Longevity, even in simple organisms, is undoubtedly regulated by multiple genetic loci. We propose to identify and functionally characterize genes that control one or more aspect of longevity in cultured human fibroblasts, the yeast Saccharomyces cerevisiae, and genetically heterogeneous and selectively bred mice. Genes that modulate the finite proliferative lifespan of human fibroblasts will be identified by a highly selective differential cloning strategy. Candidate senescence-modulating human genes will be cloned and tested for three functions: 1) inhibition of cell proliferation; 2) stimulation of DNA synthesis or mitosis; and 3) because it is unlikely that any single gene controls cellular senescence, cooperation with wild-type or mutant viral oncogenes in stimulating DNA synthesis or mitosis. In collaboration with Dr. Michal Jazwinski, S. cerevisiae will be used to 1) determine the effects of these genes on (yeast) longevity; 2) establish whether they act as dominant or recessive alleles; 3) isolate yeast genes that enhance or suppress their effects. In parallel, yeast genes shown by Jazwinski's laboratory to affect longevity will be tested for function in human fibroblasts. Yeast strains bearing or requiring viral oncogenes will be used to isolate additional yeast and human genes that control longevity or proliferative senescence. In collaboration with Dr. Richard Miller, genes that modulate senescence or longevity will be assessed for expression in young and old mice, either genetically heterogeneous or selectively bred for late reproduction or high or low immune function. mRNAs that are differentially expressed by pure cell types isolated from young, old or selectively bred animals will be identified, characterized for age- and tissue-specific expression, and a subset will be cloned and tested for function in human fibroblasts and S. cerevisiae. The long range goal is to identify strong candidates for human longevity assurance genes, and to know enough about their function to develop rational strategies for testing in transgenic animals and, ultimately, for therapeutic or interventional applications in humans.