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KMID : 0578319950050010077
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Molecules and Cells
1995 Volume.5 No. 1 p.77 ~ p.80
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Direct isolation of the gerB-narA Stretch from the Bacillus subtilis chromosome by I-SceI Site Insertion
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Song,Bang-Ho
Fuente, Veronica De La/Glaser, Philippe/Danchin, Antonie
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Abstract
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A spectionmycin cassette harboring the I-SceI meganuclease recognition site was inserted into the open rending frame of the Bacillus narA gene. This fusion vector was integrated into the narA locus of the chromosome and selected by marker rescue with spectinomycin resistance. Subsequent integration of the I-SceI site near the gerB region was performed in B. subtilis (pSO19) which already had the I-SceI site in the narA gene. The transformant B. subtilis (pSO19/20), containing I-SceI1 sites both in the locut narA and in the vicinity of gerB was isolated by selection with spectinomycin and chloramphenicol resistance. Direct isolation of the 70 kbp of narA-gerB stretch from the nested chromosome embedded in an agar plug was obtained by pulsed field gel electrophoresis after cutting both I-SceI sites. By this method, the precise location of the!ie gencs and the distance between ger and narA were determined. This unique method is very useful and applieable as a biochemical tool for isolating any intended fragments, for blocking specific loci like site directed mutagenesis or large deletion, and for precise mapping of unknown genes by determination of their chromosomal distance and orientation.
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KEYWORD
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