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.
Toh-e
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Department of Biology, Faculty of Science, University of Tokyo, Japan.
D015139:Blotting, Southern; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D008957:Models, Genetic; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase