Rudolph H, Koenig-Rauseo I, Hinnen A
Gene. 1985;36(1-2):87-95. doi: 10.1016/0378-1119(85)90072-1.
A general method to replace chromosomal DNA sequences of Saccharomyces cerevisiae by any in vitro modified DNA sequence has been developed and was applied to the PHO5 locus on chromosome II. A recipient strain was constructed in which part of the chromosomal PHO5 sequence was substituted by the URA3 gene. Replacement of this pho5-URA3 substitution by pho5 mutant alleles was achieved in one step by cotransformation with a pho5 DNA fragment and the self-replicating plasmid YEp13, which contains the LEU2 gene as a selectable marker. Leu+ transformants were selected, and the replacement events at the PHO5 locus were detected by their Ura- phenotype (1-4% of the Leu+ were Ura-). In a similar way the PHO5 coding sequence was replaced by the sequence coding for human tissue-type plasminogen activator (t-PA).
已开发出一种用任何体外修饰的DNA序列替换酿酒酵母染色体DNA序列的通用方法，并将其应用于第二条染色体上的PHO5位点。构建了一个受体菌株，其中染色体PHO5序列的一部分被URA3基因取代。通过用pho5 DNA片段和含有LEU2基因作为选择标记的自我复制质粒YEp13共转化，一步实现了用pho5突变等位基因替换该pho5-URA3替换。选择亮氨酸+转化体，并通过其尿嘧啶-表型检测PHO5位点的替换事件（亮氨酸+中有1-4%为尿嘧啶-）。以类似的方式，PHO5编码序列被编码人组织型纤溶酶原激活剂（t-PA）的序列所取代。
