Heat-inducible degron: a method for constructing temperature-sensitive mutants

Science. 1994 Mar 4;263(5151):1273-6. doi: 10.1126/science.8122109.

Abstract

A temperature-sensitive (ts) mutant retains the function of a gene at a low (permissive) temperature but not at a high (nonpermissive) temperature. Arg-DHFR, a dihydrofolate reductase bearing an amino-terminal (N-terminal) arginine, is long-lived in the yeast Saccharomyces cerevisiae, even though arginine is a destabilizing residue in the N-end rule of protein degradation. A ts derivative of Arg-DHFR was identified that is long-lived at 23 degrees C but rapidly degraded by the N-end rule pathway at 37 degrees C. Fusions of ts Arg-DHFR to either Ura3 or Cdc28 of S. cerevisiae confer ts phenotypes specific for these gene products. Thus, Arg-DHFRts is a heat-inducible degradation signal that can be used to produce ts mutants without a search for ts mutations.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • CDC28 Protein Kinase, S cerevisiae / genetics
  • CDC28 Protein Kinase, S cerevisiae / metabolism
  • Fungal Proteins / genetics*
  • Fungal Proteins / metabolism
  • Half-Life
  • Hot Temperature
  • Mutagenesis*
  • Phenotype
  • Recombinant Fusion Proteins / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Temperature
  • Tetrahydrofolate Dehydrogenase / genetics*
  • Tetrahydrofolate Dehydrogenase / metabolism
  • Ubiquitins

Substances

  • Fungal Proteins
  • Recombinant Fusion Proteins
  • Ubiquitins
  • Tetrahydrofolate Dehydrogenase
  • CDC28 Protein Kinase, S cerevisiae