttaatgtg caagtctaag agccaacttc ca
#actacatt  29520
gagcccaaga ctcatttgct agatagcagc actgatgcca gcatttcccc ct
#gcggcaat  29580
attgtctttg ctacttgttt accactgtcc tcaaccaacc agccaaaacc ac
#caggaaca  29640
accttataat ccaacaagtt acattcattg atttatcaca atgagggaga ct
#gcatgcca  29700
gtggagctgt gacacatcct accaaaaaag aaaaaaaaga ataattatta tg
#ggatttta  29760
agagaagggt acattttaag tgaaatttaa atgaagcagt gcttaaagtt tt
#tttttttt  29820
tgagacggag tctcgctctg tcaccaggct ggagcacagt ggcccaatct ca
#gctcaccg  29880
caacctctgc ctcctgggtt caagcaattc tcatgcctca gcctcccaag ta
#gctgggac  29940
tacaggcgca tgccaccacg cccagctaat ttttgtattt ttagtagaga tg
#gggtttca  30000
ccatgttggc caggatggtg tcgatctctt gacctcgtga tcctcccacc tt
#ggcctccc  30060
acagtgctgg gattacaggt gtgagccact gcgcctggcc taaatttttt tt
#tgttttat  30120
tatgctaaaa tgtgtgtaac ataaaattga ccattttaat cattttcaag tg
#tacagttc  30180
agtggcattt aagtacattc acattgttgt gtaaccatca caactattca tc
#cccagaac  30240
attttcttct tgcaaaactg aaactctgtg ccccttaaac aataactcta ta
#tttcccac  30300
tctcccaaag cctctggtga ccactattct attttctgtc tcaatacgaa tt
#tgactatt  30360
ctaggtcttt tatagaaatg gaatcatgca atatttgtcc tgtgtctggc tt
#gtttcatt  30420
tggcataatg gtgaagcagt gttttgatgg gctatatgta aaatagttca ta
#agaaatct  30480
ggacttgaag tggacctaga ctcttgttcc ttgaaattta caaagttagt tc
#ccaaatct  30540
tgatagcgtt tttttgtttt ttgtttgttt gtttgtttgt tggtttgttt ga
#gacacatt  30600
ccggctgtgt tgcccaggct ggagtgcagt ggcgtgatct tggctcactg ca
#acctctgc  30660
ctcctgggct caagcgatcc tcccacctca gcctcttggg tagctgggac ta
#caggtgca  30720
tgccaccacg cctggctagt tttgtttgtt tgtttgtttg ttttttggta ca
#gatggggt  30780
ttcaccatgt tgcccaggct gttactgaac tcttgggcta aagtgatcct cc
#catcttgg  30840
cctcccaaag tgctggaatt acaggcatga gccaccgtgt tcagcttcaa ca
#gcctcttt  30900
cagcctcata tcttgccact cttccttcgc agcatgataa actttagcac ac
#taaatgcc  30960
ctctattccc taaacatgca tgtgaatatt tgcacctact gttctttctg ct
#ggagcatt  31020
atgccatcct tcaggttttg tcttagaaac cccttcctct gggaagtctt cc
#tgaacttc  31080
ccaagactgg atgagttgcc ctttctttgt tccgctatag gatcctgacc tt
#acctacaa  31140
catagcacta atcaagcata attgtcacta tttgtttacg tgttcatctt cg
#ccggatta  31200
caaaagcaag aataattcaa cctccaagca tttggcatca tacctggcac at
#agccatta  31260
caaatgcact tttaattaat aataacaata atcaggtcca gggcagcact tt
#gggaggcc  31320
aagatgggcg gatcacttga agtctcaaaa aaaaaaaaaa aaaaaaagaa at
#aagaagta  31380
ctagctgtgc acaggcacac gcctgtaacc ccagcacttt gggaggctga gg
#caggagca  31440
ctgcttgagg ccaggagttt gagaccagcc tgggcaacat aggcagactc ca
#cctctaaa  31500
aaaagtacat atataaaaat aaattttaaa aattaggtgg ctgtggtggt gc
#acacctat  31560
aggctcagct actcgggagg ctgaggtggg aggattgctt gattccagga gg
#ccaaggct  31620
gcagtggatg atgattagtc cattgcactc cagcctgggt gacagacctc at
#ctcttaaa  31680
aaaaaaagta cagctagtac aagactttct tctagtgtgt actttcatat tg
#ctaaatat  31740
catgtttaga atggtattta ttaattgttc agtttgggct tcatctatta ag
#atttatta  31800
cttttacatt acttgcctca cacacaagca atgcccaatt ttcccaatct tt
#gtgtctat  31860
ttttttaaaa tcaatattca atgtctctgt tattatgact aggtaaaata tt
#atttgcag  31920
ctgagctcca tagtgtgttg attacatttc ctctcctttt agacattgta tt
#tatctcag  31980
cattagtaat aaccacttca tttcttcatt tgcttacttt ttgtatatct gt
#tactaatt  32040
catcccatcc tgtgtattgc acctataaaa caaatctcaa tacaggtgat ta
#gatatcag  32100
gcaatctgtt ggttcctttt gtttttggag acattgctcc tggaccctcc tg
#gcctctaa  32160
ttttactcca caccacctgc tctctggatc cactgcccag ccgcccatct ga
#gattccct  32220
tcgtgtcatc ctgggaattc ccttgcctcc ttgctgtgtt gaatccttgt gt
#actggata  32280
tgtggcttaa tcttcctttc cttacttttt tttttttttt ttgagacaga gt
#ctgactct  32340
gtcacccatg cttggagtgc aatggcgcga tctcagctcc ctgcagcctc cg
#tcttctgg  32400
gctgaagcca ttctcccctc ttcagcctcc tgagtagctg ggactacagg ca
#tgcaccac  32460
caggcctagc tactttaaaa aaatttcttt ggtagcgatg gggtcttact at
#attgccca  32520
ggctggtctt gaactcttgg gctcaagtga tttacccacc tcagcttccc aa
#agtgttgg  32580
gattacaggc ttgagccacc ttgcctggct tccttgcttt acttaatcct ct
#tttactag  32640
ggcatatttc ccagcagctt cttgagaaag ggtacacgga gaatatgaaa ga
#tagagttg  32700
tttgttgtct ctaattcttc tgagctcttt tctttctttc agtttgatct gt
#tctctgta  32760
tgttcatatt ggagcatttc ctcacatatc agttgaatca catatcctca ca
#tatcagtt  32820
gaatttgtat ctaaaagagt ttcactaaaa agttctgtat gtttgagtga gc
#ttgttgaa  32880
tgggcctcaa aggagctgaa taggtggaga actggacgat tgatagaggg at
#tcccaagt  32940
gtcagcttgt atagatcaat ggaccttttc tctcagctag ttttccccag ag
#agataatc  33000
caaacacctg cctgtaggtt atgagactgg aggcaacatt cttggcactg aa
#tggggttc  33060
atatttcagt gtgtagactc ttctttgtcc tcatattttc actccagctc cc
#catttctg  33120
ctcccagcca tacccagctc cttagcatct ttgtttcaag ccctccaggg ag
#taaacttc  33180
cagccaactg ccaggaaagg agaagagtaa ctcctcatag gggacagggc ag
#ggaatcca  33240
gtacttattc cagcacagac ttatgagcac cctctcgttt cagtcttgcc tg
#catccctg  33300
tcttcagagg taccttcagt tcccattcct ttctgaaatt cttatttctg gt
#tgggctgt  33360
ccccttgcaa gcattgggca gaacacagaa agctgacaac tcaatcagtt at
#tattcgtc  33420
catatagttt tctctgtcca aaatgttgat attgctcatc tgttgtttta tc
#atttgggt  33480
gtttttattt tttgtcctta tttacatgtt ttttaattct tttactgtga tt
#ttagtgtg  33540
atttgtggag ggattggaga aaagcttgta cattcaatct gccattttta at
#tggaactc  33600
tgtacttatt ttattttatt tacttttttg gagatagggt ctcgctctgt tg
#cccatgct  33660
ggagtgcagt ggtgcaaaca tggctcgctg cagcctcaat attccaggct ta
#agtgatcc  33720
tccaccacag cctcctgagt agctgggagt acaggtgcat ggcaccacac ct
#ggctactt  33780
ttaacatttt ttgtagagat ggggtctcgc tatgttgctc agagtggtct ct
#acacattt  33840
ttaaaaggct ttgacacatg ttaccaaatt acctaccaga aagatcttgc ct
#ctacattc  33900
ccaccaaaag tctttacccc acataattcc tgaccaatac tggataatac at
#attcaaat  33960
atttataaga atacttgaaa gcgttttttt aaaaaattca ggatgctatc ca
#ttatgtac  34020
ccaactataa attatattca gttgtatttc tagattaact tctaacatct tt
#tcaataga  34080
aaacctcaac ctctagaatg caacctctgg gagcaaagag caaagatctg tc
#tttcctgc  34140
ccacaactat aaattgccat cttctgggac agtgttggcc actcagcagg ca
#ctcagtaa  34200
ataattgttg agtaaatgca ttaagaatga aggggaggtg ccatggccag ct
#gtgtccaa  34260
ggggaatgcc tgtgccccct cctgttgcct gttggggtcc tcttcttagg tg
#acttgttt  34320
ttcacctggg attggctttt ctactgtgtt aaatcttaga agtctttttc tc
#tccgtgtg  34380
aaacttcaga atgacagcct gaggctgaaa tggacctaca gacatttgtt tg
#accctcac  34440
aacattgaaa aacaagggag gagaggccag gcccagtggc tcacacctgg aa
#tcccagaa  34500
ctttgggaag ccaagaaggg aggattgttt gagcctagga gtttgagacc ag
#cctgggca  34560
atacagtaag accctgtcta tacaaaaaat taaaaatata aaaattttaa at
#aaataagc  34620
aagggtgggg gaaggagatt tcacataaaa cctgcagctt gggttgggcg cg
#gtggctca  34680
cacctgtaat cccagcactt tgggaggccg aggccggtgg atcacgaggt ca
#agtgttcg  34740
agaccagcct agccaacata gtgaaacccc gtgtctacta aaaaataaaa aa
#tacacaaa  34800
aaattagccg ggcatgatgg caggtgcctg taatcccagc ttctcgggag gc
#tgaggcag  34860
gagaattgct tgaacccagg aggtggaggt tgcagcgagc tgagatcatg cc
#actatact  34920
ccagcctggg cgacagagcg agaccctgtc tcaaaaaaaa aaaatctgca gc
#tctctggc  34980
ttcttttgga agatgtagca gggctggact atctatctgg gttggataac at
#cactgcga  35040
gctgggtaat gatgcccctt tagttgggca tatgatctcg atttactgct gt
#gtcttcct  35100
gtcccacatc atccatttct gtgaactgtt ttgaccctgg agacactgga gc
#ttttggct  35160
tcagctttag aaagtccaaa ctatgcagaa gtggtggtgg tggtggttca tg
#gggttttg  35220
gggatcattc tgactttttg gtaagaagag aacaacttgt aagttttata ct
#acctagta  35280
agtcccatct cgttccctag gtgagtcttc ctcacactca cctttcagag tt
#tatggtcg  35340
atctagttta aacaactgtt gggagacact tatacaagaa tattttcaca tt
#tctgcaca  35400
gttcaggctt tctaagcaaa aaacactagg aaactaagtt aaaagatgac tg
#aatgtcag  35460
aaacgcctcc gaagttagtg tattgctcca gagaaattta gaggctgatt tt
#cccaaaag  35520
ctgtttgctt atattctagg gtaataaaac atagagtcat ctttctcctg ga
#ggcatttg  35580
cttacaattc atagtaaagt gctctctcct tctctggagg gaaagatggg ct
#aaagtgcc  35640
accacccaat ataccacctg agtctcatca ttccagagct ccctcctgtg at
#gcagctct  35700
gccagctgtg caggtcaaca cccggctctc atcacgttgc cctgtgagga ac
#tgggttgt  35760
ggggaactgg cattacaatg ttctgtgagt gataaatggt ctgctctctg gt
#ccagagat  35820
ctcaggtttt ctgtcagaat agagatataa atataaaaca gcaacccctg ct
#agtggcag  35880
cagcctgaag ttttgtgtga tgattccacc tctgtgtgaa ttccacaggg ga
#aacctcca  35940
atttctacaa cttttcctca gaccccttag catctgtatt actccatccc ca
#gactctgg  36000
cttgagactg ttttctttct actactaaga atatccagtt attgtttttc tt
#gttgtaga  36060
gttttcgacc tctcatatga agtacagtgg ccgaagcatc aaggtaagat ta
#gtgctagc  36120
atttttgact tgagaattaa aaccaaacaa ctctattcac taatttagaa cc
#aaatcctc  36180
agcaattaca cttgaccctt caacaatgca gggggtaggg tcactgatgt cc
#ccaacaca  36240
gtcaaaaatc cacacataag ctttgattcc cccaaaactt agctactaat ag
#cctaccgg  36300
ttgttttgtt ttgttttgtt ttgttttgtt ttgttttttg agacagagtc tc
#actctgtc  36360
acccaggcta gagtgcagtg gtgcaatctc ggctcactgc aacgctccgc ct
#cccgggtt  36420
cacgccattc tcctgcctca gcctcccgag tagctgggac tacacgcacc cg
#ccaccacg  36480
cccggctaat tttttgtatt tttagtagag acggggtttc tccatgttag cc
#aggatggt  36540
ctcgatctcc tgacctcgtg atccgcccgc ctcggcctcc caaagtgctg gg
#tgttctgt  36600
tttgttttgt ttagagacag ggtctcgcta tgttggccac gttggtcttg aa
#ctcctggc  36660
ctcaagcaat actccccctt agcctcccaa agtgctaggg ttacagatgt ta
#gccaccgc  36720
acatggctgc agtagcctac tgttgaccag agccttatag ataacataaa ca
#gttgatta  36780
acacacacat tttgtgttat atgtattata tgctgtattc ttacaataaa gc
#caagaaaa  36840
taaaatgtta ttaagaaaat cataaggggc caggtgtggt ggctcacgcc tt
#aatcccag  36900
cactttggga ggccaaggcg ggtggttcac gaggataaga gatcgaaacc at
#cctggcca  36960
acatggtgaa actccgtctc tactaaaata caaaacatta gctgggcatg gt
#ggcgggtg  37020
cctgtagtcc cagctactcg ggaggctgag gcaggagaat tgcttgaacc tg
#gaaggtgg  37080
aggttgcagt gacccgacat catgccaccg gactccagcc tggcaacaga gc
#aagactcc  37140
gtctcaaaaa taaaacaaac aaacaaccaa aaaaaaaaaa caaaaaaaga aa
#atcataag  37200
gaagagaaaa tatatttact cttcattaag tggaagtgga tcaccataaa gg
#tcttcatc  37260
ctcactgtct tcatgttgaa taggctgagg acgaggagga acaggagggc tt
#ggtcgtgc  37320
tgtcacagag gtagcagagg aggaagaaaa tccacatata ggtggacttg cg
#tagtttga  37380
agccctgttg ttcaagggtc aactgtattt cttggaaaaa caacaactca ca
#tatagttc  37440
ctagagtagc aaatcgttcc tgggaaaatt atgccttgcc atgtgcagtg ct
#tttctgga  37500
gtgtttctgt tctttacata atgagctgag tagctccctt agacattttt tt
#ttttttga  37560
gacagagtct cactctgttg cccaggctgg agtgcagttg gcacaatctc gg
#ctcactgc  37620
aaccaccacc tcctgggttc aagcgtttct cctgcctcag cctcctgagt ag
#ctgggatt  37680
acaggcacct gccaccacac ccagctaatt tttgtatttt tagtagagat gg
#ggtttcac  37740
catgttggtc aggtgggtct tgaactcctg accttgagtg atccgattgc ct
#cggcctcc  37800
caaagtgctg ggattacagg tgtgagccac cacacctggc cagacatatt tt
#aatttgtc  37860
ttttttcaac ctatttagaa attaggcaat tcttttcttt cccccagtgg tg
#gaaagatt  37920
ttcctagctg tctaatttat aagtttttgg aaagatattt gcaattctta gt
#ttctcaac  37980
tacctgaccc ttcttttcct atgagccttt gagaaatact tatgcatagg ta
#ctgcttag  38040
cattgtaaaa ggagtttatt gacctaaaaa attgtaatgg ctgttactag gc
#agatggtt  38100
aagcactgga tgaatctgcc tttatgtcct aagtcatttt tgagaaatga gg
#aaaatcat  38160
ctagacagta aaactggggt ctacactaca tctcatctac ttttaatgcc ta
#agtttcta  38220
gagtcaggtt ccatttcctt ccttcttaca cacaggtggc aatagaatga aa
#attaaaca  38280
tacatttctc aattactacc catgacccat gcctataaat attgtgatat aa
#ataggtat  38340
tgaatctgta tacacaggaa aagaccacaa tgaaaagaag cataaagtta ag
#gagtttat  38400
agcttactgc ccgcaaagtt taatattata cattgggtta cactgacctc ta
#caggatga  38460
taataaaaac tagcttagtt tgaaactaga ggagggcaaa ggagaaagga aa
#aactagct  38520
tagtttgaaa ctagagggca aaggagaaag gaaagccatc catttgcctg tc
#atccacaa  38580
aaatgaaatt ttgtacattt cattcacaaa ctaattcagc aaaacgatgg tg
#aggtagtt  38640
gtgtttcgga tatgaattct gagttagtca aataactggt aatttttgag gt
#attttaac  38700
agcaatttta aactgttttc agtgggattt caaaaatctt aaatcaattc ta
#tggaaagt  38760
aaaaagaaaa aagaagagaa ataaaatgct tcttatctta aatttttacc at
#ttacatta  38820
tagggccttc atttaaaaat atataaccat gaatatttac atctataata at
#cctggttt  38880
taaaacgtgt tgttttaaat tggttctaaa aaaaatattg ggaatgaggt tt
#taatttta  38940
aaaattgtga tctttccagg catagtggct catgcctgta attccagcat tt
#tgggaggc  39000
agaagtggga ggattgcttg aggccaggag tttgagacca gcctgggcaa ca
#tagagaga  39060
ccttgtctct actaaaattt aaacattagc cgagcatagt ggcacatgcc tg
#cagtccca  39120
gctacttggg aggctgaggt gggaggatcg cttgagccca ggaagtcaag gc
#tgcaatga  39180
gctgtgatta tgccactgca ccccagcctg ggtgacagag cgagatcttg tc
#tcaagaag  39240
aaaaaaaaga attgtgattt ccaggatagc tttgaacttt aaaagccttc ct
#taagagga  39300
tattataatc tctttagact actttaaacg agttagcgtg atatttatat at
#gtttctgc  39360
attcacagct ttttctgtct tccttttagt tccttctgcc accactgtca ct
#cttgccca  39420
cgcgatctgg tgtccttact atcccccaaa atcacaagtt tccaaaagaa aa
#agaaagaa  39480
acattccaag tctcacatct tttgtgccta agctctcagt gtctgttcgt ca
#atctgatg  39540
agctcagccc atcaaacgag cctccgggag ccctagttaa gtcgttgatg ga
#tccgactc  39600
tcaggtcttc tgatggcttc atttggtcaa gaaacatgtg ctcttttcct aa
#gactaacc  39660
atcacaggca atgcctggag aaggaggaaa actggaaatc caaggaaata ga
#agaatgta  39720
acaaaattga aatcactcac tttgaaaaag ggcagtcttt ggtgtctttt ga
#gaatttga  39780
aggaaggcaa tattcctgca gttagggaag aggatattga ctgccatggt ag
#taaaacgc  39840
gaaaacctga agaagagaac tctcaatatc tttcatcaag aaagaatgag ag
#ttcagtag  39900
ccaaaaacta tgaacaagat ccagaaatag tatgtaccat tccaagcaag tt
#ccaagaaa  39960
cccagcattc agaaataact ccaagccagg atgaagagat gagaaataat aa
#agctgctt  40020
caaaaagagt ttcattacat aaaaatgaag caatggaacc aaacaatatt tt
#agaagagt  40080
gtactgtact taaaagctta tccagtgtag tctttgatga ccccattgat aa
#actcccag  40140
aaggttgtag cagcatggag acaaacataa aaatatcaat agcagaaaga gc
#caaaccag  40200
aaatgagtag gatggtgcct cttatccaca tcaccttccc tgtggatgga ag
#ccccaagg  40260
aaccagtgat agccaaacca agcctccaaa caagaaaggg aaccattcat aa
#caaccata  40320
gtgtcaacat acctgtacac caagaaaatg acaagcataa gatgaattcc ca
#taggagta  40380
agttggattc aaagaccaag acaagtaaga agacacctca gaattttgtg at
#ttctactg  40440
aaggtcccat taagcctacc atgcataaaa ccagcataaa aacacaaatt tt
#cccggctt  40500
tgggacttgt ggaccccagg ccttggcaat tgcccaggtt tcaaaagaaa at
#gccacaga  40560
tagcaaagaa gcaatcaact caccggactc agaaacctaa aaagcaatca tt
#tccttgca  40620
tctgtaaaaa tccaggaaca cagaagtcat gtgttcctct ctctgttcaa cc
#gacagagc  40680
caagactaaa ttacctagat cttaagtata gtgatatgtt caaagaaatc aa
#ttcaactg  40740
ctaatggacc tggaatctat gaaatgtttg ggacccctgt ttattgtcat gt
#gcgagaga  40800
ctgaaaggga tgaaaacacg tattaccgtg agatatgttc ggctccatca gg
#cagacgta  40860
tcaccaataa atgtcgatct tcacacagtg agaggaagag caatatcaga ac
#aagacttt  40920
ctcagaaaaa aacacatatg aaatgcccaa agacttcatt tggcattaaa ca
#agagcaca  40980
aagtcttaat ttctaaagaa aagagttcca aggctgtaca tagcaaccta ca
#tgacattg  41040
aaaatggtga tggtatttca gaaccagact ggcagataaa gtcttcagga aa
#tgagtttc  41100
tatcttccaa agatgaaatt catcccatga acttggctca gacacctgag ca
#gtccatga  41160
aacagaatga attccctcct gtctcagatt tatccattgt tgaagaagtt tc
#tatggaag  41220
agtctactgg tgatagagac atttctaaca atcaaatact caccacaagc ct
#cagagatc  41280
tgcaagaact tgaagagcta catcaccaga tcccatttat cccttcagaa ga
#cagctggg  41340
cagtgcccag tgagaagaat tctaacaagt atgtacagca agaaaagcag aa
#tacagcat  41400
ctcttagtaa agtaaatgcc agccgaattt taactaatga tctagagttt ga
#tagtgttt  41460
cagatcactc taaaacactt acaaatttct ctttccaagc aaaacaagaa ag
#tgcatctt  41520
cccagacata tcaatattgg gtacattatt tggatcatga tagtttagca aa
#taagtcaa  41580
tcacatatca aatgtttgga aaaaccttaa gtggcacaaa ttcaatttcc ca
#agaaatta  41640
tggactctgt aaataatgaa gaattgacag atgaactatt aggttgtcta gc
#tgcagaat  41700
tattagctct tgatgagaaa gataacaact cttgccaaaa aatggcaaat ga
#aacagatc  41760
ctgaaaacct aaatcttgtc ctcagatgga gaggaagtac cccaaaagaa at
#gggcagag  41820
agacaacaaa agtcaaaata caggttggta taattagaat ccaagattca tt
#ggggtggg  41880
aaggacctca gagacaatct ggttcaaacc ccttattttc aaatgaggaa tt
#ataaaccc  41940
taaacaatta aatagttttt tcaaggtctc actgtttgat cacaaggttg ga
#aatcaggt  42000
cctctgaccc ccaggctaag atgttttcat tatattgact cccttctgga at
#ttagctag  42060
cttgacattg caatgaaatc agtttggtta aattaattta gcaaaaccat tc
#aaataggt  42120
cagtatttta ttcaatgatg acattttcaa tcaacagcat atcatttcca ac
#tatcagca  42180
gatacataat tataggcaag acattgctct aggtatgtga gatagaaaga aa
#tgaacatg  42240
gctccagaag tggctcacca ttttgttcat aggaagacat gaaatgtaca tt
#tctcagag  42300
cccctacacc tgagcatttg ctctcagatg attcactact ttaatgcaaa at
#tattattg  42360
atgcctactg tgcttctggc agtgggccaa gaactaggag catagtgctg ta
#caagactc  42420
ggccattgct ctcatggaag tgtaagcaaa aatcctgaaa taagattttt aa
#aaattttg  42480
tttggcatga gagttggcat ggagtgggga agaagatcaa cacatagtcg gg
#ttttcttt  42540
gttatcgttt tcactaaagt acacaagcct cccaaactga aattttaaag ac
#agaaacag  42600
taggtaaact gaaatattat ttattgaaca ctaactcagg tcatactgca ct
#atatccac  42660
actatatcag gatcaggaat aatttttttt tgagatggag tcttgctcta tt
#gcccaggc  42720
tggagcgcag tgctgcgatc tcggcttgct gcaaactcca cctcctgggt ta
#aagcgatt  42780
ctcctgcctc agcctcccaa gtagctggga ttacaggcac tcaccaccac gc
#ctggataa  42840
tttttgtatt tttagtagag acgggatttc accatcttgg ccaggctggt ct
#tggaactc  42900
ctgacctcgc gatccacctt cctcggcctc ccaaagtgct gggattacag gc
#gtgagcca  42960
cggcgcccag ccaggaataa ttattttaaa taattattgg tcagaagaac at
#acaaggta  43020
aataattatc ccatagcttc ctggactgtt tgctagagat actagtctga ct
#tactgcaa  43080
gtctggcttg tggatggtaa actggcttcc tgttttggtt actgtagata at
#gggttgat  43140
ttcctgggtt ggttgctgca cattgtaggt cagagttcta tttttatata tg
#atctggcc  43200
attgttggtt tgtatattat ctctcagtac atatgtgtat gtatatatat ga
#tatatatg  43260
tgtgcatgat atatatttat gtttatgtgt gtgtacattt gtgtgaacac at
#atgtgaat  43320
atgtgtgtat gagtttgtgt gtctctatgt gtgtgtccag ctctgtgtat gt
#ttctcttt  43380
ctgaacttgt ctgtgtttag gagcaagctg accacgataa tgggaatttt ga
#ggagagag  43440
ttgaggttag ggggctgagg agatggcaca cactaacata ttctgtcatg at
#agggacct  43500
tgtgaaagat aattctcaaa agacagtggt tagtagctgc aggcctatgt gg
#ggcctgag  43560
atgaacagga ctaagatctc ctcctataaa atatgcagag caagatgtgg tt
#ttaaaatg  43620
tgtataatta acaaggctga agttcacaac taagatacac tatgtggtca tt
#tgggggaa  43680
tgatgtgtct ctagaagtta cctgtaagag tggccacaga caggaacatt tg
#aaaagaag  43740
actttactct cacccctttc tctccatccc agtgacttgg tttaatggtc at
#ctttcctt  43800
ttgtctcatt cttccagagg catagtagtg ggctcaggat atatgacagg ga
#ggagaaat  43860
ttctcatctc aaatgaaaag aagatatttt ctgaaaatag tttaaagtct ga
#agaaccta  43920
tcctatggac caagggtgag attcttggaa agggagccta cggcacagta ag
#ttaaactg  43980
gaaacttgaa atcaaacctt ccccccaccc ccccacagtc cctccctcca cc
#cctcccac  44040
tcccccagtc atcctccctg cttcctctgg caagcactct tttacttaga ac
#tctttcag  44100
ttggaagtaa cagaaaatcc aaccactgag ggaaaggaca gttactgctt ta
#tccgactg  44160
aaaggtctgg aataggtctg gctctgggtc caggaggctt cagggatcag ac
#aatgtcat  44220
caggatctgg tctctctctc tctttgcctg gctttttctc aggcacatat ag
#tgactcaa  44280
tggccactgc atttctaacc tctcatcctc ccaggttcaa gtccaatggg aa
#agaaatat  44340
cttccttcaa cagctgaata tgttactgga agtttggaga atcattacta ga
#tggcaaaa  44400
acaaaagatg ttccttccat tttgtgaact gcataagaga tcttgggggg tg
#ggcgatga  44460
agagaggtgg gtacaaacat acagtcagat agaagaaata agttctagtg tt
#tgataaca  44520
cagtagggtg actatagtta acaacaatat attgtgtatt tccaattagc ta
#gaagattg  44580
aaatgccccc aacacaaaga aaatgacaaa tgtttgaggt gatggatgtc ct
#aaacacac  44640
tgtcttgatc attacacatt ctatgcatgt attaatatat cagatgtgcc tc
#ttaaatat  44700
gtacaaacat tatatatcta aaccctagca ctttagatag ttatttacat ag
#acgagtaa  44760
agaaaaggct ggcccccaaa taagacttgt gctgtctcca gatggggaca tt
#tcagaaat  44820
cagtgagaag acaggaagac acaaaaccac tgagattaca tcacaatggt ga
#tttccagg  44880
gcctgtctcc ttctcactcc agagagcttg ggagctgaac cagctctatt tt
#acatatta  44940
tcaggagctt ttccaaacca ccatctcatg tagtcatcat agaaatctgg ga
#ggcaggcc  45000
aggtgtggtg gctttcacct gtaatcccag aactttggga ggccgaggcg gg
#tggatcac  45060
ttgaggtcag gagttcgaga ctagactggc catatggtaa aaccccgtct ct
#actaaaaa  45120
tacaaaaatt agccaggtgt ggtggcacag acctgtaatc ccagctactc ag
#gaggctga  45180
ggcatgagaa ttgcttgaac cccggggcag aggttgcagt gagcccagat ca
#caccactg  45240
cactccagcc tgggcgacag agcgagaccc tgtctccaaa aaaaaaaaaa aa
#agaaaaaa  45300
atctgtgagg cagcctgggc aacatagaga gacctcgtct ccacaaaaat ac
#tttaaaaa  45360
ttagcctagt gtggtggtac atgcctgtag tcccagctac tcaggacact ga
#ggcaggag  45420
gatcgcttga gcccaggaat ttgaggctgc agtgagatat gatcagggcc ac
#tgcactcc  45480
agcctgggtg acagagagag actctgtctc caaaaaaaaa aaaaaaaaaa aa
#gaaagaaa  45540
aaggtagcac ggtggctcta caaaaagtac acacacacaa ttagccaggt gt
#ggtggcac  45600
acacctgtga tcctagctac gagctgctca ggaggctgag gtaggaggat tg
#cttgaacc  45660
caggaggttg agcctgcaat gagctgtgat tgtgccaatg cactccagcc tg
#ggcaacag  45720
agtgagaccc tgtctaaaaa caaccaaaaa aaaaaaaaaa aaaaagaaaa ga
#aatctctg  45780
aggcaagtat tgttacctca gttttacaga tgagaaaaac tgaagtcaaa ag
#attacaca  45840
tttatcccaa gttatatagc tggggaaaga tgaagccagg attctagcca at
#tcaagcca  45900
cttgacttta agccaatatg acatccatcc accatgtttc tcatacccat ct
#tggctcca  45960
ctgaaacact gaatttgctt aaacactttg catttaggaa gggaggtatc aa
#cttagaga  46020
aagacaaggg tttagaaaga gaagggaaag tcaagtgtca cctgaggcat tt
#tgtgaata  46080
agttatgtca ttaatttaat aacaaggtat tattgatttg cttctaggta ta
#ctgtggtc  46140
tcactagtca aggacagcta atagctgtaa aacaggtggc tttggatacc tc
#taataaat  46200
tagctgctga aaaggaatac cggaaactac aggaagaagt agatttgctc aa
#agcactga  46260
aacatgtcaa cattgtggcc tatttgggga catgcttgca agagaacact gt
#gagcattt  46320
tcatggagtt tgttcctggt ggctcaatct ctagtattat aaaccgtttt gg
#gccattgc  46380
ctgagatggt gttctgtaaa tatacgaaac aaatacttca aggtgttgct ta
#tctccatg  46440
agaactgtgt ggtacatcgc gatatcaaag gaaataatgt tatgctcatg cc
#aactggaa  46500
taataaagct gattgacttt ggctgtgcca ggcgtttggc ctgggcaggt tt
#aaatggca  46560
cccacagtga catgcttaag tccatgcatg ggactccata ttggatggcc cc
#agaagtca  46620
tcaatgagtc tggctatgga cggaaatcag atatctggag cattggttgt ac
#tgtgtttg  46680
agatggctac agggaagcct ccactggctt ccatggacag gatggccgcc at
#gttttaca  46740
tcggagcaca ccgagggctg atgcctcctt taccagacca cttctcagaa aa
#tgcagcag  46800
actttgtgcg catgtgcctg accaggtaag aaactgaaag caagaggagg aa
#gataaatg  46860
cccggagatt ccaagtggca gacatttccc tttcaattta tggcccatta aa
#agctctgt  46920
tttggttatg aagtcaagta gacagtgatt ttgtgccgaa agtaatcata at
#cagtcata  46980
ttgggtaatt gtgttcattg ttgtatcagg gtataggagg caatgcttca ag
#tagaaagt  47040
gcctcaatta aatgtcttat caagttctgt caatacttgc ccaaatcaat gg
#gtttgcaa  47100
aatttgttaa agatctactt atttaccaat gagacatctt cctaggaact gg
#ctagggtg  47160
aaatgacatc atcttgcatt taaagtgagg ggaaacattt tgagccaaag aa
#acaaattg  47220
gagatttcaa gcgtcaagtg ggggagtatt tggtgaatcg gaaaagcctt ag
#aaaattgc  47280
ctgttttccc cttccttatc ttctctccta tctatggaat taaattgtgg gt
#aaaatgtt  47340
agaactgtaa ctgtaatgta atggaaatta actagtgctg tgattttcaa at
#ttttagcc  47400
aggtactctg ctcatagaaa tcttaaatca aagaataaaa taaaagcaga ca
#gatggctc  47460
tagttaaagt gtgtatccat ggggcgggga agagttaagg agtagggctg tg
#ggtgctgg  47520
agcccactct aggatactgc acagcagccc caaacccacc tacctagcaa gg
#ctcaactt  47580
taattggagg acaagaaagg cctgagactc aaagtcaatt tcctgtcttt ca
#agtaagtt  47640
tgccttctta tccctagatg aaaaactcca gtgtcccatc ttttagcaag ca
#catatggc  47700
aacccccaac tcccaggggg ttcattttgc ctttctgaat aaatcttaga at
#ctacaggt  47760
ctccctctct gccaatgaat gtgcctctct ttcagtctct gtctctctct cc
#cagcacat  47820
gtgtatcagc ctgtcctggc tgatttcagg atgattacat gggccagggc ag
#gaatgcca  47880
ctccaggggt acagtttttg gcattgctag atgcagagaa cccttaggtt tc
#cagcgtgg  47940
attttgtgga cagagcccca gtcattgagc tgcccaccct ctccaaaaaa aa
#aaaaaaaa  48000
aaaaccgcat aaatgtgttg gaaaatcgta tagacaagta ctagtttgat at
#tggtgtta  48060
actgttaaaa ctattgtagt tgctttgttc cgaatttaac aattacctat at
#tattgact  48120
cacagctaga aaccacttgt tattctcatt ttctttcaag ttgtgattac ac
#acacacac  48180
acacacacac acacacacac acgaagcact ttaaagagaa agggtggaat ct
#tcttttat  48240
ggctctcctt ttgaaccgtt gcttcataaa ctaagcaata tacaattcac ac
#cactaata  48300
aaaattaaca gggttattgt gaaggttaag tgaaatggtg catgtaaatt gc
#ttagcaga  48360
gtgtggggca caaaattagg agtttacagt taataatcat taggaagaat at
#taacatac  48420
cttacctaat tagagtcata tacaagtata taattacctc ctaaaattct at
#ggcaaaga  48480
ccctgaggac cctagcatct cacctgatat caataacaat actccttgga ga
#tagggata  48540
ttcagaaaat aaagggcgag gcactcttaa agattcagaa atagagataa tc
#aggcatag  48600
actagggaaa gtctaaagaa aacagaaatg aacttgggga agctgagaga aa
#taagcatg  48660
gagggggtac tcctattgac agatcaagtt cctgggaagt caggccaagg ag
#tttagctt  48720
tgttgcaata ggcagtgagg agcagggggc tgcaaaagat ttggggtaga aa
#aggccata  48780
aagaaaaggg tctttgggaa ggcaggtcag atggcaatgt attgaagggc ct
#gggatgga  48840
tgtcgcttga gactagaaag ctctgcagaa atccagagct tggatgctga tg
#gtggtaga  48900
agcagtggga ttgtaaagga ttccagaaaa tttcagagaa aaggtgaatc aa
#gacttggt  48960
aatggagcag aatgatagga tttcacattt ttgactctgg ataatgggag aa
#atcacagt  49020
tgtgagagaa gaacagggag gcagctaaac ccttcccacc tcctgtaagg ag
#acatttga  49080
agctatggaa ttgcagctca ggaaagcaat taagattgga aggacacatt ta
#aaaataat  49140
tataacagcc aggtgcagtg gctcatgcct gtaatcccag cacttaggaa gg
#ccgaggtg  49200
gggggatcac ttaagcccag gagttcaaga tggagaccaa cctgggccac at
#gaagaaac  49260
cccatcttta caaaaaaata caaaaattag ccaggcatgg tggtgtgtgc cc
#gtagtccc  49320
agctactcag gaggctgagg tgagaggatg agaggatcgc ttgaccccgg aa
#gttgatgc  49380
tgcagtgggc tgagatggca ccactgcact ccagcctaag ggacagagtg ag
#actctgtc  49440
tcaaaaaaaa aaaaaaatca ttataaggtt gattgctaca gtcataacaa aa
#ttataggg  49500
ctgaggaaaa tattttgaaa atgctcacaa tggaagctaa cagaaatgca tg
#gcatcaag  49560
tctagcacat aactggagaa gggaagggag gaagggaagg gagttgcccc aa
#ggtgtaag  49620
aagaaacaag aggacagagt gtccctaagt ctaagcagag gtagtttcag gt
#aggaggga  49680
gtagtgaatg tttcaagcgc tacagaaatg acaaacagct cattaaatct gg
#ttaatttc  49740
aagagggcaa tttctataga ggaatgggcc aaatggttaa gaatacaggg gg
#gaagtcac  49800
cgagcttagc cttgttagag acatttggca gagacattta aaatgggatg gg
#ccaggcgc  49860
agtggtccac gcttgtaatc ccagcacttt gggaggctga ggcagaataa ct
#gattgagc  49920
gcaggagttt gagatcagcc tgggcaacat agggagaccc tgtttctaca aa
#aaatttaa  49980
aaattagccg ggcgcggtgt cacgccagta atcccagcac tttgggaggc cg
#aggcgggc  50040
ggatcacgag gtcaggagat caagaccatc ctannnnnnn nnnnnnnnnn nn
#nnnnnnnn  50100
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  50160
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  50220
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnct gg
#gtgacaga  50280
gcgagacttc atctcaaaaa aaaaaaaaaa aaaaaaaaat ttaaaaatta gc
#aagtcatg  50340
gttgtgtaca cctgtagtcc cagtgactca gaaggctgag gtgggaggat ca
#cttgagcc  50400
tggaaggttg agaccacagt gaaccgtgat catgccactg cactccagct tt
#ggcaacag  50460
aatgagaccc tgtctcaaaa aaaaaaaaaa gtgggtgggg gagcggtggt ag
#ctagaaat  50520
ggtatccagt tcaaggaaag gattttaaag gagagagatt tctgcatatt tt
#aaaggccg  50580
gagaaagggc ctccagatag tgaaagaatt tttttttttt ttttttttcc ga
#gacggagt  50640
cttgctttgt tacccaggct ggaatgcggt ggtgtgacct tggctcactg ca
#acctccgt  50700
ccatgggttc aagcaattct cctgtctcag cctcccaagt agatgggact ac
#aggcgcct  50760
gccactgggg ccagctgatg tttttgtttt tttagtagag acggggtttc ac
#catgttgg  50820
ccaggctggt ctcgaactcc tgacctcgtg atccacccac cttagcctcc ca
#aagtgctg  50880
agattacagg tgtgagccac tgtgccttgc tgtatttttt ttttttttac tt
#ttgaaatg  50940
acacaaaata taatactttt atacaaaata cttttaagag tatttatttc ca
#ttttcacc  51000
tggaaaatga tctggtggcc attgtgcttt caaaattatt aaaagaggag gg
#gcttcaag  51060
atggctgact agagacatct ggcacttact tcctccacaa agaactaaaa ta
#gcaagtag  51120
ataagcacat ttcaaatata gcatcctgag agagaacact ggatttcaac ag
#agaagtta  51180
caggaaacac ctgagacatg gaagaaaagg aaaggaagac agtcagtttg gt
#tgagattg  51240
gccgagagcc cagagagcct ccctagtgtg gggaaagggt gagcagatcc tc
#agtggtcc  51300
acattctcac agtgaactcc tgcaatccta gccatgggag aaccctttag tc
#cttgcaga  51360
cactgagact agaatatgga gctgcctgga aaccatgtga cagcattgct cc
#ggagaggg  51420
agctcacacc tgagtcctaa gcagctacag catggcacca ttttgagagt cc
#agccccca  51480
ccagactcca tcccgccctg gggtccaaca gcccctgcaa ctccatatcc tt
#ggaaccct  51540
acttacatct tcttgtgttt acctggaggg ctgcagcagt gtgatgccag tt
#gtacccag  51600
tggagtggcc agatccccag cattgtagca cacatggtgt cctgcacccc ag
#aaacaaca  51660
gtgcagcgca ccagggaggc tgctcctggg acaaagggag ccaaagcatg tg
#ctccccag  51720
tgcctaagaa ctgcctacct gaggtggcta ttacagatag caaccccacc ct
#ttctagca  51780
gcagggctgc cacacacatg ctctgaggac agactctgct gctgtccact gc
#agcttctg  51840
cttaggctga agtgtgtgcc actggcagtg accccacccg cttcagcaac ag
#ggttgcag  51900
cacatttgca tgtgccctga ggactggctt tcttggctgc agctgctgcc ac
#caccagaa  51960
gccaaaccat gagctccctg gaacctgaga gccacctgcc tgaagctgct gc
#cactgacg  52020
gcaactctgc ttccaccagt agcagggcta tagcacactt gcacatgccc ta
#atgacagg  52080
ctccccttgc ccaccaccac cggagctgca gccacccaat catcatgcca gg
#gccctggg  52140
gatcacccca ccctgcccac tactgctgac ccctgcgtgt accactggag gg
#cctgagga  52200
aaggtcaacc aagcctggcc cagcagccct gccggtgtct gagcacattg cc
#tggggcct  52260
ggggattctc tgccctatca ctgctggtat ctgtacattc ctcatgagga cc
#tgaggacc  52320
ggcccatcca gcccattgca gccactatta acaccagtgc ctgctgctat gg
#agcccaag  52380
cattatccca gtaccactat tgccattgcc catgccatgc atgctgccca gg
#agtctaag  52440
gacctatcca cccacccagc acaccactgc cactaccagg acctgagcaa gc
#cttggagg  52500
cccaagaatt ggctcatttg aacccactaa cactagtgcc catgtatgtc ac
#ccaggggc  52560
ccaaggatgg gcatgcttga cacaccactg ctaccactca gnnnnnnnnn nn
#nnnnnnnn  52620
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  52680
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  52740
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  52800
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  52860
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  52920
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  52980
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  53040
nnnnnnnacc cattcagaca aaaataaaga aaaaaaagag tgaacaaagc ct
#acatgaca  53100
tgtaggaaac tataaattgg ccagatatac aattttgatt gttccagaag gt
#gaagagaa  53160
gaccaaaggt atagaaaatc tatttaaaga cagaatagtt gaaactttcc ca
#ggtctagc  53220
aagagattta aacatccaga tacaggaagc taagagatcc acaaatagat ac
#aacctaga  53280
aaggtcttct ccagggtaca ttgtagtcaa actgtcaaaa gtcaaagaca aa
#gagaaaat  53340
tctaagaaca gcaaaagaaa aacatctagt aatgtataaa agaaccccca tc
#agactaac  53400
agtggattta tcagcagaaa tcttacaggc caggagagaa tgagataata ta
#ttaaaagt  53460
tttaggccag gcatagtggc tcgcacctgt aatcccagca ctttggaagg ct
#gaagtggg  53520
tggatcacct aaagtcggga gtttgagacc agcctgacca acatggagaa aa
#tccatctc  53580
tactaaaaat acaaaattgc ccaggtgtgg tggtacatgc ctgtaatccc ag
#ctactctg  53640
gaggctgaga caggagaatt gcttgaacct gggaggtgga gggtgcagtg ag
#ccgagatt  53700
gtgcctttgc actctagcct gggcaacaac agcaaaactc catctcaaaa aa
#acaaacaa  53760
acaaaaaaaa aagttttgaa aggcataaaa acaaaacaaa actgtcagcc aa
#gaatgcta  53820
tactcagcaa agttatcctt caaaaatgga gaaagtcttt cacagacatg ca
#aaaactga  53880
gagacttcat caccattagt ggccctacaa gaaatgctta agaaagtcct ac
#acctggaa  53940
gtgaaaggtc atatctatca tcatgaaaac atatgaaagt gtaaaactca ca
#ggtagagg  54000
aaaccacaca aaagaggtag agaaaggact caaacgttaa cactacagaa aa
#ccaccaaa  54060
ccacaatgat aaataacaag agagaaagaa agaaagaaac aaacaaacaa ac
#aaacaaac  54120
caaccagaaa acaatcaaca aaatgacagg aataagaaca taaatggatt aa
#aatttcca  54180
attaaaatgg ctgaatagat ttttaaaaag tgacccaaaa atatactgct tt
#caagaaac  54240
tcactttacc tgtaaagaca catatagact gaaagtgaaa ggatggaaaa ag
#atagttca  54300
tgcaaataga aaccaataga gagcatgagt agctatattc atatcagata aa
#acacactt  54360
tatgtcaaaa acagtaaaaa gagacaaagt cactatataa tgataaagag aa
#aaattcag  54420
ccagaggatg taacagttct gatgcaccct gcaccagagc acccaggtat at
#gaagcaaa  54480
tattattaga tctgaagaga gagataaact ctaatacaat catagatggg ga
#ctttaaca  54540
ccccactctc aacattaagc agatcatcta aacaaaacat caatagagaa ac
#ctggattt  54600
aaattgcact ttaaaccaaa cagacacaac agatacctac agaatatttt ct
#ccaacaat  54660
ggcagaataa atgttcccat taaaacatgg aacattttcc aggataggcc at
#acattagg  54720
ctgcaaaaca agtttcaaca aatttttaaa aatcaaaatc ataccaagta tt
#ctttcagc  54780
cacaatggaa taaaactaga aatcaataac aagaggaact ttggaaactg ta
#taaataca  54840
tggaaactaa acaacatgtt cctgaatggc tactggggca agaaagaaat ta
#agaagaaa  54900
attaaaaaat ttctcaaaac aaatgaaaat caaaacacaa catacccaaa tc
#tatgtgac  54960
atagtaaaag cagtgctaag agggaggttt atagcaataa aagcctacat ca
#aaaatgta  55020
tgaagattgg ctgggcatgg tggcttacac ctgtaatccc aacactgtgg ga
#ggccaagg  55080
tgggaggatc acttgaagcc aagagttcaa gaccagcctg ggtagcaatg tg
#agaccttg  55140
tctcaaaaag aaaaaaaaaa aattagctag ctaggtcact tggtaggcta gg
#gtgggagg  55200
attgcttgaa cccaagagtt cgagactgca gtaagccatg attgcaccat tg
#cattccag  55260
atggggtgac cttttaaaaa agtataaaaa tttaaataaa taatcaagga aa
#cagaagaa  55320
aaagggaaca aaccaaaccc caaattagta gaaaaaaaga aataaagatc ag
#attatgtt  55380
aagtgaaata aaccaggatc agaaagacaa acattgcatg tcctcactta tt
#tgtgggat  55440
ctaaaaataa aaacaattaa attcattaac atagagagta gaaggatggt ta
#ccagaggc  55500
tgggaatgat agtaggagga taggagtagg gcagataggg atggttaatg ga
#ttaaaaaa  55560
aaaatagaaa gcttgaataa gacctaccat ttgatagaac atcagggaga ca
#atagtcat  55620
taataactta attgtacatt ttaaaataat taaaagagtg taattagatt gt
#ttgtaaca  55680
caaaggataa atgcttgaga ggatggatac cccattctcc atgatgtaat ta
#tttgacat  55740
tgcatgcctg tatcaaaaca tctcatgtac cccataaata tatacaccat gt
#acctacaa  55800
aaattaaaaa taaaaaaata taaaaatcaa tagaaaagta ataaaggtca ga
#gtagcatt  55860
aaatgaaata cagaaaaaaa tacaaaggat cagtgaaatg agaagttggt ta
#aaaaaaaa  55920
ataaaatcaa taaactgcta gctagactaa ccaagaaaaa aaagagagat ga
#ctgaaata  55980
aaaatcagaa acaaaaaagg agacataaca actaatacca cagaaatgaa aa
#aacccacc  56040
agagaacatt atgaacaaat ataagctaac aaaatggaaa acctagagga aa
#tggataaa  56100
ttcctggaca catacaagac tgagtcagga agaaatagag aacctgaaca ga
#ccaataat  56160
gagcaataag attgaatcag taataaaata tctcctaaca aagaaaagcc ca
#ggactgga  56220
tggcttcact gccatattct accaaactca taaagaagaa ctaacaccag tt
#atcctcca  56280
actattccaa aaaattgaga aggaaggaat tctccctaac tcattcaatg aa
#gccagcat  56340
taccctgata ccaaaaccag acaaggatgc gaaaaccaca aaaaaagaaa ac
#tataggcc  56400
agtatccttg atgaacacag atacaaaatt cctgaacaaa atactagcaa ac
#ctaaccca  56460
acagcacatc aaaaagataa tacaccataa tcaagtgagt tttatactag tg
#atgcaagg  56520
atggtttaac atgcacaaat caataaacat gatacatcac attaacagaa tg
#aaggacaa  56580
aaacaatatg accatctcaa tagaaacaga aaagacattt tctaaaatcc aa
#catccctt  56640
tgtgataaaa actatcaaca aactaggcat agaaagaaca tacctcaata ta
#ataggcca  56700
tatatgacaa acccacagct aacatcatac agaatgggga aaaggtgaaa gc
#ctttcttc  56760
ttagaactgg aacaagagaa ggatgccaac tttcaccgct cctattcaac at
#agtattgg  56820
aagttctagc cagagtgatt aggcaagaga aagaataaaa ggcattcagg ct
#gggcgcag  56880
tggctcatgc ctgtaatccc agcactttgt ggggctaagg caggcagatc at
#gaggtcag  56940
aaaatcgaga ccatcctggc taacacagtg aaaccccatc tctactaaaa at
#acaaaaaa  57000
ttagccaggt gtggtggcgg gcacctgtag tcccagctac tcaggaggct ga
#ggcaggag  57060
aatggcatga acccgggagg tggagcttgc agtgagctga gatcgcacca ct
#gcactcca  57120
gcctgggcga cagagtgaga ctccatctaa aaaaaaaaaa aaaaaaaaag gc
#attcaaac  57180
tggaaaagag aaagccaaac agtgcctctt tgcagatgac gtgatcttat at
#ctagaaaa  57240
acctaaagac tccaccaaaa aactcttaga tcgattcagt aaagattcag ta
#aagttgca  57300
ggatacaaaa ttaacatacg aaaatttgtt gtgtttctat ataccaacaa tg
#aagtagct  57360
gaaaaagaaa tcaagaaggc aatcccattt aaaatggcta caaaaataaa at
#aaaatacc  57420
tgggaacaaa tgtaaccaag gaggtgaaag acctctacaa ggaaaactac aa
#aacattga  57480
tgaaaaaaat tgaagacaca aacaaatgct catgggtcac aagaatcaat at
#tgttaaag  57540
tggtcatact aaccaaagtt atttatggat tcaatgcaaa aataccaatg ta
#atttttca  57600
cagaaatata tacaaaacaa tcctaaaatt tgtgtggaac caaaaaggag ct
#caaagagc  57660
caaagcaata ctaaacaaaa agaacaaagc tggaggcatc acactatgtc ac
#ttcaaaat  57720
atacagaaaa tatatacaaa atatattaca aggctacagt aaccaaacag ca
#tggtattg  57780
gtgtaaaaat agacacataa accaatagaa cagagtagag aacccagaaa ta
#agtcccca  57840
tatgtaaacc aacttatttt tgacaaaggg accaagaaca tatactgggg aa
#ttgacacc  57900
ctcttcaata tatggtgcat attcatatgc agatgaacga agttagaccc ct
#atctcacc  57960
atatacaaaa atcaactcaa aattgattaa atacctaaac ataagactca aa
#actataaa  58020
attactagaa gaaaacatag ggaaacactc caggttattg gtctgtgcag aa
#gctcttta  58080
atatatagtt ccatttgtct atttttggtt ttgtcacctg tgcttttaag gt
#aaaggaaa  58140
gcacagtgtg aagagacgac ctgttgaatg ggagaaaata tttgcaaaat gt
#tcatccaa  58200
caagaaacat atctcaaaag aagacacaaa tagcccacag gtatatgaag aa
#atgctcaa  58260
catcactaat caacaaggaa atgcaaatta aaaccaccaa gagataccta cc
#atcttatc  58320
ccagttaaaa tgactactat taaaaacaca caaaagctct ccctctccct tt
#ccctctcc  58380
ctctcgtctc cctctcccca cggtctccct ctccctctct ttccacggtc tc
#cctctgat  58440
gccgagctga agctggactg tactgctgcc atctcggctc actgcaacct cc
#ctgcctga  58500
ttctcctgcc tcagcctgcc gagtgcctgc gattacaggc acgcgccgcc ac
#acctgact  58560
ggttttcgta tttttttttg gtggagacgg ggtttcnnnn nnnnnnnnnn nn
#nnnnnnnn  58620
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  58680
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  58740
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  58800
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  58860
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  58920
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  58980
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  59040
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  59100
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  59160
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  59220
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  59280
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  59340
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  59400
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  59460
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  59520
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  59580
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnnnnnnn  59640
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn
#nnntttgg  59700
acaatacggc gctttcaagg gcagagctcc ctgagctttc cacagtgtat gt
#tgcccctg  59760
atttattgag actggggagt ggcgatgact tttaccaagt atactgcttg ga
#aacatctt  59820
gttagcaagg cgcatcctgc acagccctag atcccttaaa ccttgatttc at
#acaacaca  59880
tgcttttgtg agcttcaggt tgggtcaaag tggtttgttc aaagtgactg gg
#gcaaagct  59940
acagattaac aacatctcag caaagaaatt gttgaaagta caggcctttt tc
#aaaatgga  60000
gtctcttatg tctttccttt ctacatagac acagtaagag tctgattgct ct
#ttctttag  60060
cctacactca ctgaactgcc cttcccctcc gctgggccat gaccatggag aa
#caggtcca  60120
ctgtcctccc tgcgtggtgc accatggagg ctcagactcc gtcctcgagg ct
#ggcaagaa  60180
gacagggtaa gacatgagcc tcctgataca ggagatgtct gtggagccca ca
#ggactgca  60240
acctcacact gcagggctgg aggcacagac tgactattta ctattctgtg gc
#ctgggggg  60300
ctcaaggcac agagctcctc attagccaaa gtcacccaag ttcccaacct ct
#aaggattt  60360
cctcataata atgcaagaag aagaaaagtg agtgcccgta gaagctttgg gg
#ctcttcct  60420
ctaatcagga gaaagctggt gtgtattctt cacttctttc ttttcttttt aa
#acatccaa  60480
ctgctttaat tttcatcttt tattatggga aaatatatca cttataaata tt
#aaaaaaaa  60540
cccacaaaaa taacagatgc tggcaagaat gtgtagataa ggaaactcac gt
#actgttgg  60600
gtgtgaatgt aaattaatac agccattatg gaaaacagta tggagatttc tc
#aaaaaaac  60660
cccaaaaaac taaaaataga actacctgcc gtgtgatcca gcaatcctcc ta
#ctgagtat  60720
ttatccaaag gaaagaaaat cattatctct aagggatacc tgcatcctca tg
#cttattgc  60780
agcactattc acaataacaa aggtatggat ccacctaagt gtccctcaac ag
#atgaatag  60840
ataaagaaaa cttagtatat atgcacaaca gaatgctact cagccataaa aa
#aaatgaag  60900
tcttatcatt ttcagcaaca gagatggatc tggagttctt tatcttaagt aa
#aataagcc  60960
aggcccagca agacaaatac cacgttctct cttatgtggg agctacgaaa gt
#agatctca  61020
tggaagtaga gagtagaatg atagttatca gaggctggga agggtgtgta tg
#tggtgggg  61080
cagggaggat aaaaagaggt tggttaatgg gtacataatt agatagaagg ag
#taagttct  61140
aatgtttgat aacagagcag ggtgactgta attaacaaca atgtattctg ta
#tttcaaat  61200
agctagaaga gaggacttga agtgttcctg acacatagaa atgacaaata ct
#cattatat  61260
atcaataaag aaagtggttg cacaatgtag cgggtagggg aagttacctg gt
#tgttaaag  61320
ccttaataaa tatttatgta tctgaaaaaa aaatcaaaag atggccaatt ta
#accaaaag  61380
aatgcctctg gaataggcca ttgcagctaa tcattgacta tttcattagc tc
#attggttc  61440
attaactggc tcattgactg atacctttct aaaatctttt gaatttcttg aa
#gaaaaaaa  61500
ctatgccaca atagtactga acaactgtct ccctctatct tacgttaatc ca
#ggagtgcc  61560
caaaacggga ttatttcaat taatcaccaa agcatatttg aatatctatt tt
#aaaaggtt  61620
ttcaattctg gattttaatg cttctgaatt ttaaaagtaa atgtaagtgt ga
#attttacc  61680
atacgtaaat tagactccaa acaaattgca caaaagtaca atgggaaagt ag
#ggcctagt  61740
tttcaatcac aatagctacc acttttcaaa caagtaccat gctattgttt aa
#aagttgta  61800
tatatattat ttaattctcc caatgagtta ggtattattg ttatctccat ct
#tactgatg  61860
aagagagttt tagtcactta gcttaaggtc acacagctaa aaattggaga ct
#ggactcaa  61920
cccaagtctg tttgactatc agaagttgta tttccgtctt taaaagttca ca
#tttaagta  61980
gatctacatt ggcagtctca ttactgagtg ctgctgcttc taatgtgttt tt
#cccttctt  62040
agggaccagc atgagcgacc ttctgctctc cagctcctga agcactcctt ct
#tggagaga  62100
agtcactgaa tatacatcaa gactttcttc ccagttccac tgcagatgct cc
#cttgctta  62160
attgtgggga atgatggcta agggatcttt gtttccccac tgaaaattca gt
#ctaaccca  62220
gtttaagcag atcctatgga gtcattaact gaaagttgca gttacatatt ag
#cctcctca  62280
agtgtcagac attattactc atagtatcag aaaacatgtt cttaataaca ac
#aaaaaact  62340
atttcagtgt ttacagtttt gattgtccag gaactacatt ctctattgtt tt
#atatgaca  62400
tttcttttta tttttggcct gtcctgtcaa ttttaatgtt gttagtttaa aa
#taaattgt  62460
aaaaacaact tatattttct tgcttggtga gtaaagatgc ttacttaatt cg
#tccaaagc  62520
agagcagagg aaggcaggaa ggtaagttaa agagattcta gattctgtac tt
#tggcagca  62580
atcttagcct aaaagattct aggaggctca aggcctaata gggaggaggt ga
#gggcctcg  62640
gcatttcatt atcagagggc ccccaaactc ctcagatgtc tctgagaaat tg
#tgctagtt  62700
aaggcggcat cataaacctt gggctctttt ctctgtaatt tatttgtagt ga
#tttgaagt  62760
ttttaatcta tttgcagtga atcaggtcat ttccatatgc agaactagct aa
#gtctaaat  62820
cagctggtag gacaaaagct aggtctggta agggaaggat gatttttcca ca
#gacctttg  62880
ctcatttcat ttgaatagtt acctctgctg aggtcatcct tcaaatactg cc
#attcccag  62940
aacattagta gacctcacaa aagtgagcat ggatgagtta gtagtattac aa
#gccatttt  63000
aagttggtgg attaagcaat atttttttta gactgagtct tactctactg cc
#ccaggctg  63060
gagtgcagtt gcgttatctt ggctcactgc aacaacctcc gcctgctggg tt
#caagtgat  63120
tcttttgcct cagcctccca agtagctggg attacagttg cccaccacca cg
#cccagcta  63180
atttttgtat tttttgtgga aatggggttt caccatgttg gccgagatgg ag
#tttcactg  63240
tgttggccag gctgtcttga actccagacc tcaagtgatc cacctgcctt gg
#cctcccaa  63300
agtgctggga ttacaggcgt gagccatcgt gcccagccag gattaagcat tt
#tttataag  63360
gtttccattg ctgttgatct cactcatcca ctaaacttcg cacctattgt tc
#tttttttt  63420
tattattatt atttgagatg gagtctcact ctgttgccca ggctggagtg ca
#gtggcgtg  63480
atcttggctc accgcaacct ctgccacctg ggttcaagca attttcctgt ct
#cagcctgc  63540
caagtagctg agattactgg gacctgccac tgtgcctggc taatttgtgt ag
#ttttagta  63600
gagatggggt ttcaccatct tggccaggct ggtcttgaac tcctgacctc at
#gatccacc  63660
cgccttggcc tcccaaagtg ttgggattac aggcgtgagc catcgcgccc ag
#ccagcacc  63720
tattgctcta agctatagcc acagatattt ttattggctg ccgtcatttc aa
#gctggtac  63780
aactaaaaat taactttagg agtattctaa tactggtatc aggatttgtc aa
#aacaaagc  63840
tggtttagtt tttatgaaat aaatgtgaaa tgctgtccag gtgaggtaaa aa
#cagatttt  63900
actctggaca tgtaacatta gatgagtctt tgtgggtata acttttctca aa
#tttttttt  63960
tcatatttaa gaaattaagg gaagaatatg tcctttattt tacttacttg ta
#tctcaaca  64020
tgaccagaaa caacataatt ttgaaaggtt agggcttatt ccttttccat tt
#tggaggga  64080
tcttcagcat tctttcaaat ctgaatatta tattggattt taaagcaact at
#ttacaatc  64140
aagcctgtta aaccctatgg ggaaagggca aagagtaaga cctgttaata ct
#gtgtatag  64200
agatcaccgt aatggacaca agaagttggt gttaacaagt ttattcctat tc
#tactgaaa  64260
tataagggta ctgaagacaa ttttggaata ttgaacagaa acttcaaaaa gc
#tgaagttt  64320
tggccaggca gggtggctca cccctgtaat cccagcactt tgggaggccg ag
#gcaggtgg  64380
atcacttgag gtcaggagtt gggagaccag cctggccaac atgctgaaac cc
#catctcta  64440
ctaaaaatac aaaaaattag ctgggca
#
#          64467
<210> SEQ ID NO 4
<211> LENGTH: 168
<212> TYPE: PRT
<213> ORGANISM: Human
<400> SEQUENCE: 4
Met Glu Phe Val Pro Gly Gly Ser Ile Ser Se
#r Ile Ile Asn Arg Phe
1               5
#                10
#                15
Gly Pro Leu Pro Glu Met Val Phe Cys Lys Ty
#r Thr Lys Gln Ile Leu
20
#            25
#            30
Gln Gly Val Ala Tyr Leu His Glu Asn Cys Va
#l Val His Arg Asp Ile
35
#        40
#        45
Lys Gly Asn Asn Val Met Leu Met Pro Thr Gl
#y Ile Ile Lys Leu Ile
50
#    55
#    60
Asp Phe Gly Cys Ala Arg Arg Leu Ala Trp Al
#a Gly Leu Asn Gly Thr
65
#70
#75
#80
His Ser Asp Met Leu Lys Ser Met His Gly Th
#r Pro Tyr Trp Met Val
85
#                90
#                95
Pro Glu Val Ile Asn Glu Ser Gly Tyr Gly Ar
#g Lys Ser Asp Ile Trp
100
#           105
#           110
Ser Ile Gly Cys Thr Val Phe Glu Met Ala Th
#r Gly Lys Pro Pro Leu
115
#       120
#       125
Ala Ser Met Asp Arg Met Ala Ala Met Phe Ty
#r Ile Gly Ala His Arg
130
#   135
#   140
Gly Leu Met Pro Pro Leu Pro Asp His Phe Se
#r Glu Asn Ala Ala Asp
145                 1
#50                 1
#55                 1
#60
Phe Val Arg Met Cys Leu Thr Arg
165
<210> SEQ ID NO 5
<211> LENGTH: 275
<212> TYPE: PRT
<213> ORGANISM: Dictyostelium discoideum
<400> SEQUENCE: 5
Ile Ile Asn Glu His Glu Glu Leu Ile Ser As
#n His Asn Ile Lys Trp
1               5
#                10
#                15
Gln Lys Gly Gln Ile Leu Gly Arg Gly Gly Ty
#r Gly Ser Val Tyr Leu
20
#            25
#            30
Gly Leu Asn Lys Asp Thr Gly Glu Leu Phe Al
#a Val Lys Gln Leu Glu
35
#        40
#        45
Ile Val Asp Ile Asn Ser Asp Pro Lys Leu Ly
#s Asn Met Ile Leu Ser
50
#    55
#    60
Phe Ser Lys Glu Ile Glu Val Met Arg Ser Le
#u Arg His Asp Asn Ile
65
#70
#75
#80
Val Arg Tyr Leu Gly Thr Ser Leu Asp Gln Se
#r Phe Leu Ser Val Phe
85
#                90
#                95
Leu Glu Tyr Ile Pro Gly Gly Ser Ile Ser Se
#r Leu Leu Gly Lys Phe
100
#           105
#           110
Gly Ala Phe Ser Glu Asn Val Ile Lys Val Ty
#r Thr Lys Gln Ile Leu
115
#       120
#       125
Gln Gly Leu Ser Phe Leu His Ala Asn Ser Il
#e Ile His Arg Asp Ile
130
#   135
#   140
Lys Gly Ala Asn Ile Leu Ile Asp Thr Lys Gl
#y Ile Val Lys Leu Ser
145                 1
#50                 1
#55                 1
#60
Asp Phe Gly Cys Ser Lys Ser Phe Ser Gly Il
#e Val Ser Gln Phe Lys
165
#               170
#               175
Ser Met Gln Gly Thr Pro Tyr Trp Met Ala Pr
#o Glu Val Ile Lys Gln
180
#           185
#           190
Thr Gly His Gly Arg Ser Ser Asp Ile Trp Se
#r Leu Gly Cys Val Ile
195
#       200
#       205
Val Glu Met Ala Thr Ala Gln Pro Pro Trp Se
#r Asn Ile Thr Glu Leu
210
#   215
#   220
Ala Ala Val Met Tyr His Ile Ala Ser Ser As
#n Ser Ile Pro Asn Ile
225                 2
#30                 2
#35                 2
#40
Pro Ser His Met Ser Gln Glu Ala Phe Asp Ph
#e Leu Asn Leu Cys Phe
245
#               250
#               255
Lys Arg Asp Pro Lys Glu Arg Pro Asp Ala As
#n Gln Leu Leu Lys His
260
#           265
#           270
Pro Phe Ile
275
<210> SEQ ID NO 6
<211> LENGTH: 277
<212> TYPE: PRT
<213> ORGANISM: Arabidopsis thaliana
<400> SEQUENCE: 6
Asn Thr Val Asp Met Ala Pro Pro Ile Ser Tr
#p Arg Lys Gly Gln Leu
1               5
#                10
#                15
Ile Gly Arg Gly Ala Phe Gly Thr Val Tyr Me
#t Gly Met Asn Leu Asp
20
#            25
#            30
Ser Gly Glu Leu Leu Ala Val Lys Gln Val Le
#u Ile Ala Ala Asn Phe
35
#        40
#        45
Ala Ser Lys Glu Lys Thr Gln Ala His Ile Gl
#n Glu Leu Glu Glu Glu
50
#    55
#    60
Val Lys Leu Leu Lys Asn Leu Ser His Pro As
#n Ile Val Arg Tyr Leu
65
#70
#75
#80
Gly Thr Val Arg Glu Asp Asp Thr Leu Asn Il
#e Leu Leu Glu Phe Val
85
#                90
#                95
Pro Gly Gly Ser Ile Ser Ser Leu Leu Glu Ly
#s Phe Gly Pro Phe Pro
100
#           105
#           110
Glu Ser Val Val Arg Thr Tyr Thr Arg Gln Le
#u Leu Leu Gly Leu Glu
115
#       120
#       125
Tyr Leu His Asn His Ala Ile Met His Arg As
#p Ile Lys Gly Ala Asn
130
#   135
#   140
Ile Leu Val Asp Asn Lys Gly Cys Ile Lys Le
#u Ala Asp Phe Gly Ala
145                 1
#50                 1
#55                 1
#60
Ser Lys Gln Val Ala Glu Leu Ala Thr Met Th
#r Gly Ala Lys Ser Met
165
#               170
#               175
Lys Gly Thr Pro Tyr Trp Met Ala Pro Glu Va
#l Ile Leu Gln Thr Gly
180
#           185
#           190
His Ser Phe Ser Ala Asp Ile Trp Ser Val Gl
#y Cys Thr Val Ile Glu
195
#       200
#       205
Met Val Thr Gly Lys Ala Pro Trp Ser Gln Gl
#n Tyr Lys Glu Val Ala
210
#   215
#   220
Ala Ile Phe Phe Ile Gly Thr Thr Lys Ser Hi
#s Pro Pro Ile Pro Asp
225                 2
#30                 2
#35                 2
#40
Thr Leu Ser Ser Asp Ala Lys Asp Phe Leu Le
#u Lys Cys Leu Gln Glu
245
#               250
#               255
Val Pro Asn Leu Arg Pro Thr Ala Ser Glu Le
#u Leu Lys His Pro Phe
260
#           265
#           270
Val Met Gly Lys His
275
<210> SEQ ID NO 7
<211> LENGTH: 277
<212> TYPE: PRT
<213> ORGANISM: Arabidopsis thaliana
<400> SEQUENCE: 7
Asn Thr Val Asp Met Ala Pro Pro Ile Ser Tr
#p Arg Lys Gly Gln Leu
1               5
#                10
#                15
Ile Gly Arg Gly Ala Phe Gly Thr Val Tyr Me
#t Gly Met Asn Leu Asp
20
#            25
#            30
Ser Gly Glu Leu Leu Ala Val Lys Gln Val Le
#u Ile Ala Ala Asn Phe
35
#        40
#        45
Ala Ser Lys Glu Lys Thr Gln Ala His Ile Gl
#n Glu Leu Glu Glu Glu
50
#    55
#    60
Val Lys Leu Leu Lys Asn Leu Ser His Pro As
#n Ile Val Arg Tyr Leu
65
#70
#75
#80
Gly Thr Val Arg Glu Asp Asp Thr Leu Asn Il
#e Leu Leu Glu Phe Val
85
#                90
#                95
Pro Gly Gly Ser Ile Ser Ser Leu Leu Glu Ly
#s Phe Gly Pro Phe Pro
100
#           105
#           110
Glu Ser Val Val Arg Thr Tyr Thr Arg Gln Le
#u Leu Leu Gly Leu Glu
115
#       120
#       125
Tyr Leu His Asn His Ala Ile Met His Arg As
#p Ile Lys Gly Ala Asn
130
#   135
#   140
Ile Leu Val Asp Asn Lys Gly Cys Ile Lys Le
#u Ala Asp Phe Gly Ala
145                 1
#50                 1
#55                 1
#60
Ser Lys Gln Val Ala Glu Leu Ala Thr Met Th
#r Gly Ala Lys Ser Met
165
#               170
#               175
Lys Gly Thr Pro Tyr Trp Met Ala Pro Glu Va
#l Ile Leu Gln Thr Gly
180
#           185
#           190
His Ser Phe Ser Ala Asp Ile Trp Ser Val Gl
#y Cys Thr Val Ile Glu
195
#       200
#       205
Met Val Thr Gly Lys Ala Pro Trp Ser Gln Gl
#n Tyr Lys Glu Val Ala
210
#   215
#   220
Ala Ile Phe Phe Ile Gly Thr Thr Lys Ser Hi
#s Pro Pro Ile Pro Asp
225                 2
#30                 2
#35                 2
#40
Thr Leu Ser Ser Asp Ala Lys Asp Phe Leu Le
#u Lys Cys Leu Gln Glu
245
#               250
#               255
Val Pro Asn Leu Arg Pro Thr Ala Ser Glu Le
#u Leu Lys His Pro Phe
260
#           265
#           270
Val Met Gly Lys His
275

Claims

1. An isolated polypeptide, wherein the amino acid sequence of said polypeptide consists of SEQ ID NO:2.

2. An isolated polypeptide, wherein the amino acid sequence of said polypeptide comprises SEQ ID NO:2.

3. An isolated MEK kinase, wherein the amino acid sequence of said MEK kinase consists of an amino sequence having at least 99% sequence identity to SEQ ID NO:2.

4. An isolated MEK kinase, wherein the amino acid sequence of said MEK kinase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:2.

5. The polypeptide of claim 1, further comprising a heterologous amino acid sequence.

6. The polypeptide of claim 2, further comprising a heterologous amino acid sequence.

7. The MEK kinase of claim 3, further comprising a heterologous amino acid sequence.

8. The MEK kinase of claim 4, further comprising a heterologous amino acid sequence.

9. A composition comprising the polypeptide of claim 1 and a carrier.

10. A composition comprising the polypeptide of claim 2 and a carrier.

11. A composition comprising the MEK kinase of claim 3 and a carrier.

12. A composition comprising the MEK kinase of claim 4 and a carrier.

13. A composition comprising the polypeptide of claim 5 and a carrier.

14. A composition comprising the polypeptide of claim 6 and a carrier.

15. A composition comprising the MEK kinase of claim 7 and a carrier.

16. A composition comprising the MEK kinase of claim 8 and a carrier.