Mori M, Tanimoto A, Yoda K, Harada S, Koyama N, Hashiguchi K, Obinata M, Yamasaki M, Tamura G
J Bacteriol. 1986 Jun;166(3):787-94. doi: 10.1128/jb.166.3.787-794.1986.
Bacillus subtilis B7, a mutant which acquired gene amplification of the amyE-tmrB region, showed, as a result, hyperproductivity (about a 5- to 10-fold increase) of alpha-amylase and tunicamycin resistance. The mutational character was transferred to recipient cells by competence transformation. A 14-kilobase (kb) EcoRI chromosomal DNA fragment of strain B7 was found to have the transforming activity. We cloned a 6.4-kb EcoRI fragment on a phage vector lambda Charon 4A through a spontaneous deletion of 7.6 kb from the 14-kb fragment and subcloned a 1.6-kb HindIII fragment on pGR71. The cloned 6.4-kb EcoRI and 1.6-kb HindIII fragments retained the transforming activity of inducing gene amplification of the amyE-tmrB region. At the junction point (J) of the repeating units (16 kb), the tmrB gene was linked to a DNA region (M) located 4 kb upstream of amyE. The essential structure of the cloned, transforming (gene amplification-inducing) DNA was deduced to be that around J. The subcloned 1.6-kb HindIII fragment that retained the transforming activity was shown to be almost solely composed of the tmrB-J-M region. In addition, the DNA sequence around J was determined.
枯草芽孢杆菌B7是一种获得了amyE - tmrB区域基因扩增的突变体，结果显示其α -淀粉酶高产（约增加5至10倍）且对衣霉素具有抗性。这种突变特征通过感受态转化转移到受体细胞中。发现菌株B7的一个14千碱基（kb）的EcoRI染色体DNA片段具有转化活性。我们通过从14 kb片段中自发缺失7.6 kb，将一个6.4 kb的EcoRI片段克隆到噬菌体载体λCharon 4A上，并将一个1.6 kb的HindIII片段亚克隆到pGR71上。克隆的6.4 kb EcoRI和1.6 kb HindIII片段保留了诱导amyE - tmrB区域基因扩增的转化活性。在重复单元（16 kb）的连接点（J）处，tmrB基因与位于amyE上游4 kb的一个DNA区域（M）相连。推断克隆的、具有转化活性（诱导基因扩增）的DNA的基本结构是J周围的结构。显示保留转化活性的亚克隆1.6 kb HindIII片段几乎完全由tmrB - J - M区域组成。此外，还测定了J周围的DNA序列。
