Heat-inducible degron and the making of conditional mutants

Methods Enzymol. 2005;399:799-822. doi: 10.1016/S0076-6879(05)99052-6.

Abstract

Conditional mutants retain the function of a specific gene under one set of conditions, called permissive, and lack that function under a different set of conditions, called nonpermissive; the latter must be still permissive for the wild-type allele of a gene. Such mutants make possible the analysis of physiological changes that follow controlled inactivation of a gene or gene product and can be used to address the function of any gene. Temperature-sensitive (ts) mutants, first used in functional studies more than half a century ago, remain a mainstay of genetic analyses. One limitation of the classical ts approach is the uncertainty as to whether a given gene can be mutated to yield a ts product. Another problem with conventional ts mutations is that they are often too leaky to be useful. In 1994, we described a new method, based on a heat-activated degradation signal (degron) that is targeted by the N-end-rule pathway in the yeast Saccharomyces cerevisiae. The corresponding mutants were termed td (temperature-activated degron) to distinguish them from conventional ts mutants. The td method requires neither a missense mutation in a gene of interest nor an alteration in its expression patterns. Arg-DHFR(ts), a ts variant of dihydrofolate reductase-bearing N-terminal Arg residue (a destabilizing residue in the N-end rule) was shown to function as a portable, heat-activated degron, in that Arg-DHFR(ts) was long-lived at 23 degrees but became short-lived at 37 degrees , owing to activation of its previously cryptic degron. Linking, in a linear fusion, this portable ts-degron to a protein of interest results in destruction of the latter at 37 degrees , thereby yielding a ts (td) mutant of a corresponding gene. Since the introduction of the td method in 1994, numerous studies have successfully used td alleles of specific genes in functional analyses.

Publication types

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

MeSH terms

  • Base Sequence
  • DNA Primers
  • Hot Temperature*
  • Mutation*
  • Plasmids
  • Polymerase Chain Reaction
  • Saccharomyces cerevisiae Proteins / biosynthesis*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Ubiquitin / metabolism

Substances

  • DNA Primers
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin