Nevzgliadova O V, Smolianitskiĭ A G
Genetika. 1984 Feb;20(2):212-8.
The plasmid mutation AntR determining multiple resistance to antibiotics--tetracycline and cycloheximide in Saccharomyces cerevisiae was earlier obtained and genetically characterized. In this work we describe experiments on cytoduction and transformation, proving the localization of this mutation in the yeast 2 mu DNA. As a result of cotransformation of the sensitive cells carrying a double mutation in the gene LEU2 with the yeast vector marked by LEU2 and 2 mu DNA obtained from the yeast AntR mutant, the Leu+ AntR clones were selected. Though the primary co-transformans contain both plasmids in an unlinked state, we managed to get clones in which the markers AntR and LEU2 were linked. The putative recombinant molecules were cloned in Escherichia coli and then introduced into the yeast recipient cells, differing by the presence of the endogenous 2 mu DNA. Retransformation of cir0 cells results in the appearance of the clones in which LEU2 and AntR markers segregate together. Thus, the result of cotransformation and selection in vivo is that the mutation of multiple resistance was included into the yeast vector plasmid, presumably, in its 2 mu part.
早前已获得酿酒酵母中决定对四环素和环己酰亚胺多重抗生素抗性的质粒突变体AntR，并对其进行了遗传学特征分析。在本研究中，我们描述了关于细胞导入和转化的实验，证实了该突变位于酵母2μm DNA中。将携带LEU2基因双突变的敏感细胞与以LEU2为标记的酵母载体以及从酵母AntR突变体获得的2μm DNA进行共转化，筛选出Leu+ AntR克隆。尽管最初的共转化子中两个质粒处于不连锁状态，但我们成功获得了AntR和LEU2标记连锁的克隆。将推定的重组分子克隆到大肠杆菌中，然后导入具有内源性2μm DNA的酵母受体细胞。cir0细胞的再转化导致出现LEU2和AntR标记一起分离的克隆。因此，体内共转化和筛选的结果是多重抗性突变大概在其2μm部分被纳入酵母载体质粒。
