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 t he present study, the S.pombe ura4(+) gene was amplified by PCR with o ligonucleotides that had short flanking regions (similar to 40 bp) to the target gene, Using this purified PCR product we were able to disru pt genes in an S.pombe strain bearing a ura4 deletion, with an efficie ncy ranging between 1 and 3% among selected transformants, The results indicated that despite S.pombe's preference for non-homologous or ill egitimate recombination, even very short stretches of homologous regio ns could be used to target genes at a defined frequency in this organi sm. The successful disruption of four independent genes (sts1(+), gcs1 (+), gsh2(+) and hmt1(+)) by this method further demonstrates that, de spite 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.