Designed gene amplification on the Bacillus subtilis chromosome.
We previously reported the cloning of a 1.6 kb HindIII fragment (containing the junction of the repeating unit) from chromosomal DNA of Bacillus subtilis strain B7 in which tandem amplification of a 16 kb region occurred, and the induction of B7-type gene amplification by competence transformation with this cloned fragment. Based on this result, we designed, on the B. subtilis chromosome, a gene amplification of the 22 kb repeating unit containing the alpha-amylase structural gene (amyE), the tunicamycin-resistance gene (tmrB) and the shikimate kinase structural gene (aroI). We cloned only two short DNA fragments from both termini of the 22 kb region, constructed a junction structure of the designed repeating unit on pBR327 and transformed a B. subtilis wild-type strain by this constructed plasmid. As a result, we succeeded in obtaining tunicamycin-resistant (Tmr) transformants in which the designed gene amplification of 22 kb occurred on the chromosome. The Tmr transformants showed high productivity of alpha-amylase and shikimate kinase. The copy number of the repeating unit was estimated to be 10-20. This system may provide an effective means of amplifying long (greater than 20 kb) DNA regions on the chromosome.
Mori
M
M
Department of Agricultural Chemistry, University of Tokyo, Japan.
Hashiguchi
K
K
Yoda
K
K
Yamasaki
M
M
D000681:Amylases / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D002874:Chromosome Mapping; D004269:DNA, Bacterial; D004587:Electrophoresis, Agar Gel; D005784:Gene Amplification; D009693:Nucleic Acid Hybridization; D010770:Phosphotransferases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D014170:Transformation, Genetic
D004269:DNA, Bacterial; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase; D000681:Amylases