During the past decade, convincing evidence has accumulated in several invertebrate systems demonstrating that longevity is a genetically determined trait. This evidence has bolstered the conviction that aging possesses a genetic determinant in vertebrates as well. Longevity is clearly a polygenic trait. The objectives of the proposed research are two-fold. First, we wish to develop and to implement a relatively simple system for the evaluation of candidate longevity-assurance genes (LAGs), genes that play a role in determining longevity. The experimental system we will utilize is the yeast Saccharomyces cerevisiae. Second, we plan to determine whether the yeast longevity-assurance gene LAG1 is involved in determining life span or maintaining """"""""youthful"""""""" function in mammals, as a model for the analysis of other yeast LAGs in higher eukaryotes. Specifically: a) A functional cloning approach will be adapted to isolate yeast homologs of the SV4O T-antigen and then of candidate nematode and fruit fly LAGs. These genes will be directly tested for LAG function in yeast; b) The human cDNA homologous to yeast LAG1 will be cloned and characterized.
This aim will include an examination of the level of expression of the human gene in a variety of human cell types and tissues. It will also involve an analysis of expression during the replicative life span of human diploid fibroblasts in tissue culture and in fibroblasts from human donors of different ages; c) The effects on the replicative life span of fibroblasts in culture of overexpressing human LAG1 and of eliminating its expression will be examined; d) The mouse cDNA homolog of LAG1 will be cloned and characterized. The expression of the gene in the mouse will be ascertained at various stages of development and during the adult life span by in situ hybridization. This analysis will form the prelude to the final specific aim; e) Transgenic mice overexpressing LAG1, as well as mice expressing dominant-negative mutations of LAG1, will be generated. The effects of these genetic alterations on the development, aging, and life span of the animal will be determined. The overall goal of the proposed studies is to pave a two-way street in which yeast LAGs can be tested in mammals and candidate LAGs from other eukaryotes can be tested rapidly in yeast. This research should provide insights into the basic biological processes of aging and thus to contribute to the quality of the later years of life in humans.
