Heat-Inducible Degron: A Method for Constructing Temperature-Sensitive Mutants

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°C but rapidly degraded by the N-end rule pathway at 37°C. Fusions of ts Arg-DHFR to either Ura3 or Cdc28 of S. cerevisiae confer ts phenotypes specific for these gene products. Thus, Arg-DHFR^ts is a heat-inducible degradation signal that can be used to produce ts mutants without a search for ts mutations.

Additional Information

© 1994 American Association for the Advancement of Science. 27 September 1993; accepted 21 January 1994. We thank N. Kleckner (Harvard University) for the gift of plasmids and members of this laboratory, especially E. Johnson, K. Madura, F. Lévy, C. Byrd, and T. Clandinin, for plasmids and strains and for advice and comments on the manuscript. Supported by grants to A.V. from the National Institutes of Health. R.J.D. was supported by a postdoctoral fellowship from the European Molecular Biology Organization.

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