PCR-mediated direct gene disruption in Schizosaccharomyces pombe.
We have examined the feasibility and efficiency of PCR-mediated direct gene disruptions in the fission yeast Schizosaccharomyces pombe. In the present study, the S.pombe ura4+ gene was amplified by PCR with oligonucleotides that had short flanking regions ( approximately 40 bp) to the target gene. Using this purified PCR product we were able to disrupt genes in an S. pombe strain bearing aura4 deletion, with an efficiency ranging between 1 and 3% among selected transformants. The results indicated that despite S.pombe's preference for non-homologous or illegitimate recombination, even very short stretches of homologous regions could be used to target genes at a defined frequency in this organism. The successful disruption of four independent genes (sts1+, gcs1+, gsh2+and hmt1+) by this method further demonstrates that, despite the relatively low efficiency, the method is very feasible, and it's simplicity, especially when coupled to phenotype-based screening, should greatly facilitate disruption of genes in S.pombe.
Kaur
R
R
Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036, India.
Ingavale
S S
SS
Bachhawat
A K
AK
D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D005240:Feasibility Studies; D005656:Fungal Proteins / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005800:Genes, Fungal; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics*
D018528:ATP-Binding Cassette Transporters; D005656:Fungal Proteins; C085810:STS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins