Struhl K
Gene. 1983 Dec;26(2-3):231-41. doi: 10.1016/0378-1119(83)90193-2.
A method that facilitates gene replacement at the HIS3 locus of Saccharomyces cerevisiae (yeast) has been developed. First, an internal region of the cloned HIS3 gene was replaced by a DNA segment containing the wild-type ribosomal protein gene, CYH2. Second, by using standard yeast transformation methods, the wild-type HIS3 locus of a cycloheximide resistant strain (cyh2r) was replaced by this his3-CYH2 substitution. The resulting strain is sensitive to cycloheximide because CYH2 is dominant to cyh2r. Third, his3 mutations cloned into integrating or replicating vectors were introduced into this strain by selecting transformants via the vector-encoded marker. Selection for cycloheximide-resistant colonies resulted in the replacement of the his3-CYH2 allele by newly introduced his3 alleles. Thus, this scheme provides for the direct selection of gene replacement events at the HIS3 locus independently of the phenotype of the cloned his3 derivatives. In principle, it can be extended to any region of the yeast genome.
一种促进酿酒酵母（酵母）HIS3位点基因替换的方法已被开发出来。首先，克隆的HIS3基因的一个内部区域被一个包含野生型核糖体蛋白基因CYH2的DNA片段所取代。其次，通过使用标准的酵母转化方法，一个抗环己酰亚胺菌株（cyh2r）的野生型HIS3位点被这个his3 - CYH2替换所取代。所得菌株对环己酰亚胺敏感，因为CYH2对cyh2r呈显性。第三，通过载体编码的标记选择转化体，将克隆到整合或复制载体中的his3突变引入该菌株。选择抗环己酰亚胺菌落导致his3 - CYH2等位基因被新引入的his3等位基因所取代。因此，该方案提供了在HIS3位点直接选择基因替换事件的方法，而与克隆的his3衍生物的表型无关。原则上，它可以扩展到酵母基因组的任何区域。
