A strategy for the generation of conditional mutations by protein destabilization.

Abstract:

:Conditional mutations such as temperature-sensitive (ts) mutations are important for the analysis of protein function but are often difficult, or impossible, to obtain. Here we present a simple method for generating conditional mutations based on the use of a protein-destabilizing genetic element in combination with systems allowing the induction and repression of gene expression. This genetic cassette can be fused to other protein-coding sequences, and once transcription is turned off and synthesis of the gene product ceases, the preexisting protein is rapidly degraded. We have applied this method to the analysis of the yeast ARD1 gene product, a subunit of an N-terminal acetyltransferase, and show that a complete loss of ARD1 product can be achieved in less than one generation. Despite the rapid loss of ARD1 protein, there is a prolonged delay in the expression of the ard1 mutant phenotype, suggesting that the acetylated substrates of ARD1 are metabolically stable and/or exert a long-lasting effect on processes such as the repression of the silent mating type cassettes.

authors

Park EC,Finley D,Szostak JW

doi

10.1073/pnas.89.4.1249

keywords:

subject

Has Abstract

pub_date

1992-02-15 00:00:00

pages

1249-52

issue

4

eissn

0027-8424

issn

1091-6490

journal_volume

89

pub_type

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