Nishikawa M, Suzuki K, Yoshida K
Botanical Institute, Faculty of Science, Hiroshima University.
Jpn J Genet. 1990 Oct;65(5):323-34. doi: 10.1266/jjg.65.323.
In order to establish a gene transfer system for yeast by promiscuous conjugation, we constructed plasmid pAY101 which contained an oriT sequence derived from RK2 (IncP) and the yeast TRP1 and ARS1 genes. A conjugation mixture consisted of yeast Saccharomyces cerevisiae, E. coli harboring pAY101, and E. coli carrying a helper plasmid with mob and tra. In the conjugation mixture a tryptophan-requiring yeast mutant (trp1) was converted to be prototrophic for tryptophan at a frequency of about 10(-5) to 10(-3) per recipient cell. This E. coli-yeast conjugation system required the mob, tra, oriT, TRP1 and ARS1 genes. The mob and tra genes were trans-acting elements as in an E. coli conjugation system. The mobilization was inhibited by nalidixic acid as in a typical bacterial conjugation. DNA analysis indicated that the plasmid pAY101 was transferred from E. coli to S. cerevisiae, and retained its original structure and function in yeast host cells.
为了通过杂乱结合建立酵母的基因转移系统，我们构建了质粒pAY101，其包含源自RK2（IncP）的oriT序列以及酵母TRP1和ARS1基因。一个结合混合物由酿酒酵母、携带pAY101的大肠杆菌以及携带具有mob和tra的辅助质粒的大肠杆菌组成。在结合混合物中，一个需要色氨酸的酵母突变体（trp1）以约每受体细胞10^(-5)至10^(-3)的频率转变为色氨酸原养型。这种大肠杆菌 - 酵母结合系统需要mob、tra、oriT、TRP1和ARS1基因。mob和tra基因如同在大肠杆菌结合系统中一样是反式作用元件。如同在典型的细菌结合中一样，萘啶酸抑制转移作用。DNA分析表明质粒pAY101从大肠杆菌转移至酿酒酵母，并在酵母宿主细胞中保留其原始结构和功能。
