Induction of Gene Amplification on the Chromosome of Bacillus subtilis and Its Application

我们在研究耐衣霉素和淀粉酶高产突变体的过程中发现了枯草芽孢杆菌染色体上发生的基因扩增。我们发现通过能力转化可以在受体细菌的染色体上诱导相同的基因扩增，并揭示了转化DNA片段的基本结构。本研究的目的是诱导外源基因在枯草芽孢杆菌染色体上扩增，并分析tunicamycin耐药基因tmrB作为基因扩增的选择标记的功能。第一年，我们的目标是诱导外源基因的扩增。我们克隆了一个6.4kb的EcoRI片段，其中包含tmrB基因和部分α -淀粉酶amyE基因。该片段可通过能力转化诱导基因扩增。我们将质粒pC194的氯霉素乙酰转移酶基因cat插入到6.4kb EcoRI片段的中间。我们用这个片段转化了一个amyE07突变体，并选择了淀粉酶阳性和tunicamycin抗性的转化体。这些转化体在染色体上有大约20个猫基因拷贝，并且对氯霉素（40微克/毫升）具有高度抗性。去年，我们的目标是分析tmrB基因可能的功能。我们通过Sl定位确定了tmrB基因的转录起始碱基，证实该基因确实被转录。我们检查了耐药突变体中是否发生了tunicamycin的降解或修饰，但没有发现任何意义。耐药细胞和敏感细胞对tunicamycin的靶酶也有差异。

We discovered a gene amplification occurred on the chromosome of Bacillus subtilis in the course of the study of a tunicamycin-resistant and amylase hyper-producing mutant. We found that the same gene amplification can be induced on the chromosome of the recipient bacteria by competence transformation and revealed the essential structure of the transforming DNA fragment. The objectives of this study were to induce amplification of foreign genes on the chromosome of B. subtilis and to analyse the function of the tunicamycin-resistant gene, tmrB,used as the selective marker of gene amplification.In the first year, we aimed to induce amplification of foreign genes. We have cloned a 6.4kb EcoRI fragment which contains the tmrB gene and a part of the amyE gene for alpha-amylase. This fragment can induce gene amplification by competence transformation. We inserted the chloramphenicol acetyltransferase gene, cat, of the plasmid pC194 in the middle of the 6.4kb EcoRI fragment. We transformed a amyE07 mutant with this fragment and selected amylase-positive and tunicamycin-resistant transformants. These transformants had about 20 copies of the cat gene on the chromosome and were highly resistant to chloramphenicol (40 mcg/ml).In the last year, we aimed to analyze the possible function of the tmrB gene. We determined the initiation base of transcription of tmrB gene by Sl mapping and confirmed that the gene is actually transcribed. We examined if any degradation or modification of tunicamycin occurred in the resistant mutants but failed to find any significance. The target enzyme of tunicamycin also had any difference between the resistant and sensitive cells.