Lagace, Mark2013-11-082013-11-0820042004Source: Dissertation Abstracts International, Volume: 66-05, Section: B, page: 2423.http://hdl.handle.net/10393/29129http://dx.doi.org/10.20381/ruor-12783This thesis documents the genetic characterization of the X-Linked Inhibitor of Apoptosis (XIAP) gene, and the subsequent discovery and characterization of a novel gene, called the Testis-specific IAP (TsIAP). Genomic library screening and bulk sequencing was used to isolate and sequence the genomic region that encompasses the XIAP gene. Following this, we screened cDNA libraries, as well as performed RT PCR and rapid amplification of cDNA ends (RACE) reactions to elucidate the full length cDNA sequence and intron/exon boundaries of XIAP. The XIAP gene spans a 33 kb region of chromosome X and is encoded in seven exons. The transcript is 10 kb in size, containing very large 5' and 3' untranslated regions. Four separate regions of the genome cross-react with probe derived from XIAP coding region sequence. These 'pseudogenes' were sequenced and characterized. Three of the pseudogenes appeared to be derived from retrotransposition events as they contained partially conserved copies of XIAP lacking any introns. One of these pseudogenes turned out to maintain a viable open reading frame and encoded a novel protein with expression limited to the testis. We employed a similar library screening strategy to clone and sequence the novel testis-specific IAP gene. FISH analysis was performed to localize the gene to chromosome 19q13.4. The TsIAP gene was found to encode a 2kb mRNA with an open reading frame encoding a region highly homologous to the carboxy-terminal end of the X-linked IAP. Attempts were made to find a murine homologue of TsIAP with no success, and later work published by another group confirmed that the TsIAP gene is a recent evolutionary event and that no murine homologue exists. Given the unusually long 5' UTR present in the TsIAP gene, we tested whether the mRNA could produce a protein by using Western blot analysis on both overexpressed TsIAP in cell culture, as well as endogenous expression in testicular protein extracts. A band was detected in both cases migrating at approximately 26kDa, the predicted size of the TsIAP protein. Having confirmed that the novel gene we isolated is capable of being expressed, we proceeded to characterize its functional properties. TsIAP was able to protect against apoptosis, however, only against a limited subset of apoptotic triggers when compared to its parent gene XIAP. TsIAP could not protect against the chemotoxic drugs etoposide or adriamycin, however, it showed a strong protective effect against BaX-induced apoptosis. Surprisingly, against Bax-induced apoptosis, TsIAP was a more potent inhibitor than the corresponding region of XIAP. Smac (second mitochondrial activator of caspases) is an important inhibitor of XIAP function in the context of the mitochondrial pathway of apoptosis induced by Bax. We determined that Smac was unable to bind to the TsIAP protein in pull-down experiments and thus could not reverse the protective effects of TsIAP in a Bax induced apoptosis model.177 p.enBiology, Molecular.Thesis