Mammalian IAP gene family, primers, probes, and detection methods

Abstract

Disclosed is substantially pure DNA encoding mammalian IAP polypeptides; substantially pure polypeptides; and methods of using such DNA to express the IAP polypeptides in cells and animals to inhibit apoptosis. Also disclosed are conserved regions characteristic of the IAP family and primers and probes for the identification and isolation of additional IAP genes. In addition, methods for treating diseases and disorders involving apoptosis are provided.

1 . A substantially pure nucleic acid encoding a mammalian inhibitor of apoptosis: protein (IAP) polypeptide, wherein said inhibitor of apoptosis protein is a protein that modulates apoptosis and comprises a ring zinc finger domain and at least one baculovirus inhibitor of apoptosis repeat (BIR) domain.  
     
     
         2 . The nucleic acid of  claim 1 , wherein said polypeptide has at least two BIR domains.  
     
     
         3 . The nucleic acid of  claim 2 , wherein said polypeptide has at least three BIR domains.  
     
     
         4 . The nucleic acid of  claim 1 , wherein said nucleic acid contains the X-linked inhibitor of apoptosis protein (xiap) gene.  
     
     
         5 . The nucleic acid of  claim 1 , wherein said nucleic acid contains the human inhibitor of apoptosis protein 2 (hiap2) gene.  
     
     
         6 . The nucleic acid of  claim 1 , wherein said nucleic acid contains the human inhibitor of apoptosis protein 1 (hiap1) gene.  
     
     
         7 . The nucleic acid of  claim 1 , wherein said nucleic acid is genomic DNA.  
     
     
         8 . The nucleic acid of  claim 1 , wherein said nucleic acid is cDNA.  
     
     
         9 . The nucleic acid of  claim 1 , wherein said nucleic acid is mammalian DNA.  
     
     
         10 . The nucleic acid of  claim 9 , wherein said mammalian DNA is human DNA.  
     
     
         11 . The nucleic acid of  claim 11 , wherein said mammalian DNA is murine DNA.  
     
     
         12 . A substantially pure nucleic acid having the sequence of  FIG. 5  (SEQ ID NO: 39), or degenerate variants thereof, and encoding the amino acid sequence of  FIG. 5  (SEQ ID NO: 40).  
     
     
         13 . A substantially pure nucleic acid having the sequence of  FIG. 6  (SEQ ID NO: 41), or degenerate variants thereof, and encoding the amino acid sequence of  FIG. 6  (SEQ ID NO: 42).  
     
     
         14 . A method of producing a mammalian IAP polypeptide comprising: 
 (a) providing a cell transformed with a nucleic acid encoding a mammalian IAP polypeptide positioned for expression in said cell, said polypeptide comprising a ring zinc finger (RZF) domain;    (b) culturing said transformed cell of step (a) under conditions for expressing said nucleic acid; and    (c) producing said IAP polypeptide.    
     
     
         15 . The method of  claim 14 , wherein said mammalian IAP polypeptide is murine human inhibitor of apoptosis protein 1 (m-HIAP1).  
     
     
         16 . The method of  claim 14 , wherein said mammalian IAP polypeptide is murine human inhibitor of apoptosis protein 2 (m-HIAP2).  
     
     
         17 . A substantially pure nucleic acid that hybridizes to a probe of at least 40 nucleotides in length, said probe derived from the nucleic acid sequence of  FIG. 5  (SEQ ID NO: 39), wherein said nucleic acid hybridizes to said probe under low stringency conditions, said conditions comprising washing with 2×SSC at 40° C., and wherein said nucleic acid encodes a mammalian LAP polypeptide, said polypeptide comprising a ring zinc finger domain and at least one BIR domain.  
     
     
         18 . A substantially pure nucleic acid that hybridizes to a probe of at least 40 nucleotides in length, said probe derived from the DNA sequence of  FIG. 6  (SEQ ID NO: 41), wherein said nucleic acid hybridizes to said probe under low stringency conditions, said conditions comprising washing with 2×SSC at 40° C., and wherein said nucleic acid encodes a mammalian IAP polypeptide, said polypeptide comprising a ring zinc finger domain and at least one BIR domain.  
     
     
         19 . A substantially pure nucleic acid encoding a BIR domain, said nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.  
     
     
         20 . A substantially pure nucleic acid encoding a ring zinc finger domain, said nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, and SEQ ID NO: 68.  
     
     
         21 . A substantially pure nucleic acid that hybridizes to a probe of at least 40 nucleotides in length, said probe derived from the DNA sequence of  FIG. 1  (SEQ ID NO: 3), wherein said nucleic acid hybridizes to said probe under low stringency conditions, said conditions comprising washing with 2×SSC at 40° C., and wherein said nucleic acid encodes a mammalian IAP polypeptide, said polypeptide comprising a ring zinc finger domain and at least one BIR domain.  
     
     
         22 . A substantially pure nucleic acid that hybridizes to a probe of at least 40 nucleotides in length, said probe derived from the DNA sequence of  FIG. 2  (SEQ ID NO: 5), wherein said nucleic acid hybridizes to said probe under low stringency conditions, said conditions comprising washing with 2×SSC at 40° C., and wherein said nucleic acid encodes a mammalian IAP polypeptide, said polypeptide comprising a ring zinc finger domain and at least one BIR domain.  
     
     
         23 . A substantially pure nucleic acid that hybridizes to a probe of at least 40 nucleotides in length, said probe derived from the DNA sequence of  FIG. 3  (SEQ ID NO: 7), wherein said nucleic acid hybridizes to said probe under low stringency conditions, said conditions comprising washing with 2×SSC at 40° C., and wherein said nucleic acid encodes a mammalian IAP polypeptide, said polypeptide comprising a ring zinc finger domain and at least one BIR domain.  
     
     
         24 . A substantially pure nucleic acid that hybridizes to a probe of at least 40 nucleotides in length, said probe derived from the DNA sequence of  FIG. 4  (SEQ ID NO: 9), wherein said nucleic acid hybridizes to said probe under low stringency conditions, said conditions comprising washing with 2×SSC at 40° C., and wherein said nucleic acid encodes a mammalian IAP polypeptide, said polypeptide comprising a ring zinc finger domain and at least one BIR domain.  
     
     
         25 . A substantially pure nucleic acid probe of at least 40 nucleotides in length, wherein said probe hybridizes to the DNA sequence of  FIG. 1  (SEQ ID NO: 3) under low stringency conditions, said conditions comprising washing with 2×SSC at 40° C.  
     
     
         26 . A substantially pure nucleic acid probe of at least 40 nucleotides in length, wherein said probe hybridizes to the DNA sequence of  FIG. 2  (SEQ ID NO: 5) under low stringency conditions, said conditions comprising washing with 2×SSC at 40° C.  
     
     
         27 . A substantially pure nucleic acid probe of at least 40 nucleotides in length, wherein said probe hybridizes to the DNA sequence of  FIG. 3  (SEQ ID NO: 7) under low stringency conditions, said conditions comprising washing with 2×SSC at 40° C.  
     
     
         28 . A substantially pure nucleic acid probe of at least 40 nucleotides in length, wherein said probe hybridizes to the DNA sequence of  FIG. 4  (SEQ ID NO: 9) under low stringency conditions, said conditions comprising washing with 2×SSC at 40° C.  
     
     
         29 . A substantially pure nucleic acid probe of at least 40 nucleotides in length, wherein said probe hybridizes to the DNA sequence of  FIG. 6  (SEQ ID NO: 41) under low stringency conditions, said conditions comprising washing with 2×SSC at 40° C.