Reporter assay systems for [URE3] detection and analysis.
The Saccharomyces cerevisiae prion [URE3] is the infectious amyloid form of the Ure2p protein. [URE3] provides a useful model system for studying amyloid formation and stability in vivo. When grown in the presence of a good nitrogen source, [URE3] cells are able to take up ureidosuccinate, an intermediate in uracil biosynthesis, while cells lacking the [URE3] prion can not. This ability to take up ureidosuccinate has been commonly used to assay for the presence of [URE3]. However, this assay has a number of practical limitations, affecting the range of experiments that can be performed with [URE3]. Here, we describe recently developed alternative selection methods for the presence or absence of [URE3]. They make use of the Ure2p-regulated DAL5 promoter in conjunction with ADE2, URA3, kanMX, and CAN1 reporter genes, and allow for higher stringency in selection both for and against [URE3], nonselective assay of prion variants, and direct transformation of prion filaments. We discuss advantages and limitations of each of these assays.
Brachmann
Andreas
A
Department Biologie I, Ludwig-Maximilians-Universität, Maria-Ward-Str. 1a, 80638 München, Germany. a.brachmann@lrz.uni-muenchen.de
Toombs
James A
JA
Ross
Eric D
ED
D026923:Amino Acid Transport Systems, Basic / Q000235:genetics; D000682:Amyloid / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D005821:Genetic Techniques; D005838:Genotype; D005979:Glutathione Peroxidase; D026901:Membrane Transport Proteins / Q000235:genetics; D010641:Phenotype; D011328:Prions / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D026923:Amino Acid Transport Systems, Basic; D000682:Amyloid; C502212:CAN1 protein, S cerevisiae; D003470:Culture Media; C503299:DAL5 protein, S cerevisiae; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011328:Prions; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae