Checkpoint-activating oligonucleotides

Abstract

The present invention provides polypeptides (Cds1) that are involved in regulating the progression of the cell cycle. The polypeptides are activated by double-stranded DNA and phosphorylated in response to the presence of double-stranded DNA. Also provided are polynucleotides encoding Cds1 polypeptidese and methods for modulating cell cycle progression in a cell.

What is claimed is:  
     
         1 . A substantially pure polypeptide characterized as: 
 (a) phosphorylating Cdc25 or a homologue thereof,    (b) having a molecular mass of about 58 kD;    (c) having about 517 amino acids,    (d) having SQ/TQ motifs at the amino terminal region;    (e) having a carboxyl terminal kinase domain; and    (f) having an amino terminal forkhead-associated domain.    
     
     
         2 . A polypeptide of  claim 1 , wherein the polypeptide has an amino acid sequence as set forth in SEQ ID NO:2.  
     
     
         3 . A substantially pure polypeptide having an amino acid sequence as set forth in SEQ ID NO:2 or conservative variants thereof.  
     
     
         4 . A substantially pure pQlypeptide having an amino acid sequence that is about 80% homologous to the polypeptide of  claim 3 .  
     
     
         5 . An isolated polynucleotide encoding a polypeptide of  claim 1 .  
     
     
         6 . An isolated polynucleotide selected from the group consisting of: 
 (a) a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:2;    (b) a polynucleotide of (a), wherein all T's are U;    (c) a polynucleotide complementary to (a) or (b);    (d) a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1; and    (e) degenerate variants of (a), (b), (c) or (d).    
     
     
         7 . An isolated polynucleotide having at least 15 continuous base pairs that hybridizes to a polynucleotide selected from the group consisting of: 
 (a) a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in amino acids 1 to 86 or amino acids 461 to 517 of SEQ ID NO:2;    (b) a polynucleotide of (a), wherein T can be U;    (c) a polynucleotide complementary to (a) or (b);    (d) a polynucleotide having a nucleotide sequence as set forth in nucleotides 224 to 481 or nucleotides 1604 to 1770 of SEQ ID NO:1; and    (e) degenerate variants of (a), (b), (c) or (d).    
     
     
         8 . An isolated polynucleotide at least 15 bases in length which hybridizes under moderately to highly stringent conditions to DNA encoding a polypeptide as set forth in SEQ ID NO:2.  
     
     
         9 . An isolated polynucleotide according to  claim 8 , wherein the polynucleotide is antisense nucleic acid.  
     
     
         10 . An isolated oligonucleotide as set forth in SEQ ID NO:3.  
     
     
         11 . An isolated oligonucleotide as set forth in SEQ ID NO:4.  
     
     
         12 . An isolated oligonucleotide as set forth in SEQ ID NO:5.  
     
     
         13 . An antibody that binds to a polypeptide of  claim 1  or binds to immunoreactive fragments thereof.  
     
     
         14 . The antibody of  claim 13 , wherein the antibody is polyclonal.  
     
     
         15 . The antibody of  claim 13 , wherein the antibody is monoclonal.  
     
     
         16 . An expression vector comprising a polynucleotide of  claim 5 .  
     
     
         17 . The expression vector of  claim 16 , wherein the vector is virus-derived.  
     
     
         18 . The expression vector of  claim 16 , wherein the vector is plasmid-derived.  
     
     
         19 . A host cell comprising a vector of  claim 16 .  
     
     
         20 . A method for producing a polypeptide comprising the steps of: 
 (a) culturing a host cell of  claim 19  under conditions suitable for the expression of the polypeptide; and    (b) recovering the polypeptide from the host cell culture.    
     
     
         21 . A transgenic non-human animal having a transgene that expresses a polypeptide of  claim 1  chromosomally integrated into the germ cells of the animal.  
     
     
         22 . The transgenic animal of  claim 25 , wherein the animal is murine.  
     
     
         23 . A method for increasing mitotic delay in a vertebrate cell comprising providing to the cell one or more oligonucleotides that form double-stranded DNA.  
     
     
         24 . The method of  claim 23 , wherein the one or more oligonucleotides have the sequence set forth in SEQ ID NO:3 and SEQ ID NO:.  
     
     
         25 . The method of  claim 23 , wherein the one or more oligonucleotide has the sequence set forth in SEQ ID NO:5.  
     
     
         26 . A method for identifying a reagent that modulates phosphorylation of a polypeptide comprising: 
 (a) incubating a reagent with the polypeptide, and one or more oligonucleotides that form double-stranded DNA, under conditions that allow the components to interact with each other; and    (b) comparing the phosphorylation of the polypeptide to phosphorylation of a polypeptide not incubated with the reagent, wherein a difference in phosphorylation is indicative of a reagent that modulates phosphorylation of the polypeptide.    
     
     
         27 . The method of  claim 26 , wherein the modulation is an increase in phosphorylation.  
     
     
         28 . The method of  claim 26 , wherein the modulation is a decrease in phosphorylation.  
     
     
         29 . The method of  claim 26 , wherein the polypeptide is Xenopus Cds1.  
     
     
         30 . The method of  claim 26 , wherein the polypeptide is human Cds1.  
     
     
         31 . The method of  claim 26 , wherein the one or more oligonucleotides have a sequence as set forth in SEQ ID NO:3 and SEQ ID NO:4.  
     
     
         32 . The method of  claim 26 , wherein the one oligonucleotide has a sequence as set forth in SEQ ID NO:5.  
     
     
         33 . The method of  claim 26 , wherein the reagent is selected a peptide, a peptidomimetic, a polypeptide, a pharmaceutical, a chemical compound, a polynucleotide or an antibody.  
     
     
         34 . A method for modulating cell cycle progression in a cell, said method comprising providing to the cell a compound that affects the activity or expression of a Cds1 polypeptide, thereby modulating cell cycle progression.  
     
     
         35 . The method of  claim 34 , wherein modulation of cell cycle progression is inhibition or a reduction in progression.  
     
     
         36 . The method of  claim 34 , wherein the compound is a peptide, a peptidomimetic, a polypeptide, a pharmaceutical, a chemical compound, a polynucleotide, or an antibody.  
     
     
         37 . The method of  claim 34 , wherein the polynucleotide is double-stranded DNA.  
     
     
         38 . A method of treating a subject having a cellular disorder associated with increased cell cycle progression compared to a subject not having the cellular disorder, comprising administering to a subject having the disorder a therapeutically effective amount of a reagent that increases a Cds1 polypeptide activity, thereby treating the cellular disorder.  
     
     
         39 . The method of  claim 38 , wherein the disorder is a cell proliferative disorder.  
     
     
         40 . The method of  claim 38 , wherein the reagent is double-stranded DNA.  
     
     
         41 . The method of  claim 38 , wherein the reagent is poly(dT) 40 .  
     
     
         42 . A method of diagnosing a Cds1-associated disorder in a subject comprising determining the level of Cds1 mRNA or protein expression in the subject, wherein a low level of Cds1 in the subject compared to the level in a subject not having a Cds-associated disorder is indicative of a Cds-associated disorder.  
     
     
         43 . A kit for activating a Cds1 polypeptide comprising 
 (a) double-stranded DNA; and    (b) a container for the DNA.    
     
     
         44 . A computer system comprising a processor and a data storage device wherein said data storage device has stored thereon a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, and sequences substantially identical thereto, or a polypeptide sequence selected from the group consisting SEQ ID NO:2, and sequences substantially identical thereto.  
     
     
         45 . The computer system of  claim 44 , further comprising a sequence comparison algorithm and a data storage device having at least one reference sequence stored thereon.  
     
     
         46 . The computer system of  claim 44 , wherein the sequence comparison algorithm comprises a computer program which indicates polymorphisms.  
     
     
         47 . A method for comparing a first sequence to a reference sequence wherein said first sequence is a nucleic acid sequence selected from the group consisting SEQ ID NO:1, and sequences substantially identical thereto, or a polypeptide sequence selected from the group consisting of SEQ ID NO:2, and sequences substantially identical thereto comprising: 
 (a) reading the first sequence and the reference sequence through use of a computer program which compares sequences; and    (b) determining differences between the first sequence and the reference sequence with the computer program.    
     
     
         48 . The method of  claim 47 , wherein determining differences between the first sequence and the reference sequence comprises identifying polymorphisms.