Because of problems associated with some of the more conventional approaches to the genes and gene products involved in mitotic recombination in mammalian cells, we have decided to use the fission yeast, Schizosaccharomyces pombe, to learn more about the proteins involved in this process. In view of the predominance of recombinational repair in S. pombe, the absence of photo-reactivation in this organism, the intimate link between recombination/repair pathways in all organisms and the radiation-sensitive phenotypes associated with mutations in many bona fide recombination genes in prokaryotes and eukaryotes, we will screen existing radiation-sensitive mutants for those that are aberrant in mitotic recombination, isolate new mutants aberrant in reciprocal and non-reciprocal mitotic recombination and characterize the genes that complement some of these mutants. The choice of S. pombe is based on the fact that it offers some special advantages over S. cerevisiae for the isolation of mammalian homologs of recombination genes, if they exist.
The specific aims are: 1.To test whether any of the existing S. pombe radiation-sensitive (rad) mutants are aberrant in mitotic recombination. 2.To isolate and characterize new S.pombe rec mutants affected in mitotic recombination. 3.To complement several (about 6) of these rad/rec mutants with S. pombe genomic libraries. 4.To obtain S. cerevisiae and mammalian homologs of these genes, if they exist,. directly (by complementation) or indirectly (using antibody or oligonucleotide probes). 5.To elucidate the function of some of the gene products using genetic (e.g. gene disruption, mutant alleles, extragenic suppressors and dominant mutations in S. pombe), immunological and biochemical techniques, with priority given to those genes that have mammalian homologs or complement hyper-rec phenotypes.
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