The goal of the proposed research is to develop diagnostic reagents and new therapeutics for the cure or prevention of degenerative diseases through the isolation and analysis of human genes implicated in the pathways responsible for cell death. The investigator's approach to the identification of these genes involves a multi-step strategy for the detection of human homologues of previously characterized genes that are known to be centrally involved in cell death processes of the nematode Caenorhabditis elegans. In Phase I, the authors' homology cloning strategy was refined through the search for human homologues of the C. elegans genes ced-4, deg-1 and med-4. The results indicated that in order to develop probes capable of discriminating human homologues from non-homologous signals, steps must be taken to obtain a greater understanding of conserved protein sequences. By analyzing cell death gene homologues in related nematodes, several remnants of primordial amino acid sequences have been identified.This analysis has provided much of the information required to set criteria to identify homologues and to refine probes. In Phase II the revised search strategy will initially be implemented on non-nematodes using degenerate oligonucleotides derived from multiple conserved regions of cell death proteins. Genomic DNA, mRNA and cDNA libraries will be screened and amplified; candidate homologues will be analyzed by DNA sequencing. Should a structural human homologue be identified, the investigators will undertake preliminary functional studies as a prelude to assay development and to the discovery of novel cell- protective therapies.
