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. Copyright © 2006 John Wiley & Sons, Ltd.