Novel CREBa isoform

Abstract

The present invention relates generally to a novel CREBa polypeptide isoform, polynucleotides encoding the polypeptide, expression constructs comprising the polynucleotides, host cell transformed or transfected with the polynucleotides, methods for producing the polypeptide, and methods to identify inhibitors of binding between the CREBa and other polypeptides or polynucleotides.

What is claimed is:  
     
         1 . A polypeptide comprising the mCREBa amino acid sequence set forth in SEQ ID NO: 2.  
     
     
         2 . A polynucleotide encoding the polypeptide according to  claim 1 .  
     
     
         3 . The polynucleotide according to  claim 2  comprising the sequence set forth in SEQ ID NO: 1.  
     
     
         4 . The polynucleotide of claims  2  or  3  which is a DNA molecule.  
     
     
         5 . The DNA of  claim 4  which is a cDNA molecule.  
     
     
         6 . The DNA of clam  4  which is a genomic DNA molecule.  
     
     
         7 . The DNA of  claim 4  which is a wholly or partially chemically synthesized DNA molecule.  
     
     
         8 . An anti-sense polynucleotide which specifically hybridizes with the polynucleotide of  claim 2 .  
     
     
         9 . A expression construct comprising the polynucleotide according to  claim 2 .  
     
     
         10 . A host cell transformed or transfected with the polynucleotide according to  claim 2  or  9 .  
     
     
         11 . A method for producing an mCREBa polypeptide comprising the steps of: 
 a) growing the host cell according to  claim 5  under conditions appropriate for expression of the mCREBa polypeptide and    b) isolating the mCREBa polypeptide from the host cell of the medium of its growth.    
     
     
         12 . An antibody specifically immunoreactive with the polypeptide according to  claim 1 .  
     
     
         13 . The antibody according to  claim 12  which is a monoclonal antibody.  
     
     
         14 . A hybridoma which secretes the antibody according to  claim 13 .  
     
     
         15 . An anti-idiotype antibody specifically immunoreactive with the antibody according to  claim 12 .  
     
     
         16 . A method to identify modulators of mCREBa binding to DNA comprising the steps of: 
 a) incubating mCREBa with a DNA sequence known to specifically bind mCREBa;    b) determining the degree of binding between mCREBa and the DNA sequence;    c) repeating step (a) in the presence of a putative modulator of mCREBa binding to the DNA sequences; and    d) comparing binding of mCREBa with the DNA sequence in the presence of the putative modulator to the interaction determined in step (b), wherein increased binding in the presence of the putative modulator is indicative of a binding enhancer and decreased binding in the presence of the putative modulator is indicative of a binding repressor.    
     
     
         17 . A method to identify an inhibitor of binding between mCREBa or binding fragment thereof and a mCREBa-binding protein or binding fragment thereof comprising the steps of: 
 a) producing host cells transformed or transfected with DNA comprising: 
 a repressor gene encoding a repressor protein, said repressor gene under transcriptional control of a promoter;  
 a selectable marker gene encoding a selectable marker protein; said selectable marker gene under transcriptional control of an operator; said operator regulated by interaction with said repressor protein;  
 a first recombinant fusion protein gene encoding mCREBa or a binding fragment thereof in frame with either a DNA binding domain of a transcriptional activating protein or a transactivating domain of a transcriptional activating protein; and  
 a second recombinant fusion protein gene encoding a mCREBa-binding protein or binding fragment thereof in frame with either a DNA binding domain of a transcriptional activating protein or a transactivating domain of a transcriptional activating protein, whichever domain is not encoded by the first fusion protein gene, said second binding protein or binding fragment thereof capable of interacting with said first binding protein or binding fragment thereof such that interaction of said second binding protein or binding fragment thereof and said first binding protein or binding fragment thereof brings into proximity a DNA binding domain and a transactivating domain forming a functional transcriptional activating protein; said functional transcriptional activating protein acting on said promoter to increase expression of said repressor gene.  
   b) growing the host cells in the absence of a test compound and under conditions which permit expression of said mCREBa or binding fragment thereof and said mCREBa-binding protein or binding fragment thereof such that said mCREBa or fragment thereof and said mCREBa binding protein or binding fragment thereof interact bringing into proximity said DNA binding domain and said transactivating domain forming said functional transcriptional activating protein; said transcriptional activating protein acting on said promoter to increase expression of said repressor protein; said repressor protein interacting with said operator such that said selectable marker protein is not expressed;    c) confirming lack of expression of said selectable marker protein in said host cell;    d) growing said host cells in the presence of a test compound; and    e) comparing expression of said selectable marker protein in the presence and absence of said test compound wherein increased expression of said selectable marker protein is indicative that the test compound is an inhibitor of binding between said first binding protein or binding fragment thereof and said second binding protein or binding fragment thereof.    
     
     
         18 . The method of  claim 17  wherein said DNA binding domain and said transactivating domain are derived from a common transcriptional activating protein.  
     
     
         19 . The method of  claim 17  wherein one or more of the repressor gene, the selectable marker gene, the first recombinant fusion protein gene, and the second recombinant fusion protein gene are encoded on distinct DNA expression constructs.  
     
     
         20 . The method of  claim 17  wherein said selectable marker protein is an enzyme in a pathway for synthesis of a nutritional requirement for said host cell such that expression of said selectable marker protein is required for growth of said host cell on media lacking said nutritional requirement.  
     
     
         21 . The method of  claim 17  wherein said host cell is a yeast cell or a mammalian.  
     
     
         22 . The method of  claim 18  wherein said selectable marker gene encodes HIS3;  
     
     
         23 . The method of  claim 18  wherein said repressor protein gene encodes a tetracycline resistance protein;  
     
     
         24 . The method of  claim 18  wherein said operator is a tet operator.  
     
     
         25 . The method of  claim 18  wherein said promoter is selected from the group consisting of the LexA promoter, the alcohol dehydrogenase promoter, the Ga14 promoter.  
     
     
         26 . The method of  claim 18  wherein said DNA binding domain derived from a protein selected from the group consisting of LexA and Ga14.  
     
     
         27 . The method of  claim 18  wherein said transactivating domain is derived from a protein selected from the group consisting of VP16 and Ga14.  
     
     
         28 . The method of  claim 18  wherein the mCREBa-binding protein is selected from the group consisting of CKI, CKII, cdc2, MAP kinase, and S6 kinase.