Premium
A ‘marker switch’ approach for targeted mutagenesis of genes in Schizosaccharomyces pombe
Author(s) -
MacIver Fiona H.,
Glover David. M.,
Hagan Iain M.
Publication year - 2003
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.983
Subject(s) - schizosaccharomyces pombe , biology , genetics , gene , locus (genetics) , mutagenesis , schizosaccharomyces , genome , computational biology , mutant
The completion of the Schizosaccharomyces pombe genome sequencing project has led to a dramatic acceleration of gene characterization in this system. Once a gene has been identified, the challenge then comes in using reverse genetics to generate a range of mutants in this gene of interest so that the powerful genetics and wealth of genetic backgrounds available in Sz. pombe can be exploited to study the function of the newly identified molecule. Beyond simple PCR‐tagging approaches, the high frequency with which illegitimate recombination occurs in Sz. pombe has made the manipulation of some loci complex, time consuming and a process of trial and error. Here we describe a simple ‘marker switch’ approach that enables the rapid selection of integration events at the locus of interest from an excessive background of integration at heterologous sites. We use the generation of temperature‐sensitive mutations in the plo1 + gene to validate this approach. Copyright © 2003 John Wiley & Sons, Ltd.