Miura-Masuda A, Ikeda H
Institute of Medical Science, University of Tokyo, Japan.
Mol Gen Genet. 1990 Feb;220(3):345-52. doi: 10.1007/BF00391737.
A system for detecting a spontaneous deletion in Escherichia coli was developed and the role of DNA gyrase in deletion formation was studied. A derivative of lambda plac5, lambda AM36, was isolated in which whole pBR322 DNA was inserted in the lacZ gene and 227 bp of the lac gene duplicated at both sides of the pBR322 DNA. E. coli lac- strains lysogenized by lambda AM36 had a Lac- phenotype and segregated Lac+ revertants. Sequence analyses showed that the revertant was formed by a deletion that eliminated the inserted pBR322 DNA and one copy of the duplicated segments. The frequency of lac+ revertant formation was independent of recA function, was increased by oxolinic acid, an inhibitor of DNA gyrase, but was not increased in a lysogen of a nalidixic acid-resistant derivative. The reversion frequencies of temperature sensitive mutants of gyrA gene are 10 to 100 times lower than that of the wild-type strain. These results indicate that the DNA gyrase of E. coli participated in the in vivo deletion formation resulting from the direct repeats.
开发了一种用于检测大肠杆菌中自发缺失的系统,并研究了DNA促旋酶在缺失形成中的作用。分离出λplac5的衍生物λAM36,其中整个pBR322 DNA插入到lacZ基因中,并且在pBR322 DNA两侧重复了227 bp的lac基因。被λAM36溶原化的大肠杆菌lac-菌株具有Lac-表型,并分离出Lac+回复体。序列分析表明,回复体是由一个缺失形成的,该缺失消除了插入的pBR322 DNA和重复片段的一个拷贝。lac+回复体形成的频率与recA功能无关,被DNA促旋酶抑制剂奥索利酸增加,但在耐萘啶酸衍生物的溶原菌中没有增加。gyrA基因温度敏感突变体的回复频率比野生型菌株低10至100倍。这些结果表明,大肠杆菌的DNA促旋酶参与了由直接重复导致的体内缺失形成。