US20030045491A1

US20030045491A1 - TTK in diagnosis and as a therapeutic target in cancer

Info

Publication number: US20030045491A1
Application number: US10/081,119
Authority: US
Inventors: Christoph Reinhard; Anne Jefferson; Vivien Chan
Original assignee: Christoph Reinhard; Jefferson Anne B.; Chan Vivien W.
Current assignee: Novartis Vaccines and Diagnostics Inc
Priority date: 2001-02-23
Filing date: 2002-02-21
Publication date: 2003-03-06
Also published as: US7501244B2; US20050063974A1

Abstract

The present invention provides methods for identification of cancerous cells by detection of expression levels of TTK, as well as diagnostic, prognostic and therapeutic methods that take advantage of the differential expression of these genes in mammalian cancer. Such methods can be useful in determining the ability of a subject to respond to a particular therapy, e.g., as the basis of rational therapy. In addition, the invention provides assays for identifying pharmaceuticals that modulate activity of these genes in cancers in which these genes are involved, as well as methods of inhibiting tumor growth by inhibiting activity of TTK.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application serial No. 60/289,813, filed Feb. 21, 2001, which application is hereby incorporated by reference.[0001]

FIELD OF THE INVENTION

The field of the present invention relates to disease diagnosis and treatment of cancer and identification of anti-cancer agents.

BACKGROUND OF THE INVENTION

Mitotic checkpoint genes have become widely studied for their roles in development as well as for their potential role in disease such as cancer. The mitotic checkpoint involves a number of different mechanisms to ensure proper cellular division. For example, the spindle assembly checkpoint modulates the timing of anaphase initiation in response to the improper alignment of chromosomes at the metaphase plate. If defects are detected, a signal is transduced to halt further progression of the cell cycle until correct bipolar attachment to the spindle is achieved. Initially identified in budding yeast, several mammalian spindle checkpoint-associated proteins have recently been identified and partially characterized. These proteins associate with all active human centromeres, including neocentromeres, in the early stages of mitosis prior to the commencement of anaphase. The proteins associated with the checkpoint protein complex (BUB1, BUBR1, BUB3, MAD2), the anaphase-promoting complex (Tsg24, p55CDC), and other proteins associated with mitotic checkpoint control (ERK1, 3F3/2 epitope, hZW10), were found to specifically associate with only the active centromere, suggesting that only active centromeres participate in the spindle checkpoint. Saffery R et al., Hum Genet. 107:376-84 (2000).

Tyrosine threonine kinase (TTK), a protein kinase, phosphorylates serine, threonine, and tyrosine hydroxyamino acids (Mills et al,. Biol. Chem. 267:16000-6 (1992)). The kinases most closely related to TTK include SPK1 serine, threonine, and tyrosine kinase, the Pim1, PBS2, and CDC2 serine/threonine kinases, and the TIK kinase (Mills et al. J. Biol. Chem. 267:16000 -6 (1992)). The nucleotide and amino acid sequences of human TTK are provided at, for example, GenBank Accession No. M86699. Expression of TTK is markedly reduced or absent in resting cells and in tissues with a low proliferative index (Hogg et al. Oncogene 9:89-96 (1994)). TTK mRNA is expressed in human testis, thymus, bone marrow, and other tissues that contain a large number of proliferating cells and is not detected in tissues that contain few or no dividing cells. TTK expression was detected in several rapidly proliferating cells lines, including various cancer cell lines. TTK expression was also detected and in samples tissue samples from two patients with malignant ovarian cancer (Mills et al., ibid.; Schmandt et al. J. Immunol. 152:96-105 (1994)). TTK expression is correlated with cell proliferation, and plays a role in cell cycle control (Hogg et al., ibid.). Very low levels of TTK mRNA and protein are present in starved cells. When cells are induced to enter the cell cycle, levels of TTK mRNA, protein, and kinase activity increase at the G1/S phase of the cell cycle and peak in G2/M. TTK mRNA levels, as well as kinase activity, drop sharply in early G1, whereas protein levels are largely maintained. TTK is a human homologue of the S. cerevesiae kinase mps1 and the S. pombe protein mph1, both of which are involved in cell cycle spindle assembly checkpoint, thus indicating that TTK is a spindle checkpoint gene (see, e.g., Cahill et al. Genomics 58:181-7 (1999).

Although mitotic checkpoint impairment has been detected in human cancers (e.g., such impairment is present in about 40% of human lung cancer cell lines) mutations in the MAD mitotic checkpoint genes and the BUB gene family are infrequent. Haruki N et al., Cancer Lett. 162:201-205 (2001); Mimori K et al., Oncol Rep. 8:39-42 (2001); Cahill et al., ibid.). There is thus a need for identification of mitotic checkpoint genes that have a role in human cancers, as they can serve as informative diagnostic and/or prognostic indicators, and therapeutic targets.

SUMMARY OF THE INVENTION

In a first embodiment, the present invention provides a method for identifying TTK levels in a sample of a subject suspected of having cancer (e.g., a lung, colon, prostrate or breast tissue biopsy) comprising quantifying the level of TTK in the sample. The identification of increased levels of TTK in the sample provides an indication of impairment of the cell cycle checkpoint in the sampled cells.

In another embodiment, the invention provides a method for determining the characteristics of a malignant or pre-malignant growth comprising determining (either qualitatively or quantitatively) the level of TTK in the cells of the growth, and comparing levels with known levels in various stages of cancer and/or normal tissue. For example, to determine the characteristics of a particular subject's colon cancer, a sample of the cancer may be removed, the levels of TTK in the cancer determined, and the levels compared to normal tissue and/or levels in various stage colon cancers derived from the same cell type. The levels of TTK identified in the sample can thus be indicative of various characteristics of the malignant or pre-malignant growth, as determined by the characteristics of known tissue and cancers. The TTK levels can be compared directly to the levels in other single samples, or may be compared to a standard that is derived from the data of multiple samples.

In another embodiment, the TTK levels of a sample can be used as one index for determining the appropriate therapeutic intervention for a subject with a malignant or pre-malignant growth. Highly increased levels of TTK, for example, can be indicative of the need for more aggressive therapy, as it is indicative of a later stage cancer. Alternatively, the level of TTK expression may be indicative of the responsiveness of a subject to a particular pharmaceutical, and in particular to a therapeutic intervention that affects the cancer via the mitotic checkpoint.

In another embodiment, the invention features a method for identifying agents for inhibiting growth of a tumor, particular by a breast or colon tumor, by contacting a cell expressing TTK with a candidate agent, and assessing the effect of the agent upon TTK activity.

Accordingly, in one aspect the invention features a method of diagnosing cancer in a subject, the method comprising detection of TTK polynucleotide or polypeptide in a test sample obtained from a subject so as to determine a level of expression of the gene product; and comparing the level of expression of the TTK in the test sample to a level of expression in a normal cell corresponding to the same tissue; wherein detection of an expression level of TTK in the test sample that is significantly increased from the level of expression in a normal cell indicates that the test cell is cancerous. In specific embodiments, the cancer is other than ovarian cancer, with colon cancer and breast cancer being of particular interest.

In another aspect, the invention features a method for determining the prognosis of a cancerous disease in a subject, the method comprising detecting expression of TTK in a test cell from the subject; and comparing a level of expression of TTK in the test cell with a level of TTK expression in a control cell; wherein the level of expression of TTK in the test cell relative to the level of expression in the control cell is indicative of the prognosis of the cancerous disease. For example, where the control cell is a normal cell, an elevated level of TTK expression in the test cell relative to the normal cell is indicative of the continued presence of cancerous cells in the subject and thus a relatively poorer prognosis than where the level of TTK expression in the test cell is at a level comparable to that found in an normal (non-cancer) cell. In specific embodiments, progress of a cancer other than ovarian cancer is of particular interest, especially colon and breast cancer.

In another aspect, the invention features a method for inhibiting growth of a cancerous cell comprising introducing into a cell an antisense polynucleotide for inhibition of TTK expression, wherein inhibition of TTK expression inhibits replication of the cancerous cell.

In still another aspect, the invention features a method for assessing the tumor burden of a subject, the method comprising detecting a level of TTK expression in a test sample from a subject, the test sample suspected of comprising increased TTK expression; wherein detection of the level of TTK expression in the test sample is indicative of the tumor burden in the subject, with an increased level of TTK expression in the test sample relative to a control non-cancer cell indicates the presence of a tumor in the subject.

In yet another aspect, the invention features a method of identifying an agent having anti-TTK activity, the method comprising contacting a cancerous cell displaying elevated expression of TTK with a candidate agent; and determining the effect of the candidate agent on TTK activity; wherein a decrease in TTK activity indicates that the agent has anti-TTK activity. In specific embodiments, TTK activity is detected by detecting TTK expression or by detecting a biological activity of TTK

In yet another aspect, the invention features an assay for identifying a candidate agent that inhibits growth of a cancerous cell, comprising contacting a cell expressing TTK polypeptide with a candidate agent; and detecting activity of the TTK polypeptide, comparing the activity of the TTK polypeptide in the cell in the presence of the candidate agent to activity of a TTK polypeptide in a cell in the absence of the candidate agent; wherein reduction of TTK activity in the presence of the candidate agent relative to TTK activity in the absence of the candidate agent indicates that the candidate agent reduces TTK activity and inhibits growth of a cancerous cell.

A primary object of the invention is to exploit TTK as a therapeutic target, e.g. by identifying candidate agents that modulate, usually that decrease, TTK activity in a target cell in order to, for example, inhibit cell growth.

An object of the present invention is to inhibit tumor growth by inhibition of activity of a mitotic checkpoint gene product, particularly though inhibition of TTK activity in the target tumor cell.

Another object of the invention is to facilitate rational cancer therapy. For example, where the cancer in the subject is associated with increased TTK activity levels, a therapeutic agent is selected accordingly so as to facilitate reduction of TTK activity levels.

Another object of the present invention is to design clinical trials based on levels of TTK expression in a cancer, and more particularly to design clinical trials based on TTK expression in combination with other patient attributes.

Yet another object of the invention is to identify the association of TTK expression and intervention attributes that yield efficacious changes in selected disease progression measures.

An advantage of the invention is the ability to project disease progression based on expression of TTK in a malignant or pre-malignant growth.

Another advantage of the present invention is that it allows a more systematic approach for intervention of a cancerous disease based upon objective indicia.

These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the methods as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph illustrating expression of TTK in various normal tissue types as detected by PCR. [0025]
FIG. 2 is a bar graph illustrating expression of TTK in various tumor cell lines as detected by PCR. [0026]
FIGS. [0027] 3-6 are graphs illustrating expression profiles for IGF2, MAPKAPK2, TTK, and MARCKS in patients with colorectal carcinoma.
FIGS. 7 and 8 are graphs illustrating growth suppression of MDA-MB-231 cells following antisense suppression of TTK expression. [0028]
FIG. 9 is a graph illustrating growth suppression of SW620 cells following antisense suppression of TTK expression. [0029]
FIG. 10 is a graph illustrating suppression of colony formation of SW620 cells in soft agar following antisense suppression of TTK expression. [0030]
FIG. 11 is a graph illustrating that antisense suppression of TTK has no detectable effect on normal immortal fibroblasts. [0031]
FIG. 12 is a bar graph illustrating induction of cell death upon depletion of TTK from SW620 cells.[0032]

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Before the present invention is described, it is to be understood that this invention is not limited to particular methodologies described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. [0033]
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0034]
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. [0035]
It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth. [0036]
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. [0037]

DEFINITIONS

The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, refer to a polymeric forms of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, these terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. These terms further include, but are not limited to, mRNA or cDNA that comprise intronic sequences (see, e.g., Niwa et al. (1999) [0038] Cell 99(7):691-702). The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidites and thus can be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996) Nucl. Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucl. Acids Res. 24:2318-2323. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.
The terms “polypeptide” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like. [0039]
As used herein “TTK polynucleotide” and “TTK polypeptide” encompass polynucleotides and polypeptides having sequence similarity or sequence identity to the human TTK (having GenBank accession number M86699; SEQ ID NO:13 and 14), or the [0040] S. cerevesiae kinase mps1 gene and gene products (SEQ ID NO:29 and 30), the S. pombe protein mph1 gene and gene products (SEQ ID NO:31 and 32), and other genes and gene products related to TTK, such as SPK1 (SEQ ID NO:15 and 16), Pim1 (SEQ ID NO:17 and 18), PBS2 (SEQ ID NO:19 and 20), CDC2 (SEQ ID NO:21 and 22), and TIK (SEQ ID NO:23 and 24) of at least about 65%, preferably at least about 80%, more preferably at least about 85%, and can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Sequence similarity and sequence identity are calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. In general, percent sequence identity is calculated by counting the number of residue matches (e.g., nucleotide residue or amino acid residue) between the query and test sequence and dividing total number of matches by the number of residues of the individual sequences found in the region of strongest alignment. Thus, where 10 residues of an 11 residue query sequence matches a test sequence, the percent identity above would be 10 divided by 11, or approximately, 90.9%. Algorithms for computer-based sequence analysis are known in the art, such as BLAST (see, e.g., Altschul et al., J. Mol. Biol., 215:403-10 (1990)), particularly the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular). For the purposes of this invention, a preferred method of calculating percent identity is the Smith-Waterman algorithm, using the following. Global DNA sequence identity must be greater than 65% as determined by the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular) using an affine gap search with the following search parameters: gap open penalty, 12; and gap extension penalty, 1. The human TTK cDNA is represented by the polynucleotide sequence of SEQ ID NO:13 and the human TTK polypeptide is represented by the sequence of SEQ ID NO:14.
“Antisense polynucleotide” or “antisense oligonucleotide” are used interchangeably herein to mean an unmodified or modified nucleic acid having a nucleotide sequence complementary to a given polynucleotide sequence (e.g., a polynucleotide sequence encoding TTK) including polynucleotide sequences associated with the transcription or translation of the given polynucleotide sequence (e.g., a promoter of a polynucleotide encoding TTK), where the antisense polynucleotide is capable of hybridizing to a TTK-encoding polynucleotide sequence. Of particular interest are antisense polynucleotides capable of inhibiting transcription and/or translation of a TTK-encoding polynucleotide either in vitro or in vivo. [0041]
The term “cDNA” as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons (e.g., sequences encoding open reading frames of the encoded polypeptide) and 3′ and 5′ non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns removed by nuclear RNA splicing to create a continuous open reading frame encoding TTK. [0042]
A “variant” as used in the context of a “variant polypeptide” refers to an amino acid sequence that is altered by one or more amino acids relative to a reference amino acid sequence. The variant can have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant can have “nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations can also include amino acid deletions or insertions, or both. Guidance in determining which and how many amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity can be found using computer programs well known in the art, for example, DNAStar software. [0043]
A “deletion” is defined as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to reference amino acid sequence or nucleotide sequence. Deletions can be of any length, but are preferably approximately 50, 20, 15, 10, 5 or 3 amino acids or nucleotides in length. [0044]
An “insertion” or “addition” is that change in an amino acid or nucleotide sequence which has resulted in the addition of one or more amino acid or nucleotide residues, respectively, as compared to a reference amino acid sequence or nucleotide sequence. Insertions or additions can be of any length, but are preferably approximately 50, 20, 15, 10, 5 or 3 amino acids or nucleotides in length. [0045]
A “substitution” results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively, as compared to a reference amino acid sequence or nucleotide sequence. Substitutions can be of any length, but are preferably approximately 50, 20, 15, 10, 5 or 3 amino acids or nucleotides in length. [0046]
The terms “single nucleotide polymorphism” and “SNP” refer to polymorphisms of a single base change relative to a reference sequence. [0047]
The term “biologically active” refers to gene product, usually a polypeptide, having structural, regulatory, or biochemical functions of a naturally occurring gene product, e.g., protein. “Immunologically active” defines the capability of the natural, recombinant, or synthetic polypeptide, or any oligopeptide thereof, to elicit a specific immune response in appropriate animals or cells and to bind with specific antibodies. [0048]
The term “derivative” as used herein refers to the chemical modification of a nucleic acid or amino acid sequence relative to a reference nucleic acid or amino acid sequence. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivative generally encodes a polypeptide which retains essential biological characteristics of the polypeptide encoded by the reference nucleic acid (e.g., the “parent” molecule). [0049]
As used herein the term “isolated” is meant to describe a compound of interest (e.g., either a polynucleotide or a polypeptide) that is in an environment different from that in which the compound naturally occurs. “Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified. [0050]
As used herein, the term “substantially purified” refers to a compound (e.g., either a polynucleotide or a polypeptide) that is removed from its natural environment and is at least 60% free, preferably 75% free, and most preferably 90% free from other components with which it is naturally associated. [0051]
“Stringency” typically occurs in a range from about Tm −5° C. (5° C. below the Tm of the probe or antibody) to about 20° C. to 25° C. below Tm. As will be understood by those of skill in the art, a stringency hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. [0052]
The term “hybridization” as used herein shall include “any process by which a strand of nucleic acid joins with a complementary strand through base pairing” (Coombs, Dictionary of Biotechnology, Stockton Press, New York N.Y. (1994)). Amplification as carried out in the polymerase chain reaction technologies is described in Dieffenbach et al., PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y. (1995). [0053]
The term “transformation” as used herein refers to a permanent or transient genetic change, induced in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell). Genetic change can be accomplished either by incorporation of the new DNA into the genome of the host cell, or by transient or stable maintenance of the new DNA as an episomal element. Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. [0054]
The term “construct” as used herein refers to a recombinant nucleic acid, generally recombinant DNA, that has been generated for the purpose of the expression of a specific nucleotide sequence(s), or is to be used in the construction of other recombinant nucleotide sequences. [0055]
As used herein, the term “differentially expressed” generally refers to a polynucleotide that is expressed at levels in a test cell that differ significantly from levels in a reference cell, e.g., mRNA is found at levels at least about 25%, at least about 50% to about 75%, at least about 90% increased or decreased, generally at least about 1.2-fold, at least about 1.5-fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, or at least about 50-fold or more increased or decreased in a cancerous cell when compared with a cell of the same type that is not cancerous. The comparison can be made between two tissues, for example, if one is using in situ hybridization or another assay method that allows some degree of discrimination among cell types in the tissue. The comparison may also be made between cells removed from their tissue source. “Differential expression” refers to both quantitative, as well as qualitative, differences in the genes' temporal and/or cellular expression patterns among, for example, normal and neoplastic tumor cells, and/or among tumor cells which have undergone different tumor progression events. [0056]
The terms “correspond to” or “represents” as used in, for example, the phrase “polynucleotide corresponds to a differentially expressed gene” are used to refer to the relationship between a given polynucleotide and the gene from which the polynucleotide sequence is derived (e.g., a polynucleotide that is derived from a coding region of the gene, a splice variant of the gene, an exon, and the like) or to which the polynucleotide hybridizes to under stringer conditions. [0057]
“Differentially expressed polynucleotide” as used herein refers to a nucleic acid molecule (RNA or DNA) comprising a sequence that represents or corresponds to a differentially expressed gene, e.g., the differentially expressed polynucleotide comprises a sequence (e.g., an open reading frame encoding a gene product; a non-coding sequence) that uniquely identifies a differentially expressed gene so that detection of the differentially expressed polynucleotide in a sample is correlated with the presence of a differentially expressed gene in a sample. “Differentially expressed polynucleotides” is also meant to encompass fragments of the disclosed polynucleotides, e.g., fragments retaining biological activity, as well as nucleic acids homologous, substantially similar, or substantially identical (e.g., having about 90% sequence identity) to the disclosed polynucleotides. [0058]
“Diagnosis” as used herein generally includes determination of a subject's susceptibility to a disease or disorder, determination as to whether a subject is presently affected by a disease or disorder, prognosis of a subject affected by a disease or disorder (e.g., identification of pre-metastatic or metastatic cancerous states, stages of cancer, or responsiveness of cancer to therapy), and therametrics (e.g., monitoring a subject's condition to provide information as to the effect or efficacy of therapy). [0059]
As used herein, the term “a polypeptide associated with cancer” (e.g., as in polypeptide associated with colon cancer) refers to a polypeptide that is present at relatively higher or lower levels in a cancer cell relative to a normal cell of the same type. [0060]
The term “biological sample” encompasses a variety of sample types obtained from an organism and can be used in a diagnostic or monitoring assay. The term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components. The term encompasses a clinical sample, and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. [0061]
The terms “treatment”, “treating”, “treat” and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or relieving the disease symptom, i.e., causing regression of the disease or symptom. Thus “treatment of cancer” thus encompasses one or more of inhibition of cellular proliferation, inhibition of metastasis, and the like. [0062]
The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on. [0063]
The phrase “specific binding pair” as used herein comprises a specific binding member and a binding partner which have a particular specificity for each other and which bind to each other in preference to other molecules under stringent conditions. Examples of specific binding pairs are antigens and antibodies, molecules and receptors and complementary nucleotide sequences. Other examples of binding pairs will be apparent to one skilled in the art upon reading the present disclosure. Further, the term “specific binding pair” is also applicable where either or both of the specific binding member and the binding partner comprise a part of a larger molecule. In embodiments in which the specific binding pair are nucleic acid sequences, they are preferably between 10 to 200 nucleotides long, more preferably greater than 15 to 100 nucleotides long. [0064]
By “antibody” is meant an immunoglobulin protein which is capable of binding an antigen. Antibody as used herein is meant to include the entire antibody as well as any antibody fragments (e.g., F(ab′)[0065] ₂, Fab′, Fab, Fv) capable of binding the epitope, antigen, or antigenic fragment of interest.
Antibodies of the invention are immunoreactive or immunospecific for and therefore specifically and selectively bind to a protein of interest, e.g., human TTK protein. Antibodies which are immunoreactive and immunospecific for human TTK are preferred. Antibodies for human TTK are preferably immunospecific—i.e., not substantially cross-reactive with related materials, although they may recognize TTK homologs across species. The term “antibody” encompasses all types of antibodies (e.g., monoclonal and polyclonal). [0066]
By “binds specifically” is meant high avidity and/or high affinity binding of an antibody to a specific polypeptide, e.g., epitope of a TTK protein. Antibody binding to its epitope on this specific polypeptide is stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest. Antibodies which bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to the compound or polypeptide of interest, e.g., by use of appropriate controls. [0067]
The terms “cancer”, “neoplasm”, “tumor”, and the like are used interchangeably herein to refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In general, cells of interest for detection or treatment in the present application include pre-malignant (e.g., benign hyperplasiac), malignant, metastatic, and non-metastatic cells. [0068]
“TTK activity” as used herein refers to activity of the TTK polypeptide in phosphorylation of a recipient substrate. [0069]
“Modulation of TTK activity” as used herein refers to an increase or decrease in TTK activity that can be a result of, for example, interaction of an agent with a TTK polypeptide (e.g., reversible or irreversible binding of an inhibitory agent so as to interfere with TTK polypeptide interaction with a donor molecule or a recipient (acceptor) molecule in the phosphorylation activity of TTK), inhibition of TTK transcription and/or translation (e.g., through antisense interaction with the TTK gene or TTK transcript, through modulation of transcription factors that facilitate TTK expression), and the like. Modulation of TTK activity that results in a decrease of TTK activity is of particular interest in the invention. In this context, TTK activity can be decreased by an inhibitory agent at least 10%, 25%, 50%, 75%, 85%, 90%, up to 100% relative to TTK activity in the absence of an agent. TTK activity can be assessed by assaying enzymatic activity, by assessing TTK polypeptide levels, or by assessing TTK transcription levels. Comparisons of TTK activity can also be accomplished by comparing TTK activity assessed (either qualitatively or quantitatively) in a test sample to a standard TTK activity (e.g., a level of TTK activity in the absence of an inhibitory agent or agonist, that is associated with a normal cell, a level of TTK activity of a cancerous cell of a selected tissue type, and the like). [0070]
Overview [0071]
Human TTK is a mitotic checkpoint gene which encodes an 857 amino acid protein that exhibits activity of a mixed specificity (tyr/thr) kinase. TTK is expressed in rapidly proliferating tissues such as testis and thymus. See, e.g., Mills G B et al., [0072] J Biol Chem. 267:16000-6 (1992). The present invention is based upon the finding that TTK is differentially expressed in colon tumor cells relative to normal colon cells as detected by microarray analysis. Differential expression was confirmed in cell lines derived from various forms of cancer, indicating that the involvement of TTK in cancer as a more general mechanism. In addition, disruption of TTK function using antisense oligonucleotides to “knock-out” TTK message decreased proliferation, inhibited anchorage independent growth, and induced apoptosis of cancer cell lines, including a metastatic breast cancer cell line (MDA-MB-213) and a colorectal carcinoma cell line (SW620). These data indicate that TTK can be a therapeutic target for chemotherapy in cancers in which TTK is overexpressed.
The identification of the association of TTK with cancer, and the confirmation that inhibition of TTK activity (e.g., by reducing TTK expression) serves as the basis for the materials and methods of the invention, such as are disclosed and discussed herein, for use in, for example, diagnosing cancer of a patient, particularly a cancer that is susceptible to treatment by decreasing activity of TTK. The invention also provides for planning and selection of appropriate therapeutic and/or prophylactic treatment, permitting streamlining of treatment by targeting those most likely to benefit. The invention also provides for treatment of a cancer associated with aberrant TTK levels (e.g., associated with overexpression or overproduction of TTK), e.g. by inhibition of gene product production (e.g., decreasing levels of transcription and/or translation), by decreasing TTK activity (e.g., by decreasing TTK gene product production (e.g., at the level of transcription or translation) and/or by reducing one or more of TTK's kinase activities). [0073]
Various aspects of the invention will now be described in more detail. [0074]
Diagnostic Methods [0075]
In one aspect the invention is based on the discovery that TTK activity is present at higher levels in cancerous cells (particularly in colon cancer and breast cancer) than in normal cells of the same cell type. This discovery serves as the basis for identification of cancerous cells, as well as identification of tumors that are susceptible to therapy by inhibiting activity of TTK, e.g., by inhibiting TTK expression at the level of transcription or translation or both, by inhibiting TTK activity, and the like. [0076]
TTK gene products e.g. TTK encoding mRNA or TTK polypeptides are of particular interest as markers (e.g., in bodily fluids (such as blood) or in tissues) to detect the earliest changes along the carcinogenesis pathway (e.g., to differentiate cancerous tissue from non-cancerous tissue) and/or to monitor the efficacy of various therapies and preventive interventions. For example, a relatively increased level of expression of TTK compared to normal cells or tissues of the same type can be indicative of a poorer prognosis, and therefore warrant more aggressive therapy (e.g., chemo- or radio-therapy) for a patient or vice versa. The correlation of surrogate tumor specific features with response to treatment and outcome in patients can define prognostic indicators that allow the design of tailored therapy based on the molecular profile of the tumor. These therapies include antibody targeting, antagonists (e.g., small molecules), and gene therapy. Determining TTK expression and comparison of a patient's profile with known expression in normal tissue and variants of the disease allows a determination of the best possible treatment for a patient, both in terms of specificity of treatment and in terms of comfort level of the patient. Surrogate tumor markers, such as polynucleotide expression, can also be used to better classify, and thus diagnose and treat, different forms and disease states of cancer. Two classifications widely used in oncology that can benefit from identification of TTK expression levels are staging of the cancerous disorder, and grading the nature of the cancerous tissue. [0077]
TTK polynucleotides, as well as their encoded gene products, can be useful to monitor patients having or susceptible to cancer to detect potentially malignant events at a molecular level before they are detectable at a gross morphological level. In addition, detection of TTK gene products can be useful as therametrics, e.g., to assess the effectiveness of therapy by using the polynucleotides or their encoded gene products, to assess, for example, tumor burden in the patient before, during, and after therapy. [0078]
Furthermore, a polynucleotide identified as corresponding to a gene that is differentially expressed in, and thus is important for, one type of cancer can also have implications for development or risk of development of other types of cancer, e.g., where a polynucleotide represents a gene differentially expressed across various cancer types. Thus, for example, expression of a polynucleotide corresponding to a gene that has clinical implications for metastatic colon cancer can also have clinical implications for stomach cancer or endometrial cancer. [0079]
In making a diagnosis, prognosis, risk assessment, or measurement of tumor burden based on the enzymatic activity of TTK or the expression levels of TTK polypeptide or TTK encoding polynucleotides, activity or expression levels may be compared to those of suitable cancerous or non-cancerous control samples. For example, a diagnosis of cancer can be made if TTK activity is increased at by 25%, 50%, 75%, 90%, up to 100%, or, alternatively by 5-fold, 10-fold, 50-fold, or more than 100-fold relative to a normal non-cancerous cell of the same tissue type. [0080]
Other gene products that are differentially expressed in cancerous cells relative to, for example, non-cancer cells of between cancer cells of differing malignant potential (e.g., non-malignant tumor cells versus cells of high potential malignancy) can also be assayed in addition to TTK for differential expression in a test cell. Such exemplary gene products include, but are not necessarily limited to MAPKAP kinase 2 (SEQ ID. No. 33 and 34), MARCKS (SEQ ID NO:35 and 36) and/or IGF2 (SEQ ID NO:37 and 38). [0081]
Staging. Staging is a process used by physicians to describe how advanced the cancerous state is in a patient. Staging assists the physician in determining a prognosis, planning treatment and evaluating the results of such treatment. Staging systems vary with the types of cancer, but generally involve the following “TNM” system: the type of tumor, indicated by T; whether the cancer has metastasized to nearby lymph nodes, indicated by N; and whether the cancer has metastasized to more distant parts of the body, indicated by M. Generally, if a cancer is only detectable in the area of the primary lesion without having spread to any lymph nodes it is called Stage I. If it has spread only to the closest lymph nodes, it is called Stage II. In Stage II, the cancer has generally spread to the lymph nodes in near proximity to the site of the primary lesion. Cancers that have spread to a distant part of the body, such as the liver, bone, brain or other site, are Stage IV, the most advanced stage. [0082]
The differential expression level of TTK can facilitate fine-tuning of the staging process by identifying markers for the aggressiveness of a cancer, e.g. the metastatic potential, as well as the presence in different areas of the body. Thus, a Stage II cancer with a large differential level of expression of TTK can signify a cancer with a high metastatic potential and can be used to change a borderline Stage II tumor to a Stage III tumor, justifying more aggressive therapy. [0083]
Grading of cancers. Grade is a term used to describe how closely a tumor resembles normal tissue of its same type. The microscopic appearance of a tumor is used to identify tumor grade based on parameters such as cell morphology, cellular organization, and other markers of differentiation. As a general rule, the grade of a tumor corresponds to its rate of growth or aggressiveness, with undifferentiated or high-grade tumors generally being more aggressive than well differentiated or low-grade tumors. The following guidelines are generally used for grading tumors: 1) GX Grade cannot be assessed; 2) G1 Well differentiated; G2 Moderately well differentiated; 3) G3 Poorly differentiated; 4) G4 Undifferentiated. TTK activity levels (e.g., expression levels) can be especially valuable in determining the grade of the tumor, as they not only can aid in determining the differentiation status of the cells of a tumor, they can also identify factors other than differentiation that are valuable in determining the aggressiveness of a tumor, such as metastatic potential. [0084]
Detection of colon cancer. Polynucleotides and polypeptides corresponding to TTK can be used to detect colon cancer in a subject. Colorectal cancer is one of the most common neoplasms in humans and perhaps the most frequent form of hereditary neoplasia. Prevention and early detection are key factors in controlling and curing colorectal cancer. Colorectal cancer begins as polyps, which are small, benign growths of cells that form on the inner lining of the colon. Over a period of several years, some of these polyps accumulate additional mutations and become cancerous. Multiple familial colorectal cancer disorders have been identified, which are summarized as follows: 1) Familial adenomatous polyposis (FAP); 2) Gardner's syndrome; 3) Hereditary nonpolyposis colon cancer (HNPCC); and 4) Familial colorectal cancer in Ashkenazi Jews. The expression of appropriate polypeptide andpolynucleotides can be used in the diagnosis, prognosis and management of colorectal cancer. Detection of colon cancer can be determined using expression levels of TTK alone or in combination with the levels of expression of other genes differentially expressed in colon cancer. Determination of the aggressive nature and/or the metastatic potential of a colon cancer can be determined by comparing levels of TTK with a level associated with a normal cell, and comparing total levels of another sequence known to be differentially expressed, or otherwise be a marker of, cancerous tissue, e.g., expression of p53, DCC, ras, FAP (see, e.g., Fearon E R, et al., [0085] Cell (1990) 61(5):759; Hamilton S R et al., Cancer (1993) 72:957; Bodmer W, et al., Nat Genet. (1994) 4(3):217; Fearon E R, Ann N Y Acad Sci. (1995) 768:101)or MAPKAP kinase 2 (SEQ ID. No. 33 and 34), MARCKS (SEQ ID NO:35 and 36) and/or IGF2 (SEQ ID NO:37 and 38). For example, development of colon cancer can be detected by examining the level of expression of a gene corresponding to a polynucleotides described herein to the levels of oncogenes (e.g. ras) or tumor suppressor genes (e.g. FAP or p53). Thus expression of specific marker polynucleotides can be used to discriminate between normal and cancerous colon tissue, to discriminate between colon cancers with different cells of origin, to discriminate between colon cancers with different potential metastatic rates, etc. For a review of markers of cancer, see, e.g., Hanahan et al. (2000) Cell 100:57-70.
Detection of breast cancer. The majority of breast cancers are adenocarcinomas subtypes, which can be summarized as follows: 1) ductal carcinoma in situ (DCIS), including comedocarcinoma; 2) infiltrating (or invasive) ductal carcinoma (IDC); 3) lobular carcinoma in situ (LCIS); 4) infiltrating (or invasive) lobular carcinoma (ILC); 5) inflammatory breast cancer; 6) medullary carcinoma; 7) mucinous carcinoma; 8) Paget's disease of the nipple; 9) Phyllodes tumor; and 10) tubular carcinoma. [0086]
The expression levels of TTK can be used in the diagnosis and management of breast cancer, as well as to distinguish between types of breast cancer. Detection of breast cancer can be determined using expression levels of TTK, either alone or in combination with expression of other gene known to be differentially expressed in breast cancer. Determination of the aggressive nature and/or the metastatic potential of a breast cancer can also be determined by comparing levels of TTK and comparing levels of another sequence known to vary in cancerous tissue, e.g. ER expression. In addition, development of breast cancer can be detected by examining the ratio of expression of TTK to the levels of steroid hormones (e.g., testosterone or estrogen) or to other hormones (e.g., growth hormone, insulin). Thus expression of specific marker polynucleotides and polypeptides can be used to discriminate between normal and cancerous breast tissue, to discriminate between breast cancers with different cells of origin, to discriminate between breast cancers with different potential metastatic rates, etc. [0087]
Detection Methods [0088]
A number of methods are known in the art for analyzing biological samples from individuals to determine whether the individual has increased expression of a TTK gene product (e.g., RNA or protein) by detecting the TTK gene product in a biological sample from that subject. As discussed above, the purpose of such analysis may be used for diagnosis, to detect the presence of an existing cancer, to help identify the type of cancer, to assist a physician in determining the severity or likely course of the cancer, and/or to optimize treatment of it. In specific non-limiting embodiments, the methods are useful for detecting cancer cells, facilitating diagnosis of cancer and the severity of a cancer (e.g., tumor grade, tumor burden, and the like) in a subject, facilitating a determination of the prognosis of a subject, and assessing the responsiveness of the subject to therapy (e.g., by providing a measure of therapeutic effect through, for example, assessing tumor burden during or following a chemotherapeutic regimen). In additional embodiments, the methods are useful for classification or stratification of cancer cells, e.g., for the purpose of selecting patients to be included in a clinical trial population, for selecting an appropriate therapy (e.g., selecting therapy according to an expression profile of the cancerous cells), and the like. [0089]
Kits [0090]
The detection methods can be provided as part of a kit. Thus, the invention further provides kits for detecting the presence and/or a level of TTK activity e.g., by detection of a TTK-encoding mRNA and/or a polypeptide encoded thereby or by measuring TTK activity, in a biological sample. Procedures using these kits can be performed by clinical laboratories, experimental laboratories, medical practitioners, or private individuals. The kits of the invention for detecting TTK polypeptide that is differentially expressed in cancer cells comprise a moiety that specifically binds the polypeptide, which may be a specific antibody. The kits of the invention for detecting a TTK-encoding polynucleotide that is differentially expressed in cancer cells comprise a moiety that specifically hybridizes to such a polynucleotide such as a primer. The kits of the invention for detecting TTK activity comprise a recipient substrate capable of being phosphorylated by TTK, and a labeled donor substrate. The kits may optionally provide additional components that are useful in the procedure, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, standards, instructions, and interpretive information. [0091]
Screening for TTK Nucleic Acid or Polypeptide [0092]
Methods for detection of TTK activity include screening for the presence of TTK nucleic acid sequences representing an expressed TTK gene or alleles or variants thereof, and detecting the TTK polypeptide. The methods make use of biological samples from individuals that are suspected of contain the nucleic acid sequences or polypeptide. Examples of biological samples include blood, plasma, serum, tissue samples, tumor samples, saliva and urine. [0093]
Exemplary approaches for detecting TTK nucleic acid or polypeptides include: (a) determining the presence of the polypeptide encoded by the TTK gene; (b) using a specific binding member capable of binding to a TTK nucleic acid sequence (e.g., a known complementary sequence), the specific binding member comprising a nucleic acid that hybridizes with the TTK sequence under stringent conditions (c) using a substance comprising an antibody domain with specificity for a TTK nucleic acid sequence or the polypeptide encoded by it, the specific binding member being labeled to allow detection of the specific binding member to its binding partner is detectable; (d) using PCR involving one or more primers to determine relative levels of TTK in a sample from a patient; and (e) using an assay for TTK activity, e.g., phosphorylation of a TTK substrate. [0094]
The determination of TTK levels can include both levels of normal TTK and/or variant forms of TTK. A variant form of the gene may contain one or more insertions, deletions, substitutions and/or additions of one or more nucleotides compared with the wild-type sequence which may or may not alter the gene function. Differences at the nucleic acid level are not necessarily reflected by a difference in the amino acid sequence of the encoded polypeptide due to the degeneracy of the genetic code. However, a mutation or other difference in a gene may result in a frame-shift or stop codon, which could seriously affect the nature of the polypeptide produced (if any), or a point mutation or gross mutational change to the encoded polypeptide, including insertion, deletion, substitution and/or addition of one or more amino acids or regions in the polypeptide. [0095]
A mutation in a promoter sequence or other regulatory region may alter (e.g., reduce or enhance) expression from the gene or affect the processing or stability of the mRNA transcript. [0096]
There are various methods for detecting a particular nucleic acid sequence in a test sample. Tests may be carried out on preparations containing mRNA or cDNA generated from isolated mRNA in a manner that reflects the relative levels of mRNA transcripts in the sample. Levels of RNA can be determined specific amplification reaction such as PCR using one or more pairs of primers may be employed to amplify a region of the nucleic acid, and preferably a region with less homology to other genes. Nucleic acid for testing may be prepared from nucleic acid removed from cells or in a library using a variety of other techniques such as restriction enzyme digest and electrophoresis. [0097]
Nucleic acid may be screened using a TTK-specific probe. Such a probe corresponds in sequence to a region of the TTK gene, or its complement. Under stringent conditions, specific hybridization of such a probe to test nucleic acid is indicative of the presence of the TTK nucleic acid in a sample. For efficient screening purposes, more than one probe may be used on the same test sample. The probe may contain as few as 15, 20, 50 or 100 nucleotides of the TTK gene of SEQ ID. No. 13 or may be as long as or 500, 1 kb or as much as 3.8 kb or longer in length. [0098]
Allele- or variant-specific oligonucleotides may similarly be used in PCR to specifically amplify particular sequences if present in a test sample. Assessment of whether a PCR band contains a gene variant may be carried out in a number of ways familiar to those skilled in the art. The PCR product may for instance be treated in a way that enables one to display the mutation or polymorphism on a denaturing polyacrylamide DNA sequencing gel, with specific bands that are linked to the gene variants being selected. This can be done simultaneous to or sequentially to determining the level of a normal TTK sequence, e.g., to determine the combinatory levels of total TTK. [0099]
The presence of absence of a lesion in a promoter or other regulatory sequence may also be assessed by determining the level of mRNA production by transcription or the level of polypeptide production by translation from the mRNA. The presence of differences in sequence of nucleic acid molecules may be detected by means of restriction enzyme digestion, such as in a method of DNA fingerprinting where the restriction pattern produced when one or more restriction enzymes are used to cut a sample of nucleic acid is compared with the pattern obtained when a sample containing the normal gene or a variant or allele is digested with the same enzyme or enzymes. [0100]
A test sample of nucleic acid may be provided for example by extracting nucleic acid from cells, e.g., cells from a tumor biopsy. [0101]
Detection of TTK Polypeptides [0102]
There are various methods for determining the presence or absence in a test sample of a TTK polypeptide. A sample may be tested for the presence of a binding partner for a specific binding member such as an antibody (or mixture of antibodies), specific for wild-type TTK and/or one or more particular variants (e.g., allelic variants) of the TTK polypeptide. In such cases, the sample may be tested by being contacted with a specific binding member such as an antibody under appropriate conditions for specific binding. Where a panel of antibodies is used, different reporting labels may be employed for each antibody so that binding of each can be determined. In addition to detection of TTK polypeptides using anti-TTK antibodies, TTK polypeptide can also be identified using TTK-specific activity assays. [0103]
Arrays [0104]
Binding agents (such as antibodies or nucleic acid sequences) can also be immobilized in small, discrete locations and/or as arrays on solid supports or on diagnostic chips. These approaches can be particularly valuable as they can provide great sensitivity, particularly through the use of fluorescently labeled reagents, require only very small amounts of biological sample from individuals being tested and allow a variety of separate assays can be carried out simultaneously. This latter advantage can be useful as it provides an assay for different proteins (e.g., an oncogene or tumor suppressor) in tandem with the assay for TTK. Thus, in a further aspect, the present invention provides a support or diagnostic chip having immobilized thereon one or more binding agents capable of specifically binding TTK nucleic acid or polypeptides, optionally in combination with other reagents needed to carrying out an assay. [0105]
Methods for Expression of TTK Polypeptide [0106]
The full-length or partial polypeptides encoded by TTK may be expressed in any expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems. Suitable vectors and host cells for which are described in U.S. Pat. No. 5,654,173. Appropriate polynucleotide constructs are purified using standard recombinant DNA techniques as described in, for example, Sambrook et al., (1989) [0107] Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), and under current regulations described in United States Dept. of HHS, National Institute of Health (NIH) Guidelines for Recombinant DNA Research.
Bacteria. Expression systems in bacteria include those described in Chang et al., [0108] Nature (1978) 275:615, Goeddel et al., Nature (1979) 281:544, Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0 036,776, U.S. Pat. No. 4,551,433, DeBoer et al., Proc. Natl. Acad. Sci. (USA) (1983) 80:21-25, and Siebenlist et al., Cell (1980) 20:269.
Yeast. Expression systems in yeast include those described in Hinnen et al., [0109] Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J. Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142; Kunze et al., J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen. Microbiol. (1986) 132:3459, Roggenkamp et al., Mol. Gen. Genet. (1986) 202:302) Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt et al., J. Bacteriol. (1983) 154:737, Van den Berg et al., Bio/Technology (1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg et al., Mol. Cell. Biol. (1985) 5:3376, U.S. Pat. Nos. 4,837,148 and 4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr. Genet. (1985) 10:380, Gaillardin et al., Curr. Genet. (1985) 10:49, Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284-289; Tilburn et al., Gene (1983) 26:205-221, Yelton et al., Proc. Natl. Acad. Sci. (USA) (1984) 81:1470-1474, Kelly and Hynes, EMBO J. (1985) 4:475479; EP 0 244,234, and WO 91/00357.
Insect Cells. Expression of heterologous genes in insects is accomplished as described in U.S. Pat. No. 4,745,051, Friesen et al. (1986) “The Regulation of Baculovirus Gene Expression” in: The Molecular Biology Of Baculoviruses (W. Doerfler, ed.), [0110] EP 0 127,839, EP 0 155,476, and Vlak et al., J. Gen. Virol. (1988) 69:765-776, Miller et al., Ann. Rev. Microbiol. (1988) 42:177, Carbonell et al., Gene (1988) 73:409, Maeda et al., Nature (1985) 315:592-594, Lebacq-Verheyden et al., Mol. Cell Biol. (1988) 8:3129; Smith et al., Proc. Natl. Acad. Sci. (USA) (1985) 82:8404, Miyajima et al., Gene (1987) 58:273; and Martin et al., DNA (1988) 7:99. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts are described in Luckow et al., Bio/Technology (1988) 6:47-55, Miller et al., Generic Engineering (Setlow, J. K. et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al., Nature, (1985) 315:592-594.
Mammalian Cells. Mammalian expression is accomplished as described in Dijkema et al., [0111] EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad. Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41:521 and U.S. Pat. No. 4,399,216. Other features of mammalian expression are facilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44, Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985.
Screening Assays to Identify Chemotherapeutic Agents [0112]
The invention also encompasses screening assays to identify agents that modulate TTK activity, specifically that decrease aberrant TTK activity in an affected cell, e.g., a cancerous or pre-cancerous cell in which TTK is differentially expressed. Such assays may be performed either in vitro or in vivo. [0113]
Candidate Agents [0114]
The term “agent” as used herein describes any molecule with the capability of altering the expression or physiological function of a gene product of a differentially expressed gene. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection. [0115]
Candidate agents encompass numerous chemical classes, including, but not limited to, organic molecules (e.g., small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons), peptides, monoclonal antibodies, antisense polynucleotides, and ribozymes, and the like. Candidate agents can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited to: polynucleotides, peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. [0116]
Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Candidate agents can be assessed for modulation of TTK activity either singly or in pools. [0117]
Screening of Candidate Agents In Vitro [0118]
A wide variety of in vitro assays may be used to screen candidate agents for the desired biological activity, including, but not limited to, labeled in vitro protein-protein binding assays, protein-DNA binding assays (e.g., to identify agents that affect expression), electrophoretic mobility shift assays, immunoassays for protein binding, and the like. For example, by providing for the production of large amounts of a differentially expressed polypeptide, one can identify ligands or substrates that bind to, modulate or mimic the action of the polypeptide. Further methods for identifying these ligands and substrates are provided below. The purified polypeptide may also be used for determination of three-dimensional crystal structure, which can be used for modeling intermolecular interactions, transcriptional regulation, etc. [0119]
The screening assay can be a binding assay, wherein one or more of the molecules may be joined to a label, and the label directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g.,magnetic particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures. [0120]
A variety of other reagents may be included in the screening assays described herein. Where the assay is a binding assay, these include reagents like salts, neutral proteins, e.g.,albumin, detergents, etc that are used to facilitate optimal protein-protein binding, protein-DNA binding, and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40° C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hours will be sufficient. [0121]
Many mammalian genes have homologs in yeast and lower animals. The study of such homologs physiological role and interactions with other proteins in vivo or in vitro can facilitate understanding of biological function. In addition to model systems based on genetic complementation, yeast has been shown to be a powerful tool for studying protein-protein interactions through the two hybrid system described in Chien et al. 1991 [0122] Proc. Natl. Acad. Sci. USA 88:9578-9582.
Screening of Candidate Agents In Vivo [0123]
Candidate agents can be screened in a non-human animal model of cancer (e.g., in animals into which have been injected cancerous cells; in animals that are transgenic for an alteration in expression of a differentially expressed gene as described herein, e.g., a transgenic “knock-out,” or a transgenic “knock-in,” a polynucleotide encoding all or a portion of a differentially expressed gene product and comprising an operably linked reporter gene, and the like). [0124]
In general, the candidate agent is administered to the animal, and the effects of the candidate agent determined. The candidate agent can be administered in any manner desired and/or appropriate for delivery of the agent in order to effect a desired result. For example, the candidate agent can be administered by injection (e.g., by injection intravenously, intramuscularly, subcutaneously, or directly into the tissue in which the desired affect is to be achieved), orally, or by any other desirable means. Normally, the in vivo screen will involve a number of animals receiving varying amounts and concentrations of the candidate agent (from no agent to an amount of agent hat approaches an upper limit of the amount that can be delivered successfully to the animal), and may include delivery of the agent in different formulation. The agents can be administered singly or can be combined in combinations of two or more, especially where administration of a combination of agents may result in a synergistic effect. [0125]
The effect of agent administration upon the transgenic animal can be monitored by assessing expression of the gene product, growth of the injected tumor cells, and the like. [0126]
Identified Candidate Agents [0127]
Compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host for treatment of a condition that is amenable to treatment by modulation of expression of a differentially expressed gene product. The therapeutic agents may be administered in a variety of ways, orally, topically, parenterally e.g., subcutaneously, intraperitoneally, by viral infection, intravascularly, etc. Oral and inhaled treatments are of particular interest. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt. %. The therapeutic agents can be administered in a single dose, or as multiple doses over a course of treatment. [0128]
The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents. [0129]
Methods of Screening for Drugs that Modulate TTK Activity [0130]
A TTK polypeptide or TTK-encoding nucleic acid according to the present invention may be used in screening for molecules which affect or modulate TTK activity or function. Such molecules may be useful in a therapeutic and/or prophylactic context. Means for screening for substances potentially useful in treating or preventing cancer is provided by the present invention. In general, the methods of the invention are to facilitate identification of modulators of TTK activity (e.g., by modulating activity of TTK polypeptide or other TTK gene product, or by affecting TTK activity by targeting activity of gene products that act either upstream or downstream of TTK in a cascade that leads to TTK activity), with agents that decrease TTK activity generally being of particular interest. Substances identified as modulators of the TTK activity represent an advance in the fight against cancer since they provide basis for design and investigation of pharmaceuticals for in vivo use. [0131]
A method of screening for a substance which modulates activity of a polypeptide may include contacting one or more test substances with the polypeptide in a suitable reaction medium, testing the activity of the treated polypeptide (e.g., the ability to phosphorylate its substrate) and comparing that activity with the activity of the polypeptide in comparable reaction medium untreated with the test substance or substances. A difference in activity between the treated and untreated polypeptides is indicative of a modulating effect of the relevant test substance or substances. [0132]
Combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a polypeptide. Such libraries and their use are known in the art. The use of peptide libraries is preferred. Test substances may also be screened for ability to interact with the polypeptide, e.g., in a yeast two-hybrid system. This may be used as a coarse screen prior to testing a substance for actual ability to modulate activity of the polypeptide. Alternatively, the screen could be used to screen test substances for binding to a TTK specific binding partner. [0133]
A substance identified using as a modulator of TTK polypeptide function may be peptide or non-peptide in nature. Non-peptide “small molecules” are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use. [0134]
TTK activity assays [0135]
The activity of the TTK may be measured using any suitable kinase assay known in the art. For example, and not by way of limitation, the methods described in Hogg et al (Oncogene 1994 9:98-96), Mills et al (J. Biol. Chem. 1992 267:16000-006) and Tomizawa et al 2001 (FEBS Lett. 2001 492: 221-7), Schmandt et al, (J. Immunol. 1994, 152:96-105) may be used. Further serine, threonine and tyrosine kinase assays are described in Ausubel et al. (Short Protocols in Molecular Biology, 1999, unit 17.6). [0136]
TTK assays generally use TTK polypeptide, a labeled donor substrate, and a receptor substrate that is either specific or non-specific for TTK. In such assays TTK transfers a labeled moiety from the donor substrate to the receptor substrate, and kinase activity is measured by the amount of labeled moiety transferred from the donor substrate to the receptor substrate. [0137]
TTK polypeptide may be produced using various expression systems as detailed above, may be purified from cells, may be in the form of a cleaved or uncleaved recombinant fusion protein and may have non-TTK polypeptide sequences, for example a His tag or β-galactosidase at its N- or C-terminus. TTK activity may be assayed in cancerous cells lines if the cancerous cell lines are used as a source of the TTK to be assayed. Suitable donor substrates for TTK assays include any molecule that is susceptible to dephosphorylation by TTK include γ-labeled ATP and ATP analogs, wherein the label is [0138] ³³P, ³²P, ³⁵S or any other radioactive isotope or a suitable fluorescent marker. Suitable recipient substrates for TTK assays include any polypeptide or other molecule that is susceptible to phosphorylation by TTK. Recipient substrates are usually derived from fragments of in vivo targets of TTK. Recipient substrates fragments may be 8 to 50 amino acids in length, usually 10 to 30 amino acids and preferably of about 10, 12, 15, 18, 20 and 25 amino acids in length Further recipient substrates can be determined empirically using a set of different polypeptides or other molecules. Targets of TTK suitable for TTK assays include tau and cdc25. Recipient substrates for TTK are typically capable of being purified from other components of the reaction once the reaction has been performed. This purification is usually done through a molecular interaction, where the recipient substrates is biotinylated and purified through its interaction with streptavidin, or a specific antibody is available that can specifically recognize the recipient substrates. The reaction can be performed in a variety of conditions, such as on a solid support, in a gel, in solution or in living cells.
One exemplary recipient substrate for TTK phosphorylation is the human protein cdc25, SEQ ID NO:26, which is phosphorylated by TTK at the serine residues of amino acid position 214 and 216. Two fragments of cdc25 are used as substrates in the kinase assay described below. These fragments comprise peptides A (SEQ ID NO:27), corresponding to amino acids 209 to 225 of the cdc25 polypeptide sequence or peptide B (SEQ ID NO:28), corresponds to amino acids 210 to 223 of the cdc25 polypeptide. In this assay, two biotinylated polypeptides of comprising either SEQ ID NO:27 (Biotin-SGSGSGLYRSPSMPENLNRPR-NH2) or SEQ ID NO:28 (Biotin-GGGGLYRSPSMPENLNRK-OH) are used. [0139]
The choice of detection methods depends on type of label used for the donor molecule and may include, for example, measurement of incorporated radiation or fluorescence by autoradiography, scintillation, scanning or fluorography. [0140]
Methods of Inhibiting Tumor Growth and Other Treatment Goals [0141]
The invention further provides methods for reducing growth of cancer cells, particular breast or colon cancer cells. In general, the methods comprise contacting a cancer cell that expresses TTK at an aberrant level relative to normal cells with a substance that (1) modulates, generally decreases, expression of TTK (e.g., a antisense polynucleotide corresponding to TTK); or (2) otherwise modulates, generally decreases, TTK polypeptide levels and/or TTK activity in a cancerous cell having aberrant TTK activity. [0142]
“Reducing growth of a cancer cell” includes, but is not limited to, reducing proliferation of cancer cells, and reducing the incidence of a normal cell from developing a cancerous phenotype or morphology. Whether a reduction in cancer cell growth has been achieved can be readily determined using any known assay, including, but not limited to, [[0143] ³H]-thymidine incorporation; counting cell number over a period of time; detecting, measuring a marker associated with colon cancer (e.g., CEA, CA19-9, and LASA), and/or methods well known in the art for assessing tumor burden.
The present invention provides methods for treating cancer (particularly breast and colon cancer or other cancer that is associated with aberrantly high TTK activity) which methods generally comprise administering to an individual an agent that reduces TTK activity in an amount sufficient to reduce cancer cell growth to treat the cancer. Whether a substance, or a specific amount of the substance, is effective in treating cancer can be assessed using any of a variety of known diagnostic assays, e.g. in the case of colon cancer, sigmoidoscopy, proctoscopy, rectal examination, colonoscopy with biopsy, contrast radiographic studies, CAT scans, angiography, and detection of a tumor marker associated with colon cancer in the blood of the individual. The substance can be administered systemically or locally. Thus, in some embodiments, the substance is administered locally, and colon cancer growth is decreased at the site of administration. Local administration may be useful in treating, e.g., a solid tumor. [0144]
In one embodiment, the invention features polynucleotides that act as antisense polynucleotides and decrease TTK activity. Antisense TTK polynucleotides generally comprise a polynucleotide of at least about 20 to 3000 nucleotides, usually at least about 20 to 1000 nucleotides and more usually at least about 8 to 50 nucleotides, and preferably about 26, 20, 18, 17, 15, 10 and 8 nucleotides. Exemplary TTK polynucleotides are provided in the Examples and in SEQ ID NO:1-12, although any antisense fragment of SEQ ID NO:13 will suffice. [0145]
The therapeutic regimen is selected according to the expression profile. For example, if a patient's tumor indicates that the tumor produces aberrantly high level of TTK relative to normal cells, then a drug having efficacy in the treatment of such TTK-expressing tumors is selected for therapy of that patient. [0146]
Pharmaceutical Compositions [0147]
Pharmaceutical compositions of the invention can comprise a therapeutically effective amount of a polypeptide, antibody, polynucleotide (including antisense nucleotides and ribozymes), or small molecule or other compound identified as modulating activity of TTK, preferably decreasing TTK activity. The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature, and/or in the effect upon tumor load in the subject (e.g., decrease in tumor size or inhibition in tumor growth). The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation is determined by routine experimentation and is within the judgment of the clinician. For purposes of the present invention, an effective dose will generally be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered. [0148]
A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier. Pharmaceutically acceptable salts can also be present in the pharmaceutical composition, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in [0149] Remington 's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). The precise nature of the carrier or other material may depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. [0150]
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is has suitable pH, isotonicity and stability. Suitable solutions, for example, optionally include but are not limited to isotonic vehicles such as sodium chloride, preservatives, stabilizers, buffers, antioxidants and/or other additives as required. [0151]
Administration of the pharmaceutical is administered in a prophylactically effective amount or a therapeutically effective amount. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Decisions on dosage etc, can be determined by one skilled in the art based upon the disclosed methods, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980. [0152]
Alternatively, targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells. Targeting can be accomplished by, for example, administering a drug-antibody complex to a subject, wherein the antibody is specific for a cancer-associated antigen, and the drug is one that reduces cancer cell growth. Targeting can be accomplished by coupling (e.g., linking, directly or via a linker molecule, either covalently or non-covalently, so as to form a drug-antibody complex) a drug to an antibody specific for a cancer-associated antigen. Methods of coupling a drug to an antibody are well known in the art and need not be elaborated upon herein. [0153]
Pharmaceutical agents can also be produced in the target cells by expression from an encoding gene introduced into the cells, e.g., in a viral or liposomal vector. The vector could be targeted to the specific cells to be treated, or it could contain regulatory elements which are switched on more or less selectively by the target cells. [0154]
Alternatively, the agent could be administered in a precursor form, for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. [0155]
Delivery Methods for Therapy [0156]
Once formulated, the compositions of the invention or identified using the methods of the invention can be administered directly to the subject (e.g., as polynucleotide or polypeptides). Direct delivery of the compositions will generally be accomplished by parenteral injection, e.g., subcutaneously, intraperitoneally, intravenously or intramuscularly, intratumoral or to the interstitial space of a tissue. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications, needles, and gene guns or hyposprays. Dosage treatment can be a single dose schedule or a multiple dose schedule. [0157]
Once a gene corresponding to a polynucleotide of the invention has been found to correlate with a proliferative disorder, such as neoplasia, dysplasia, and hyperplasia, the disorder can be amenable to treatment by administration of a therapeutic agent based on the provided polynucleotide, corresponding polypeptide or other corresponding molecule (e.g., antisense, ribozyme, etc.). [0158]
The dose and the means of administration are determined based on the specific qualities of the therapeutic composition, the condition, age, and weight of the patient, the progression of the disease, and other relevant factors. For example, administration of polynucleotide therapeutic compositions agents of the invention includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. Preferably, the therapeutic polynucleotide composition contains an expression construct comprising a promoter operably linked to a polynucleotide of at least 12, 15, 17, 18, 22, 25, 30, or 35 contiguous-nucleotides of the polynucleotide disclosed herein. Various methods can be used to administer the therapeutic composition directly to a specific site in the body. For example, a small metastatic lesion is located and the therapeutic composition injected several times in several different locations within the body of tumor. Alternatively, arteries which serve a tumor are identified, and the therapeutic composition injected into such an artery, in order to deliver the composition directly into the tumor. A tumor that has a necrotic center is aspirated and the composition injected directly into the now empty center of the tumor. The antisense composition is directly administered to the surface of the tumor, for example, by topical application of the composition. X-ray imaging is used to assist in certain of the above delivery methods. [0159]
Receptor-mediated targeted delivery of therapeutic compositions containing an antisense polynucleotide, subgenomic polynucleotides, or antibodies to specific tissues can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., [0160] Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during a gene therapy protocol. Factors such as method of action (e.g., for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy of the antisense subgenomic polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of antisense subgenomic polynucleotides or the same amounts readministered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of, for example, a tumor site, may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect. For polynucleotide related genes encoding polypeptides or proteins with anti-inflammatory activity, suitable use, doses, and administration are described in U.S. Pat. No. 5,654,173.
The therapeutic polynucleotides and polypeptides of the present invention can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, [0161] Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.
Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5, 219,740; WO 93/11230; WO 93/10218; U.S. Pat. No. 4,777,127; GB Patent No. 2,200,651; [0162] EP 0 345 242; and WO 91/02805), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), and adeno-associated virus (AAV) vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.
Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, [0163] Hum. Gene Ther. (1992) 3:147); ligand-linked DNA(see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.
Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al., [0164] Proc. Natl. Acad. Sci. USA (1994) 91(24): 11581. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials or use of ionizing radiation (see, e.g., U.S. Pat. No. 5,206,152 and WO 92/11033). Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun (see, e.g., U.S. Pat. No. 5,149,655); use of ionizing radiation for activating transferred gene (see, e.g., U.S. Pat. No. 5,206,152 and WO 92/11033).
As an alternative to the use of viral vectors other known methods of introducing nucleic acid into cells includes electroporation, calcium phosphate co-precipitation, mechanical techniques such as microinjection, transfer mediated by liposomes and direct DNA uptake and receptor-mediated DNA transfer. Gene transfer techniques which selectively target the TTK nucleic acid to the affected cell type are preferred. Examples of this included receptor-mediated gene transfer, in which the nucleic acid is linked to a protein ligand via polylysine, with the ligand being specific for a receptor present on the surface of the target cells. [0165]
Screening for Substances Affecting TTK Expression [0166]
The present invention also provides the use of all or part of the nucleic acid sequence of the TTK promoter and/or enhancer regions in methods of screening for substances which modulate the activity of the promoter and increase or decrease the level of TTK expression. This assay can be performed to identify anti-cancer agents for therapeutic and/or prophylactic purposes. The level of promoter activity, i.e., the ability to initiate transcription, is quantifiable for instance by assessment of the amount of mRNA produced by transcription from the promoter or by assessment of the amount of protein product produced by translation of mRNA produced by transcription from the promoter. The amount of a specific mRNA present in an expression system may be determined for example using specific oligonucleotides which are able to hybridize with the mRNA and which are labeled or may be used in a specific amplification reaction such as PCR. Use of a reporter gene facilitates determination of promoter activity by reference to protein production. [0167]
Generally, a reporter gene under control of the TTK promoter and/or enhancers may be transcribed into mRNA which may be translated into a peptide or polypeptide product which may be detected and preferably quantitated following expression. The reporter gene preferably encodes an enzyme which catalyses a reaction which produces a detectable signal, preferably a visually detectable signal, such as a coloured product. Many examples are known, including β-galactosidase and luciferase. β-galactosidase activity may be assayed by production of blue color on substrate, the assay being by eye or by use of a spectrophotometer to measure absorbance. Fluorescence, for example that produced as a result of luciferase activity, may be quantitated using a spectrophotometer. Radioactive assays may be used, for instance using choloramphenicol acetyltransferase, which may also be used in non-radioactive assays. The presence and/or amount of gene product resulting from expression from the reporter gene may be determined using a molecule able to bind the product, such as an antibody or fragment thereof. The binding molecule may be labeled directly or indirectly using any standard technique. [0168]
Those skilled in the art are well aware of a multitude of possible reporter genes and assay techniques which may be used to determine gene activity according to the presently disclosed methods. Any suitable reporter/assay may be used and the present invention is intended to encompass such systems. [0169]
Following identification of a substance which modulates or affects promoter activity, the substance may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. [0170]
Integrated Disease Information System [0171]
The levels of TTK in a sample can be used in an integrated disease information system to aid in analysis such as proposed patient interventions, designing clinical trials, performing pharmacoeconomic analysis, and illustrating disease progression for various patients over time. For example, TTK information determined according to the methods of the invention can be used in a system such as that described in U.S. Pat. No. 6,108,635 issued to Herren, et al. on Aug. 22, 2000. Such a system can be for collecting the results of medical treatments given to patients in a plurality of locations. See, e.g., U.S. Pat. No. 5,713,350 issued to Yokota, et al. on Feb. 3, 1998. [0172]

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. [0173]

Example 1

Source of Patient Tissue Samples

Normal and cancerous tissues were collected from patients using laser capture microdissection (LCM) techniques, which techniques are well known in the art (see, e.g., Ohyama et al. (2000) [0174] Biotechniques 29:530-6; Curran et al. (2000) Mol. Pathol. 53:64-8; Suarez-Quian et al. (1999) Biotechniques 26:328-35; Simone et al (1998) Trends Genet 14:272-6; Conia et al. (1997) J. Clin. Lab. Anal. 11:28-38; Emmert-Buck et al. (1996) Science 274:998-1001). Table 1 (inserted following the last page of the Examples ) provides information about each patient from which the samples were isolated, including: the Patient ID and Path ReportID, numbers assigned to the patient and the pathology reports for identification purposes; the anatomical location of the tumor (AnatomicalLoc); The Primary Tumor Size; the Primary Tumor Grade; the Histopathologic Grade; a description of local sites to which the tumor had invaded (Local Invasion); the presence of lymph node metastases (Lymph Node Metastasis); incidence of lymph node metastases (provided as number of lymph nodes positive for metastasis over the number of lymph nodes examined) (Incidence Lymphnode Metastasis); the Regional Lymphnode Grade; the identification or detection of metastases to sites distant to the tumor and their location (Distant Met & Loc);a description of the distant metastases (Description Distant Met); the grade of distant metastasis (Distant Met Grade); and general comments about the patient or the tumor (Comments). Adenoma was not described in any of the patients; adenoma dysplasia (described as hyperplasia by the pathologist) was described in Patient ID No. 695. Extranodal extensions were described in two patients, Patient ID Nos. 784 and 791. Lymphovascular invasion was described in seven patients, Patient ID Nos. 128, 278, 517, 534, 784, 786, and 791. Crohn's-like infiltrates were described in seven patients, Patient ID Nos. 52, 264, 268, 392, 393, 784, and 791.

Example 2

Differential Expression of TTK

cDNA probes were prepared from total RNA isolated from the patient cells described in Example 1. Since LCM provides for the isolation of specific cell types to provide a substantially homogenous cell sample, this provided for a similarly pure RNA sample. [0175]
Total RNA was first reverse transcribed into cDNA using a primer containing a T7 RNA polymerase promoter, followed by second strand DNA synthesis. cDNA was then transcribed in vitro to produce antisense RNA using the T7 promoter-mediated expression (see, e.g., Luo et al. (1999) [0176] Nature Med 5:117-122), and the antisense RNA was then converted into cDNA. The second set of cDNAs were again transcribed in vitro, using the T7 promoter, to provide antisense RNA. Optionally, the RNA was again converted into cDNA, allowing for up to a third round of T7-mediated amplification to produce more antisense RNA. Thus the procedure provided for two or three rounds of in vitro transcription to produce the final RNA used for fluorescent labeling. Fluorescent probes were generated by first adding control RNA to the antisense RNA mix, and producing fluorescently labeled cDNA from the RNA starting material. Fluorescently labeled cDNAs prepared from the tumor RNA sample were compared to fluorescently labeled cDNAs prepared from normal cell RNA sample. For example, the cDNA probes from the normal cells were labeled with Cy3 fluorescent dye (green) and the cDNA probes prepared from the tumor cells were labeled with Cy5 fluorescent dye (red).
Each array used had an identical spatial layout and control spot set. Each microarray was divided into two areas, each area having an array with, on each half, twelve groupings of 32×12 spots for a total of about 9,216 spots on each array. The two areas are spotted identically which provide for at least two duplicates of each clone per array. Spotting was accomplished using PCR amplified products from 0.5 kb to 2.0 kb and spotted using a Molecular Dynamics Gen III spotter according to the manufacturer's recommendations. The first row of each of the 24 regions on the array had about 32 control spots, including 4 negative control spots and 8 test polynucleotides. The test polynucleotides were spiked into each sample before the labeling reaction with a range of concentrations from 2-600 pg/slide and ratios of 1:1 . For each array design, two slides were hybridized with the test samples reverse-labeled in the labeling reaction. This provided for about 4 duplicate measurements for each clone, two of one color and two of the other, for each sample. [0177]
The differential expression assay was performed by mixing equal amounts of probes from tumor cells and normal cells of the same patient. The arrays were prehybridized by incubation for about 2 hrs at 60° C. in 5×SSC/0.2% SDS/1 mM EDTA, and then washed three times in water and twice in isopropanol. Following prehybridization of the array, the probe mixture was then hybridized to the array under conditions of high stringency (overnight at 42° C. in 50% formamide, 5×SSC, and 0.2% SDS. After hybridization, the array was washed at 55° C. three times as follows: 1) first wash in 1×SSC/0.2% SDS; 2) second wash in 0.1×SSC/0.2% SDS; and 3) third wash in 0.1×SSC. [0178]
The arrays were then scanned for green and red fluorescence using a Molecular Dynamics Generation III dual color laser-scanner/detector. The images were processed using BioDiscovery Autogene software, and the data from each scan set normalized to provide for a ratio of expression relative to normal. Data from the microarray experiments was analyzed according to the algorithms described in U.S. application Ser. No. 60/252,358, filed Nov. 20, 2000, by E. J. Moler, M. A. Boyle, and F. M. Randazzo, and entitled “Precision and accuracy in cDNA microarray data,” which application is specifically incorporated herein by reference. [0179]
The experiment was repeated, this time labeling the two probes with the opposite color in order to perform the assay in both “color directions.” Each experiment was sometimes repeated with two more slides (one in each color direction). The level fluorescence for each sequence on the array expressed as a ratio of the geometric mean of 8 replicate spots/genes from the four arrays or 4 replicate spots/gene from 2 arrays or some other permutation. The data were normalized using the spiked positive controls present in each duplicated area, and the precision of this normalization was included in the final determination of the significance of each differential. The fluorescent intensity of each spot was also compared to the negative controls in each duplicated area to determine which spots have detected significant expression levels in each sample. [0180]
A statistical analysis of the fluorescent intensities was applied to each set of duplicate spots to assess the precision and significance of each differential measurement, resulting in a p-value testing the null hypothesis that there is no differential in the expression level between the tumor and normal samples of each patient. During initial analysis of the microarrays, the hypothesis was accepted if p>10[0181] ⁻³, and the differential ratio was set to 1.000 for those spots. All other spots have a significant difference in expression between the tumor and normal sample. If the tumor sample has detectable expression and the normal does not, the ratio is truncated at 1000 since the value for expression in the normal sample would be zero, and the ratio would not be a mathematically useful value (e.g., infinity). If the normal sample has detectable expression and the tumor does not, the ratio is truncated to 0.001, since the value for expression in the tumor sample would be zero and the ratio would not be a mathematically useful value. These latter two situations are referred to herein as “on/off.” Database tables were populated using a 95% confidence level (p>0.05).
The difference in the expression level of TTK in the colon tumor cells relative to the matched normal colon cells was greater than or equal to 2 fold (“>=2×”) in 39% of the patients, greater than or equal to 2.5 fold in 36% of the patients, and greater than or equal to 5 fold in 27% of the patients examined. [0182]

Quantitative PCR of a number of normal tissues and tumor cell lines, particularly colorectal carcinoma cell lines was used to analyze expression of TTK. Quantitative real-time PCR was performed by first isolating RNA from cells using a Roche RNA Isolation kit according to manufacturer's directions. One microgram of RNA was used to synthesize a first-strand cDNA using MMLV reverse transcriptase (Ambion) using the manufacturers buffer and recommended concentrations of oligo dT, nucleotides, and Rnasin. This first-strand cDNA served as a template for quantitative real-time PCR using the Roche light-cycler as recommended in the machine manual. TTK was amplified with the forward primer CGGAATCAAGTCTTCTAGCT (SEQ ID NO: 1) and reverse primer GGTTGCTCAAAAGTTGGTATG (SEQ ID NO:2) PCR product was quantified based on the cycle at which the amplification entered the linear phase of amplification in comparison to an internal standard and using the software supplied by the manufacturer. Small differences in amounts or total template in the first-strand cDNA reaction were eliminated by normalizing to amount of actin amplified in a separate quantitative PCR reaction using the forward primer 5′-CGGGAAATCGTGCGTGACATTAAG-3′ (SEQ ID NO:3) and the reverse primer: 5′-TGATCTCCTTCTGCATCCTGTCGG-3′ (SEQ ID NO:4). The results for TTK mRNA levels in normal tissues are shown in FIG. 1; the results for TTK mRNA levels in tumor cell lines are shown in FIG. 2. A brief description of the cell lines analyzed is provided in the table below.



Cell Line	Tissue Source	Cell Line	Tissue Source

MDA-MB-231	Human breast; high	Caco-2	Human colorectal
	metastatic potential		adenocarcinoma
	(micromets in lung;
	adenocarcinoma;
	pleural effusion
MDA-MB-435	Human breast, high	SW620	Human colorectal
	metastatic potential		adenocarcinoma;
	(macrometastases in		from metastatic
	lung)		site (lymph node)
MCF-7	Human breast; non-	LS174T	High metastatic
	metastatic		potential human
			colorectal
			adenocarcinoma
MDA-MB-468	Human breast;	LOVO	Human colorectal
	adenocarcinoma		adenocarcinoma;
			colon; from
			metastatic site
			(colon)
Alab	Human breast,	HT29	Human colorectal
	metastatic		adenocarcinoma;
			colon
SKOV3	Human ovarian	SW480	Human colorectal
	adenocarcinoma		adenocarcinoma;
			colon
OVCAR3	Human ovarian	HCT116	Human colorectal
	adenocarcinoma		carcinoma; colon
KM12C	Human colon; low	Colo 320DN	Human colorectal
	metastatic potential		adenocarcinoma;
			colon
KM12L4	Human colon; high	T84	Human colorectal
	metastatic potential		carcinoma; colon;
	(derived from		from metastatic site
	Km12C)		(lung)
DU 145	Human prostate;	HCT15	Human colorectal
	carcinoma; from		adenocarcinoma;
	metastatic site: brain		colon
HT1080	Human sarcoma cell	CCD112	Human colorectal
	line;		adenocarcinoma,
			low metastatic
			potential
HMVEC	Primary human	DLD1	Human colon;
	microvascular		colorectal
	endothelial cells		adenocarcinoma
184B5	normal breast	293	kidney epithelial
	epithelial cells;		cells
	chemically trans-
	formed
LNCAP	prostate carcinoma;	GRDP2	primary prostate
	metastasis to left		epithelium
	supraclavicular
	lymph
U373MG	glioblastoma cell	IMR90	primary lung
			fibroblast
WOCA	primary prostate	PC3	prostate cancer;
	epithelium		androgen receptor
			negative

TTK was expressed in normal cells (FIG. 1), with thymus and testis identified as the normal tissues that most highly expressed the gene for TTK. Numerous cancer cells, however, displayed a significantly elevated level of TTK expression (FIG. 2) as compared to most wild-type tissues. [0184]

Example 3

Hierarchical Clustering and Stratification of Colon Cancers Using Differential Expression Data

Differential expression patterns from Example 2 were analyzed by applying hierarchical clustering methods to the data sets (see Eisen et al. (1998) [0185] PNAS 95:14863-14868). In short, hierarchical clustering algorithms are based on the average-linkage method of Sokal and Michener (Sokal, R R & Michener, C D (1958) Univ. Kans. Sci. Bull. 38, 1409-1438), which was developed for clustering correlation matrixes. The object of this algorithm is to compute a dendrogram that assembles all elements into a single tree. For any set of n genes, an upper-diagonal similarity matrix is computed which contains similarity scores for all pairs of genes. The matrix is scanned to identify the highest value (representing a similar pair of genes). Using this technique, four groups of differential expression patterns were identified and assigned to clusters.
Application of hierarchical clustering to the data from Example 2 revealed that IGF2 (insulin-like growth factor 2), TTK (serine, threonine, tyrosine kinase implicated in the cell cycle), MAPKAPK2 (mitogen-activated protein (MAP) kinase-activated protein kinase), and MARCKS (myristoylated alanine-rich C kinase substrate, which is a substrate of protein kinase C) are concurrently upregulated as detected in 9 out of the 33 colon cancer patient samples examined. The data for these experiments is presented in graphical form in FIGS. [0186] 3-6. The concurrent upregulation suggests that these genes are co-regulated and that patients with an elevated serum level of IGF2 may be candidates for treatment with inhibitors to TTK, MAPKAP kinase 2, MARCKS and/or IGF2.

Example 4

Antisense Regulation of TTK Expression

Additional functional information on TTK was generated using antisense knockout technology. TTK expression in cancerous cells was further analyzed to confirm the role and function of the gene product in tumorgenesis, e.g., in promoting a metastatic phenotype. [0187]
A number of different oligonucleotides complementary to TTK mRNA were designed as potential antisense oligonucleotides, and tested for their ability to suppress expression of TTK. The ability of each designed antisense oligonucleotide to inhibit gene expression was tested through transfection into SW620 colon colorectal carcinoma cells. For each transfection mixture, a carrier molecule, preferably a lipitoid or cholesteroid, was prepared to a working concentration of 0.5 mM in water, sonicated to yield a uniform solution, and filtered through a 0.45 μm PVDF membrane. The antisense or control oligonucleotide was then prepared to a working concentration of 100 μM in sterile Millipore water. The oligonucleotide was further diluted in OptiMEM™ (Gibco/BRL), in a microfuge tube, to 2 μM, or approximately 20 μg oligo/ml of OptiMEM™. In a separate microfuge tube, lipitoid or cholesteroid, typically in the amount of about 1.5-2 nmol lipitoid/pg antisense oligonucleotide, was diluted into the same volume of OptiMEM™ used to dilute the oligonucleotide. The diluted antisense oligonucleotide was immediately added to the diluted lipitoid and mixed by pipetting up and down. Oligonucleotide was added to the cells to a final concentration of 30 nM. [0188]
The level of target mRNA (TTK) in the transfected cells was quantitated in the cancer cell lines using the Roche LightCycler™ real-time PCR machine. Values for the target mRNA were normalized versus an internal control (e.g., beta-actin). For each 20 μl reaction, extracted RNA (generally 0.2-1 μg total) was placed into a sterile 0.5 or 1.5 ml microcentrifuge tube, and water was added to a total volume of 12.5 μl. To each tube was added 7.5 μl of a buffer/enzyme mixture, prepared by mixing (in the order listed) 2.5 μl H[0189] ₂O, 2.0 μl 10×reaction buffer, 10 μl oligo dT (20 pmol), 1.0 μl dNTP mix (10 mM each), 0.5 μl RNAsin® (20 u) (Ambion, Inc., Hialeah, Fla.), and 0.5 μl MMLV reverse transcriptase (50 u) (Ambion, Inc.). The contents were mixed by pipetting up and down, and the reaction mixture was incubated at 42° C. for 1 hour. The contents of each tube were centrifuged prior to amplification.
An amplification mixture was prepared by mixing in the following order: 1×PCR buffer II, 3 mM MgCl[0190] ₂, 140 μM each dNTP, 0.175 pmol each oligo, 1:50,000 dil of SYBR® Green, 0.25 mg/ml BSA, 1 unit Taq polymerase, and H₂O to 20 μl. (PCR buffer II is available in 10×concentration from Perkin-Elmer, Norwalk, Conn.). In 1×concentration it contains 10 mM Tris pH 8.3 and 50 mM KCl. SYBR® Green (Molecular Probes, Eugene, Oreg.) is a dye which fluoresces when bound to double stranded DNA. As double stranded PCR product is produced during amplification, the fluorescence from SYBR® Green increases. To each 20 μl aliquot of amplification mixture, 2 μl of template RT was added, and amplification was carried out according to standard protocols.
The following antisense oligonucleotides were shown to effectively deplete TTK RNA in the transfection assays: [0191]
Oligo 79-5AS: GGGACTCTTCCAAATGGGCATGACT (SEQ ID NO:5) [0192]
Oligo 79-9AS: TCCAGTAACTCTTGCGTTCCCATGG (SEQ ID NO:6) [0193]
The reverse control of each of these antisense oligonucleotides were synthesized, as were oligonucleotides with the identical sequence of the antisense oligonucleotides in reverse orientation (Reverse Control): [0194]
Oligo 79-5RC: TCAGTACGGGTAAACCTTCTCAGGG (SEQ ID NO:7) [0195]
Oligo 79-9RC: GGTACCCTTGCGTTCTCAATGACCT (SEQ ID NO:8) [0196]
The antisense oligonucleotides were introduced into a test cell and the effect upon TTK expression of the corresponding gene, as well as the effect induction of the cancerous phenotype, was examined as described below. [0197]

Example 5

Effect of TTK Expression on Proliferation

The effect of TTK on proliferation was assessed in metastatic breast cancer cell lines (MDA-MB-231 (“231”)), SW620 colon colorectal carcinoma cells, or 847 human immortal fibroblast cells. Transfection was carried out as described above in Example 4. [0198]
Cells were plated to approximately 60-80% confluency in 96-well dishes. Antisense or reverse control oligonucleotide was diluted to 2 μM in OptiMEM™ and added to OptiMEM™ into which the delivery vehicle, lipitoid 116-6 in the case of SW620 cells or 1:1 lipitoid 1:cholesteroid 1 in the case of MDA-MB-231 cells, had been diluted. The oligo/ delivery vehicle mixture was then further diluted into medium with serum on the cells. The final concentration of oligonucleotide for all experiments was 300 nM, and the final ratio of oligo to delivery vehicle for all experiments was 1.5 nmol lipitoid/μg oligonucleotide. Cells were transfected overnight at 37° C. and the transfection mixture was replaced with fresh medium the next morning. [0199]
Transfection of the antisense oligonucleotides into both SW620 colorectal carcinoma cells (FIG. 7) and 231 cells (FIG. 8) resulted in a decreased rate of proliferation compared to matched reverse control (RC) and oligonucleotides, but no inhibition of growth of 847 human immortal fibroblast cells (FIG. 11), suggesting possible tissue or transformation specificity in the functional role for the TTK protein. [0200]

Example 6

Effect of TTK Expression on Colony Formation

The effect of TTK expression upon colony formation was tested in a soft agar assay. Soft agar assays were conducted by first establishing a bottom layer of 2 ml of 0.6% agar in media plated fresh within a few hours of layering on the cells. The cell layer was formed on the bottom layer by removing cells transfected as described above from plates using 0.05% trypsin and washing twice in media. The cells were counted in a Coulter counter, and resuspended to 106 per ml in media. 10 μl aliquots are placed with media in 96-well plates (to check counting with WST1), or diluted further for soft agar assay. 2000 cells are plated in 800 μl 0.4% agar in duplicate wells above 0.6% agar bottom layer. After the cell layer agar solidifies, 2 ml of media is dribbled on top and antisense or reverse control oligo is added without delivery vehicles. Fresh media and oligos are added every 3-4 days. Colonies are formed in 10 days to 3 weeks. Fields of colonies were counted by eye. Wst-1 metabolism values can be used to compensate for small differences in starting cell number. Larger fields can be scanned for visual record of differences. [0201]
As shown in FIG. 9, antisense oligonucleotides to TTK (79-9AS) led to decreased colony size and number compared to control reverse control oligonucleotides (79-9RC) or to control oligonucleotides (52-3AS: TAGGTCTTTGGCCGGTGATGGGTCG (SEQ ID NO:9) and 52-3RC: GCTGGGTAGTGGCCGGTTTCTGGAT (SEQ ID NO:10)). The 52-3 antisense oligonucleotide is directed to the hD53 mRNA, and serves as a negative control in the experiment. [0202]

Example 7

Induction of Cell Death Upon Depletion of TTK (“Antisense Knockout”)

SW620 cells were transfected as described for proliferation assays. For cytotoxic effect in the presence of cisplatin (cis), the same protocol was followed but cells were left in the presence of 2 μM drug. Each day, cytotoxicity was monitored by measuring the amount of LDH enzyme released in the medium due to membrane damage. The activity of LDH was measured using the Cytotoxicity Detection Kit from Roche Molecular Biochemicals. The data is provided as a ratio of LDH released in the medium vs. the total LDH present in the well at the same time point and treatment (rLDH/tLDH). A positive control using antisense and reverse control oligonucleotides for BCL2 (a known anti-apoptotic gene) shows that loss of message for BCL2 leads to an increase in cell death compared with treatment with the control oligonucleotide (background cytotoxicity due to transfection). [0203]

The following antisense oligonucleotides were tested for the ability to deplete the message levels of the gene corresponding to the indicated cluster. Oligo Name: AS or RC provides the name of the target gene or name of the oligo, and whether the oligo is antisense (AS) or a reverse control (RC).



Oligo Name: Antisense
(AS) or Reverse
Control (RC)	Oligo Sequence	SEQ ID NO:

Chir39-5:AS	ACTCATCTGGCTGGGCTATGGTGGT	SEQ ID NO:11

Chir39-5:RC	TGGTGGTATCGGGTCGGTCTACTCA	SEQ ID NO:12

Chir79-9:AS	TCCAGTAACTCTTGCGTTCCCATGG	SEQ ID NO:6

Chir79-9:RC	GGTACCCTTGCGTTCTCAATGACCT	SEQ ID NO:8

As shown in FIG. 12, Chiron 79-9 (TTK) antisense does not sensitize the cells to treatment by cisplatin at a detectable level, but leads to increased death compared to control oligo at [0205] day 3.

Example 8

Sample Assay for Agents that Modulate TTK Activity

This assay may be performed in microtitre plates. TTK was purified as a 6×IIis tagged fusion protein using a baculovirus expression system. Essentially 20 ul of 20 nM TTK (100 k Da) in TTK kinase buffer comprising 50 mM Hepes pH 7.4, 2mM MgCl[0206] ₂, 10 mM MnCl₂, 1 mM NaF, 50 mM NaCl, 1 mM DTT and 1 mg/ml BSA was added to 5 ul of a candidate agent diluted in 20% DMSO, 10 ul of a 2.8 uM solution of a biotinylated substrate peptide derived from cdc25, such as Biotin-SGSGSGLYRSPSMPENLNRPR-NH2 (SEQ ID NO:27) or Biotin-GGGGLYRSPSMPENLNRK-OH (SEQ ID NO:28) and 5 ul of 80 nM ³³P-γATP in a well of a microtitre plate. Samples were mixed, incubated for 2 hours and each reaction is terminated using 20 ul of 0.5 M EDTA pH 8.0. 50 ul of the sample is transferred to a 96 well flat bottom Streptavidin coated flash plate, and the sample is incubated with the plate for 1 hr at room temperature. The wells of the plate are washed four times with 250 ul of calcium and magnesium-free phosphate buffered saline, and scintillation fluid is added to the sample. Activity of TTK was measured by calculating the emission of ³³P, transferred by TTK from ³³P-γATP to a substrate peptide, by scintillation.
Agents modulating TTK activity can be identified by comparing the activity of TTK in the presence of a candidate agent to the activity of TTK in the absence of a candidate agent. [0207]
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the invention. [0208]

0

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 38

<210> SEQ ID NO 1

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: synthesized oligonucleotide

<400> SEQUENCE: 1

cggaatcaag tcttctagct 20

<210> SEQ ID NO 2

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: synthesized oligonucleotide

<400> SEQUENCE: 2

ggttgctcaa aagttggtat g 21

<210> SEQ ID NO 3

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: synthesized oligonucleotide

<400> SEQUENCE: 3

cgggaaatcg tgcgtgacat taag 24

<210> SEQ ID NO 4

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: synthesized oligonucleotide

<400> SEQUENCE: 4

tgatctcctt ctgcatcctg tcgg 24

<210> SEQ ID NO 5

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: synthesized oligonucleotide

<400> SEQUENCE: 5

gggactcttc caaatgggca tgact 25

<210> SEQ ID NO 6

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: synthesized oligonucleotide

<400> SEQUENCE: 6

tccagtaact cttgcgttcc catgg 25

<210> SEQ ID NO 7

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: synthesized oligonucleotide

<400> SEQUENCE: 7

tcagtacggg taaaccttct caggg 25

<210> SEQ ID NO 8

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: synthesized oligonucleotide

<400> SEQUENCE: 8

ggtacccttg cgttctcaat gacct 25

<210> SEQ ID NO 9

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: synthesized oligonucleotide

<400> SEQUENCE: 9

taggtctttg gccggtgatg ggtcg 25

<210> SEQ ID NO 10

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: synthesized oligonucleotide

<400> SEQUENCE: 10

gctgggtagt ggccggtttc tggat 25

<210> SEQ ID NO 11

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: synthesized oligonucleotide

<400> SEQUENCE: 11

actcatctgg ctgggctatg gtggt 25

<210> SEQ ID NO 12

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: synthesized oligonucleotide

<400> SEQUENCE: 12

tggtggtatc gggtcggtct actca 25

<210> SEQ ID NO 13

<211> LENGTH: 3866

<212> TYPE: DNA

<213> ORGANISM: Homo sapien

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (1026)...(3551)

<221> NAME/KEY: misc_feature

<222> LOCATION: (0)...(0)

<223> OTHER INFORMATION: TTK

<400> SEQUENCE: 13

ggaattcctt tttttttttt tttgagatgg agtttcactc ttgttggcca ggctggagtg 60

caatggcaca atctcagctt actgcaacct ccgcctcccg ggttcaagcg attctcctgc 120

ctcagcctct caagtagctg ggattacagg catgtgccac cacccctggc taactaattt 180

cttttctatt tagtagagat ggggtttcac catgttggtc aggctggtct tgaactcctg 240

acctcaggtg atccacttgc cttggcctcc caaagtgcta ggattacagc cgtgaaactg 300

tgcctggctg attctttttt tgttgttgga tttttgaaac agggtctccc ttggtcgccc 360

aggctggagt gcagtggtgc gatcttggct cactataacc tccacctcct ggtttcaagt 420

gatcctccca ctttagcctc ctgagtagct gtgattacag gcgtgcacca ccacacccgg 480

ctaatttttg tatttttatt agagacaggg tttcaccatg ttggccaggc tgttctcaaa 540

ctcctggact caagggatcc gcctgcctcc acttcccaaa gtcccgagat tacaggtgtg 600

agtcaccatg cctgacctta taattcttaa gtcatttttt ctggtccatt tcttccttag 660

ggtcctcaca acaaatctgc attaggcggt acaataatcc ttaacttcat gattcacaaa 720

aggaagatga agtgattcat gatttagaaa ggggaagtag taagcccact gcacactcct 780

ggatgatgat cctaaatcca gatacagtaa aaatggggta tgggaaggta gaatacaaaa 840

tttggtttaa attaattatc taaatatcta aaaacatttt tggatacatt gttgatgtga 900

atgtaagact gtacagactt cctagaaaac agtttgggtt ccatcttttc atttccccag 960

tgcagttttc tgtagaaatg gaatccgagg atttaagtgg cagagaattg acaattgatt 1020

ccata atg aac aaa gtg aga gac att aaa aat aag ttt aaa aat gaa gac 1070

Met Asn Lys Val Arg Asp Ile Lys Asn Lys Phe Lys Asn Glu Asp

1 5 10 15

ctt act gat gaa cta agc ttg aat aaa att tct gct gat act aca gat 1118

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

20 25 30

aac tcg gga act gtt aac caa att atg atg atg gca aac aac cca gag 1166

Asn Ser Gly Thr Val Asn Gln Ile Met Met Met Ala Asn Asn Pro Glu

35 40 45

gac tgg ttg agt ttg ttg ctc aaa cta gag aaa aac agt gtt ccg cta 1214

Asp Trp Leu Ser Leu Leu Leu Lys Leu Glu Lys Asn Ser Val Pro Leu

50 55 60

agt gat gct ctt tta aat aaa ttg att ggt cgt tac agt caa gca att 1262

Ser Asp Ala Leu Leu Asn Lys Leu Ile Gly Arg Tyr Ser Gln Ala Ile

65 70 75

gaa gcg ctt ccc cca gat aaa tat ggc caa aat gag agt ttt gct aga 1310

Glu Ala Leu Pro Pro Asp Lys Tyr Gly Gln Asn Glu Ser Phe Ala Arg

80 85 90 95

att caa gtg aga ttt gct gaa tta aaa gct att caa gag cca gat gat 1358

Ile Gln Val Arg Phe Ala Glu Leu Lys Ala Ile Gln Glu Pro Asp Asp

100 105 110

gca cgt gac tac ttt caa atg gcc aga gca aac tgc aag aaa ttt gct 1406

Ala Arg Asp Tyr Phe Gln Met Ala Arg Ala Asn Cys Lys Lys Phe Ala

115 120 125

ttt gtt cat ata tct ttt gca caa ttt gaa ctg tca caa ggt aat gtc 1454

Phe Val His Ile Ser Phe Ala Gln Phe Glu Leu Ser Gln Gly Asn Val

130 135 140

aaa aaa agt aaa caa ctt ctt caa aaa gct gta gaa cgt gga gca gta 1502

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

145 150 155

cca cta gaa atg ctg gaa att gcc ctg cgg aat tta aac ctc caa aaa 1550

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

160 165 170 175

aag cag ctg ctt tca gag gag gaa aag aag aat tta tca gca tct acg 1598

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

180 185 190

gta tta act gcc caa gaa tca ttt tcc ggt tca ctt ggg cat tta cag 1646

Val Leu Thr Ala Gln Glu Ser Phe Ser Gly Ser Leu Gly His Leu Gln

195 200 205

aat agg aac aac agt tgt gat tcc aga gga cag act act aaa gcc agg 1694

Asn Arg Asn Asn Ser Cys Asp Ser Arg Gly Gln Thr Thr Lys Ala Arg

210 215 220

ttt tta tat gga gag aac atg cca cca caa gat gca gaa ata ggt tac 1742

Phe Leu Tyr Gly Glu Asn Met Pro Pro Gln Asp Ala Glu Ile Gly Tyr

225 230 235

cgg aat tca ttg aga caa act aac aaa act aaa cag tca tgc cca ttt 1790

Arg Asn Ser Leu Arg Gln Thr Asn Lys Thr Lys Gln Ser Cys Pro Phe

240 245 250 255

gga aga gtc cca gtt aac ctt cta aat agc cca gat tgt gat gtg aag 1838

Gly Arg Val Pro Val Asn Leu Leu Asn Ser Pro Asp Cys Asp Val Lys

260 265 270

aca gat gat tca gtt gta cct tgt ttt atg aaa aga caa acc tct aga 1886

Thr Asp Asp Ser Val Val Pro Cys Phe Met Lys Arg Gln Thr Ser Arg

275 280 285

tca gaa tgc cga gat ttg gtt gtg cct gga tct aaa cca agt gga aat 1934

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

290 295 300

gat tcc tgt gaa tta aga aat tta aag tct gtt caa aat agt cat ttc 1982

Asp Ser Cys Glu Leu Arg Asn Leu Lys Ser Val Gln Asn Ser His Phe

305 310 315

aag gaa cct ctg gtg tca gat gaa aag agt tct gaa ctt att att act 2030

Lys Glu Pro Leu Val Ser Asp Glu Lys Ser Ser Glu Leu Ile Ile Thr

320 325 330 335

gat tca ata acc ctg aag aat aaa acg gaa tca agt ctt cta gct aaa 2078

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

340 345 350

tta gaa gaa act aaa gag tat caa gaa cca gag gtt cca gag agt aac 2126

Leu Glu Glu Thr Lys Glu Tyr Gln Glu Pro Glu Val Pro Glu Ser Asn

355 360 365

cag aaa cag tgg caa gct aag aga aag tca gag tgt att aac cag aat 2174

Gln Lys Gln Trp Gln Ala Lys Arg Lys Ser Glu Cys Ile Asn Gln Asn

370 375 380

cct gct gca tct tca aat cac tgg cag att ccg gag tta gcc cga aaa 2222

Pro Ala Ala Ser Ser Asn His Trp Gln Ile Pro Glu Leu Ala Arg Lys

385 390 395

gtt aat aca gag cag aaa cat acc act ttt gag caa cct gtc ttt tca 2270

Val Asn Thr Glu Gln Lys His Thr Thr Phe Glu Gln Pro Val Phe Ser

400 405 410 415

gtt tca aaa cag tca cca cca ata tca aca tct aaa tgg ttt gac cca 2318

Val Ser Lys Gln Ser Pro Pro Ile Ser Thr Ser Lys Trp Phe Asp Pro

420 425 430

aaa tct att tgt aag aca cca agc agc aat acc ttg gat gat tac atg 2366

Lys Ser Ile Cys Lys Thr Pro Ser Ser Asn Thr Leu Asp Asp Tyr Met

435 440 445

agc tgt ttt aga act cca gtt gta aag aat gac ttt cca cct gct tgt 2414

Ser Cys Phe Arg Thr Pro Val Val Lys Asn Asp Phe Pro Pro Ala Cys

450 455 460

cag ttg tca aca cct tat ggc caa cct gcc tgt ttc cag cag caa cag 2462

Gln Leu Ser Thr Pro Tyr Gly Gln Pro Ala Cys Phe Gln Gln Gln Gln

465 470 475

cat caa ata ctt gcc act cca ctt caa aat tta cag gtt tta gca tct 2510

His Gln Ile Leu Ala Thr Pro Leu Gln Asn Leu Gln Val Leu Ala Ser

480 485 490 495

tct tca gca aat gaa tgc att tcg gtt aaa gga aga att tat tcc ata 2558

Ser Ser Ala Asn Glu Cys Ile Ser Val Lys Gly Arg Ile Tyr Ser Ile

500 505 510

tta aag cag ata gga agt gga ggt tca agc aag gta ttt cag gtg tta 2606

Leu Lys Gln Ile Gly Ser Gly Gly Ser Ser Lys Val Phe Gln Val Leu

515 520 525

aat gaa aag aaa cag ata tat gct ata aaa tat gtg aac tta gaa gaa 2654

Asn Glu Lys Lys Gln Ile Tyr Ala Ile Lys Tyr Val Asn Leu Glu Glu

530 535 540

gca gat aac caa act ctt gat agt tac cgg aac gaa ata gct tat ttg 2702

Ala Asp Asn Gln Thr Leu Asp Ser Tyr Arg Asn Glu Ile Ala Tyr Leu

545 550 555

aat aaa cta caa caa cac agt gat aag atc atc cga ctt tat gat tat 2750

Asn Lys Leu Gln Gln His Ser Asp Lys Ile Ile Arg Leu Tyr Asp Tyr

560 565 570 575

gaa atc acg gac cag tac atc tac atg gta atg gag tgt gga aat att 2798

Glu Ile Thr Asp Gln Tyr Ile Tyr Met Val Met Glu Cys Gly Asn Ile

580 585 590

gat ctt aat agt tgg ctt aaa aag aaa aaa tcc att gat cca tgg gaa 2846

Asp Leu Asn Ser Trp Leu Lys Lys Lys Lys Ser Ile Asp Pro Trp Glu

595 600 605

cgc aag agt tac tgg aaa aat atg tta gag gca gtt cac aca atc cat 2894

Arg Lys Ser Tyr Trp Lys Asn Met Leu Glu Ala Val His Thr Ile His

610 615 620

caa cat ggc att gtt cac agt gat ctt aaa cca gct aac ttt ctg ata 2942

Gln His Gly Ile Val His Ser Asp Leu Lys Pro Ala Asn Phe Leu Ile

625 630 635

gtt gat gga atg cta aag cta att gat ttt ggg att gca aac caa atg 2990

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

640 645 650 655

caa cca gat aca aca agt gtt gtt aaa gat tct cag gtt ggc aca gtt 3038

Gln Pro Asp Thr Thr Ser Val Val Lys Asp Ser Gln Val Gly Thr Val

660 665 670

aat tat atg cca cca gaa gca atc aaa gat atg tct tcc tcc aga gag 3086

Asn Tyr Met Pro Pro Glu Ala Ile Lys Asp Met Ser Ser Ser Arg Glu

675 680 685

aat ggg aaa tct aag tca aag ata agc ccc aaa agt gat gtt tgg tcc 3134

Asn Gly Lys Ser Lys Ser Lys Ile Ser Pro Lys Ser Asp Val Trp Ser

690 695 700

tta gga tgt att ttg tac tat atg act tac ggg aaa aca cca ttt cag 3182

Leu Gly Cys Ile Leu Tyr Tyr Met Thr Tyr Gly Lys Thr Pro Phe Gln

705 710 715

cag ata att aat cag att tct aaa tta cat gcc ata att gat cct aat 3230

Gln Ile Ile Asn Gln Ile Ser Lys Leu His Ala Ile Ile Asp Pro Asn

720 725 730 735

cat gaa att gaa ttt ccc gat att cca gag aaa gat ctt caa gat gtg 3278

His Glu Ile Glu Phe Pro Asp Ile Pro Glu Lys Asp Leu Gln Asp Val

740 745 750

tta aag tgt tgt tta aaa agg gac cca aaa cag agg ata tcc att cct 3326

Leu Lys Cys Cys Leu Lys Arg Asp Pro Lys Gln Arg Ile Ser Ile Pro

755 760 765

gag ctc ctg gct cat cca tat gtt caa att caa act cat cca gtt aac 3374

Glu Leu Leu Ala His Pro Tyr Val Gln Ile Gln Thr His Pro Val Asn

770 775 780

caa atg gcc aag gga acc act gaa gaa atg aaa tat gtt ctg ggc caa 3422

Gln Met Ala Lys Gly Thr Thr Glu Glu Met Lys Tyr Val Leu Gly Gln

785 790 795

ctt gtt ggt ctg aat tct cct aac tcc att ttg aaa gct gct aaa act 3470

Leu Val Gly Leu Asn Ser Pro Asn Ser Ile Leu Lys Ala Ala Lys Thr

800 805 810 815

tta tat gaa cac tat agt ggt ggt gaa agt cat aat tct tca tcc tcc 3518

Leu Tyr Glu His Tyr Ser Gly Gly Glu Ser His Asn Ser Ser Ser Ser

820 825 830

aag act ttt gaa aaa aaa agg gga aaa aaa tga tttgcagtta ttcgtaatgt 3571

Lys Thr Phe Glu Lys Lys Arg Gly Lys Lys *

835 840

cagataggag gtataaaata tattggactg ttatactctt gaatccctgt ggaaatctac 3631

atttgaagac aacatcactc tgaagtgtta tcagcaaaaa aaattcagtg agattatctt 3691

taaaagaaaa ctgtaaaaat agcaaccact tatggcactg tatatattgt agacttgttt 3751

tctctgtttt atgctcttgt gtaatctact tgacatcatt ttactcttgg aatagtgggt 3811

ggatagcaag tatattctaa aaaactttgt aaataaagtt ttgtggctaa aatga 3866

<210> SEQ ID NO 14

<211> LENGTH: 841

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 14

Met Asn Lys Val Arg Asp Ile Lys Asn Lys Phe Lys Asn Glu Asp Leu

1 5 10 15

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

20 25 30

Ser Gly Thr Val Asn Gln Ile Met Met Met Ala Asn Asn Pro Glu Asp

35 40 45

Trp Leu Ser Leu Leu Leu Lys Leu Glu Lys Asn Ser Val Pro Leu Ser

50 55 60

Asp Ala Leu Leu Asn Lys Leu Ile Gly Arg Tyr Ser Gln Ala Ile Glu

65 70 75 80

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

85 90 95

Gln Val Arg Phe Ala Glu Leu Lys Ala Ile Gln Glu Pro Asp Asp Ala

100 105 110

Arg Asp Tyr Phe Gln Met Ala Arg Ala Asn Cys Lys Lys Phe Ala Phe

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Leu Thr Ala Gln Glu Ser Phe Ser Gly Ser Leu Gly His Leu Gln Asn

195 200 205

Arg Asn Asn Ser Cys Asp Ser Arg Gly Gln Thr Thr Lys Ala Arg Phe

210 215 220

Leu Tyr Gly Glu Asn Met Pro Pro Gln Asp Ala Glu Ile Gly Tyr Arg

225 230 235 240

Asn Ser Leu Arg Gln Thr Asn Lys Thr Lys Gln Ser Cys Pro Phe Gly

245 250 255

Arg Val Pro Val Asn Leu Leu Asn Ser Pro Asp Cys Asp Val Lys Thr

260 265 270

Asp Asp Ser Val Val Pro Cys Phe Met Lys Arg Gln Thr Ser Arg Ser

275 280 285

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

290 295 300

Ser Cys Glu Leu Arg Asn Leu Lys Ser Val Gln Asn Ser His Phe Lys

305 310 315 320

Glu Pro Leu Val Ser Asp Glu Lys Ser Ser Glu Leu Ile Ile Thr Asp

325 330 335

Ser Ile Thr Leu Lys Asn Lys Thr Glu Ser Ser Leu Leu Ala Lys Leu

340 345 350

Glu Glu Thr Lys Glu Tyr Gln Glu Pro Glu Val Pro Glu Ser Asn Gln

355 360 365

Lys Gln Trp Gln Ala Lys Arg Lys Ser Glu Cys Ile Asn Gln Asn Pro

370 375 380

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

385 390 395 400

Asn Thr Glu Gln Lys His Thr Thr Phe Glu Gln Pro Val Phe Ser Val

405 410 415

Ser Lys Gln Ser Pro Pro Ile Ser Thr Ser Lys Trp Phe Asp Pro Lys

420 425 430

Ser Ile Cys Lys Thr Pro Ser Ser Asn Thr Leu Asp Asp Tyr Met Ser

435 440 445

Cys Phe Arg Thr Pro Val Val Lys Asn Asp Phe Pro Pro Ala Cys Gln

450 455 460

Leu Ser Thr Pro Tyr Gly Gln Pro Ala Cys Phe Gln Gln Gln Gln His

465 470 475 480

Gln Ile Leu Ala Thr Pro Leu Gln Asn Leu Gln Val Leu Ala Ser Ser

485 490 495

Ser Ala Asn Glu Cys Ile Ser Val Lys Gly Arg Ile Tyr Ser Ile Leu

500 505 510

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

515 520 525

Glu Lys Lys Gln Ile Tyr Ala Ile Lys Tyr Val Asn Leu Glu Glu Ala

530 535 540

Asp Asn Gln Thr Leu Asp Ser Tyr Arg Asn Glu Ile Ala Tyr Leu Asn

545 550 555 560

Lys Leu Gln Gln His Ser Asp Lys Ile Ile Arg Leu Tyr Asp Tyr Glu

565 570 575

Ile Thr Asp Gln Tyr Ile Tyr Met Val Met Glu Cys Gly Asn Ile Asp

580 585 590

Leu Asn Ser Trp Leu Lys Lys Lys Lys Ser Ile Asp Pro Trp Glu Arg

595 600 605

Lys Ser Tyr Trp Lys Asn Met Leu Glu Ala Val His Thr Ile His Gln

610 615 620

His Gly Ile Val His Ser Asp Leu Lys Pro Ala Asn Phe Leu Ile Val

625 630 635 640

Asp Gly Met Leu Lys Leu Ile Asp Phe Gly Ile Ala Asn Gln Met Gln

645 650 655

Pro Asp Thr Thr Ser Val Val Lys Asp Ser Gln Val Gly Thr Val Asn

660 665 670

Tyr Met Pro Pro Glu Ala Ile Lys Asp Met Ser Ser Ser Arg Glu Asn

675 680 685

Gly Lys Ser Lys Ser Lys Ile Ser Pro Lys Ser Asp Val Trp Ser Leu

690 695 700

Gly Cys Ile Leu Tyr Tyr Met Thr Tyr Gly Lys Thr Pro Phe Gln Gln

705 710 715 720

Ile Ile Asn Gln Ile Ser Lys Leu His Ala Ile Ile Asp Pro Asn His

725 730 735

Glu Ile Glu Phe Pro Asp Ile Pro Glu Lys Asp Leu Gln Asp Val Leu

740 745 750

Lys Cys Cys Leu Lys Arg Asp Pro Lys Gln Arg Ile Ser Ile Pro Glu

755 760 765

Leu Leu Ala His Pro Tyr Val Gln Ile Gln Thr His Pro Val Asn Gln

770 775 780

Met Ala Lys Gly Thr Thr Glu Glu Met Lys Tyr Val Leu Gly Gln Leu

785 790 795 800

Val Gly Leu Asn Ser Pro Asn Ser Ile Leu Lys Ala Ala Lys Thr Leu

805 810 815

Tyr Glu His Tyr Ser Gly Gly Glu Ser His Asn Ser Ser Ser Ser Lys

820 825 830

Thr Phe Glu Lys Lys Arg Gly Lys Lys

835 840

<210> SEQ ID NO 15

<211> LENGTH: 2735

<212> TYPE: DNA

<213> ORGANISM: Saccharomyces cerevisiae

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (34)...(2499)

<400> SEQUENCE: 15

agaaaagata gtgttacaca acatcaacta aaa atg gaa aat att aca caa ccc 54

Met Glu Asn Ile Thr Gln Pro

1 5

aca cag caa tcc acg cag gct act caa agg ttt ttg att gag aag ttt 102

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

10 15 20

tct caa gaa cag atc ggc gaa aac att gtg tgc agg gtc att tgt acc 150

Ser Gln Glu Gln Ile Gly Glu Asn Ile Val Cys Arg Val Ile Cys Thr

25 30 35

acg ggt caa att ccc atc cga gat ttg tca gct gat att tca caa gtg 198

Thr Gly Gln Ile Pro Ile Arg Asp Leu Ser Ala Asp Ile Ser Gln Val

40 45 50 55

ctt aag gaa aaa cga tcc ata aag aaa gtt tgg aca ttt ggt aga aac 246

Leu Lys Glu Lys Arg Ser Ile Lys Lys Val Trp Thr Phe Gly Arg Asn

60 65 70

cca gcc tgt gac tat cat tta gga aac att tca aga ctg tca aat aag 294

Pro Ala Cys Asp Tyr His Leu Gly Asn Ile Ser Arg Leu Ser Asn Lys

75 80 85

cat ttc caa ata cta cta gga gaa gac ggt aac ctt tta ttg aat gac 342

His Phe Gln Ile Leu Leu Gly Glu Asp Gly Asn Leu Leu Leu Asn Asp

90 95 100

att tcc act aat ggg acc tgg tta aat ggg caa aaa gtc gag aag aac 390

Ile Ser Thr Asn Gly Thr Trp Leu Asn Gly Gln Lys Val Glu Lys Asn

105 110 115

agc aat cag tta ctg tct caa ggt gat gaa ata acc gtt ggt gta ggc 438

Ser Asn Gln Leu Leu Ser Gln Gly Asp Glu Ile Thr Val Gly Val Gly

120 125 130 135

gtg gaa tca gat att tta tct ctg gtc att ttc ata aac gac aaa ttt 486

Val Glu Ser Asp Ile Leu Ser Leu Val Ile Phe Ile Asn Asp Lys Phe

140 145 150

aag cag tgc ctc gag cag aac aaa gtt gat cgc ata aga tct aac ctg 534

Lys Gln Cys Leu Glu Gln Asn Lys Val Asp Arg Ile Arg Ser Asn Leu

155 160 165

aaa aat acc tct aaa ata gct tct cct ggt ctt aca tca tct act gca 582

Lys Asn Thr Ser Lys Ile Ala Ser Pro Gly Leu Thr Ser Ser Thr Ala

170 175 180

tca tca atg gtg gcc aac aag act ggt att ttt aag gat ttt tcg att 630

Ser Ser Met Val Ala Asn Lys Thr Gly Ile Phe Lys Asp Phe Ser Ile

185 190 195

att gac gaa gtg gtg ggc cag ggt gca ttt gcc aca gta aag aaa gcc 678

Ile Asp Glu Val Val Gly Gln Gly Ala Phe Ala Thr Val Lys Lys Ala

200 205 210 215

att gaa aga act act ggg aaa aca ttc gcg gtg aag att ata agt aaa 726

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

220 225 230

cgc aaa gta ata ggc aat atg gat ggt gtg aca aga gag tta gaa gta 774

Arg Lys Val Ile Gly Asn Met Asp Gly Val Thr Arg Glu Leu Glu Val

235 240 245

ttg caa aag ctc aat cat cca agg ata gta cga ttg aaa gga ttt tat 822

Leu Gln Lys Leu Asn His Pro Arg Ile Val Arg Leu Lys Gly Phe Tyr

250 255 260

gaa gat act gag agt tat tat atg gtg atg gag ttc gtt tct ggt ggt 870

Glu Asp Thr Glu Ser Tyr Tyr Met Val Met Glu Phe Val Ser Gly Gly

265 270 275

gac tta atg gat ttt gtt gct gct cat ggt gcg gtt gga gaa gat gct 918

Asp Leu Met Asp Phe Val Ala Ala His Gly Ala Val Gly Glu Asp Ala

280 285 290 295

ggg agg gag ata tcc agg cag ata ctc aca gca ata aaa tac att cac 966

Gly Arg Glu Ile Ser Arg Gln Ile Leu Thr Ala Ile Lys Tyr Ile His

300 305 310

tct atg ggc atc agc cat cgt gac cta aag ccc gat aat att ctt att 1014

Ser Met Gly Ile Ser His Arg Asp Leu Lys Pro Asp Asn Ile Leu Ile

315 320 325

gaa caa gac gat cct gta ttg gta aag ata acc gac ttt ggt ctg gca 1062

Glu Gln Asp Asp Pro Val Leu Val Lys Ile Thr Asp Phe Gly Leu Ala

330 335 340

aaa gta caa gga aat ggg tct ttt atg aaa acc ttc tgt ggc act ttg 1110

Lys Val Gln Gly Asn Gly Ser Phe Met Lys Thr Phe Cys Gly Thr Leu

345 350 355

gca tat gtg gca cct gaa gtc atc aga ggt aaa gat aca tcc gta tct 1158

Ala Tyr Val Ala Pro Glu Val Ile Arg Gly Lys Asp Thr Ser Val Ser

360 365 370 375

cct gat gaa tac gaa gaa agg aat gag tac tct tcg tta gtg gat atg 1206

Pro Asp Glu Tyr Glu Glu Arg Asn Glu Tyr Ser Ser Leu Val Asp Met

380 385 390

tgg tca atg gga tgt ctt gtg tat gtt atc cta acg ggc cac tta cct 1254

Trp Ser Met Gly Cys Leu Val Tyr Val Ile Leu Thr Gly His Leu Pro

395 400 405

ttt agt ggt agc aca cag gac caa tta tat aaa cag att gga aga ggc 1302

Phe Ser Gly Ser Thr Gln Asp Gln Leu Tyr Lys Gln Ile Gly Arg Gly

410 415 420

tca tat cat gaa ggg ccc ctc aaa gat ttc cgg ata tct gaa gaa gca 1350

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

425 430 435

aga gat ttc ata gat tca ttg tta cag gtg gat cca aat aat agg tcg 1398

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

440 445 450 455

aca gct gca aaa gcc ttg aat cat ccc tgg atc aag atg agt cca ttg 1446

Thr Ala Ala Lys Ala Leu Asn His Pro Trp Ile Lys Met Ser Pro Leu

460 465 470

ggc tca caa tca tat ggt gat ttt tca caa ata tcc tta tca caa tcg 1494

Gly Ser Gln Ser Tyr Gly Asp Phe Ser Gln Ile Ser Leu Ser Gln Ser

475 480 485

ttg tcg cag cag aaa tta tta gaa aat atg gac gat gct caa tac gaa 1542

Leu Ser Gln Gln Lys Leu Leu Glu Asn Met Asp Asp Ala Gln Tyr Glu

490 495 500

ttt gtc aaa gcg caa agg aaa tta caa atg gag caa caa ctt caa gaa 1590

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

505 510 515

cag gat cag gaa gac caa gat gga aaa att caa gga ttt aaa ata ccc 1638

Gln Asp Gln Glu Asp Gln Asp Gly Lys Ile Gln Gly Phe Lys Ile Pro

520 525 530 535

gca cac gcc cct att cga tat aca cag ccc aaa agc att gaa gca gaa 1686

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

540 545 550

act aga gaa caa aaa ctt tta cat tcc aat aat act gag aat gtc aag 1734

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

555 560 565

agc tca aag aaa aag ggt aat ggt agg ttt tta act tta aaa cca ttg 1782

Ser Ser Lys Lys Lys Gly Asn Gly Arg Phe Leu Thr Leu Lys Pro Leu

570 575 580

cct gac agc att att caa gaa agc ctg gag att cag caa ggt gtg aat 1830

Pro Asp Ser Ile Ile Gln Glu Ser Leu Glu Ile Gln Gln Gly Val Asn

585 590 595

cca ttt ttc att ggt aga tcc gag gat tgc aat tgt aaa att gaa gac 1878

Pro Phe Phe Ile Gly Arg Ser Glu Asp Cys Asn Cys Lys Ile Glu Asp

600 605 610 615

aat agg ttg tct cga gtt cat tgc ttc att ttc aaa aag agg cat gct 1926

Asn Arg Leu Ser Arg Val His Cys Phe Ile Phe Lys Lys Arg His Ala

620 625 630

gta ggc aaa agc atg tat gaa tct ccg gca caa ggt tta gat gat att 1974

Val Gly Lys Ser Met Tyr Glu Ser Pro Ala Gln Gly Leu Asp Asp Ile

635 640 645

tgg tat tgc cac acc gga act aac gtg agc tat tta aat aat aac cgc 2022

Trp Tyr Cys His Thr Gly Thr Asn Val Ser Tyr Leu Asn Asn Asn Arg

650 655 660

atg ata cag ggt acg aaa ttc ctt tta caa gac gga gat gaa atc aag 2070

Met Ile Gln Gly Thr Lys Phe Leu Leu Gln Asp Gly Asp Glu Ile Lys

665 670 675

atc att tgg gat aaa aac aat aaa ttt gtc att ggc ttt aaa gtg gaa 2118

Ile Ile Trp Asp Lys Asn Asn Lys Phe Val Ile Gly Phe Lys Val Glu

680 685 690 695

att aac gat act aca ggt ctg ttt aac gag gga tta ggt atg tta caa 2166

Ile Asn Asp Thr Thr Gly Leu Phe Asn Glu Gly Leu Gly Met Leu Gln

700 705 710

gaa caa aga gta gta ctt aag caa aca gcc gaa gaa aaa gat ttg gtg 2214

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

715 720 725

aaa aag tta acc cag atg atg gca gct caa cgt gca aat caa ccc tcg 2262

Lys Lys Leu Thr Gln Met Met Ala Ala Gln Arg Ala Asn Gln Pro Ser

730 735 740

gct tct tct tca tca atg tcg gct aag aag ccg cca gtt agc gat aca 2310

Ala Ser Ser Ser Ser Met Ser Ala Lys Lys Pro Pro Val Ser Asp Thr

745 750 755

aat aat aac ggc aat aat tcg gta cta aac gac ttg gta gag tca ccg 2358

Asn Asn Asn Gly Asn Asn Ser Val Leu Asn Asp Leu Val Glu Ser Pro

760 765 770 775

att aat gcg aat acg ggg aac att ttg aag aga ata cat tcg gta agt 2406

Ile Asn Ala Asn Thr Gly Asn Ile Leu Lys Arg Ile His Ser Val Ser

780 785 790

tta tcg caa tca caa att gat cct agt aag aag gtt aaa agg gca aaa 2454

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

795 800 805

ttg gac caa acc tca aaa ggc ccc gag aat ttg caa ttt tcg taa 2499

Leu Asp Gln Thr Ser Lys Gly Pro Glu Asn Leu Gln Phe Ser *

810 815 820

ccaaggacaa atacccatag aaaatgctgc ccctttttaa gagagaagat ggtagatacc 2559

aatactcaga attcccagta caaagaacca atatcggagt caataaacag tatgatgaac 2619

ttgctttcgc aaataaaaga tatcactcag aagcacccag taataaagga tgcagatagc 2679

tcgagatttg gtaaggttga gtttagggac ttttatgacg aagtttcacg gaattc 2735

<210> SEQ ID NO 16

<211> LENGTH: 821

<212> TYPE: PRT

<213> ORGANISM: Saccharomyces cerevisiae

<400> SEQUENCE: 16

Met Glu Asn Ile Thr Gln Pro Thr Gln Gln Ser Thr Gln Ala Thr Gln

1 5 10 15

Arg Phe Leu Ile Glu Lys Phe Ser Gln Glu Gln Ile Gly Glu Asn Ile

20 25 30

Val Cys Arg Val Ile Cys Thr Thr Gly Gln Ile Pro Ile Arg Asp Leu

35 40 45

Ser Ala Asp Ile Ser Gln Val Leu Lys Glu Lys Arg Ser Ile Lys Lys

50 55 60

Val Trp Thr Phe Gly Arg Asn Pro Ala Cys Asp Tyr His Leu Gly Asn

65 70 75 80

Ile Ser Arg Leu Ser Asn Lys His Phe Gln Ile Leu Leu Gly Glu Asp

85 90 95

Gly Asn Leu Leu Leu Asn Asp Ile Ser Thr Asn Gly Thr Trp Leu Asn

100 105 110

Gly Gln Lys Val Glu Lys Asn Ser Asn Gln Leu Leu Ser Gln Gly Asp

115 120 125

Glu Ile Thr Val Gly Val Gly Val Glu Ser Asp Ile Leu Ser Leu Val

130 135 140

Ile Phe Ile Asn Asp Lys Phe Lys Gln Cys Leu Glu Gln Asn Lys Val

145 150 155 160

Asp Arg Ile Arg Ser Asn Leu Lys Asn Thr Ser Lys Ile Ala Ser Pro

165 170 175

Gly Leu Thr Ser Ser Thr Ala Ser Ser Met Val Ala Asn Lys Thr Gly

180 185 190

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

195 200 205

Phe Ala Thr Val Lys Lys Ala Ile Glu Arg Thr Thr Gly Lys Thr Phe

210 215 220

Ala Val Lys Ile Ile Ser Lys Arg Lys Val Ile Gly Asn Met Asp Gly

225 230 235 240

Val Thr Arg Glu Leu Glu Val Leu Gln Lys Leu Asn His Pro Arg Ile

245 250 255

Val Arg Leu Lys Gly Phe Tyr Glu Asp Thr Glu Ser Tyr Tyr Met Val

260 265 270

Met Glu Phe Val Ser Gly Gly Asp Leu Met Asp Phe Val Ala Ala His

275 280 285

Gly Ala Val Gly Glu Asp Ala Gly Arg Glu Ile Ser Arg Gln Ile Leu

290 295 300

Thr Ala Ile Lys Tyr Ile His Ser Met Gly Ile Ser His Arg Asp Leu

305 310 315 320

Lys Pro Asp Asn Ile Leu Ile Glu Gln Asp Asp Pro Val Leu Val Lys

325 330 335

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

340 345 350

Lys Thr Phe Cys Gly Thr Leu Ala Tyr Val Ala Pro Glu Val Ile Arg

355 360 365

Gly Lys Asp Thr Ser Val Ser Pro Asp Glu Tyr Glu Glu Arg Asn Glu

370 375 380

Tyr Ser Ser Leu Val Asp Met Trp Ser Met Gly Cys Leu Val Tyr Val

385 390 395 400

Ile Leu Thr Gly His Leu Pro Phe Ser Gly Ser Thr Gln Asp Gln Leu

405 410 415

Tyr Lys Gln Ile Gly Arg Gly Ser Tyr His Glu Gly Pro Leu Lys Asp

420 425 430

Phe Arg Ile Ser Glu Glu Ala Arg Asp Phe Ile Asp Ser Leu Leu Gln

435 440 445

Val Asp Pro Asn Asn Arg Ser Thr Ala Ala Lys Ala Leu Asn His Pro

450 455 460

Trp Ile Lys Met Ser Pro Leu Gly Ser Gln Ser Tyr Gly Asp Phe Ser

465 470 475 480

Gln Ile Ser Leu Ser Gln Ser Leu Ser Gln Gln Lys Leu Leu Glu Asn

485 490 495

Met Asp Asp Ala Gln Tyr Glu Phe Val Lys Ala Gln Arg Lys Leu Gln

500 505 510

Met Glu Gln Gln Leu Gln Glu Gln Asp Gln Glu Asp Gln Asp Gly Lys

515 520 525

Ile Gln Gly Phe Lys Ile Pro Ala His Ala Pro Ile Arg Tyr Thr Gln

530 535 540

Pro Lys Ser Ile Glu Ala Glu Thr Arg Glu Gln Lys Leu Leu His Ser

545 550 555 560

Asn Asn Thr Glu Asn Val Lys Ser Ser Lys Lys Lys Gly Asn Gly Arg

565 570 575

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

580 585 590

Glu Ile Gln Gln Gly Val Asn Pro Phe Phe Ile Gly Arg Ser Glu Asp

595 600 605

Cys Asn Cys Lys Ile Glu Asp Asn Arg Leu Ser Arg Val His Cys Phe

610 615 620

Ile Phe Lys Lys Arg His Ala Val Gly Lys Ser Met Tyr Glu Ser Pro

625 630 635 640

Ala Gln Gly Leu Asp Asp Ile Trp Tyr Cys His Thr Gly Thr Asn Val

645 650 655

Ser Tyr Leu Asn Asn Asn Arg Met Ile Gln Gly Thr Lys Phe Leu Leu

660 665 670

Gln Asp Gly Asp Glu Ile Lys Ile Ile Trp Asp Lys Asn Asn Lys Phe

675 680 685

Val Ile Gly Phe Lys Val Glu Ile Asn Asp Thr Thr Gly Leu Phe Asn

690 695 700

Glu Gly Leu Gly Met Leu Gln Glu Gln Arg Val Val Leu Lys Gln Thr

705 710 715 720

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

725 730 735

Gln Arg Ala Asn Gln Pro Ser Ala Ser Ser Ser Ser Met Ser Ala Lys

740 745 750

Lys Pro Pro Val Ser Asp Thr Asn Asn Asn Gly Asn Asn Ser Val Leu

755 760 765

Asn Asp Leu Val Glu Ser Pro Ile Asn Ala Asn Thr Gly Asn Ile Leu

770 775 780

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

785 790 795 800

Lys Lys Val Lys Arg Ala Lys Leu Asp Gln Thr Ser Lys Gly Pro Glu

805 810 815

Asn Leu Gln Phe Ser

820

<210> SEQ ID NO 17

<211> LENGTH: 2525

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (289)...(1230)

<400> SEQUENCE: 17

tcctgccccg cggcgctgcc gcacgagccc cacgagccgc tcaccccgcc gttctcagcg 60

ctgcccgacc ccgctggcgc gccctcccgc cgccagtccc ggcagcgccc tcagttgtcc 120

tccgactcgc cctcggcctt ccgcgccagc cgcagccaca gccgcaacgc cacccgcagc 180

cacagccaca gccacagccc caggcatagc cttcggcaca gccccggctc cggctcctgc 240

ggcagctcct ctgggcaccg tccctgcgcc gacatcctgg aggttggg atg ctc ttg 297

Met Leu Leu

1

tcc aaa atc aac tcg ctt gcc cac ctg cgc gcc gcg ccc tgc aac gac 345

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

5 10 15

ctg cac gcc acc aag ctg gcg ccc ggc aag gag aag gag ccc ctg gag 393

Leu His Ala Thr Lys Leu Ala Pro Gly Lys Glu Lys Glu Pro Leu Glu

20 25 30 35

tcg cag tac cag gtg ggc ccg cta ctg ggc agc ggc ggc ttc ggc tcg 441

Ser Gln Tyr Gln Val Gly Pro Leu Leu Gly Ser Gly Gly Phe Gly Ser

40 45 50

gtc tac tca ggc atc cgc gtc tcc gac aac ttg ccg gtg gcc atc aaa 489

Val Tyr Ser Gly Ile Arg Val Ser Asp Asn Leu Pro Val Ala Ile Lys

55 60 65

cac gtg gag aag gac cgg att tcc gac tgg gga gag ctg cct aat ggc 537

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

70 75 80

act cga gtg ccc atg gaa gtg gtc ctg ctg aag aag gtg agc tcg ggt 585

Thr Arg Val Pro Met Glu Val Val Leu Leu Lys Lys Val Ser Ser Gly

85 90 95

ttc tcc ggc gtc att agg ctc ctg gac tgg ttc gag agg ccc gac agt 633

Phe Ser Gly Val Ile Arg Leu Leu Asp Trp Phe Glu Arg Pro Asp Ser

100 105 110 115

ttc gtc ctg atc ctg gag agg ccc gag ccg gtg caa gat ctc ttc gac 681

Phe Val Leu Ile Leu Glu Arg Pro Glu Pro Val Gln Asp Leu Phe Asp

120 125 130

ttc atc acg gaa agg gga gcc ctg caa gag gag ctc gcc cgc agc ttc 729

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

135 140 145

ttc tgg cag gtg ctg gag gcc gtg cgg cac tgc cac aac tgc ggg gtg 777

Phe Trp Gln Val Leu Glu Ala Val Arg His Cys His Asn Cys Gly Val

150 155 160

ctc cac cgc gac atc aag gac gaa aac atc ctt atc gac ctc aat cgc 825

Leu His Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp Leu Asn Arg

165 170 175

ggc gag ctc aag ctc atc gac ttc ggg tcg ggg gcg ctg ctc aaa gac 873

Gly Glu Leu Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu Leu Lys Asp

180 185 190 195

acc gtc tac acg gac ttc gat ggg acc cga gtg tat agc cct cca gag 921

Thr Val Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr Ser Pro Pro Glu

200 205 210

tgg atc cgc tac cat cgc tac cat ggc agg tcg gcg gca gtc tgg tcc 969

Trp Ile Arg Tyr His Arg Tyr His Gly Arg Ser Ala Ala Val Trp Ser

215 220 225

ctg ggg atc ctg ctg tat gat atg gtg tgt gga gat att cct ttc gag 1017

Leu Gly Ile Leu Leu Tyr Asp Met Val Cys Gly Asp Ile Pro Phe Glu

230 235 240

cat gac gaa gag atc atc agg ggc cag gtt ttc ttc agg cag agg gtc 1065

His Asp Glu Glu Ile Ile Arg Gly Gln Val Phe Phe Arg Gln Arg Val

245 250 255

tct tca gaa tgt cag cat ctc att aga tgg tgc ttg gcc ctg aga cca 1113

Ser Ser Glu Cys Gln His Leu Ile Arg Trp Cys Leu Ala Leu Arg Pro

260 265 270 275

tca gat agg cca acc ttc gaa gaa atc cag aac cat cca tgg atg caa 1161

Ser Asp Arg Pro Thr Phe Glu Glu Ile Gln Asn His Pro Trp Met Gln

280 285 290

gat gtt ctc ctg ccc cag gaa act gct gag atc cac ctc cac agc ctg 1209

Asp Val Leu Leu Pro Gln Glu Thr Ala Glu Ile His Leu His Ser Leu

295 300 305

tcg ccg ggg ccc agc aaa tag cagcctttct ggcaggtcct cccctctctt 1260

Ser Pro Gly Pro Ser Lys *

310

gtcagatgcc cgagggaggg gaagcttctg tctccagctt cccgagtacc agtgacacgt 1320

ctcgccaagc aggacagtgc ttgatacagg aacaacattt acaactcatt ccagatccca 1380

ggcccctgga ggctgcctcc caacagtgag gaagagtgac tctccagggg tcctaggcct 1440

caactcctcc catagatact ctcttcttct cataggtgtc cagcattgct ggactgctga 1500

aatatcccgg gggtgggggg tgggggtggg tcagaaccct gccatggaac tgtttccttc 1560

atcatgagtt ctgctgaatg ccgcgatggg tcaggtaggg gggaaacagg ttgggatggg 1620

ataggactag caccatttta agtccctgtc acctcttccg actctttctg agtgccttct 1680

gtggggactc cggctgtgct gggagaaata cttgaacttg cctcttttac ctgctgcttc 1740

tccaaaaatc tgcctgggtt ttgttcccta tttttctctc ctgtcctccc tcaccccctc 1800

cttcatatga aaggtgccat ggaagaggct acagggccaa acgctgagcc acctgccctt 1860

ttttctgcct cctttagtaa aactccgagt gaactggtct tcctttttgg tttttactta 1920

actgtttcaa agccaagacc tcacacacag aaaaaatgca caaacaatgc aatcaacaga 1980

aaagctgtaa atgtgtgtac agttggcatg gtagtataca aaaagattgt agtggatcta 2040

atttttcaga aattttgcct ttaagttatt ttacctgttt ttgtttcttg ttttgaaaga 2100

tgcgcattct aacctggagg tcaatgttat gtatttattt atttatttat ttggttccct 2160

tcctattcca agcttccata gctgctgccc tagttttctt tcctcctttc ctcctctgac 2220

ttggggacct tttgggggag ggctgcgacg cttgctctgt ttgtggggtg acgggactca 2280

ggcgggacag tgctgcagct ccctggcttc tgtggggccc ctcacctact tacccaggtg 2340

ggtcccggct ctgtgggtga tggggagggg cattgctgac tgtgtatata ggataattat 2400

gaaaagcagt tctggatggt gtgccttcca gatcctctct ggggctgtgt tttgagcagc 2460

aggtagcctg gctggtttta tctgagtgaa atactgtaca ggggaataaa agagatctta 2520

ttttt 2525

<210> SEQ ID NO 18

<211> LENGTH: 313

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 18

Met Leu Leu Ser Lys Ile Asn Ser Leu Ala His Leu Arg Ala Ala Pro

1 5 10 15

Cys Asn Asp Leu His Ala Thr Lys Leu Ala Pro Gly Lys Glu Lys Glu

20 25 30

Pro Leu Glu Ser Gln Tyr Gln Val Gly Pro Leu Leu Gly Ser Gly Gly

35 40 45

Phe Gly Ser Val Tyr Ser Gly Ile Arg Val Ser Asp Asn Leu Pro Val

50 55 60

Ala Ile Lys His Val Glu Lys Asp Arg Ile Ser Asp Trp Gly Glu Leu

65 70 75 80

Pro Asn Gly Thr Arg Val Pro Met Glu Val Val Leu Leu Lys Lys Val

85 90 95

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

100 105 110

Pro Asp Ser Phe Val Leu Ile Leu Glu Arg Pro Glu Pro Val Gln Asp

115 120 125

Leu Phe Asp Phe Ile Thr Glu Arg Gly Ala Leu Gln Glu Glu Leu Ala

130 135 140

Arg Ser Phe Phe Trp Gln Val Leu Glu Ala Val Arg His Cys His Asn

145 150 155 160

Cys Gly Val Leu His Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp

165 170 175

Leu Asn Arg Gly Glu Leu Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu

180 185 190

Leu Lys Asp Thr Val Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr Ser

195 200 205

Pro Pro Glu Trp Ile Arg Tyr His Arg Tyr His Gly Arg Ser Ala Ala

210 215 220

Val Trp Ser Leu Gly Ile Leu Leu Tyr Asp Met Val Cys Gly Asp Ile

225 230 235 240

Pro Phe Glu His Asp Glu Glu Ile Ile Arg Gly Gln Val Phe Phe Arg

245 250 255

Gln Arg Val Ser Ser Glu Cys Gln His Leu Ile Arg Trp Cys Leu Ala

260 265 270

Leu Arg Pro Ser Asp Arg Pro Thr Phe Glu Glu Ile Gln Asn His Pro

275 280 285

Trp Met Gln Asp Val Leu Leu Pro Gln Glu Thr Ala Glu Ile His Leu

290 295 300

His Ser Leu Ser Pro Gly Pro Ser Lys

305 310

<210> SEQ ID NO 19

<211> LENGTH: 3401

<212> TYPE: DNA

<213> ORGANISM: Saccharomyces cerevisiae

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (789)...(2795)

<400> SEQUENCE: 19

atcgatacaa ctccccagaa atatcactgt tgagagttca tgtcaagacg tagtatagta 60

ttgcaacagg aaaaaaaaat cttattgcgt agcactctgt atctatttta tatatctgtt 120

tctatatttg attaatctct tgttatcttg gtgatgatcg cacaagtatg tactcctgta 180

tctgcaagaa tatctgtttt aaacttttca aagcaaggaa accccgtctt atataggtta 240

tccgcaaagg tcacattttc ttgcaaatag aagaaaaagc acccacaagc acactaacac 300

agtgccagag caaaactata tcctttgcat ccgatctcaa acgctgttct tatcgcatct 360

gtcttcgtcc tttcatctgc atttaccttt tctttttcat cctctatttg ccttttcatt 420

agtggcaatt tttccagttt tttccctctg cgtcccgttg cacctgaaag gatctttcta 480

acgtgtgttg tctactagtg agcgatttcg tgagccatac acgttctata gaaaattgaa 540

taaactttac ttcaaaggga tctggacaca gagataactg cttacctgct tgccggaaga 600

aaagaattac taaaaaagaa gacaagggta gctgctattg tgggtacacg tttcacagaa 660

ctactttttc cttgtccttc tccagacatc aacgtcatac aactaaaact gataaagtac 720

ccgtttttcc gtacatttct atagatacat tattatatta agcagatcga gacgttaatt 780

tctcaaag atg gaa gac aag ttt gct aac ctc agt ctc cat gag aaa act 830

Met Glu Asp Lys Phe Ala Asn Leu Ser Leu His Glu Lys Thr

1 5 10

ggt aag tca tct atc caa tta aac gag caa aca ggc tca gat aat ggc 878

Gly Lys Ser Ser Ile Gln Leu Asn Glu Gln Thr Gly Ser Asp Asn Gly

15 20 25 30

tct gct gtc aag aga aca tct tcg acg tcc tcg cac tac aat aac atc 926

Ser Ala Val Lys Arg Thr Ser Ser Thr Ser Ser His Tyr Asn Asn Ile

35 40 45

aac gct gac ctt cat gct cgt gta aaa gct ttt caa gaa caa cgt gca 974

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

50 55 60

ttg aaa agg tct gcc agc gtg ggc agt aat caa agc gag caa gac aaa 1022

Leu Lys Arg Ser Ala Ser Val Gly Ser Asn Gln Ser Glu Gln Asp Lys

65 70 75

ggc agt tca caa tca cct aaa cat att cag cag att gtt aat aag cca 1070

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

80 85 90

ttg ccg cct ctt ccc gta gca gga agt tct aag gtt tca caa aga atg 1118

Leu Pro Pro Leu Pro Val Ala Gly Ser Ser Lys Val Ser Gln Arg Met

95 100 105 110

agt agc caa gtc gtg caa gcg tcc tcc aag agc act ctt aag aac gtt 1166

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

115 120 125

ctg gac aat caa gaa aca caa aac att acc gac gta aat att aac atc 1214

Leu Asp Asn Gln Glu Thr Gln Asn Ile Thr Asp Val Asn Ile Asn Ile

130 135 140

gat aca acc aaa att acc gcc aca aca att ggt gta aat act ggc cta 1262

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

145 150 155

cct gct act gac att acg ccg tca gtt tct aat act gca tca gca aca 1310

Pro Ala Thr Asp Ile Thr Pro Ser Val Ser Asn Thr Ala Ser Ala Thr

160 165 170

cat aag gcg caa ttg ctg aat cct aac aga agg gca cca aga agg ccg 1358

His Lys Ala Gln Leu Leu Asn Pro Asn Arg Arg Ala Pro Arg Arg Pro

175 180 185 190

ctt tct acc cag cac cct aca aga cca aat gtt gcc ccg cat aag gcc 1406

Leu Ser Thr Gln His Pro Thr Arg Pro Asn Val Ala Pro His Lys Ala

195 200 205

cct gct ata atc aac aca cca aaa caa agt tta agt gcc cgt cga ggg 1454

Pro Ala Ile Ile Asn Thr Pro Lys Gln Ser Leu Ser Ala Arg Arg Gly

210 215 220

ctc aaa tta cca cca gga gga atg tca tta aaa atg ccc act aaa aca 1502

Leu Lys Leu Pro Pro Gly Gly Met Ser Leu Lys Met Pro Thr Lys Thr

225 230 235

gct caa cag ccg cag cag ttt gcc cca agc cct tca aac aaa aaa cat 1550

Ala Gln Gln Pro Gln Gln Phe Ala Pro Ser Pro Ser Asn Lys Lys His

240 245 250

ata gaa acc tta tca aac agc aaa gtt gtt gaa ggg aaa aga tcg aat 1598

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

255 260 265 270

ccg ggt tct ttg ata aat ggt gtg caa agc aca tcc acc tca tca agt 1646

Pro Gly Ser Leu Ile Asn Gly Val Gln Ser Thr Ser Thr Ser Ser Ser

275 280 285

acc gaa ggc cca cat gac act gta ggc act aca ccc aga act gga aac 1694

Thr Glu Gly Pro His Asp Thr Val Gly Thr Thr Pro Arg Thr Gly Asn

290 295 300

agc aac aac tct tca aat tct ggt agt agt ggt ggt ggt ggt ctt ttc 1742

Ser Asn Asn Ser Ser Asn Ser Gly Ser Ser Gly Gly Gly Gly Leu Phe

305 310 315

gca aat ttc tcg aaa tac gtg gat atc aaa tcc ggc tct ttg aat ttt 1790

Ala Asn Phe Ser Lys Tyr Val Asp Ile Lys Ser Gly Ser Leu Asn Phe

320 325 330

gca ggc aaa cta tcg cta tcc tct aaa gga ata gat ttc agc aat ggt 1838

Ala Gly Lys Leu Ser Leu Ser Ser Lys Gly Ile Asp Phe Ser Asn Gly

335 340 345 350

tct agt tcg aga att aca ttg gac gaa cta gaa ttt ttg gat gaa ctg 1886

Ser Ser Ser Arg Ile Thr Leu Asp Glu Leu Glu Phe Leu Asp Glu Leu

355 360 365

ggt cat ggt aac tat ggt aac gtc tca aag gta ctg cat aag ccc aca 1934

Gly His Gly Asn Tyr Gly Asn Val Ser Lys Val Leu His Lys Pro Thr

370 375 380

aat gtt att atg gcg acg aag gaa gtc cgt ttg gag cta gat gag gct 1982

Asn Val Ile Met Ala Thr Lys Glu Val Arg Leu Glu Leu Asp Glu Ala

385 390 395

aaa ttt aga caa att tta atg gaa cta gaa gtt ttg cat aaa tgc aat 2030

Lys Phe Arg Gln Ile Leu Met Glu Leu Glu Val Leu His Lys Cys Asn

400 405 410

tct ccc tat att gtg gat ttt tat ggt gca ttc ttt att gag ggc gcc 2078

Ser Pro Tyr Ile Val Asp Phe Tyr Gly Ala Phe Phe Ile Glu Gly Ala

415 420 425 430

gtc tac atg tgt atg gaa tac atg gat ggt ggt tcc ttg gat aaa ata 2126

Val Tyr Met Cys Met Glu Tyr Met Asp Gly Gly Ser Leu Asp Lys Ile

435 440 445

tac gac gaa tca tct gaa atc ggc ggc att gat gaa cct cag cta gcg 2174

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

450 455 460

ttt att gcc aat gct gtc att cat gga cta aaa gaa ctc aaa gag cag 2222

Phe Ile Ala Asn Ala Val Ile His Gly Leu Lys Glu Leu Lys Glu Gln

465 470 475

cat aat atc ata cac aga gat gtc aaa cca aca aat att tta tgt tca 2270

His Asn Ile Ile His Arg Asp Val Lys Pro Thr Asn Ile Leu Cys Ser

480 485 490

gcc aac caa ggc acc gta aag ctg tgc gat ttc ggt gtt tct ggt aat 2318

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

495 500 505 510

ttg gtg gca tct tta gcg aag act aat att ggt tgt cag tca tac atg 2366

Leu Val Ala Ser Leu Ala Lys Thr Asn Ile Gly Cys Gln Ser Tyr Met

515 520 525

gca cct gaa cga atc aaa tcg ttg aat cca gat aga gcc acc tat acc 2414

Ala Pro Glu Arg Ile Lys Ser Leu Asn Pro Asp Arg Ala Thr Tyr Thr

530 535 540

gta cag tca gac atc tgg tct tta ggt tta agc att ctg gaa atg gca 2462

Val Gln Ser Asp Ile Trp Ser Leu Gly Leu Ser Ile Leu Glu Met Ala

545 550 555

cta ggt aga tat ccg tat cca cca gaa aca tac gac aac att ttc tct 2510

Leu Gly Arg Tyr Pro Tyr Pro Pro Glu Thr Tyr Asp Asn Ile Phe Ser

560 565 570

caa ttg agc gct att gtt gat ggg ccg cca ccg aga tta cct tca gat 2558

Gln Leu Ser Ala Ile Val Asp Gly Pro Pro Pro Arg Leu Pro Ser Asp

575 580 585 590

aaa ttc agt tct gac gca caa gat ttt gtt tct tta tgt cta caa aag 2606

Lys Phe Ser Ser Asp Ala Gln Asp Phe Val Ser Leu Cys Leu Gln Lys

595 600 605

att ccg gaa aga aga cct aca tac gca gct tta aca gag cat cct tgg 2654

Ile Pro Glu Arg Arg Pro Thr Tyr Ala Ala Leu Thr Glu His Pro Trp

610 615 620

tta gta aaa tac aga aac cag gat gtc cac atg agt gag tat atc act 2702

Leu Val Lys Tyr Arg Asn Gln Asp Val His Met Ser Glu Tyr Ile Thr

625 630 635

gaa cga tta gaa agg cgc aac aaa atc tta cgg gaa cgt ggt gag aat 2750

Glu Arg Leu Glu Arg Arg Asn Lys Ile Leu Arg Glu Arg Gly Glu Asn

640 645 650

ggt tta tct aaa aat gta ccg gca tta cat atg ggt ggt tta tag 2795

Gly Leu Ser Lys Asn Val Pro Ala Leu His Met Gly Gly Leu *

655 660 665

cgttaatatc caaataaaag caaacaggca cgtgaatata acaacaaaaa aaaagcagac 2855

gaaaagctac tgtggaaatg atgcggcgaa tacaaaaaaa ccttacatat acatatgttt 2915

attgtaataa acttgcatta tactcgttat agacatatat atatatatat attcatatat 2975

atatatcgtc tgacttcctt ttgtcgaacc taaaaaaggg cacgaattat gacagagtat 3035

tgaggggatg ttatttcaag caccggcaag tgaagcgatg tggacgtcaa tatattgtgt 3095

tattcgatta ttgctacggc catcgactcc tcgaaattat ttacgttcgg ggctgacaac 3155

gcaagaaaga aaaaatgctc tggaattgtc tgatggtttt tccgctcttt acggctcaag 3215

gctaggaaag aaaaaaaagt ccaaaatcat cgagaaaata aaaggtgttt tgaaagttca 3275

aatccacgtt attgagagta gatgtggagt ctggaccagg aactatacct gtatcttacc 3335

ctaacttcta aattttgcta ctttcacgga aaacagtaaa taattaccta tcaagataaa 3395

gagctc 3401

<210> SEQ ID NO 20

<211> LENGTH: 668

<212> TYPE: PRT

<213> ORGANISM: Saccharomyces cerevisiae

<400> SEQUENCE: 20

Met Glu Asp Lys Phe Ala Asn Leu Ser Leu His Glu Lys Thr Gly Lys

1 5 10 15

Ser Ser Ile Gln Leu Asn Glu Gln Thr Gly Ser Asp Asn Gly Ser Ala

20 25 30

Val Lys Arg Thr Ser Ser Thr Ser Ser His Tyr Asn Asn Ile Asn Ala

35 40 45

Asp Leu His Ala Arg Val Lys Ala Phe Gln Glu Gln Arg Ala Leu Lys

50 55 60

Arg Ser Ala Ser Val Gly Ser Asn Gln Ser Glu Gln Asp Lys Gly Ser

65 70 75 80

Ser Gln Ser Pro Lys His Ile Gln Gln Ile Val Asn Lys Pro Leu Pro

85 90 95

Pro Leu Pro Val Ala Gly Ser Ser Lys Val Ser Gln Arg Met Ser Ser

100 105 110

Gln Val Val Gln Ala Ser Ser Lys Ser Thr Leu Lys Asn Val Leu Asp

115 120 125

Asn Gln Glu Thr Gln Asn Ile Thr Asp Val Asn Ile Asn Ile Asp Thr

130 135 140

Thr Lys Ile Thr Ala Thr Thr Ile Gly Val Asn Thr Gly Leu Pro Ala

145 150 155 160

Thr Asp Ile Thr Pro Ser Val Ser Asn Thr Ala Ser Ala Thr His Lys

165 170 175

Ala Gln Leu Leu Asn Pro Asn Arg Arg Ala Pro Arg Arg Pro Leu Ser

180 185 190

Thr Gln His Pro Thr Arg Pro Asn Val Ala Pro His Lys Ala Pro Ala

195 200 205

Ile Ile Asn Thr Pro Lys Gln Ser Leu Ser Ala Arg Arg Gly Leu Lys

210 215 220

Leu Pro Pro Gly Gly Met Ser Leu Lys Met Pro Thr Lys Thr Ala Gln

225 230 235 240

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

245 250 255

Thr Leu Ser Asn Ser Lys Val Val Glu Gly Lys Arg Ser Asn Pro Gly

260 265 270

Ser Leu Ile Asn Gly Val Gln Ser Thr Ser Thr Ser Ser Ser Thr Glu

275 280 285

Gly Pro His Asp Thr Val Gly Thr Thr Pro Arg Thr Gly Asn Ser Asn

290 295 300

Asn Ser Ser Asn Ser Gly Ser Ser Gly Gly Gly Gly Leu Phe Ala Asn

305 310 315 320

Phe Ser Lys Tyr Val Asp Ile Lys Ser Gly Ser Leu Asn Phe Ala Gly

325 330 335

Lys Leu Ser Leu Ser Ser Lys Gly Ile Asp Phe Ser Asn Gly Ser Ser

340 345 350

Ser Arg Ile Thr Leu Asp Glu Leu Glu Phe Leu Asp Glu Leu Gly His

355 360 365

Gly Asn Tyr Gly Asn Val Ser Lys Val Leu His Lys Pro Thr Asn Val

370 375 380

Ile Met Ala Thr Lys Glu Val Arg Leu Glu Leu Asp Glu Ala Lys Phe

385 390 395 400

Arg Gln Ile Leu Met Glu Leu Glu Val Leu His Lys Cys Asn Ser Pro

405 410 415

Tyr Ile Val Asp Phe Tyr Gly Ala Phe Phe Ile Glu Gly Ala Val Tyr

420 425 430

Met Cys Met Glu Tyr Met Asp Gly Gly Ser Leu Asp Lys Ile Tyr Asp

435 440 445

Glu Ser Ser Glu Ile Gly Gly Ile Asp Glu Pro Gln Leu Ala Phe Ile

450 455 460

Ala Asn Ala Val Ile His Gly Leu Lys Glu Leu Lys Glu Gln His Asn

465 470 475 480

Ile Ile His Arg Asp Val Lys Pro Thr Asn Ile Leu Cys Ser Ala Asn

485 490 495

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

500 505 510

Ala Ser Leu Ala Lys Thr Asn Ile Gly Cys Gln Ser Tyr Met Ala Pro

515 520 525

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

530 535 540

Ser Asp Ile Trp Ser Leu Gly Leu Ser Ile Leu Glu Met Ala Leu Gly

545 550 555 560

Arg Tyr Pro Tyr Pro Pro Glu Thr Tyr Asp Asn Ile Phe Ser Gln Leu

565 570 575

Ser Ala Ile Val Asp Gly Pro Pro Pro Arg Leu Pro Ser Asp Lys Phe

580 585 590

Ser Ser Asp Ala Gln Asp Phe Val Ser Leu Cys Leu Gln Lys Ile Pro

595 600 605

Glu Arg Arg Pro Thr Tyr Ala Ala Leu Thr Glu His Pro Trp Leu Val

610 615 620

Lys Tyr Arg Asn Gln Asp Val His Met Ser Glu Tyr Ile Thr Glu Arg

625 630 635 640

Leu Glu Arg Arg Asn Lys Ile Leu Arg Glu Arg Gly Glu Asn Gly Leu

645 650 655

Ser Lys Asn Val Pro Ala Leu His Met Gly Gly Leu

660 665

<210> SEQ ID NO 21

<211> LENGTH: 1883

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (121)...(1203)

<400> SEQUENCE: 21

gtgagccacc gcccccagcc tggcctggca tttctttgag ttcaggaagt gtgacaagga 60

tttggacacc cagaaataag cgtgtcgaga agagcacaag cagaggatcc agcgctcggc 120

atg gcg gag cca gat ctg gag tgc gag cag atc cgt ctg aag tgt att 168

Met Ala Glu Pro Asp Leu Glu Cys Glu Gln Ile Arg Leu Lys Cys Ile

1 5 10 15

cgt aag gag ggc ttc ttc acg gtg cct ccg gaa cac agg ctg gga cga 216

Arg Lys Glu Gly Phe Phe Thr Val Pro Pro Glu His Arg Leu Gly Arg

20 25 30

tgc cgg agt gtg aag gag ttt gag aag ctg aac cgc att gga gag ggt 264

Cys Arg Ser Val Lys Glu Phe Glu Lys Leu Asn Arg Ile Gly Glu Gly

35 40 45

acc tac ggc att gtg tat cgg gcc cgg gac acc cag aca gat gag att 312

Thr Tyr Gly Ile Val Tyr Arg Ala Arg Asp Thr Gln Thr Asp Glu Ile

50 55 60

gtc gca ctg aag aag gtg cgg atg gac aag gag aag gat ggc atc ccc 360

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

65 70 75 80

atc agc agc ttg cgg gag atc acg ctg ctg ctc cgc ctg cgt cat ccg 408

Ile Ser Ser Leu Arg Glu Ile Thr Leu Leu Leu Arg Leu Arg His Pro

85 90 95

aac atc gtg gag ctg aag gag gtg gtt gtg ggg aac cac ctg gag agc 456

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

100 105 110

atc ttc ctg gtg atg ggt tac tgt gag cag gac ctg gcc agc ctc ctg 504

Ile Phe Leu Val Met Gly Tyr Cys Glu Gln Asp Leu Ala Ser Leu Leu

115 120 125

gag aat atg cca aca ccc ttc tcg gag gct cag gtc aag tgc atc gtg 552

Glu Asn Met Pro Thr Pro Phe Ser Glu Ala Gln Val Lys Cys Ile Val

130 135 140

ctg cag gtg ctc cgg ggc ctc cag tat ctg cac agg aac ttc att atc 600

Leu Gln Val Leu Arg Gly Leu Gln Tyr Leu His Arg Asn Phe Ile Ile

145 150 155 160

cac agg gac ctg aag gtt tcc aac ttg ctc atg acc gac aag ggt tgt 648

His Arg Asp Leu Lys Val Ser Asn Leu Leu Met Thr Asp Lys Gly Cys

165 170 175

gtg aag aca gcg gat ttc ggc ctg gcc cgg gcc tat ggt gtc cca gta 696

Val Lys Thr Ala Asp Phe Gly Leu Ala Arg Ala Tyr Gly Val Pro Val

180 185 190

aag cca atg acc ccc aag gtg gtc act ctc tgg tac cga gcc cct gaa 744

Lys Pro Met Thr Pro Lys Val Val Thr Leu Trp Tyr Arg Ala Pro Glu

195 200 205

ctg ctg ttg gga acc acc acg cag acc acc agc atc gac atg tgg gct 792

Leu Leu Leu Gly Thr Thr Thr Gln Thr Thr Ser Ile Asp Met Trp Ala

210 215 220

gtg ggc tgc ata ctg gcc gag ctg ctg gcg cac agg cct ctt ctc ccc 840

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

225 230 235 240

ggc act tcc gag atc cac cag atc gac ttg atc gtg cag ctg ctg ggc 888

Gly Thr Ser Glu Ile His Gln Ile Asp Leu Ile Val Gln Leu Leu Gly

245 250 255

acg ccc agt gag aac atc tgg ccg ggc ttt tcc aag ctg cca ctg gtc 936

Thr Pro Ser Glu Asn Ile Trp Pro Gly Phe Ser Lys Leu Pro Leu Val

260 265 270

ggc cag tac agc ctc cgg aag cag ccc tac aac aac ctg aag cac aag 984

Gly Gln Tyr Ser Leu Arg Lys Gln Pro Tyr Asn Asn Leu Lys His Lys

275 280 285

ttc cca tgg ctg tcg gag gcc ggg ctg cgc ctg ctg cac ttc ctg ttc 1032

Phe Pro Trp Leu Ser Glu Ala Gly Leu Arg Leu Leu His Phe Leu Phe

290 295 300

atg tac gac cct aag aaa agg gcg acg gcc ggg gac tgc ctg gag agc 1080

Met Tyr Asp Pro Lys Lys Arg Ala Thr Ala Gly Asp Cys Leu Glu Ser

305 310 315 320

tcc tat ttc aag gag aag ccc cta ccc tgt gag ccg gag ctc atg ccg 1128

Ser Tyr Phe Lys Glu Lys Pro Leu Pro Cys Glu Pro Glu Leu Met Pro

325 330 335

acc ttt ccc cac cac cgc aac aag cgg gcc gcc cca gcc acc tcc gag 1176

Thr Phe Pro His His Arg Asn Lys Arg Ala Ala Pro Ala Thr Ser Glu

340 345 350

ggc cag agc aag cgc tgt aaa ccc tga cggtgggcct ggcacacgcc 1223

Gly Gln Ser Lys Arg Cys Lys Pro *

355 360

tgtattccca caccaggtct tccgatcagt ggtgtctgtg aagggtgccg cgagccaggc 1283

tgaccaggcg cccgggatcc agctcatccc cttggctggg aacatcctcc actgacttcc 1343

tcccactgtc tgccctgaac ccactgctgc ccccagaaaa aggccgggtg acaccggggg 1403

ctcccagccc gtgcaccctg gaagggcagg tctggcggct ccatccgtgg ctgcaggggt 1463

ctcatgtggt cctcctcgct atgttggaaa tgtgcaacca ctgcttcttg ggaggagtgg 1523

tgggtgcagt ccccccgctg tctttgagtt gtggtggacc gctggcctgg gatgagaggg 1583

cccagaagac cttcgtatcc cctctcagtc gcccggggct gtcccgtgca tgggttggct 1643

gtggggaccc caggtgggcc tggcaggact ccagatgagg acaagaggga caaggtatgg 1703

ggtgggagcc acaattgagg ataccccgag ctaccaggag agccctgggc tggaggctga 1763

gctggatccc tgctccccac acggaggacc caacaggagg ccgtggctct gatgctgagc 1823

gaagctatag gctcttgttg gataaaagct tttttaacag aaaaaaaaaa aaaaaaaaaa 1883

<210> SEQ ID NO 22

<211> LENGTH: 360

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 22

Met Ala Glu Pro Asp Leu Glu Cys Glu Gln Ile Arg Leu Lys Cys Ile

1 5 10 15

Arg Lys Glu Gly Phe Phe Thr Val Pro Pro Glu His Arg Leu Gly Arg

20 25 30

Cys Arg Ser Val Lys Glu Phe Glu Lys Leu Asn Arg Ile Gly Glu Gly

35 40 45

Thr Tyr Gly Ile Val Tyr Arg Ala Arg Asp Thr Gln Thr Asp Glu Ile

50 55 60

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

65 70 75 80

Ile Ser Ser Leu Arg Glu Ile Thr Leu Leu Leu Arg Leu Arg His Pro

85 90 95

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

100 105 110

Ile Phe Leu Val Met Gly Tyr Cys Glu Gln Asp Leu Ala Ser Leu Leu

115 120 125

Glu Asn Met Pro Thr Pro Phe Ser Glu Ala Gln Val Lys Cys Ile Val

130 135 140

Leu Gln Val Leu Arg Gly Leu Gln Tyr Leu His Arg Asn Phe Ile Ile

145 150 155 160

His Arg Asp Leu Lys Val Ser Asn Leu Leu Met Thr Asp Lys Gly Cys

165 170 175

Val Lys Thr Ala Asp Phe Gly Leu Ala Arg Ala Tyr Gly Val Pro Val

180 185 190

Lys Pro Met Thr Pro Lys Val Val Thr Leu Trp Tyr Arg Ala Pro Glu

195 200 205

Leu Leu Leu Gly Thr Thr Thr Gln Thr Thr Ser Ile Asp Met Trp Ala

210 215 220

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

225 230 235 240

Gly Thr Ser Glu Ile His Gln Ile Asp Leu Ile Val Gln Leu Leu Gly

245 250 255

Thr Pro Ser Glu Asn Ile Trp Pro Gly Phe Ser Lys Leu Pro Leu Val

260 265 270

Gly Gln Tyr Ser Leu Arg Lys Gln Pro Tyr Asn Asn Leu Lys His Lys

275 280 285

Phe Pro Trp Leu Ser Glu Ala Gly Leu Arg Leu Leu His Phe Leu Phe

290 295 300

Met Tyr Asp Pro Lys Lys Arg Ala Thr Ala Gly Asp Cys Leu Glu Ser

305 310 315 320

Ser Tyr Phe Lys Glu Lys Pro Leu Pro Cys Glu Pro Glu Leu Met Pro

325 330 335

Thr Phe Pro His His Arg Asn Lys Arg Ala Ala Pro Ala Thr Ser Glu

340 345 350

Gly Gln Ser Lys Arg Cys Lys Pro

355 360

<210> SEQ ID NO 23

<211> LENGTH: 2299

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (142)...(1698)

<400> SEQUENCE: 23

ccggacttcc atgggcagca gcagcggcag ggaacggagg gcgaatagat ttcagagcct 60

gcacctgaag tacaattcga atcctgctcc agggagcgag ccactgtccg gatccagaaa 120

ctttggccac tgggaggaaa a atg gcc agt gat acc cca ggt ttc tac atg 171

Met Ala Ser Asp Thr Pro Gly Phe Tyr Met

1 5 10

gac aaa ctt aat aaa tac cgc cag atg cac gga gta gcc att acg tat 219

Asp Lys Leu Asn Lys Tyr Arg Gln Met His Gly Val Ala Ile Thr Tyr

15 20 25

aaa gaa ctt agt act tcg gga cct cca cat gac aga agg ttt aca ttt 267

Lys Glu Leu Ser Thr Ser Gly Pro Pro His Asp Arg Arg Phe Thr Phe

30 35 40

caa gtt tta ata gat gag aag gaa ttt gga gaa gcc aaa ggt aga tca 315

Gln Val Leu Ile Asp Glu Lys Glu Phe Gly Glu Ala Lys Gly Arg Ser

45 50 55

aag acg gag gca aga aac gct gca gcc aaa tta gct gtt gat ata ctt 363

Lys Thr Glu Ala Arg Asn Ala Ala Ala Lys Leu Ala Val Asp Ile Leu

60 65 70

gat aac gaa aac aag gtg gat tgt cac acg agt gca tgt gag caa ggc 411

Asp Asn Glu Asn Lys Val Asp Cys His Thr Ser Ala Cys Glu Gln Gly

75 80 85 90

ttg ttc gtt ggt aac tac ata ggc ctt gtc aat agc ttt gcc cag aag 459

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

95 100 105

aaa aag ctg tct gta aat tat gaa cag tgt gag ccc aac tct gag ttg 507

Lys Lys Leu Ser Val Asn Tyr Glu Gln Cys Glu Pro Asn Ser Glu Leu

110 115 120

cct caa aga ttt att tgt aaa tgc aaa att ggg cag aca atg tat ggt 555

Pro Gln Arg Phe Ile Cys Lys Cys Lys Ile Gly Gln Thr Met Tyr Gly

125 130 135

act ggt tca ggt gtc acc aaa cag gag gca aag cag ttg gct gcg aaa 603

Thr Gly Ser Gly Val Thr Lys Gln Glu Ala Lys Gln Leu Ala Ala Lys

140 145 150

gaa gcc tat cag aag ctg tta aag agc ccg ccg aaa act gcc gga aca 651

Glu Ala Tyr Gln Lys Leu Leu Lys Ser Pro Pro Lys Thr Ala Gly Thr

155 160 165 170

tcc tct agc gtt gtc aca tct aca ttc agt ggc ttt tcc agc agc tcg 699

Ser Ser Ser Val Val Thr Ser Thr Phe Ser Gly Phe Ser Ser Ser Ser

175 180 185

tct atg aca agt aat ggt gtt tcc cag tca gca cct gga agt ttt tcc 747

Ser Met Thr Ser Asn Gly Val Ser Gln Ser Ala Pro Gly Ser Phe Ser

190 195 200

tca gag aac gtg ttt acg aac ggt ctc gga gaa aat aaa agg aaa tca 795

Ser Glu Asn Val Phe Thr Asn Gly Leu Gly Glu Asn Lys Arg Lys Ser

205 210 215

gga gta aaa gta tcc cct gat gat gtg caa aga aat aaa tat acc ttg 843

Gly Val Lys Val Ser Pro Asp Asp Val Gln Arg Asn Lys Tyr Thr Leu

220 225 230

gac gcc agg ttt aac agc gat ttt gaa gac ata gaa gaa att ggc tta 891

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

235 240 245 250

ggt gga ttt ggt caa gtt ttc aaa gcg aaa cac aga att gat gga aag 939

Gly Gly Phe Gly Gln Val Phe Lys Ala Lys His Arg Ile Asp Gly Lys

255 260 265

aga tac gct att aag cgc gtt aaa tat aac acg gag aag gcg gag cac 987

Arg Tyr Ala Ile Lys Arg Val Lys Tyr Asn Thr Glu Lys Ala Glu His

270 275 280

gaa gta caa gcg ctg gca gaa ctc aat cac gtc aac att gtc caa tac 1035

Glu Val Gln Ala Leu Ala Glu Leu Asn His Val Asn Ile Val Gln Tyr

285 290 295

cat agt tgt tgg gag gga gtt gac tat gat cct gag cac agc atg agt 1083

His Ser Cys Trp Glu Gly Val Asp Tyr Asp Pro Glu His Ser Met Ser

300 305 310

gat aca agt cga tac aaa acc cgg tgc ctc ttt att caa atg gaa ttc 1131

Asp Thr Ser Arg Tyr Lys Thr Arg Cys Leu Phe Ile Gln Met Glu Phe

315 320 325 330

tgt gat aaa gga act ttg gag caa tgg atg aga aac aga aat cag agt 1179

Cys Asp Lys Gly Thr Leu Glu Gln Trp Met Arg Asn Arg Asn Gln Ser

335 340 345

aaa gtg gac aaa gct ttg att ttg gac tta tat gaa caa atc gtg acc 1227

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

350 355 360

gga gtg gag tat ata cac tcg aaa ggg tta att cac aga gat ctt aag 1275

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

365 370 375

cca ggt aat ata ttt tta gta gat gaa aga cac att aag atc gga gac 1323

Pro Gly Asn Ile Phe Leu Val Asp Glu Arg His Ile Lys Ile Gly Asp

380 385 390

ttt ggc ctt gca aca gcc ctg gaa aat gat gga aaa tcc cga aca agg 1371

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

395 400 405 410

aga aca gga act ctt caa tac atg agt cca gaa cag tta ttt tta aag 1419

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

415 420 425

cac tat gga aaa gaa gtg gac atc ttt gct ttg ggc ctt att cta gct 1467

His Tyr Gly Lys Glu Val Asp Ile Phe Ala Leu Gly Leu Ile Leu Ala

430 435 440

gaa ctt ctt cac acg tgc ttc acg gag tca gag aaa ata aag ttt ttc 1515

Glu Leu Leu His Thr Cys Phe Thr Glu Ser Glu Lys Ile Lys Phe Phe

445 450 455

gaa agt cta aga aaa ggc gac ttc tct aat gat ata ttc gac aac aaa 1563

Glu Ser Leu Arg Lys Gly Asp Phe Ser Asn Asp Ile Phe Asp Asn Lys

460 465 470

gaa aaa agc ctt cta aaa aaa cta ctc tca gag aaa ccc aag gac cga 1611

Glu Lys Ser Leu Leu Lys Lys Leu Leu Ser Glu Lys Pro Lys Asp Arg

475 480 485 490

cct gag aca tct gaa atc ctg aag acc ttg gct gaa tgg agg aac atc 1659

Pro Glu Thr Ser Glu Ile Leu Lys Thr Leu Ala Glu Trp Arg Asn Ile

495 500 505

tca gag aaa gaa aag aaa cac atg tta ggg cct ttc tga gaaaacattc 1708

Ser Glu Lys Glu Lys Lys His Met Leu Gly Pro Phe *

510 515

cttctgccgt ggttttcctt taacgatctg cagtctgagg ggagtatcag tgaatattat 1768

ccttcttttc ttaataccac tctcccagac aggttttggt tagggtgacc cacagacatt 1828

gtatttatta ggctatgaaa aagtatgccc atttcctcaa ttgttaattg ctgggcctgt 1888

ggctggctag ctagccaaat atgtaaatgc ttgtttctcg tctgcccaaa gagaaaggca 1948

ggctcctgtg tgggaagtca cagagccccc aaagccaact ggatgaggaa ggactctggc 2008

ttttggcata aaaaagagct ggtagtcaga gctggggcag aaggtcctgc agacagacag 2068

acagacagac agacagagac acaaagacat ggactagaat ggaggaggga gggaggaagg 2128

gagggaggga gagagagaga gagaaagaaa gagagagaga ggacatggag acaaaatggc 2188

ttaagttagc tgggctacct gagagactgt cccagaaaac aggccaacaa ccttccttat 2248

gctatataga tgtctcagtg tctttatcat taaacaccaa gcaggagtgc t 2299

<210> SEQ ID NO 24

<211> LENGTH: 518

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQUENCE: 24

Met Ala Ser Asp Thr Pro Gly Phe Tyr Met Asp Lys Leu Asn Lys Tyr

1 5 10 15

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

20 25 30

Gly Pro Pro His Asp Arg Arg Phe Thr Phe Gln Val Leu Ile Asp Glu

35 40 45

Lys Glu Phe Gly Glu Ala Lys Gly Arg Ser Lys Thr Glu Ala Arg Asn

50 55 60

Ala Ala Ala Lys Leu Ala Val Asp Ile Leu Asp Asn Glu Asn Lys Val

65 70 75 80

Asp Cys His Thr Ser Ala Cys Glu Gln Gly Leu Phe Val Gly Asn Tyr

85 90 95

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

100 105 110

Tyr Glu Gln Cys Glu Pro Asn Ser Glu Leu Pro Gln Arg Phe Ile Cys

115 120 125

Lys Cys Lys Ile Gly Gln Thr Met Tyr Gly Thr Gly Ser Gly Val Thr

130 135 140

Lys Gln Glu Ala Lys Gln Leu Ala Ala Lys Glu Ala Tyr Gln Lys Leu

145 150 155 160

Leu Lys Ser Pro Pro Lys Thr Ala Gly Thr Ser Ser Ser Val Val Thr

165 170 175

Ser Thr Phe Ser Gly Phe Ser Ser Ser Ser Ser Met Thr Ser Asn Gly

180 185 190

Val Ser Gln Ser Ala Pro Gly Ser Phe Ser Ser Glu Asn Val Phe Thr

195 200 205

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

210 215 220

Asp Asp Val Gln Arg Asn Lys Tyr Thr Leu Asp Ala Arg Phe Asn Ser

225 230 235 240

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

245 250 255

Phe Lys Ala Lys His Arg Ile Asp Gly Lys Arg Tyr Ala Ile Lys Arg

260 265 270

Val Lys Tyr Asn Thr Glu Lys Ala Glu His Glu Val Gln Ala Leu Ala

275 280 285

Glu Leu Asn His Val Asn Ile Val Gln Tyr His Ser Cys Trp Glu Gly

290 295 300

Val Asp Tyr Asp Pro Glu His Ser Met Ser Asp Thr Ser Arg Tyr Lys

305 310 315 320

Thr Arg Cys Leu Phe Ile Gln Met Glu Phe Cys Asp Lys Gly Thr Leu

325 330 335

Glu Gln Trp Met Arg Asn Arg Asn Gln Ser Lys Val Asp Lys Ala Leu

340 345 350

Ile Leu Asp Leu Tyr Glu Gln Ile Val Thr Gly Val Glu Tyr Ile His

355 360 365

Ser Lys Gly Leu Ile His Arg Asp Leu Lys Pro Gly Asn Ile Phe Leu

370 375 380

Val Asp Glu Arg His Ile Lys Ile Gly Asp Phe Gly Leu Ala Thr Ala

385 390 395 400

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

405 410 415

Tyr Met Ser Pro Glu Gln Leu Phe Leu Lys His Tyr Gly Lys Glu Val

420 425 430

Asp Ile Phe Ala Leu Gly Leu Ile Leu Ala Glu Leu Leu His Thr Cys

435 440 445

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

450 455 460

Asp Phe Ser Asn Asp Ile Phe Asp Asn Lys Glu Lys Ser Leu Leu Lys

465 470 475 480

Lys Leu Leu Ser Glu Lys Pro Lys Asp Arg Pro Glu Thr Ser Glu Ile

485 490 495

Leu Lys Thr Leu Ala Glu Trp Arg Asn Ile Ser Glu Lys Glu Lys Lys

500 505 510

His Met Leu Gly Pro Phe

515

<210> SEQ ID NO 25

<211> LENGTH: 2055

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (211)...(1632)

<400> SEQUENCE: 25

caggaagact ctgagtccga cgttggccta cccagtcgga aggcagagct gcaatctagt 60

taactacctc ctttccccta gatttccttt cattctgctc aagtcttcgc ctgtgtccga 120

tccctatcta ctttctctcc tcttgtagca agcctcagac tccaggcttg agctaggttt 180

tgtttttctc ctggtgagaa ttcgaagacc atg tct acg gaa ctc ttc tca tcc 234

Met Ser Thr Glu Leu Phe Ser Ser

1 5

aca aga gag gaa gga agc tct ggc tca gga ccc agt ttt agg tct aat 282

Thr Arg Glu Glu Gly Ser Ser Gly Ser Gly Pro Ser Phe Arg Ser Asn

10 15 20

caa agg aaa atg tta aac ctg ctc ctg gag aga gac act tcc ttt acc 330

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

25 30 35 40

gtc tgt cca gat gtc cct aga act cca gtg ggc aaa ttt ctt ggt gat 378

Val Cys Pro Asp Val Pro Arg Thr Pro Val Gly Lys Phe Leu Gly Asp

45 50 55

tct gca aac cta agc att ttg tct gga gga acc cca aaa tgt tgc ctc 426

Ser Ala Asn Leu Ser Ile Leu Ser Gly Gly Thr Pro Lys Cys Cys Leu

60 65 70

gat ctt tcg aat ctt agc agt ggg gag ata act gcc act cag ctt acc 474

Asp Leu Ser Asn Leu Ser Ser Gly Glu Ile Thr Ala Thr Gln Leu Thr

75 80 85

act tct gca gac ctt gat gaa act ggt cac ctg gat tct tca gga ctt 522

Thr Ser Ala Asp Leu Asp Glu Thr Gly His Leu Asp Ser Ser Gly Leu

90 95 100

cag gaa gtg cat tta gct ggg atg aat cat gac cag cac cta atg aaa 570

Gln Glu Val His Leu Ala Gly Met Asn His Asp Gln His Leu Met Lys

105 110 115 120

tgt agc cca gca cag ctt ctt tgt agc act ccg aat ggt ttg gac cgt 618

Cys Ser Pro Ala Gln Leu Leu Cys Ser Thr Pro Asn Gly Leu Asp Arg

125 130 135

ggc cat aga aag aga gat gca atg tgt agt tca tct gca aat aaa gaa 666

Gly His Arg Lys Arg Asp Ala Met Cys Ser Ser Ser Ala Asn Lys Glu

140 145 150

aat gac aat gga aac ttg gtg gac agt gaa atg aaa tat ttg ggc agt 714

Asn Asp Asn Gly Asn Leu Val Asp Ser Glu Met Lys Tyr Leu Gly Ser

155 160 165

ccc att act act gtt cca aaa ttg gat aaa aat cca aac cta gga gaa 762

Pro Ile Thr Thr Val Pro Lys Leu Asp Lys Asn Pro Asn Leu Gly Glu

170 175 180

gac cag gca gaa gag att tca gat gaa tta atg gag ttt tcc ctg aaa 810

Asp Gln Ala Glu Glu Ile Ser Asp Glu Leu Met Glu Phe Ser Leu Lys

185 190 195 200

gat caa gaa gca aag gtg agc aga agt ggc cta tat cgc tcc ccg tcg 858

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

205 210 215

atg cca gag aac ttg aac agg cca aga ctg aag cag gtg gaa aaa ttc 906

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

220 225 230

aag gac aac aca ata cca gat aaa gtt aaa aaa aag tat ttt tct ggc 954

Lys Asp Asn Thr Ile Pro Asp Lys Val Lys Lys Lys Tyr Phe Ser Gly

235 240 245

caa gga aag ctc agg aag ggc tta tgt tta aag aag aca gtc tct ctg 1002

Gln Gly Lys Leu Arg Lys Gly Leu Cys Leu Lys Lys Thr Val Ser Leu

250 255 260

tgt gac att act atc act cag atg ctg gag gaa gat tct aac cag ggg 1050

Cys Asp Ile Thr Ile Thr Gln Met Leu Glu Glu Asp Ser Asn Gln Gly

265 270 275 280

cac ctg att ggt gat ttt tcc aag gta tgt gcg ctg cca acc gtg tca 1098

His Leu Ile Gly Asp Phe Ser Lys Val Cys Ala Leu Pro Thr Val Ser

285 290 295

ggg aaa cac caa gat ctg aag tat gtc aac cca gaa aca gtg gct gcc 1146

Gly Lys His Gln Asp Leu Lys Tyr Val Asn Pro Glu Thr Val Ala Ala

300 305 310

tta ctg tcg ggg aag ttc cag ggt ctg att gag aag ttt tat gtc att 1194

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

315 320 325

gat tgt cgc tat cca tat gag tat ctg gga gga cac atc cag gga gcc 1242

Asp Cys Arg Tyr Pro Tyr Glu Tyr Leu Gly Gly His Ile Gln Gly Ala

330 335 340

tta aac tta tat agt cag gaa gaa ctg ttt aac ttc ttt ctg aag aag 1290

Leu Asn Leu Tyr Ser Gln Glu Glu Leu Phe Asn Phe Phe Leu Lys Lys

345 350 355 360

ccc atc gtc cct ttg gac acc cag aag aga ata atc atc gtg ttc cac 1338

Pro Ile Val Pro Leu Asp Thr Gln Lys Arg Ile Ile Ile Val Phe His

365 370 375

tgt gaa ttc tcc tca gag agg ggc ccc cga atg tgc cgc tgt ctg cgt 1386

Cys Glu Phe Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Cys Leu Arg

380 385 390

gaa gag gac agg tct ctg aac cag tat cct gca ttg tac tac cca gag 1434

Glu Glu Asp Arg Ser Leu Asn Gln Tyr Pro Ala Leu Tyr Tyr Pro Glu

395 400 405

cta tat atc ctt aaa ggc ggc tac aga gac ttc ttt cca gaa tat atg 1482

Leu Tyr Ile Leu Lys Gly Gly Tyr Arg Asp Phe Phe Pro Glu Tyr Met

410 415 420

gaa ctg tgt gaa cca cag agc tac tgc cct atg cat cat cag gac cac 1530

Glu Leu Cys Glu Pro Gln Ser Tyr Cys Pro Met His His Gln Asp His

425 430 435 440

aag act gag ttg ctg agg tgt cga agc cag agc aaa gtg cag gaa ggg 1578

Lys Thr Glu Leu Leu Arg Cys Arg Ser Gln Ser Lys Val Gln Glu Gly

445 450 455

gag cgg cag ctg cgg gag cag att gcc ctt ctg gtg aag gac atg agc 1626

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

460 465 470

cca tga taacattcca gccactggct gctaacaagt caccaaaaag acactgcaga 1682

Pro *

aaccctgagc agaaagaggc cttctggatg gccaaaccca agattattaa aagatgtctc 1742

tgcaaaccaa caggctacca acttgtatcc aggcctggga atggattagg tttcagcaga 1802

gctgaaagct ggtggcagag tcctggagct ggctctataa ggcagccttg agttgcatag 1862

agatttgtat tggttcaggg aactctggca ttccttttcc caactcctca tgtcttctca 1922

caagccagcc aactctttct ctctgggctt cgggctatgc aagagcgttg tctaccttct 1982

ttctttgtat tttccttctt tgtttccccc tctttctttt ttaaaaatgg aaaaataaac 2042

actacagaat gag 2055

<210> SEQ ID NO 26

<211> LENGTH: 473

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 26

Met Ser Thr Glu Leu Phe Ser Ser Thr Arg Glu Glu Gly Ser Ser Gly

1 5 10 15

Ser Gly Pro Ser Phe Arg Ser Asn Gln Arg Lys Met Leu Asn Leu Leu

20 25 30

Leu Glu Arg Asp Thr Ser Phe Thr Val Cys Pro Asp Val Pro Arg Thr

35 40 45

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

50 55 60

Gly Gly Thr Pro Lys Cys Cys Leu Asp Leu Ser Asn Leu Ser Ser Gly

65 70 75 80

Glu Ile Thr Ala Thr Gln Leu Thr Thr Ser Ala Asp Leu Asp Glu Thr

85 90 95

Gly His Leu Asp Ser Ser Gly Leu Gln Glu Val His Leu Ala Gly Met

100 105 110

Asn His Asp Gln His Leu Met Lys Cys Ser Pro Ala Gln Leu Leu Cys

115 120 125

Ser Thr Pro Asn Gly Leu Asp Arg Gly His Arg Lys Arg Asp Ala Met

130 135 140

Cys Ser Ser Ser Ala Asn Lys Glu Asn Asp Asn Gly Asn Leu Val Asp

145 150 155 160

Ser Glu Met Lys Tyr Leu Gly Ser Pro Ile Thr Thr Val Pro Lys Leu

165 170 175

Asp Lys Asn Pro Asn Leu Gly Glu Asp Gln Ala Glu Glu Ile Ser Asp

180 185 190

Glu Leu Met Glu Phe Ser Leu Lys Asp Gln Glu Ala Lys Val Ser Arg

195 200 205

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

210 215 220

Arg Leu Lys Gln Val Glu Lys Phe Lys Asp Asn Thr Ile Pro Asp Lys

225 230 235 240

Val Lys Lys Lys Tyr Phe Ser Gly Gln Gly Lys Leu Arg Lys Gly Leu

245 250 255

Cys Leu Lys Lys Thr Val Ser Leu Cys Asp Ile Thr Ile Thr Gln Met

260 265 270

Leu Glu Glu Asp Ser Asn Gln Gly His Leu Ile Gly Asp Phe Ser Lys

275 280 285

Val Cys Ala Leu Pro Thr Val Ser Gly Lys His Gln Asp Leu Lys Tyr

290 295 300

Val Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe Gln Gly

305 310 315 320

Leu Ile Glu Lys Phe Tyr Val Ile Asp Cys Arg Tyr Pro Tyr Glu Tyr

325 330 335

Leu Gly Gly His Ile Gln Gly Ala Leu Asn Leu Tyr Ser Gln Glu Glu

340 345 350

Leu Phe Asn Phe Phe Leu Lys Lys Pro Ile Val Pro Leu Asp Thr Gln

355 360 365

Lys Arg Ile Ile Ile Val Phe His Cys Glu Phe Ser Ser Glu Arg Gly

370 375 380

Pro Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu Asn Gln

385 390 395 400

Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr Ile Leu Lys Gly Gly Tyr

405 410 415

Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gln Ser Tyr

420 425 430

Cys Pro Met His His Gln Asp His Lys Thr Glu Leu Leu Arg Cys Arg

435 440 445

Ser Gln Ser Lys Val Gln Glu Gly Glu Arg Gln Leu Arg Glu Gln Ile

450 455 460

Ala Leu Leu Val Lys Asp Met Ser Pro

465 470

<210> SEQ ID NO 27

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 27

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

1 5 10 15

Arg

<210> SEQ ID NO 28

<211> LENGTH: 14

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 28

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

1 5 10

<210> SEQ ID NO 29

<211> LENGTH: 2295

<212> TYPE: DNA

<213> ORGANISM: Saccharomyces cerevisiae

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (1)...(2295)

<400> SEQUENCE: 29

atg tca aca aac tca ttc cat gat tat gtg gat tta aaa tcg aga act 48

Met Ser Thr Asn Ser Phe His Asp Tyr Val Asp Leu Lys Ser Arg Thr

1 5 10 15

aat aca cga cag ttt tca gat gac gaa gag ttc act acg cct cca aaa 96

Asn Thr Arg Gln Phe Ser Asp Asp Glu Glu Phe Thr Thr Pro Pro Lys

20 25 30

cta agc aat ttc gga tca gct tta ctt tcc cac aca gaa aaa act tca 144

Leu Ser Asn Phe Gly Ser Ala Leu Leu Ser His Thr Glu Lys Thr Ser

35 40 45

gca tca gag ata tta tca agt cat aat aat gac aag atc gca aat cga 192

Ala Ser Glu Ile Leu Ser Ser His Asn Asn Asp Lys Ile Ala Asn Arg

50 55 60

tta gaa gaa atg gac agg agt tca tca agg agt cac ccc cca ccg tca 240

Leu Glu Glu Met Asp Arg Ser Ser Ser Arg Ser His Pro Pro Pro Ser

65 70 75 80

atg ggc aat ttg aca tcc ggt cat act agt acc tca tcg cat tca acc 288

Met Gly Asn Leu Thr Ser Gly His Thr Ser Thr Ser Ser His Ser Thr

85 90 95

ttg ttc gga cga tat ctg aga aat aat cac cag act agc atg acg acg 336

Leu Phe Gly Arg Tyr Leu Arg Asn Asn His Gln Thr Ser Met Thr Thr

100 105 110

atg aac act agt gac ata gag ata aat gtt gga aat agt ctt gat aag 384

Met Asn Thr Ser Asp Ile Glu Ile Asn Val Gly Asn Ser Leu Asp Lys

115 120 125

agt ttt gaa agg ata agg aat ttg cga caa aat atg aaa gaa gat att 432

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

130 135 140

acg gca aag tat gct gaa agg aga agt aag aga ttt tta ata tcc aat 480

Thr Ala Lys Tyr Ala Glu Arg Arg Ser Lys Arg Phe Leu Ile Ser Asn

145 150 155 160

agg aca acg aag ctg ggt cct gca aag aga gcg atg act ttg aca aat 528

Arg Thr Thr Lys Leu Gly Pro Ala Lys Arg Ala Met Thr Leu Thr Asn

165 170 175

atc ttt gat gag gat gtg cct aac tct cca aac cag cca ata aat gca 576

Ile Phe Asp Glu Asp Val Pro Asn Ser Pro Asn Gln Pro Ile Asn Ala

180 185 190

agg gag aca gtg gaa tta cca ctt gag gat tct cac caa aca aac ttt 624

Arg Glu Thr Val Glu Leu Pro Leu Glu Asp Ser His Gln Thr Asn Phe

195 200 205

aaa gaa cga aga gag aat acg gat tat gat tca att gat ttt gga gat 672

Lys Glu Arg Arg Glu Asn Thr Asp Tyr Asp Ser Ile Asp Phe Gly Asp

210 215 220

ttg aat cct atc cag tat att aaa aaa cat aat ctt ccc aca agt gac 720

Leu Asn Pro Ile Gln Tyr Ile Lys Lys His Asn Leu Pro Thr Ser Asp

225 230 235 240

ctt cca cta ata tct caa atc tac ttt gat aaa caa aga gaa gaa aat 768

Leu Pro Leu Ile Ser Gln Ile Tyr Phe Asp Lys Gln Arg Glu Glu Asn

245 250 255

aga caa gca gca ctc cga aaa cat agt tcc aga gaa ttg ctt tat aaa 816

Arg Gln Ala Ala Leu Arg Lys His Ser Ser Arg Glu Leu Leu Tyr Lys

260 265 270

agt agg tct tct tcc tct tca ctt tct agt aac aac tta ttg gca aac 864

Ser Arg Ser Ser Ser Ser Ser Leu Ser Ser Asn Asn Leu Leu Ala Asn

275 280 285

aag gac aat tct ata aca tcc aat aat ggt tct caa ccc agg cga aaa 912

Lys Asp Asn Ser Ile Thr Ser Asn Asn Gly Ser Gln Pro Arg Arg Lys

290 295 300

gtt tct act gga tca tct tca tct aag tca tcg atc gaa ata aga aga 960

Val Ser Thr Gly Ser Ser Ser Ser Lys Ser Ser Ile Glu Ile Arg Arg

305 310 315 320

gct ctc aag gag aat att gat act agc aat aac agc aat ttc aac agc 1008

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

325 330 335

cca att cat aaa att tat aaa gga att tcc aga aat aaa gat tcc gac 1056

Pro Ile His Lys Ile Tyr Lys Gly Ile Ser Arg Asn Lys Asp Ser Asp

340 345 350

tcc gaa aaa aga gaa gta ctg cga aac ata agc ata aat gca aat cac 1104

Ser Glu Lys Arg Glu Val Leu Arg Asn Ile Ser Ile Asn Ala Asn His

355 360 365

gct gat aat ctc ctt caa caa gag aat aag aga cta aaa cga tca ttg 1152

Ala Asp Asn Leu Leu Gln Gln Glu Asn Lys Arg Leu Lys Arg Ser Leu

370 375 380

gat gat gca ata acg aat gag aat ata aac agt aaa aat cta gaa gta 1200

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

385 390 395 400

ttt tac cat cga cct gct ccc aaa cct cca gtc acc aag aaa gtt gaa 1248

Phe Tyr His Arg Pro Ala Pro Lys Pro Pro Val Thr Lys Lys Val Glu

405 410 415

att gtt gaa cct gca aag tcc gct tct tta tcg aat aat aga aat ata 1296

Ile Val Glu Pro Ala Lys Ser Ala Ser Leu Ser Asn Asn Arg Asn Ile

420 425 430

att aca gta aat gac tcc cag tac gaa aaa ata gaa ctt ttg ggt aga 1344

Ile Thr Val Asn Asp Ser Gln Tyr Glu Lys Ile Glu Leu Leu Gly Arg

435 440 445

ggt gga tcc tcc aga gtt tac aag gtg aaa gga tct ggc aat agg gta 1392

Gly Gly Ser Ser Arg Val Tyr Lys Val Lys Gly Ser Gly Asn Arg Val

450 455 460

tac gcg ctc aaa agg gtg tct ttt gac gct ttt gac gat tca agt att 1440

Tyr Ala Leu Lys Arg Val Ser Phe Asp Ala Phe Asp Asp Ser Ser Ile

465 470 475 480

gat gga ttc aaa gga gaa ata gaa ctt ctg gaa aaa ttg aaa gac caa 1488

Asp Gly Phe Lys Gly Glu Ile Glu Leu Leu Glu Lys Leu Lys Asp Gln

485 490 495

aag cgt gta atc caa cta cta gat tat gaa atg ggg gat ggt tta ttg 1536

Lys Arg Val Ile Gln Leu Leu Asp Tyr Glu Met Gly Asp Gly Leu Leu

500 505 510

tat ttg ata atg gaa tgt ggt gat cat gat ttg tca caa atc ctt aac 1584

Tyr Leu Ile Met Glu Cys Gly Asp His Asp Leu Ser Gln Ile Leu Asn

515 520 525

caa aga agc ggc atg cca ctg gat ttt aat ttt gtt aga ttc tat aca 1632

Gln Arg Ser Gly Met Pro Leu Asp Phe Asn Phe Val Arg Phe Tyr Thr

530 535 540

aag gaa atg ttg ctg tgc att aaa gta gtt cat gat gcg ggc ata gtt 1680

Lys Glu Met Leu Leu Cys Ile Lys Val Val His Asp Ala Gly Ile Val

545 550 555 560

cat tcg gat tta aaa cct gca aat ttt gtt tta gtg aaa ggt atc tta 1728

His Ser Asp Leu Lys Pro Ala Asn Phe Val Leu Val Lys Gly Ile Leu

565 570 575

aaa atc att gat ttt ggt ata gca aac gcg gta ccg gaa cat acg gtg 1776

Lys Ile Ile Asp Phe Gly Ile Ala Asn Ala Val Pro Glu His Thr Val

580 585 590

aat ata tat cgt gaa act caa att ggg act cca aat tat atg gca cca 1824

Asn Ile Tyr Arg Glu Thr Gln Ile Gly Thr Pro Asn Tyr Met Ala Pro

595 600 605

gaa gca cta gtt gct atg aat tac aca caa aat agt gag aac caa cat 1872

Glu Ala Leu Val Ala Met Asn Tyr Thr Gln Asn Ser Glu Asn Gln His

610 615 620

gag gga aac aag tgg aaa gtg ggg aga cca tct gat atg tgg tca tgc 1920

Glu Gly Asn Lys Trp Lys Val Gly Arg Pro Ser Asp Met Trp Ser Cys

625 630 635 640

ggt tgt att ata tat cag atg att tac ggg aaa ccc cca tat ggc agt 1968

Gly Cys Ile Ile Tyr Gln Met Ile Tyr Gly Lys Pro Pro Tyr Gly Ser

645 650 655

ttc caa ggc caa aat agg ctg ttg gct att atg aat cct gat gtg aaa 2016

Phe Gln Gly Gln Asn Arg Leu Leu Ala Ile Met Asn Pro Asp Val Lys

660 665 670

atc cca ttt cct gaa cat act agc aat aat gaa aag att cca aag tct 2064

Ile Pro Phe Pro Glu His Thr Ser Asn Asn Glu Lys Ile Pro Lys Ser

675 680 685

gcc att gaa tta atg aaa gca tgt ctg tac agg aac cca gac aaa aga 2112

Ala Ile Glu Leu Met Lys Ala Cys Leu Tyr Arg Asn Pro Asp Lys Arg

690 695 700

tgg act gtg gat aaa gtc ctg agt agc act ttc ctt caa cct ttt atg 2160

Trp Thr Val Asp Lys Val Leu Ser Ser Thr Phe Leu Gln Pro Phe Met

705 710 715 720

ata tcc gga tcg att atg gaa gac ctt att agg aat gcc gtt aga tat 2208

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

725 730 735

ggc tct gag aag cct cat ata tca caa gat gat ctc aat gat gtg gta 2256

Gly Ser Glu Lys Pro His Ile Ser Gln Asp Asp Leu Asn Asp Val Val

740 745 750

gac act gtt tta agg aaa ttt gca gat tac aaa att tag 2295

Asp Thr Val Leu Arg Lys Phe Ala Asp Tyr Lys Ile *

755 760

<210> SEQ ID NO 30

<211> LENGTH: 764

<212> TYPE: PRT

<213> ORGANISM: Saccharomyces cerevisiae

<400> SEQUENCE: 30

Met Ser Thr Asn Ser Phe His Asp Tyr Val Asp Leu Lys Ser Arg Thr

1 5 10 15

Asn Thr Arg Gln Phe Ser Asp Asp Glu Glu Phe Thr Thr Pro Pro Lys

20 25 30

Leu Ser Asn Phe Gly Ser Ala Leu Leu Ser His Thr Glu Lys Thr Ser

35 40 45

Ala Ser Glu Ile Leu Ser Ser His Asn Asn Asp Lys Ile Ala Asn Arg

50 55 60

Leu Glu Glu Met Asp Arg Ser Ser Ser Arg Ser His Pro Pro Pro Ser

65 70 75 80

Met Gly Asn Leu Thr Ser Gly His Thr Ser Thr Ser Ser His Ser Thr

85 90 95

Leu Phe Gly Arg Tyr Leu Arg Asn Asn His Gln Thr Ser Met Thr Thr

100 105 110

Met Asn Thr Ser Asp Ile Glu Ile Asn Val Gly Asn Ser Leu Asp Lys

115 120 125

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

130 135 140

Thr Ala Lys Tyr Ala Glu Arg Arg Ser Lys Arg Phe Leu Ile Ser Asn

145 150 155 160

Arg Thr Thr Lys Leu Gly Pro Ala Lys Arg Ala Met Thr Leu Thr Asn

165 170 175

Ile Phe Asp Glu Asp Val Pro Asn Ser Pro Asn Gln Pro Ile Asn Ala

180 185 190

Arg Glu Thr Val Glu Leu Pro Leu Glu Asp Ser His Gln Thr Asn Phe

195 200 205

Lys Glu Arg Arg Glu Asn Thr Asp Tyr Asp Ser Ile Asp Phe Gly Asp

210 215 220

Leu Asn Pro Ile Gln Tyr Ile Lys Lys His Asn Leu Pro Thr Ser Asp

225 230 235 240

Leu Pro Leu Ile Ser Gln Ile Tyr Phe Asp Lys Gln Arg Glu Glu Asn

245 250 255

Arg Gln Ala Ala Leu Arg Lys His Ser Ser Arg Glu Leu Leu Tyr Lys

260 265 270

Ser Arg Ser Ser Ser Ser Ser Leu Ser Ser Asn Asn Leu Leu Ala Asn

275 280 285

Lys Asp Asn Ser Ile Thr Ser Asn Asn Gly Ser Gln Pro Arg Arg Lys

290 295 300

Val Ser Thr Gly Ser Ser Ser Ser Lys Ser Ser Ile Glu Ile Arg Arg

305 310 315 320

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

325 330 335

Pro Ile His Lys Ile Tyr Lys Gly Ile Ser Arg Asn Lys Asp Ser Asp

340 345 350

Ser Glu Lys Arg Glu Val Leu Arg Asn Ile Ser Ile Asn Ala Asn His

355 360 365

Ala Asp Asn Leu Leu Gln Gln Glu Asn Lys Arg Leu Lys Arg Ser Leu

370 375 380

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

385 390 395 400

Phe Tyr His Arg Pro Ala Pro Lys Pro Pro Val Thr Lys Lys Val Glu

405 410 415

Ile Val Glu Pro Ala Lys Ser Ala Ser Leu Ser Asn Asn Arg Asn Ile

420 425 430

Ile Thr Val Asn Asp Ser Gln Tyr Glu Lys Ile Glu Leu Leu Gly Arg

435 440 445

Gly Gly Ser Ser Arg Val Tyr Lys Val Lys Gly Ser Gly Asn Arg Val

450 455 460

Tyr Ala Leu Lys Arg Val Ser Phe Asp Ala Phe Asp Asp Ser Ser Ile

465 470 475 480

Asp Gly Phe Lys Gly Glu Ile Glu Leu Leu Glu Lys Leu Lys Asp Gln

485 490 495

Lys Arg Val Ile Gln Leu Leu Asp Tyr Glu Met Gly Asp Gly Leu Leu

500 505 510

Tyr Leu Ile Met Glu Cys Gly Asp His Asp Leu Ser Gln Ile Leu Asn

515 520 525

Gln Arg Ser Gly Met Pro Leu Asp Phe Asn Phe Val Arg Phe Tyr Thr

530 535 540

Lys Glu Met Leu Leu Cys Ile Lys Val Val His Asp Ala Gly Ile Val

545 550 555 560

His Ser Asp Leu Lys Pro Ala Asn Phe Val Leu Val Lys Gly Ile Leu

565 570 575

Lys Ile Ile Asp Phe Gly Ile Ala Asn Ala Val Pro Glu His Thr Val

580 585 590

Asn Ile Tyr Arg Glu Thr Gln Ile Gly Thr Pro Asn Tyr Met Ala Pro

595 600 605

Glu Ala Leu Val Ala Met Asn Tyr Thr Gln Asn Ser Glu Asn Gln His

610 615 620

Glu Gly Asn Lys Trp Lys Val Gly Arg Pro Ser Asp Met Trp Ser Cys

625 630 635 640

Gly Cys Ile Ile Tyr Gln Met Ile Tyr Gly Lys Pro Pro Tyr Gly Ser

645 650 655

Phe Gln Gly Gln Asn Arg Leu Leu Ala Ile Met Asn Pro Asp Val Lys

660 665 670

Ile Pro Phe Pro Glu His Thr Ser Asn Asn Glu Lys Ile Pro Lys Ser

675 680 685

Ala Ile Glu Leu Met Lys Ala Cys Leu Tyr Arg Asn Pro Asp Lys Arg

690 695 700

Trp Thr Val Asp Lys Val Leu Ser Ser Thr Phe Leu Gln Pro Phe Met

705 710 715 720

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

725 730 735

Gly Ser Glu Lys Pro His Ile Ser Gln Asp Asp Leu Asn Asp Val Val

740 745 750

Asp Thr Val Leu Arg Lys Phe Ala Asp Tyr Lys Ile

755 760

<210> SEQ ID NO 31

<211> LENGTH: 2079

<212> TYPE: DNA

<213> ORGANISM: Schizosaccharomyces pombe

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (30)...(2066)

<400> SEQUENCE: 31

ggaatgctcg ttttttagta actgtgttt atg tct aag cgc aat cct cct gtg 53

Met Ser Lys Arg Asn Pro Pro Val

1 5

act aat atc gcg gac ttg gtg tca gat tct tcc tta gat gaa gac tcg 101

Thr Asn Ile Ala Asp Leu Val Ser Asp Ser Ser Leu Asp Glu Asp Ser

10 15 20

ctt tct ttt ctc gaa gag ctt cag gat cca gaa tta tac ttc aaa aac 149

Leu Ser Phe Leu Glu Glu Leu Gln Asp Pro Glu Leu Tyr Phe Lys Asn

25 30 35 40

gac act ttc tct tcc aag agt agc cat tct gat ggc acc gtt act ggg 197

Asp Thr Phe Ser Ser Lys Ser Ser His Ser Asp Gly Thr Val Thr Gly

45 50 55

gat acg ttg cgt agg cag tca agc ggt gca act gct tta gag aga ttg 245

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

60 65 70

gtc tca cat cct cgt act aaa aat ttt gat ttg caa gga aat gga gga 293

Val Ser His Pro Arg Thr Lys Asn Phe Asp Leu Gln Gly Asn Gly Gly

75 80 85

caa aat tct gct ttg aag gaa gtg aat act cca gca tat cag tca atg 341

Gln Asn Ser Ala Leu Lys Glu Val Asn Thr Pro Ala Tyr Gln Ser Met

90 95 100

cac cat ttc gag cat tta ata aca ccc ttg ccc tct act aat gcg tct 389

His His Phe Glu His Leu Ile Thr Pro Leu Pro Ser Thr Asn Ala Ser

105 110 115 120

cac agt gaa gtt tca ctc agt gca gga gtg aat gat ctc aat tct aat 437

His Ser Glu Val Ser Leu Ser Ala Gly Val Asn Asp Leu Asn Ser Asn

125 130 135

tcg gag cat gat ttg tta cct aaa agt gta aac aaa acc ccc ggt tct 485

Ser Glu His Asp Leu Leu Pro Lys Ser Val Asn Lys Thr Pro Gly Ser

140 145 150

tta tca att tca aga cga cga aga atc ggc aga att gga tta ggc cct 533

Leu Ser Ile Ser Arg Arg Arg Arg Ile Gly Arg Ile Gly Leu Gly Pro

155 160 165

cca aag cgt gct gag tac acg ttg acg gat ccc tcg aag act tcc gat 581

Pro Lys Arg Ala Glu Tyr Thr Leu Thr Asp Pro Ser Lys Thr Ser Asp

170 175 180

acc aaa aac tct aca gaa gca gat gag gat att gaa atg aaa tct cga 629

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

185 190 195 200

gaa gta tca cca gct tcc aac tct gtt gct gca aca act tta aaa cct 677

Glu Val Ser Pro Ala Ser Asn Ser Val Ala Ala Thr Thr Leu Lys Pro

205 210 215

ctg cag ctg cat aac act cct ttg caa aca tcc cag gag cat ccc aaa 725

Leu Gln Leu His Asn Thr Pro Leu Gln Thr Ser Gln Glu His Pro Lys

220 225 230

cct tct ttt cat cct tct cag ttt gag agc tct ttt tct cct agg gtg 773

Pro Ser Phe His Pro Ser Gln Phe Glu Ser Ser Phe Ser Pro Arg Val

235 240 245

cag ttt gat cac gat gtt gaa aga aga gct agt gaa ctt cat tct cgt 821

Gln Phe Asp His Asp Val Glu Arg Arg Ala Ser Glu Leu His Ser Arg

250 255 260

cca gtc acc gtt ttc caa gag cct cag cgt tct gct tct caa cca tat 869

Pro Val Thr Val Phe Gln Glu Pro Gln Arg Ser Ala Ser Gln Pro Tyr

265 270 275 280

gaa tct cat gct ctt tct cca aag gtg gct ccg tta ttt gat aac agt 917

Glu Ser His Ala Leu Ser Pro Lys Val Ala Pro Leu Phe Asp Asn Ser

285 290 295

caa gct act ccc ata ccc aag cgt cag cag gac gtt gtt act gtt gcc 965

Gln Ala Thr Pro Ile Pro Lys Arg Gln Gln Asp Val Val Thr Val Ala

300 305 310

aat cta caa ttt atc aaa tta gga gtt gtt gga aag ggt gga agt agt 1013

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

315 320 325

atg gta tat cgc ata ttt tcc ccc gat aac agt cgt tta tac gct ttg 1061

Met Val Tyr Arg Ile Phe Ser Pro Asp Asn Ser Arg Leu Tyr Ala Leu

330 335 340

aaa gag gtg aac ttt att aat gca gac caa act act ata caa gga tac 1109

Lys Glu Val Asn Phe Ile Asn Ala Asp Gln Thr Thr Ile Gln Gly Tyr

345 350 355 360

aag aac gaa att gca tta tta aga aag ctt tca ggc aat gat cgc ata 1157

Lys Asn Glu Ile Ala Leu Leu Arg Lys Leu Ser Gly Asn Asp Arg Ile

365 370 375

att aaa tta tat gct gcc gaa gtt aat gat act tta ggg caa ctc aat 1205

Ile Lys Leu Tyr Ala Ala Glu Val Asn Asp Thr Leu Gly Gln Leu Asn

380 385 390

atg gtg atg gaa tgc gga gaa acg gat tta gca aac ctt tta atg aaa 1253

Met Val Met Glu Cys Gly Glu Thr Asp Leu Ala Asn Leu Leu Met Lys

395 400 405

aac atg aag aaa ccc att aat ctt aat ttc atc aga atg tat tgg gag 1301

Asn Met Lys Lys Pro Ile Asn Leu Asn Phe Ile Arg Met Tyr Trp Glu

410 415 420

caa atg cta gag gcg gtc cag gta gtt cat gat caa aat ata gtg cat 1349

Gln Met Leu Glu Ala Val Gln Val Val His Asp Gln Asn Ile Val His

425 430 435 440

tcg gat ttg aag ccg gcc aat ttc ctg ctt gta gaa ggg aat ttg aag 1397

Ser Asp Leu Lys Pro Ala Asn Phe Leu Leu Val Glu Gly Asn Leu Lys

445 450 455

ctg att gat ttt ggc att gcc aaa gca att ggt aat gac acc act aat 1445

Leu Ile Asp Phe Gly Ile Ala Lys Ala Ile Gly Asn Asp Thr Thr Asn

460 465 470

atc cat cgt gat tcc cac atc ggt act att aat tat atg gca cct gaa 1493

Ile His Arg Asp Ser His Ile Gly Thr Ile Asn Tyr Met Ala Pro Glu

475 480 485

gct ttg aca gac atg aat gct cac aca aac tct ggc gtg aaa ctc gta 1541

Ala Leu Thr Asp Met Asn Ala His Thr Asn Ser Gly Val Lys Leu Val

490 495 500

aag ttg ggc agg ccc agc gac gtg tgg agt ttg gga tgt ata tta tat 1589

Lys Leu Gly Arg Pro Ser Asp Val Trp Ser Leu Gly Cys Ile Leu Tyr

505 510 515 520

cag atg gtg tat ggg agg gcc ccg ttt gct cat cta aaa atg atc caa 1637

Gln Met Val Tyr Gly Arg Ala Pro Phe Ala His Leu Lys Met Ile Gln

525 530 535

gct ata gca gct atc cct aat gaa caa tat cac att cat ttc ccc gaa 1685

Ala Ile Ala Ala Ile Pro Asn Glu Gln Tyr His Ile His Phe Pro Glu

540 545 550

gtt gcc tta cct gct aat gct gtc cag gag aaa gag gga tcg ttg cca 1733

Val Ala Leu Pro Ala Asn Ala Val Gln Glu Lys Glu Gly Ser Leu Pro

555 560 565

ggt gta act gtc ggg cct gat cta atg gat gtt atg aaa aga tgc ctg 1781

Gly Val Thr Val Gly Pro Asp Leu Met Asp Val Met Lys Arg Cys Leu

570 575 580

gaa agg gat caa cgg aag aga ctt aca ata ccg gaa ttg ctg gtt cat 1829

Glu Arg Asp Gln Arg Lys Arg Leu Thr Ile Pro Glu Leu Leu Val His

585 590 595 600

ccc ttt tta aac cct ttg cca tcc tat ttg aca cct ttg gcc aaa aag 1877

Pro Phe Leu Asn Pro Leu Pro Ser Tyr Leu Thr Pro Leu Ala Lys Lys

605 610 615

ccg tta cct gtt tct ggg cac acc aat aat gct cat cca ctt aga ctc 1925

Pro Leu Pro Val Ser Gly His Thr Asn Asn Ala His Pro Leu Arg Leu

620 625 630

agc aca gaa atc tca gct tct caa tta tca atg att ata gaa agg tcg 1973

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

635 640 645

gtg gag ttg agt aag cac aag cga tta aat aag gaa ctt att gat agc 2021

Val Glu Leu Ser Lys His Lys Arg Leu Asn Lys Glu Leu Ile Asp Ser

650 655 660

atg gct tat gat tgc gtt agc aat tta cga aaa atg cca gaa tag 2066

Met Ala Tyr Asp Cys Val Ser Asn Leu Arg Lys Met Pro Glu *

665 670 675

aggcactaaa ttt 2079

<210> SEQ ID NO 32

<211> LENGTH: 678

<212> TYPE: PRT

<213> ORGANISM: Schizosaccharomyces pombe

<400> SEQUENCE: 32

Met Ser Lys Arg Asn Pro Pro Val Thr Asn Ile Ala Asp Leu Val Ser

1 5 10 15

Asp Ser Ser Leu Asp Glu Asp Ser Leu Ser Phe Leu Glu Glu Leu Gln

20 25 30

Asp Pro Glu Leu Tyr Phe Lys Asn Asp Thr Phe Ser Ser Lys Ser Ser

35 40 45

His Ser Asp Gly Thr Val Thr Gly Asp Thr Leu Arg Arg Gln Ser Ser

50 55 60

Gly Ala Thr Ala Leu Glu Arg Leu Val Ser His Pro Arg Thr Lys Asn

65 70 75 80

Phe Asp Leu Gln Gly Asn Gly Gly Gln Asn Ser Ala Leu Lys Glu Val

85 90 95

Asn Thr Pro Ala Tyr Gln Ser Met His His Phe Glu His Leu Ile Thr

100 105 110

Pro Leu Pro Ser Thr Asn Ala Ser His Ser Glu Val Ser Leu Ser Ala

115 120 125

Gly Val Asn Asp Leu Asn Ser Asn Ser Glu His Asp Leu Leu Pro Lys

130 135 140

Ser Val Asn Lys Thr Pro Gly Ser Leu Ser Ile Ser Arg Arg Arg Arg

145 150 155 160

Ile Gly Arg Ile Gly Leu Gly Pro Pro Lys Arg Ala Glu Tyr Thr Leu

165 170 175

Thr Asp Pro Ser Lys Thr Ser Asp Thr Lys Asn Ser Thr Glu Ala Asp

180 185 190

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

195 200 205

Val Ala Ala Thr Thr Leu Lys Pro Leu Gln Leu His Asn Thr Pro Leu

210 215 220

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

225 230 235 240

Glu Ser Ser Phe Ser Pro Arg Val Gln Phe Asp His Asp Val Glu Arg

245 250 255

Arg Ala Ser Glu Leu His Ser Arg Pro Val Thr Val Phe Gln Glu Pro

260 265 270

Gln Arg Ser Ala Ser Gln Pro Tyr Glu Ser His Ala Leu Ser Pro Lys

275 280 285

Val Ala Pro Leu Phe Asp Asn Ser Gln Ala Thr Pro Ile Pro Lys Arg

290 295 300

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

305 310 315 320

Val Val Gly Lys Gly Gly Ser Ser Met Val Tyr Arg Ile Phe Ser Pro

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Asn Asp Thr Leu Gly Gln Leu Asn Met Val Met Glu Cys Gly Glu Thr

385 390 395 400

Asp Leu Ala Asn Leu Leu Met Lys Asn Met Lys Lys Pro Ile Asn Leu

405 410 415

Asn Phe Ile Arg Met Tyr Trp Glu Gln Met Leu Glu Ala Val Gln Val

420 425 430

Val His Asp Gln Asn Ile Val His Ser Asp Leu Lys Pro Ala Asn Phe

435 440 445

Leu Leu Val Glu Gly Asn Leu Lys Leu Ile Asp Phe Gly Ile Ala Lys

450 455 460

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

465 470 475 480

Thr Ile Asn Tyr Met Ala Pro Glu Ala Leu Thr Asp Met Asn Ala His

485 490 495

Thr Asn Ser Gly Val Lys Leu Val Lys Leu Gly Arg Pro Ser Asp Val

500 505 510

Trp Ser Leu Gly Cys Ile Leu Tyr Gln Met Val Tyr Gly Arg Ala Pro

515 520 525

Phe Ala His Leu Lys Met Ile Gln Ala Ile Ala Ala Ile Pro Asn Glu

530 535 540

Gln Tyr His Ile His Phe Pro Glu Val Ala Leu Pro Ala Asn Ala Val

545 550 555 560

Gln Glu Lys Glu Gly Ser Leu Pro Gly Val Thr Val Gly Pro Asp Leu

565 570 575

Met Asp Val Met Lys Arg Cys Leu Glu Arg Asp Gln Arg Lys Arg Leu

580 585 590

Thr Ile Pro Glu Leu Leu Val His Pro Phe Leu Asn Pro Leu Pro Ser

595 600 605

Tyr Leu Thr Pro Leu Ala Lys Lys Pro Leu Pro Val Ser Gly His Thr

610 615 620

Asn Asn Ala His Pro Leu Arg Leu Ser Thr Glu Ile Ser Ala Ser Gln

625 630 635 640

Leu Ser Met Ile Ile Glu Arg Ser Val Glu Leu Ser Lys His Lys Arg

645 650 655

Leu Asn Lys Glu Leu Ile Asp Ser Met Ala Tyr Asp Cys Val Ser Asn

660 665 670

Leu Arg Lys Met Pro Glu

675

<210> SEQ ID NO 33

<211> LENGTH: 2263

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (379)...(1491)

<400> SEQUENCE: 33

gatatcacag caacattgaa atgctaaaaa gtttttaaac actctcaatt tctaattcac 60

catgtcacag actggtgaaa aaaaaaaaaa aagcggccgc ttccccccgg ccgggccccc 120

gccgccccgc ggtccccaga gcgccaggcc cccgggggga gggagggagg gcgccgggcc 180

ggtgggagcc agcggcgcgc ggtgggaccc acggagcccc gcgacccgcc gagcctggag 240

ccgggccggc tcggggaagc cggctccagc ccggagcgaa cttcgcagcc cgtcgggggg 300

cggcggggag ggggcccgga gccggaggag ggggcggccg cgggcacccc cgcctgtgcc 360

ccggcgtccc cgggcacc atg ctg tcc aac tcc cag ggc cag agc ccg ccg 411

Met Leu Ser Asn Ser Gln Gly Gln Ser Pro Pro

1 5 10

gtg ccg ttc ccc gcc ccg gcc ccg ccg ccg cag ccc ccc acc cct gcc 459

Val Pro Phe Pro Ala Pro Ala Pro Pro Pro Gln Pro Pro Thr Pro Ala

15 20 25

ctg ccg cac ccc ccg gcg cag ccg ccg ccg ccg ccc ccg cag cag ttc 507

Leu Pro His Pro Pro Ala Gln Pro Pro Pro Pro Pro Pro Gln Gln Phe

30 35 40

ccg cag ttc cac gtc aag tcc ggc ctg cag atc aag aag aac gcc atc 555

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

45 50 55

atc gat gac tac aag gtc acc agc cag gtc ctg ggg ctg ggc atc aac 603

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

60 65 70 75

ggc aaa gtt ttg cag atc ttc aac aag agg acc cag gag aaa ttc gcc 651

Gly Lys Val Leu Gln Ile Phe Asn Lys Arg Thr Gln Glu Lys Phe Ala

80 85 90

ctc aaa atg ctt cag gac tgc ccc aag gcc cgc agg gag gtg gag ctg 699

Leu Lys Met Leu Gln Asp Cys Pro Lys Ala Arg Arg Glu Val Glu Leu

95 100 105

cac tgg cgg gcc tcc cag tgc ccg cac atc gta cgg atc gtg gat gtg 747

His Trp Arg Ala Ser Gln Cys Pro His Ile Val Arg Ile Val Asp Val

110 115 120

tac gag aat ctg tac gca ggg agg aag tgc ctg ctg att gtc atg gaa 795

Tyr Glu Asn Leu Tyr Ala Gly Arg Lys Cys Leu Leu Ile Val Met Glu

125 130 135

tgt ttg gac ggt gga gaa ctc ttt agc cga atc cag gat cga gga gac 843

Cys Leu Asp Gly Gly Glu Leu Phe Ser Arg Ile Gln Asp Arg Gly Asp

140 145 150 155

cag gca ttc aca gaa aga gaa gca tcc gaa atc atg aag agc atc ggt 891

Gln Ala Phe Thr Glu Arg Glu Ala Ser Glu Ile Met Lys Ser Ile Gly

160 165 170

gag gcc atc cag tat ctg cat tca atc aac att gcc cat cgg gat gtc 939

Glu Ala Ile Gln Tyr Leu His Ser Ile Asn Ile Ala His Arg Asp Val

175 180 185

aag cct gag aat ctc tta tac acc tcc aaa agg ccc aac gcc atc ctg 987

Lys Pro Glu Asn Leu Leu Tyr Thr Ser Lys Arg Pro Asn Ala Ile Leu

190 195 200

aaa ctc act gac ttt ggc ttt gcc aag gaa acc acc agc cac aac tct 1035

Lys Leu Thr Asp Phe Gly Phe Ala Lys Glu Thr Thr Ser His Asn Ser

205 210 215

ttg acc act cct tgt tat aca ccg tac tat gtg gct cca gaa gtg ctg 1083

Leu Thr Thr Pro Cys Tyr Thr Pro Tyr Tyr Val Ala Pro Glu Val Leu

220 225 230 235

ggt cca gag aag tat gac aag tcc tgt gac atg tgg tcc ctg ggt gtc 1131

Gly Pro Glu Lys Tyr Asp Lys Ser Cys Asp Met Trp Ser Leu Gly Val

240 245 250

atc atg tac atc ctg ctg tgt ggg tat ccc ccc ttc tac tcc aac cac 1179

Ile Met Tyr Ile Leu Leu Cys Gly Tyr Pro Pro Phe Tyr Ser Asn His

255 260 265

ggc ctt gcc atc tct ccg ggc atg aag act cgc atc cga atg ggc cag 1227

Gly Leu Ala Ile Ser Pro Gly Met Lys Thr Arg Ile Arg Met Gly Gln

270 275 280

tat gaa ttt ccc aac cca gaa tgg tca gaa gta tca gag gaa gtg aag 1275

Tyr Glu Phe Pro Asn Pro Glu Trp Ser Glu Val Ser Glu Glu Val Lys

285 290 295

atg ctc att cgg aat ctg ctg aaa aca gag ccc acc cag aga atg acc 1323

Met Leu Ile Arg Asn Leu Leu Lys Thr Glu Pro Thr Gln Arg Met Thr

300 305 310 315

atc acc gag ttt atg aac cac cct tgg atc atg caa tca aca aag gtc 1371

Ile Thr Glu Phe Met Asn His Pro Trp Ile Met Gln Ser Thr Lys Val

320 325 330

cct caa acc cca ctg cac acc agc cgg gtc ctg aag gag gac aag gag 1419

Pro Gln Thr Pro Leu His Thr Ser Arg Val Leu Lys Glu Asp Lys Glu

335 340 345

cgg tgg gag gat gtc aag ggg tgt ctt cat gac aag aac agc gac cag 1467

Arg Trp Glu Asp Val Lys Gly Cys Leu His Asp Lys Asn Ser Asp Gln

350 355 360

gcc act tgg ctg acc agg ttg tga gcagaggatt ctgtgttcct gtccaaactc 1521

Ala Thr Trp Leu Thr Arg Leu *

365 370

agtgctgttt cttagaatcc ttttattccc tgggtctcta atgggacctt aaagaccatc 1581

tggtatcatc ttctcatttt gcagaagaga aactgaggcc cagaggcgga gggcagtctg 1641

ctcaaggtca cgcagctggt gactggttgg ggcagaccgg acccaggttt cctgactcct 1701

ggcccaagtc tcttcctcct atcctgcggg atcactgggg ggctctcagg gaacagcagc 1761

agtgccatag ccaggctctc tgctgcccag cgctggggtg aggctgccgt tgtcagcgtg 1821

gaccactaac cagcccgtct tctctctctg ctcccacccc tgccgccctc accctgccct 1881

tgttgtctct gtctctcacg tctctcttct gctgtctctc ctacctgtct tctggctctc 1941

tctgtaccct tcctggtgct gccgtgcccc caggaggaga tgaccagtgc cttggccaca 2001

atgcgcgttg actacgagca gatcaagata aaaaagattg aagatgcatc caaccctctg 2061

ctgctgaaga ggcggaagaa agctcgggcc ctggaggctg cggctctggc ccactgagcc 2121

accgcgccct cctgcccacg ggaggacaag caataactct ctacaggaat atatttttta 2181

aacgaagaga cagaactgtc cacatctgcc tcctctcctc ctcagctgca tggagcctgg 2241

aactgcatca gtgactgaat tc 2263

<210> SEQ ID NO 34

<211> LENGTH: 370

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 34

Met Leu Ser Asn Ser Gln Gly Gln Ser Pro Pro Val Pro Phe Pro Ala

1 5 10 15

Pro Ala Pro Pro Pro Gln Pro Pro Thr Pro Ala Leu Pro His Pro Pro

20 25 30

Ala Gln Pro Pro Pro Pro Pro Pro Gln Gln Phe Pro Gln Phe His Val

35 40 45

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

50 55 60

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

65 70 75 80

Ile Phe Asn Lys Arg Thr Gln Glu Lys Phe Ala Leu Lys Met Leu Gln

85 90 95

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

100 105 110

Gln Cys Pro His Ile Val Arg Ile Val Asp Val Tyr Glu Asn Leu Tyr

115 120 125

Ala Gly Arg Lys Cys Leu Leu Ile Val Met Glu Cys Leu Asp Gly Gly

130 135 140

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

145 150 155 160

Arg Glu Ala Ser Glu Ile Met Lys Ser Ile Gly Glu Ala Ile Gln Tyr

165 170 175

Leu His Ser Ile Asn Ile Ala His Arg Asp Val Lys Pro Glu Asn Leu

180 185 190

Leu Tyr Thr Ser Lys Arg Pro Asn Ala Ile Leu Lys Leu Thr Asp Phe

195 200 205

Gly Phe Ala Lys Glu Thr Thr Ser His Asn Ser Leu Thr Thr Pro Cys

210 215 220

Tyr Thr Pro Tyr Tyr Val Ala Pro Glu Val Leu Gly Pro Glu Lys Tyr

225 230 235 240

Asp Lys Ser Cys Asp Met Trp Ser Leu Gly Val Ile Met Tyr Ile Leu

245 250 255

Leu Cys Gly Tyr Pro Pro Phe Tyr Ser Asn His Gly Leu Ala Ile Ser

260 265 270

Pro Gly Met Lys Thr Arg Ile Arg Met Gly Gln Tyr Glu Phe Pro Asn

275 280 285

Pro Glu Trp Ser Glu Val Ser Glu Glu Val Lys Met Leu Ile Arg Asn

290 295 300

Leu Leu Lys Thr Glu Pro Thr Gln Arg Met Thr Ile Thr Glu Phe Met

305 310 315 320

Asn His Pro Trp Ile Met Gln Ser Thr Lys Val Pro Gln Thr Pro Leu

325 330 335

His Thr Ser Arg Val Leu Lys Glu Asp Lys Glu Arg Trp Glu Asp Val

340 345 350

Lys Gly Cys Leu His Asp Lys Asn Ser Asp Gln Ala Thr Trp Leu Thr

355 360 365

Arg Leu

370

<210> SEQ ID NO 35

<211> LENGTH: 1074

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (628)...(831)

<400> SEQUENCE: 35

ttttactttt tttaagcaca aaattttgtt ttttttctcc cctccccaca gatcccatct 60

caaatcattc tgttaaccac cattccaaca ggtcgaggag agcttaaaca ccttcttcct 120

ctgccttgtt tctatttttt tatttttttg catcagtatt aatgtttttg catactctcc 180

atctttatcc aaaaatgtaa acttcctttg tcaatctatg gatatgccca tatatgaaag 240

agatgggtgg gtcaaaaagg gatatcaaat gaagtgatag gggtcacaat ggggaaatgg 300

aagtggtaca taacattgcc aaaataatgt gccactagaa atggtgtaaa ggctgtcttt 360

ttttttaaga aaagttatta ccatgtattt tgtgaggcag gtttacaaca ctacaagtct 420

tgactaagaa ggaaagagga aaaaagaaaa aacaccaata cccatattta aaaaaaaaaa 480

aatgatcata gtcttaggag ttcatttaaa ccataggaac ttttcactta tctcatgtta 540

ggtgtaccag tcagtgatta agtagaacta caagttatat aggctgtatt gtttattgct 600

ggtttatgac cttaataaag tgtaatt atg tat tac cag cag ggt gtt ttt aac 654

Met Tyr Tyr Gln Gln Gly Val Phe Asn

1 5

tgt gac tat tgt ata aaa aca aat ctt gat atc cag aag cac atg aag 702

Cys Asp Tyr Cys Ile Lys Thr Asn Leu Asp Ile Gln Lys His Met Lys

10 15 20 25

ttt gcg act ttc cac cct gcc cat ttt tgt aaa act gca gtc atc ttg 750

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

30 35 40

gac ctt tta aac aca aat ttt aaa ctc aac caa gct gtg ata agc gga 798

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

45 50 55

atg gtt act gtt tat act gtg gta tgt ttt tga ttacagcaga taatgctttc 851

Met Val Thr Val Tyr Thr Val Val Cys Phe *

60 65

ttttccagtc atctttgaga ataaaggaaa aaaaaaatct tcagatgcaa tggttttgtg 911

tagcatcttg tctatcatgt tttgtaaatg ctggagaagc gtcgaccaat ttgacttaga 971

gatggaatgt aactttgctt acaaaaattg ctattaaact cctacttaag gtgttctaat 1031

tttctgtgag cacactaaaa acaaaaatat atgtgaataa aat 1074

<210> SEQ ID NO 36

<211> LENGTH: 67

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 36

Met Tyr Tyr Gln Gln Gly Val Phe Asn Cys Asp Tyr Cys Ile Lys Thr

1 5 10 15

Asn Leu Asp Ile Gln Lys His Met Lys Phe Ala Thr Phe His Pro Ala

20 25 30

His Phe Cys Lys Thr Ala Val Ile Leu Asp Leu Leu Asn Thr Asn Phe

35 40 45

Lys Leu Asn Gln Ala Val Ile Ser Gly Met Val Thr Val Tyr Thr Val

50 55 60

Val Cys Phe

65

<210> SEQ ID NO 37

<211> LENGTH: 1356

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (553)...(1095)

<400> SEQUENCE: 37

ttctcccgca accttccctt cgctccctcc cgtccccccc agctcctagc ctccgactcc 60

ctccccccct cacgcccgcc ctctcgcctt cgccgaacca aagtggatta attacacgct 120

ttctgtttct ctccgtgctg ttctctcccg ctgtgcgcct gcccgcctct cgctgtcctc 180

tctccccctc gccctctctt cggccccccc ctttcacgtt cactctgtct ctcccactat 240

ctctgccccc ctctatcctt gatacaacag ctgacctcat ttcccgatac cttttccccc 300

ccgaaaagta caacatctgg cccgccccag cccgaagaca gcccgtcctc cctggacaat 360

cagacgaatt ctcccccccc ccccaaaaaa aaaagccatc cccccgctct gccccgtcgc 420

acattcggcc cccgcgactc ggccagagcg gcgctggcag aggagtgtcc ggcaggaggg 480

ccaacgcccg ctgttcggtt tgcgacacgc agcagggagg tgggcggcag cgtcgccggc 540

ttccagacac ca atg gga atc cca atg ggg aag tcg atg ctg gtg ctt ctc 591

Met Gly Ile Pro Met Gly Lys Ser Met Leu Val Leu Leu

1 5 10

acc ttc ttg gcc ttc gcc tcg tgc tgc att gct gct tac cgc ccc agt 639

Thr Phe Leu Ala Phe Ala Ser Cys Cys Ile Ala Ala Tyr Arg Pro Ser

15 20 25

gag acc ctg tgc ggc ggg gag ctg gtg gac acc ctc cag ttc gtc tgt 687

Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys

30 35 40 45

ggg gac cgc ggc ttc tac ttc agc agg ccc gca agc cgt gtg agc cgt 735

Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Ser Arg

50 55 60

cgc agc cgt ggc atc gtt gag gag tgc tgt ttc cgc agc tgt gac ctg 783

Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu

65 70 75

gcc ctc ctg gag acg tac tgt gct acc ccc gcc aag tcc gag agg gac 831

Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu Arg Asp

80 85 90

gtg tcg acc cct ccg acc gtg ctt ccg gac aac ttc ccc aga tac ccc 879

Val Ser Thr Pro Pro Thr Val Leu Pro Asp Asn Phe Pro Arg Tyr Pro

95 100 105

gtg ggc aag ttc ttc caa tat gac acc tgg aag cag tcc acc cag cgc 927

Val Gly Lys Phe Phe Gln Tyr Asp Thr Trp Lys Gln Ser Thr Gln Arg

110 115 120 125

ctg cgc agg ggc ctg cct gcc ctc ctg cgt gcc cgc cgg ggt cac gtg 975

Leu Arg Arg Gly Leu Pro Ala Leu Leu Arg Ala Arg Arg Gly His Val

130 135 140

ctc gcc aag gag ctc gag gcg ttc agg gag gcc aaa cgt cac cgt ccc 1023

Leu Ala Lys Glu Leu Glu Ala Phe Arg Glu Ala Lys Arg His Arg Pro

145 150 155

ctg att gct cta ccc acc caa gac ccc gcc cac ggg ggc gcc ccc cca 1071

Leu Ile Ala Leu Pro Thr Gln Asp Pro Ala His Gly Gly Ala Pro Pro

160 165 170

gag atg gcc agc aat cgg aag tga gcaaaactgc cgcaagtctg cagcccggcg 1125

Glu Met Ala Ser Asn Arg Lys *

175 180

ccaccatcct gcagcctcct cctgaccacg gacgtttcca tcaggttcca tcccgaaaat 1185

ctctcggttc cacgtccccc tggggcttct cctgacccag tccccgtgcc ccgcctcccc 1245

gaaacaggct actctcctcg gccccctcca tcgggctgag gaagcacagc agcatcttca 1305

aacatgtaca aaatcgattg gctttaaaca cccttcacat accctccccc c 1356

<210> SEQ ID NO 38

<211> LENGTH: 180

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQUENCE: 38

Met Gly Ile Pro Met Gly Lys Ser Met Leu Val Leu Leu Thr Phe Leu

1 5 10 15

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

20 25 30

Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys Gly Asp Arg

35 40 45

Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Ser Arg Arg Ser Arg

50 55 60

Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu

65 70 75 80

Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu Arg Asp Val Ser Thr

85 90 95

Pro Pro Thr Val Leu Pro Asp Asn Phe Pro Arg Tyr Pro Val Gly Lys

100 105 110

Phe Phe Gln Tyr Asp Thr Trp Lys Gln Ser Thr Gln Arg Leu Arg Arg

115 120 125

Gly Leu Pro Ala Leu Leu Arg Ala Arg Arg Gly His Val Leu Ala Lys

130 135 140

Glu Leu Glu Ala Phe Arg Glu Ala Lys Arg His Arg Pro Leu Ile Ala

145 150 155 160

Leu Pro Thr Gln Asp Pro Ala His Gly Gly Ala Pro Pro Glu Met Ala

165 170 175

Ser Asn Arg Lys

180

Claims

That which is claimed is:

1. A method for reducing growth of a cancerous cell comprising:

contacting a cancerous cell with an amount of an agent effective to redue tyrosine threonine kinase (TTK) polypeptide activity in the cell;

wherein reduction of TTK polypeptide activity in the cancerous cell reduces growth of the cell.

2. The method of claim 1 wherein said reduction of TTK activity is a result of a reduction of TTK polypeptide levels.

3. The method of claim 2 wherein the agent is a TTK antisense polynucleotide.

4. The method of claim 3 wherein the TTK antisense polynucleotide is contained in a viral-based vector.

5. The method of claim 1 wherein said reduction of TTK activity is a result of a reduction of TTK polynucleotide levels.

6. The method of claim 1 wherein the agent is a monoclonal antibody that specifically binds TTK.

7. The method of claim 1 wherein the TTK polypeptide comprises the amino acid sequence of SEQ ID NO:14.

8. An assay for identifying a candidate agent that reduces growth of a cancerous cell, comprising:

detecting the activity of a TTK polypeptide in the presence of a candidate agent; and

comparing the activity of the TTK polypeptide in the presence of the candidate agent relative to TTK polypeptide activity in the absence of the candidate agent;

wherein a reduction of TTK activity in the presence of the candidate agent relative to TTK activity in the absence of the candidate agent indicates the candidate agent reduces growth of a cancerous cell.

9. The assay of claim 8, wherein said detecting step utilizes the polypeptide of SEQ ID NO:26 as a substrate.

10. The assay of claim 8, wherein said detecting step uses a fragment of SEQ ID NO:26 susceptible to TTK phosphorylation as a substrate.

11. The assay of claim 10, wherein said fragment comprises the polypeptide of SEQ ID NO:27 or 28.

12. The assay of claim 10 wherein the polypeptide fragment is biotinylated.

13. The assay of claim 8 wherein the TTK polypeptide is a product of expression using a system selected from the group of baculovirus, bacteria, yeast and mammalian systems.

14. The assay of claim 13 wherein the TTK polypeptide is a product of expression using a baculovirus system.

15. The method of claim 8 wherein the TTK polypeptide comprises the amino acid sequence of SEQ ID NO:14.

16. A method of identifying an agent that reduces TTK activity, the method comprising:

contacting a cancerous cell displaying elevated expression of a TTK-encoding polynucleotide with a candidate agent; and

determining the effect of the candidate agent on TTK polypeptide activity;

wherein a decrease in TTK activity indicates that the agent reduces TTK activity and inhibits growth of the cancerous cell.

17. The method of claim 16 wherein said reduction of TTK activity is a result of a reduction of TTK polypeptide levels.

18. The method of claim 16 wherein said reduction of TTK activity is a result of a reduction of TTK mRNA levels.

19. The method of claim 17 wherein the candidate agent is a TTK antisense polynucleotide.

20. The method of claim 19, wherein the TTK antisense polynucleotide is contained in a viral-based vector.

21. The method of claim 16 wherein the cancerous cell is a breast cancer cell.

22. The method of claim 16 wherein the cancerous cell is a colon cancer cell.

23. The method of claim 16 wherein TTK polypeptide comprises the amino acid sequence of SEQ ID NO:14.

24. The method according to claim 18, wherein TTK activity is detected by detecting expression of a TTK-encoding polynucleotide.

25. A method of detecting cancer other than ovarian cancer in a subject, the method comprising:

detecting a level of expression of a TTK polypeptide in a test cell obtained from a subject suspected of having cancer; and

comparing the level of expression of the TTK polypeptide in the test cell to a level of expression in a normal non-cancer cell of the same tissue type;

wherein detection of an expression level of TTK polypeptide in the test cell that is significantly increased relative to the level of expression in the normal non-cancer cell indicates that the subject has cancer other than ovarian cancer.

26. The method of claim 25, wherein the test cell is a colon cell.

27. The method of claim 25, wherein the test cell is a breast cell.

28. A method of detecting cancer other than ovarian cancer in a subject, the method comprising:

detecting a level of expression of a TTK polynucleotide in a test cell obtained from a subject suspected of having cancer; and

comparing the level of expression of the TTK polynucleotide in the test cell to a level of expression in a normal non-cancer cell of the same tissue type;

wherein detection of an expression level of TTK polynucleotide in the test cell that is significantly increased relative to the level of expression in the normal non-cancer cell indicates that the subject has cancer other than ovarian cancer.

29. The method of claim 29, wherein the test cell is a colon cell.

30. The method of claim 29, wherein the test cell is a breast cell.

31. A method for assessing the prognosis of a cancerous disease other than ovarian cancer in a subject, the method comprising:

detecting expression of a TTK-encoding polynucleotide in a test cancer cell of a subject; and

comparing a level of expression of a TTK-encoding polynucleotide in the test cancer cell with a level of expression the polynucleotide in a control non-cancer cell;

wherein the level of expression of TTK in the test cancer cell relative to the level of expression in the control non-cancer cell is indicative of the prognosis of the cancerous disease

32. The method of claim 31, wherein said detecting expression is by detection of a TTK-encoding transcript.

33. The method of claim 31, wherein the test cell is a colon cell.

34. The method of claim 31, wherein the test cell is a breast cell.