| Apoptosis is a form of cell death executed by a class of cysteine proteases called caspases. Though caspases are well-conserved, the mechanisms by which caspases are regulated vary remarkably. This dissertation addresses three independent aspects of apoptosis and its regulation.; In the developing Drosophila eye, apoptosis is activated to remove extra cells that are initially present between ommatidia. Mutants for the gene echinus have a disorganized eye structure due to a failure of these cell deaths to occur. We demonstrate that echinus resembles a deubiquitinating enzyme, that it is expressed in the pupal eye during the time of cell death, and that echinus acts genetically upstream or independently of the death-inducing genes head involution defective, reaper, and grim. Based on in vitro assays and the fact that the Echinus enzyme lacks a catalytic cysteine residue, we propose that echinus and its orthologs constitute a novel class of inactive deubiquitinating enzymes, perhaps functioning in a dominant-negative manner to inhibit deubiquitination of specific substrates.; In C. elegans, the model for caspase inhibition is quite different from that in Drosophila and in mammals. To look for genes that directly inhibit the CED-3 caspase, we screened a C. elegans cDNA library for CED-3 suppressors in the yeast S. cerevisiae and found several suppressors. Experiments in yeast suggest that one of these genes, Y39B6A.12, requires the prodomain of CED-3 for suppression, and ectopic expression in the Drosophila eye shows that it can suppress apoptosis induced by the Bcl2 family member Debcl.; In Drosophila, DIAP1 is the focal point in the regulation of apoptosis. To identify novel regulators of DIAP1, deficiency chromosomes spanning the Drosophila genome were screened for dominant modifiers of a diap1 knockdown phenotype. Nine deficiencies were isolated that cover no known regulators, and two modifiers were mapped to small genomic regions. This screen has provided a starting point for identifying some of the many uncharacterized genes that are involved in regulating apoptosis. |
