The N-degron approach to create temperature-sensitive mutants in Schizosaccharomyces pombe.
Conditional mutants are a vital tool for analysis of gene function. The use of temperature-sensitive mutants in Schizosaccharomyces pombe has significantly promoted understanding of many cellular processes. A portable heat-inducible amino-terminal degron (N-degron) for conditional degradation of a gene product has been previously described in Saccharomyces cerevisiae. This paper describes the adaptation of the N-degron method to create temperature-sensitive (ts) mutants in S. pombe. A ts derivative of the mouse dihydrofolate reductase with an amino-terminal arginine (Arg-DHFR(ts)) previously described in S. cerevisiae was fused to the N-terminus of Bir1p, a nuclear protein involved in mitotic chromosome segregation in S. pombe. This fusion allele, referred to as bir1-td, conferred a chromosome segregation defect at 36 degrees C, as with previously described alleles of bir1. Deletion of the S. pombe E3 ubiquitin ligase (N-recognin), Ubr11p, reversed the temperature-dependent lethality of bir1-td, providing evidence for N-end rule mediated destruction of Bir1p. The methods we describe should therefore facilitate analysis of essential genes in fission yeast for which conditionally lethal mutants are unavailable.
Rajagopalan
Srividya
S
Laboratory of Cell Division, Temasek Life Sciences Laboratory 1, Research Link, National University of Singapore, Singapore 117604, Singapore.
Liling
Zheng
Z
Liu
Jianhua
J
Balasubramanian
Mohan
M
D009154:Mutation / Q000235:genetics*; D010446:Peptide Fragments / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics*; D013696:Temperature
D010446:Peptide Fragments