Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168

Mitsuhiro Itaya, Teruo Tanaka

研究成果: Article査読

30 被引用数 (Scopus)


We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB: : cat, leuB-, and pro: : neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB+ pro+)→(leuB: : cat pro+)→ (leuB-pro: : neo)→(leuB-pro+) resulted in a leuB- single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD- and pro-, as well as the double mutation leuB-pro- can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.

ジャーナルMGG Molecular & General Genetics
出版ステータスPublished - 1990 9月 1

ASJC Scopus subject areas

  • 遺伝学


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