An Engineered Nonsense \u3cem\u3eURA3\u3c/em\u3e Allele Provides a Versatile System to Detect the Presence, Absence and Appearance of the [em\u3ePSI\u3c/em\u3e\u3csup\u3e+\u3c/sup\u3e] Prion in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e

Abstract

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 readthrough of certain nonsense mutations in a few auxotrophic markers, for example, ade1-14. This readthrough 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-fluoorotic 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