huBUB3 gene involved in human cancers

Abstract

Methods are provided for assessing mutations and/or loss of the huBUB3 gene in human tumors. Loss of wild-type huBUB3 genes is involved in neoplastic development. Therapeutic regimens can be planned on the basis of the mutational status of huBUB3.

1 . An isolated and purified huBUB3 protein having an amino acid sequence which is at least 85% identical to SEQ ID NO: 2, wherein percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.  
     
     
         2 . The isolated and purified huBUB3 protein of  claim 1  which has the amino acid sequence shown in SEQ ID NO: 2.  
     
     
         3 . An isolated and purified polypeptide comprising at least 8 contiguous amino acids as shown in SEQ ID NO: 2.  
     
     
         4 . A huBUB3 fusion protein comprising a first protein segment and a second protein segment fused together by means of a peptide bond, wherein the first protein segment consists of at least 8 contiguous amino acids of a huBUB3 protein as shown in SEQ ID NO: 2.  
     
     
         5 . A preparation of antibodies which specifically bind to a huBUB3 protein having an amino acid sequence as shown in SEQ ID NO: 2.  
     
     
         6 . A cDNA molecule which encodes a huBUB3 protein having an amino acid sequence which is at least 85% identical to SEQ ID NO: 2, wherein percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.  
     
     
         7 . A cDNA molecule which encodes at least 8 contiguous amino acids of SEQ ID NO: 2.  
     
     
         8 . The cDNA molecule of  claim 7  which encodes SEQ ID NO: 2.  
     
     
         9 . The cDNA molecule of  claim 8  which comprises SEQ ID NO: 1.  
     
     
         10 . A cDNA molecule comprising at least 12 contiguous nucleotides of SEQ ID NO: 1.  
     
     
         11 . A cDNA molecule which is at least 85% identical to the nucleotide sequence shown in SEQ ID NO: 1, wherein percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.  
     
     
         12 . An isolated and purified subgenomic polynucleotide comprising a nucleotide sequence which hybridizes to SEQ ID NO: 1 after washing with 0.2×SSC at 65° C., wherein the nucleotide sequence encodes a huBUB3 protein having the amino acid sequence of SEQ ID NO: 2.  
     
     
         13 . A construct comprising: 
 a promoter; and    a polynucleotide segment encoding at least 8 contiguous amino acids of a huBUB3 protein as shown in SEQ ID NO: 2, wherein the polynucleotide segment is located downstream from the promoter, wherein transcription of the polynucleotide segment initiates at the promoter.    
     
     
         14 . A host cell comprising a construct which comprises: 
 a promoter and:    a polynucleotide segment encoding at least 8 contiguous amino acids of a huBUB3 protein having an amino acid sequence as shown in SEQ ID NO: 2.    
     
     
         15 . A recombinant host cell comprising a new transcription initiation unit, wherein the new transcription initiation unit comprises in 5′ to 3′ order: 
 (a) an exogenous regulatory sequence;  
 (b) an exogenous exon; and  
 (c) a splice donor site,  
 wherein the new transcription initiation unit is located upstream of a coding sequence of a huBUB3 gene as shown in SEQ ID NO: 1, wherein the exogenous regulatory sequence controls transcription of the coding sequence of the huBUB3 gene.  
 
     
     
         16 . A pair of single-stranded DNA primers, said set allowing synthesis of all or part of a huBUB3 gene coding sequence.  
     
     
         17 . The pair of  claim 16  wherein the primers have restriction enzyme sites at each 5′ end.  
     
     
         18 . A nucleic acid probe complementary to a wild-type huBUB3 gene as shown in SEQ ID NO: 1.  
     
     
         19 . A method of diagnosing a neoplastic tissue of a human, comprising the step of: 
 detecting loss of a wild-type huBUB3 gene or an expression product of the wild-type huBUB3 gene from a tissue suspected of being neoplastic, wherein the wild-type huBUB3 gene has the coding sequence shown in SEQ ID NO: 1, wherein the loss indicates neoplasia of the tissue.    
     
     
         20 . The method of  claim 19  wherein the expression product is an mRNA molecule.  
     
     
         21 . The method of  claim 19  wherein the expression product is a protein molecule.  
     
     
         22 . The method of  claim 19  wherein the loss of the wild-type huBUB3 gene is detected by sequencing all or part of a huBUB3 gene.  
     
     
         23 . The method of  claim 19  wherein the loss of the wild-type huBUB3 gene is detected by amplification of huBUB3 gene sequences and hybridization of the amplified huBUB3 sequences to nucleic acid probes which are complementary to mutant huBUB3 alleles.  
     
     
         24 . The method of  claim 19  wherein the loss of the wild-type huBUB3 gene is detected by sequencing all or part of a huBUB3 gene.  
     
     
         25 . The method of  claim 21  wherein the loss of the wild-type huBUB3 protein molecule is detected by detecting a loss of ability of a huBUB3 protein to complex with a BUB1 protein.  
     
     
         26 . The method of  claim 19  wherein detection of the loss of the wild-type huBUB3 gene comprises screening for a point mutation.  
     
     
         27 . The method of  claim 26  wherein the point mutation is a missense mutation.  
     
     
         28 . The method of  claim 19  wherein detection of the loss of the wild-type huBUB3 gene comprises screening for a frameshift mutation.  
     
     
         29 . The method of  claim 19  wherein the detection of the loss of the wild-type huBUB3 gene comprises screening for a deletion mutation.  
     
     
         30 . The method of  claim 19  wherein the tissue suspected of being neoplastic is selected from the group consisting of lung, breast, brain, colorectal, bladder, prostate, liver, and stomach.  
     
     
         31 . A method of identifying a neoplastic tissue of a human, comprising the step of: 
 comparing expression of a first huBUB3 gene in a first tissue of a human suspected of being neoplastic with expression of a second huBUB3 gene in a second tissue of the human which is normal, wherein the second huBUB3 gene has the coding sequence shown in SEQ ID NO: 1, wherein decreased expression of the first huBUB3 gene relative to the second huBUB3 gene identifies the first tissue as being neoplastic.    
     
     
         32 . A method to aid in the diagnosis or prognosis of neoplasia in a human, comprising the step of: 
 comparing a first huBUB3 gene, mRNA, or protein in a first tissue of a human suspected of being neoplastic with a second huBUB3 gene, mRNA, or protein in a second tissue of a human which is normal, wherein a difference between the first and second huBUB3 genes, mRNAs, or proteins indicates the presence of neoplastic cells in the first tissue.    
     
     
         33 . A method to aid in detecting a genetic predisposition to neoplasia in a human, comprising the step of: 
 comparing a huBUB3 gene, mRNA, or protein in the fetal tissue of a human with a wild-type huBUB3 gene, mRNA, or protein, wherein a difference between the huBUB3 gene, mRNA, or protein in the fetal tissue of the human and the wild-type human huBUB3 gene, mRNA, or protein indicates a genetic predisposition to neoplasia in the human.    
     
     
         34 . A method of screening test compounds for the ability to interfere with the binding of a huBUB3 protein to a huBUB1 protein, comprising the steps of: 
 (a) contacting a test compound with at least a huBUB3-binding domain of a huBUB1 protein as shown in SEQ ID NO: 4 and at least a huBUB1-binding domain of a huBUB3 protein as shown in SEQ ID NO: 2, wherein the huBUB3-binding domain binds to the huBUB1-binding domain in the absence of the test compound; and    (b) determining the amount of the huBUB1-binding domain which is bound or unbound to the huBUB3-binding domain or determining the amount of the huBUB3-binding domain which is bound or unbound to the huBUB1-binding domain in the presence of the test compound, wherein a test compound which decreases the amount of bound huBUB1- or huBUB3-binding domains or which increases the amount of unbound huBUB1- and huBUB3-binding domains is a potential inducer of mitosis or cell cycle progression.    
     
     
         35 . The method of  claim 34  wherein the huBUB1- and the huBUB3-binding domains are prebound prior to the step of contacting.  
     
     
         36 . The method of  claim 34  wherein the test compound is contacted with either of the huBUB1- or huBUB3-binding domains prior to the step of contacting.  
     
     
         37 . A method of screening test compounds for the ability to interfere with the binding of a huBUB1 protein to a huBUB3 protein, comprising the steps of: 
 (a) contacting a cell with a test compound, wherein the cell comprises: 
 I) a first fusion protein comprising (1) at least a huBUB1-binding domain of a huBUB3 protein as shown in SEQ ID NO: 2 and (2) either a DNA binding domain or a transcriptional activating domain;  
 ii) a second fusion protein comprising at least a huBUB3-binding domain of a huBUB1 protein as shown in SEQ ID NO: 4, wherein the huBUB1-binding domain binds to the huBUB3-binding domain, wherein if the first fusion protein comprises a DNA binding domain, then the second fusion protein comprises a transcriptional activating domain, wherein if the first fusion protein comprises a transcriptional activating domain, then the second fusion protein comprises a DNA binding domain, wherein the interaction of the first and second fusion proteins reconstitutes a sequence-specific transcription activating factor; and  
 iii) a reporter gene comprising a DNA sequence to which the DNA binding domain specifically binds; and  
   (b) measuring the expression of the reporter gene, wherein a test compound which decreases the expression of the reporter gene is a potential inducer of mitosis or cell cycle progression.    
     
     
         38 . A method of identifying compounds which interfere with the binding of a huBUB3 protein to a huBUB1 protein, comprising the steps of: 
 providing a cell which comprises three recombinant DNA constructs, wherein a first construct encodes a first polypeptide fused to a sequence-specific DNA-binding domain, wherein a second construct encodes a second polypeptide fused to a transcriptional activation domain, and wherein a third construct comprises a reporter gene downstream from a DNA element which is recognized by the sequence-specific DNA-binding domain, wherein the first polypeptide comprises a huBUB1-binding domain of a huBUB3 protein as shown in SEQ ID NO: 2 and the second polypeptide comprises a huBUB3-binding domain of a huBUB1 protein as shown in SEQ ID NO: 4 or the first polypeptide comprises a huBUB3-binding domain of a huBUB1 protein as shown in SEQ ID NO: 4 and the second polypeptide comprises a huBUB1-binding domain of a huBUB3 protein as shown in SEQ ID NO: 2;    contacting the cell with a test compound; and    determining expression of the reporter gene in the presence of the test compound, wherein a test compound which decreases expression of the reporter gene is identified as a candidate therapeutic agent.    
     
     
         39 . A cell which comprises three recombinant DNA constructs, wherein a first construct encodes a first polypeptide fused to a sequence-specific DNA-binding domain, wherein a second construct encodes a second polypeptide fused to a transcriptional activation domain, and wherein a third construct comprises a reporter gene downstream from a DNA element which is recognized by the sequence-specific DNA-binding domain, wherein the first polypeptide comprises a a huBUB1-binding domain of a huBUB3 protein as shown in SEQ ID NO: 2 and the second polypeptide comprises a huBUB3-binding domain of a huBUB1 protein as shown in SEQ ID NO: 4, or the first polypeptide comprises a huBUB3-binding domain of a huBUB1 protein as shown in SEQ ID NO: 4 and the second polypeptide comprises a huBUB1-binding domain of a huBUB3 protein as shown in SEQ ID NO: 2.  
     
     
         40 . A method of determining the quantity of huBUB1 which binds to huBUB3, or of huBUB3 which binds to huBUB1, comprising the steps of: 
 contacting a first protein and a second protein, wherein if the first protein is huBUB3 the the second protein is huBUB1 and if the first protein is huBUB1 the the second protein is huBUB3; and    determining the quantity of the first protein which is bound to the second protein.    
     
     
         41 . A method for identifying compounds which decrease the kinase activity of a huBUB1-huBUB3 complex, comprising the steps of: 
 contacting a huBUB1-huBUB3 complex with a test compound; and    determining the kinase activity of the huBUB1-huBUB3 complex, wherein a compound which decreases kinase activity of the huBUB1-huBUB3 complex is identified as a candidate therapeutic agent.