The objectives of the research described in this proposal are to generate a variety of stress-resistant mutants in the yeast S. cerevisiae and in the nematode C. elegans, with an emphasis on mutants resistant to oxidative stress. Detailed biochemical and genetic characterization of these mutants will be followed by their use to address the mechanisms of aging of a multicellular organism such as C. elegans and of the mother cells of a unicellular organism such as S. cerevisiae. In particular, we shall test, using a generally applicable approach, a hypothesis according to which the species-specific life spans of iteroparous organisms are an indirect consequence of optimization rather than maximization of the repair/defense systems of a species in the course of its evolution.
Specific Aims 1) Isolation and analysis of yeast (S. cerevisiae) mutants hyperresistant to an acute (short-term) exposure to hydrogen peroxide. 2) Determination of mother cell life spans and generation times in the hyperresistant mutants, and comparison with life spans of mother cells in congenic wild-type strains. 3) Isolation, using a turbidostat, and analysis of S. cerevisiae mutants hyperresistant to a chronic (continuous) exposure to either hydrogen peroxide or buthionine sulfoximine, an inhibitor of glutathione biosynthesis. 4) Direct selection for S. cerevisiae strains with an increased mother cell longevity by repeated enrichments for older mothers, using a cell sorter-based experimental approach. 5) Isolation and analysis of nematode (C. elegans) mutants hyperresistant to an acute exposure to hydrogen peroxide. 6) Isolation, using a modified chemostat technique, and analysis of C. elegans mutants hyperresistant to a chronic exposure to either hydrogen peroxide or buthionine sulfoximine. Examination of life spans of the above C. elegans mutants and parental strains. Detailed mechanistic and functional understanding of the stress response systems, in particular of those that underlie the resistance to oxidative stress, is expected to illuminate a number of biological problems, and especially the problem of aging.
