Construction of a marker gene cassette which is repeatedly usable for gene disruption in yeast

A disruption cassette has been constructed containing the LEU2 gene flanked by directly repeated site-specific recombination sites of the yeast plasmid, pSB3, which resembles the 2 microns DNA of Saccharomyces cerevisiae. A disruption constructed by inserting this DNA fragment acquires a Leu+ phenotype, which can be easily removed by expressing the FLP-PSB3 gene encoding the site-specific recombinase of pSB3. A test was made using a Schizosaccharomyces pombe host. The ura4+ gene of S. pombe was replaced with the ura4::LEU2 gene constructed by inserting the disruption cassette into the ura4+ gene. Then, the FLP-pSB3 gene driven by the nmt1+ promoter was introduced into this disruptant. Upon de-repression of the nmt1 promoter by removing thiamine from the medium, the rate of appearance of Leu- was increased. As expected the ura4+ locus underwent a structural change. Thus, the FLP-pSB3 protein and its target site can function adequately in S. pombe.