Toh-e A
Department of Biology, Faculty of Science, University of Tokyo, Japan.
Curr Genet. 1995 Mar;27(4):293-7. doi: 10.1007/BF00352095.
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.
构建了一个破坏盒，其中包含LEU2基因，其两侧是酵母质粒pSB3的直接重复位点特异性重组位点，该质粒类似于酿酒酵母的2微米DNA。通过插入该DNA片段构建的破坏株获得亮氨酸+表型，通过表达编码pSB3位点特异性重组酶的FLP-PSB3基因可以轻松去除该表型。使用粟酒裂殖酵母宿主进行了一项测试。将破坏盒插入粟酒裂殖酵母的ura4+基因中构建ura4::LEU2基因，从而取代ura4+基因。然后，将由nmt1+启动子驱动的FLP-pSB3基因导入该破坏株。通过从培养基中去除硫胺素使nmt1启动子去阻遏后，亮氨酸-的出现率增加。正如预期的那样，ura4+位点发生了结构变化。因此，FLP-pSB3蛋白及其靶位点在粟酒裂殖酵母中能够充分发挥作用。
