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.
Department of Biological Sciences, Laboratory for Molecular Biology, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, IL 60607, USA.
Anita L
AL
Manogaran
Kathryn T
KT
Kirkland
Susan W
SW
Liebman
D000483:Alleles; D018389:Codon, Nonsense / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D016254:Mutagenesis, Insertional; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010454:Peptide Termination Factors; D011328:Prions / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
D018389:Codon, Nonsense; D004271:DNA, Fungal; D005656:Fungal Proteins; D010454:Peptide Termination Factors; D011328:Prions; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D009963:Orotic Acid; C001242:5-fluoroorotic acid