Inducible degron and its application to creating conditional mutants.

Abstract

Conditional mutants are important tools particularly in the analysis of essential genes. In this chapter, a method is described that allows for a rapid design-based generation of temperature-sensitive alleles of many Saccharomyces cerevisiae genes. The method employs a temperature-inducible degron, denoted as td, which, when linked to the N-terminus of proteins to be studied, targets them for rapid degradation via the ubiquitin-dependent N-end rule pathway. Targeting, however, occurs only at elevated (restrictive) temperatures, whereas at lower (permissive) temperatures the degron is inactive. Strategies to generate td alleles are described, and the limitations of the method are discussed.