Targeted isolation of a designated region of the Bacillus subtilis genome by recombinational transfer.
A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.
Tomita
Satoshi
S
Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan.
Tsuge
Kenji
K
Kikuchi
Yo
Y
Itaya
Mitsuhiro
M
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005227:Fatty Acids / Q000096:biosynthesis; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D016680:Genome, Bacterial; D009842:Oligopeptides / Q000096:biosynthesis; D010456:Peptides, Cyclic; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D005227:Fatty Acids; D009842:Oligopeptides; D010456:Peptides, Cyclic; C049901:plipastatin