Roitgrund C, Steinlauf R, Kupiec M
Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel.
Mol Gen Genet. 1993 Feb;237(1-2):306-10. doi: 10.1007/BF00282812.
We describe here a new method for the introduction of non-selectable alleles into Saccharomyces cerevisiae, gene replacement by donation. This method only requires the availability of an autonomously replicating, selectable plasmid containing the allele to be introduced into yeast. The plasmid is digested at a restriction site (or sites) within this allele, and introduced into yeast by transformation. In the course of double-strand break repair, the entering plasmid donates genetic information to the chromosome, replacing the chromosomal allele in a gene conversion-like event. Gene replacement events are identified by a phenotypic screen of the transformants. When necessary, the transforming plasmid may be subsequently lost by segregation during permissive growth. We have studied several parameters affecting the utility of this protocol as a method of gene replacement. Together with our previous results, the results show gene replacement by donation to be a useful, facile method, yielding gene replacement in up to 1.5% of transformants.
我们在此描述一种将非选择性等位基因导入酿酒酵母的新方法——通过供体进行基因置换。该方法仅需一个含有待导入酵母的等位基因的自主复制、可选择质粒。将该质粒在该等位基因内的一个或多个限制酶切位点处消化，然后通过转化导入酵母。在双链断裂修复过程中，导入的质粒将遗传信息捐赠给染色体，在类似基因转换的事件中取代染色体等位基因。通过对转化子进行表型筛选来鉴定基因置换事件。必要时，在允许生长期间，转化质粒可能随后通过分离而丢失。我们研究了影响该方案作为基因置换方法实用性的几个参数。与我们之前的结果一起，这些结果表明通过供体进行基因置换是一种有用且简便的方法，在高达1.5%的转化子中产生基因置换。
