An engineered nonsense URA3 allele provides a versatile system to detect the presence, absence and appearance of the [PSI+] prion in Saccharomyces cerevisiae.

Common methods to identify yeast cells containing the prion form of the Sup35 translation termination factor, [PSI+], involve a nonsense suppressor phenotype. Decreased function of Sup35p in [PSI+] cells leads to read-through of certain nonsense mutations in a few auxotrophic markers, e.g. ade1-14. This read-through results in growth on adenine-deficient media. While this powerful tool has dramatically facilitated the study of [PSI+], it is limited to a narrow range of laboratory strains and cannot easily be used to screen for cells that have lost the [PSI+] prion. Therefore we have engineered a nonsense mutation in the widely used URA3 gene, termed the ura3-14 allele. Introduction of the ura3-14 allele into an array of genetic backgrounds, carrying a loss-of-function URA3 mutation and [PSI+], allows for growth on media lacking uracil, indicative of decreased translational termination efficiency. This ura3-14 allele is able to distinguish various forms of the [PSI+] prion, called variants, and is able to detect the de novo appearance of [PSI+] in strains carrying the prion form of Rnq1p, [PIN+]. Furthermore, 5-fluoroorotic acid, which kills cells making functional Ura3p, provides a means to select for [psi-] derivatives in a population of [PSI+] cells marked with the ura3-14 allele, making this system much more versatile than previous methods.

Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients.

Calorie restriction increases life span in many organisms, including the budding yeast Saccharomyces cerevisiae. From a large-scale analysis of 564 single-gene-deletion strains of yeast, we identified 10 gene deletions that increase replicative life span. Six of these correspond to genes encoding components of the nutrient-responsive TOR and Sch9 pathways. Calorie restriction of tor1D or sch9D cells failed to further increase life span and, like calorie restriction, deletion of either SCH9 or TOR1 increased life span independent of the Sir2 histone deacetylase. We propose that the TOR and Sch9 kinases define a primary conduit through which excess nutrient intake limits longevity in yeast.

Genes determining yeast replicative life span in a long-lived genetic background.

Here we describe the replicative life spans of more than 50 congenic Saccharomyces cerevisiae strains, each carrying a mutation previously implicated in yeast aging. This analysis provides a direct comparison, in a single, long-lived strain background, of a majority of reported yeast aging genes. Of the eleven deletion mutations previously reported to increase yeast life span, we find that deletion of FOB1, deletion of SCH9, and deletion of GPA2, GPR1, or HXK2 (three genetic models of calorie restriction) significantly enhanced longevity. In addition, over-expression of SIR2 or growth on low glucose increased life span. These results define a limited number of genes likely to regulate replicative life span in a strain-independent manner, and create a basis for future epistasis analysis to determine genetic pathways of aging.

Sir2-independent life span extension by calorie restriction in yeast.

Calorie restriction slows aging and increases life span in many organisms. In yeast, a mechanistic explanation has been proposed whereby calorie restriction slows aging by activating Sir2. Here we report the identification of a Sir2-independent pathway responsible for a majority of the longevity benefit associated with calorie restriction. Deletion of FOB1 and overexpression of SIR2 have been previously found to increase life span by reducing the levels of toxic rDNA circles in aged mother cells. We find that combining calorie restriction with either of these genetic interventions dramatically enhances longevity, resulting in the longest-lived yeast strain reported thus far. Further, calorie restriction results in a greater life span extension in cells lacking both Sir2 and Fob1 than in cells where Sir2 is present. These findings indicate that Sir2 and calorie restriction act in parallel pathways to promote longevity in yeast and, perhaps, higher eukaryotes.