Reporter assay systems for [URE3] detection and analysis

Methods. 2006 May;39(1):35-42. doi: 10.1016/j.ymeth.2006.04.008.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Transport Systems, Basic / genetics
  • Amyloid / analysis
  • Amyloid / genetics
  • Amyloid / metabolism
  • Culture Media / chemistry
  • Fungal Proteins / genetics
  • Gene Expression / genetics
  • Genes, Reporter / genetics*
  • Genetic Techniques
  • Genotype
  • Glutathione Peroxidase
  • Membrane Transport Proteins / genetics
  • Phenotype
  • Prions / analysis*
  • Prions / genetics
  • Prions / metabolism
  • Promoter Regions, Genetic / genetics
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / analysis*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • Amino Acid Transport Systems, Basic
  • Amyloid
  • CAN1 protein, S cerevisiae
  • Culture Media
  • DAL5 protein, S cerevisiae
  • Fungal Proteins
  • Membrane Transport Proteins
  • Prions
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Glutathione Peroxidase
  • URE2 protein, S cerevisiae