Mori M, Hashiguchi K, Yoda K, Yamasaki M
Department of Agricultural Chemistry, University of Tokyo, Japan.
J Gen Microbiol. 1988 Jan;134(1):85-95. doi: 10.1099/00221287-134-1-85.
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.
我们之前报道过，从枯草芽孢杆菌B7菌株的染色体DNA中克隆出一个1.6 kb的HindIII片段（包含重复单元的连接处），该菌株中发生了16 kb区域的串联扩增，并且用这个克隆片段进行感受态转化可诱导B7型基因扩增。基于这一结果，我们在枯草芽孢杆菌染色体上设计了一个基因扩增，其重复单元为22 kb，包含α-淀粉酶结构基因（amyE）、衣霉素抗性基因（tmrB）和莽草酸激酶结构基因（aroI）。我们仅从22 kb区域的两端克隆了两个短DNA片段，在pBR327上构建了设计好的重复单元的连接结构，并通过这个构建好的质粒转化枯草芽孢杆菌野生型菌株。结果，我们成功获得了衣霉素抗性（Tmr）转化子，其染色体上发生了设计好的22 kb基因扩增。Tmr转化子表现出α-淀粉酶和莽草酸激酶的高产量。重复单元的拷贝数估计为10 - 20。该系统可能为在染色体上扩增长（大于20 kb）DNA区域提供一种有效的方法。
