Dolzani L, Lagatolla C, Monti-Bragadin C
Institute of Microbiology, University of Trieste, Italy.
Mutat Res. 1991 Nov;264(3):127-34. doi: 10.1016/0165-7992(91)90129-r.
Plasmid DNA modified by in vitro treatments was transformed in E. coli bacterial cells. A streptomycin-resistant strain, carrying the peculiar rpsL421 mutation, was used as a recipient for the cloning vector pNO1523, which carries the wild-type (streptomycin-sensitive) rpsL allele. Transformants were streptomycin-sensitive unless a change in plasmid sequence had occurred. The analysis of the MaeI restriction pattern of plasmids isolated from streptomycin-resistant transformants, together with the detection of the phenotype that they conferred to a streptomycin-dependent strain, allowed us to identify plasmids that had undergone recombination with the host chromosome. The number of these plasmids exceeded by far that of plasmids resulting from mutational events.
经体外处理修饰的质粒DNA被转化到大肠杆菌细胞中。使用携带特殊rpsL421突变的链霉素抗性菌株作为克隆载体pNO1523的受体,该克隆载体携带野生型(链霉素敏感)rpsL等位基因。除非质粒序列发生变化,否则转化体对链霉素敏感。对从链霉素抗性转化体中分离的质粒的MaeI限制性图谱进行分析,以及检测它们赋予链霉素依赖性菌株的表型,使我们能够鉴定出与宿主染色体发生重组的质粒。这些质粒的数量远远超过由突变事件产生的质粒数量。