Anti-proliferation domain of human Bcl-2 and DNA encoding the same

Abstract

A domain of Bcl-2 that suppresses apoptosis by allowing cell survival permits cell proliferation when mutated. The wild type domain includes amino acid residues 51 to 97 (SEQ ID NO:13) of Bcl-2. Peptides including the domain and nucleotides encoding the domain are useful in molecular screening of human tumors for the presence of mutations that allow proliferation of cells that were otherwise marked for apoptosis. The peptides are also useful to screen for proteins that play a role in the modulation of cellular proliferation.

What is claimed:  
     
         1 . A truncated bcl-2 gene comprising nucleotides 151 to any one of nucleotides 255-291 (SEQ ID NOS:14-50), and fragments of the truncated bcl-2 gene that contain at least one codon encoding an amino acid needed to maintain full or partial anti-proliferation activity of the AP domain.  
     
     
         2 . The truncated bcl-2 gene of  claim 1 , wherein the fragments contain at least one of nucleotides 151-153, nucleotides 169-171, nucleotides 184-186, nucleotides 204-210, nucleotides 220-222, and nucleotides 223-225.  
     
     
         3 . The truncated bcl-2 gene of  claim 1 , which comprises nucleotides 151 to any one of nucleotides 255-291 (SEQ ID NO: 14-50).  
     
     
         4 . The truncated bcl-2 gene of  claim 1 , which comprises nucleotides 151-255. (SEQ ID NO:14).  
     
     
         5 . The truncated bcl-2 gene of  claim 1 , which consists of nucleotides 151 to any one of nucleotides 255-291 (SEQ ID NO:14-50).  
     
     
         6 . The truncated bcl-2 gene of  claim 1 , which consists of nucleotides 151-255 (SEQ ID NO:14).  
     
     
         7 . RNA complementary to the truncated bcl-2 gene of anyone of claims  1 - 6 .  
     
     
         8 . A truncated Bcl-2 protein comprising residues 51 to any of 85-97 (SEQ ID NOS: 1-13) and fragments of the truncated Bcl-2 protein that contain at least one amino acid needed to maintain full or partial anti-proliferation activity of the AP domain.  
     
     
         9 . The truncated Bcl-2 protein of  claim 8 , wherein the fragments contain at least one of Ser residue 51, Pro residue 57, Ser residue 62, Thr and Ser residues 69 and 70, Thr residue 74, and Pro residue 75.  
     
     
         10 . The truncated Bcl-2 protein of  claim 8  comprising residues 51-85 (SEQ ID NO:1) and fragments of the truncated protein comprising residues 51-85 that contain at least one of Ser residue 51, Pro residue 57, Ser residue 62,.Thr and Ser residues 69 and 70, Thr residue 74, and Pro residue 75.  
     
     
         11 . The truncated Bcl-2 protein of  claim 8  which consists of residues 51 to any of 85-97 (SEQ ID NOS:1-13).  
     
     
         12 . The truncated Bcl-2 protein of  claim 8  which consists of residues 51-85 (SEQ ID NO:1).  
     
     
         13 . A method of screening for mutations in the AP domain of Bcl-2, said method comprising: 
 (a) isolating genomic DNA, cDNA or mRNA from a specimen to be screened;    (b) amplifying DNA fragments encoding the AP domain or portions thereof from the genomic DNA, cDNA or mRNA    (c) denaturing the amplified product;    (d) subjecting the denatured product to electrophoresis; and    (e) detecting mutations by comparing the mobility of the denatured amplified product to a control DNA encoding the AP domain or portions thereof corresponding positionally to the DNA fragments amplified in step (b);    wherein said control DNA encoding the AP domain or portions thereof is selected from the truncated bcl-2 gene and fragments thereof claimed in  claim 1 .    
     
     
         14 . The method of  claim 13 , wherein truncated bcl-2 gene extends from nucleotide no. 151—nucleotide no. 255 (SEQ ID NO:14).  
     
     
         15 . A method of screening for mutations in the AP domain of Bcl-2, said method comprising: 
 (a) isolating genomic DNA, cDNA or mRNA from a specimen to be screened;    (b) amplifying DNA fragments encoding the AP domain or portions thereof from the genomic DNA, cDNA or mRNA;    (c) mixing the amplified product with labeled PCR product from the corresponding position in a control AP domain or portion thereof;    (d) denaturing and annealing the mixed PCR products; and    (e) analyzing for mismatched nucleotides by electrophoresis following chemical modification;    wherein the control DNA encoding the AP domain or portion thereof is selected from the truncated bcl-2 gene and fragments thereof claimed in  claim 1 .    
     
     
         16 . The method of  claim 15 , wherein truncated bcl-2 gene extends from nucleotide no. 151—nucleotide no. 255 (SEQ ID NO:14).  
     
     
         17 . A method of screening for mutations in the AP domain of Bcl-2, said method comprising: 
 (a) isolating genomic DNA, cDNA or mRNA from a specimen to be screened;    (b) amplifying DNA fragments encoding the AP domain or portions thereof from the genomic DNA, cDNA or mRNA; and    (c) sequencing the amplified DNA product.    
     
     
         18 . An isolated cDNA comprising a sequence that encodes a polypeptide that binds in a double transformation to a truncated Bcl-2 protein defined by residues 51 to any of residues 85-97 (SEQ ID NOS:1-13) and fragments thereof that contain at least one amino acid needed to maintain full or partial anti-proliferation activity of the AP domain.  
     
     
         19 . The polypeptide encoded by the isolated cDNA of  claim 18 .  
     
     
         20 . A method for screening for a polypeptide that binds the AP domain of Bcl-2 protein, said method comprising: 
 (a) conducting a double transformation wherein one vector expresses a fusion protein comprising the AP domain or a fragment thereof and a reporter molecule and the other vector expresses a fusion protein comprising a complementary protein for the reporter molecule and said polypeptide to be screened;    (b) monitoring for activation of the reporter molecule; and    (c) isolating cDNA that encodes the protein that binds to said AP domain or said fragment thereof,    wherein said AP domain or fragment thereof is a truncated Bcl-2 protein comprising residues 51 to any of 85-97 (SEQ ID NOS:1-13) or a fragment thereof that contains at least one amino acid needed to maintain full or partial anti-proliferation activity of the AP domain.    
     
     
         21 . An isolated cDNA comprising a sequence that encodes a polypeptide that binds wt bcl-2 in a double transformation and does not bind Bcl-2Δ51 to 85-97 or deletions of a fragment of a bcl-2 gene that contains at least one codon encoding an amino acid needed to maintain full or partial anti-proliferation activity of the AP domain.  
     
     
         22 . A polypeptide encoded by the isolated cDNA of  claim 21 .  
     
     
         23 . A method of screening for a polypeptide that binds the AP domain of Bcl-2 protein, said method comprising: 
 (a) conducting a first double transformation wherein one vector expresses a fusion protein comprising the AP domain and a reporter molecule and the other vector expresses a fusion protein comprising a complementary protein for the reporter molecule and said polypeptide to be screened;    (b) conducting a second double transformation wherein one vector expresses a fusion protein comprising Bcl-2 with the AP domain or fragments of Bcl-2 that contain at least one amino acid needed to maintain full or partial anti-proliferation activity of the AP domain deleted and a reporter molecule and the other vector expresses a fusion protein comprising a complementary protein for the reporter molecule and said polypeptide to be screened;    (c) monitoring for activation of the reporter molecule in both double transformations; and    (d) isolating cDNA that encodes a polypeptide that binds in step (a) but not in step (b).    
     
     
         24 . A method for screening for a polypeptide that interacts with the AP domain of Bcl-2 protein, the method comprising: 
 (a) expressing cDNA that encodes a polypeptide to be screened;    (b) immobilizing the expressed polypeptide; and    (c) detecting interaction with a polypeptide comprising the AP domain or fragment thereof    wherein the AP domain or fragment thereof is a truncated Bcl-2 protein comprising residues 51 to any of residues 85-97 (SEQ ID NOS:1-13) or a fragment thereof that contains at least one amino acid needed to maintain full or partial anti-proliferation activity of the AP domain.    
     
     
         25 . A method for screening for a polypeptide that interacts with the AP domain of Bcl-2 protein, the method comprising: 
 (a) immobilizing a polypeptide comprising the AP domain or fragment of the AP domain;    (b) contacting the immobilized polypeptide with putative interacting protein; and    (c) identifying interacting protein;    wherein the AP domain or fragment thereof is a truncated Bcl-2 protein comprising residues 51 to any of residues 85-97 (SEQ ID NOS:1-13) or a fragment thereof that contains at least one amino acid needed to maintain full or partial anti-proliferation activity of the AP domain.    
     
     
         26 . An isolated antibody that binds to the AP domain of Bcl-2 or fragments of Bcl-2 that contain at least one amino acid needed to maintain full or partial anti-proliferation activity of the AP domain.  
     
     
         27 . The isolated antibody of  claim 27 , wherein the AP domain consists of Bcl-2 residue 51 to any of residues 85-97 (SEQ ID NO:1-13) or fragments of the AP domain that contain at least one of Ser residue 51, Pro residue 57, Ser residue 62, Thr and Ser residues 69 and 70, Thr residue 74, and Pro residue 75.  
     
     
         28 . The isolated antibody of  claim 26 , wherein the AP domain consists of Bcl-2 residues 51-85 (SEQ ID NO:1).  
     
     
         29 . The isolated antibody of any one of claims  26 ,  27  or  28 , wherein the antibody is a monoclonal antibody that specifically binds to the AP domain.  
     
     
         30 . A hybridoma that makes the monoclonal antibody of  claim 29 .  
     
     
         31 . A method of producing isolated AP domain or fragments thereof, said method comprising: 
 (a) constructing a vector comprising DNA encoding the AP domain or fragments thereof containing at least one codon encoding an amino acid needed to maintain full or partial anti-proliferation activity of the AP domain;    (b) transforming a suitable host cell with said vector of step (a);    (c) culturing said host cell under conditions that allow expression of said domain or fragments thereof by said host cell; and    (d) isolating said domain or fragment thereof expressed by said host cell of step (c).    
     
     
         32 . The method of  claim 31 , wherein said host cell is a mammalian cell.