| Popis: |
Apoptosis is characterized by a series of well defined morphological and biochemical features that allow cells to initiate self-destruction in response to a variety of stimuli. CD4⁺CD8⁺ is a sub-population of immature thymocytes that are especially prone to the action of apoptosis-inducing agents and are sensitive to glucocorticoid-induced apoptosis, an event that plays a critical role in eliciting the antigen-specific thymocyte repertoire. Glucocorticoids induce apoptosis through activation of the GR, a ligand-induced transcription factor that transduces the hormonal signals into the regulated expression of target genes. While much is known about the structure and function of GR, key steroid-regulated genes believed to be required for thymocyte apoptosis have not been found. Based on the transcriptional-regulation of apoptosis by ecdysone-mediated induction of reaper gene expression in Drosophila, and p53-mediated transcriptional-activation of Bax gene expression in mammalian cells, our laboratory has set out to identify GR-regulated genes in a glucocorticoid-sensitive mouse thymocyte cell line called WEHI 7.2. Identification of such genes involved in this process, and elucidation of the underlying mechanisms by which they mediate apoptosis, have prime importance because malfunctioning of this system has been shown to cause severe human diseases and developmental abnormalities. I have isolated and characterized the full-length nucleotide sequence of mGIG1, a novel gene product that had previously been shown to be up-regulated in thymocytes undergoing glucocorticoid-induced apoptosis. The human homolog of the mouse GIG1 was also identified and mapped to chromosome 8, band q21. A peptide segment of GIG1 protein was expressed in bacteria and used to raise antibodies in rabbits in order to dissect its role in thymocyte apoptosis. Results of DNA sequence analyses suggest that mGIG1 may encode a DNA-binding protein that could function as a transcription factor in thymocytes. In a second project, I established a transient expression cloning system in mouse thymoma cells and isolated a cDNA sequence called mArg-2 on the basis of a functional cloning strategy. However, determination of the possible involvement of both GIG1 and Arg-2 in thymocyte apoptosis will require further experimentation. Finally, spatial and temporal coordination of abrupt changes in mismatch repair (MMR) gene expression with occurrence of apoptosis in mouse thymocytes prompted me to study apoptosis in MMR-deficient mice. My findings indicate that glucocorticoid and anti-Fas antibody-induced cell death machinery is intact and functional in MMR⁻/⁻ mice. |
