@article{mbs:/content/journal/micro/10.1099/00221287-134-1-85, author = "Mori, Masaki and Hashiguchi, Ken-Ichi and Yoda, Koji and Yamasaki, Makari", title = "Designed Gene Amplification on the Bacillus subtilis Chromosome", journal= "Microbiology", year = "1988", volume = "134", number = "1", pages = "85-95", doi = "https://doi.org/10.1099/00221287-134-1-85", url = "https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-134-1-85", publisher = "Microbiology Society", issn = "1465-2080", type = "Journal Article", abstract = "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 a-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 α-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 (> 20 kb) DNA regions on the chromosome.", }