Itaya M
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Mol Gen Genet. 1993 Nov;241(3-4):287-97. doi: 10.1007/BF00284680.
The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions. The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location. It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B. subtilis chromosome by transformation. It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus. Similarly, a third pBR322 sequence could be introduced. By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B. subtilis genome.
大肠杆菌质粒pBR322序列（4363 bp）整合到枯草芽孢杆菌染色体的met、pro或leuB位点，侧翼染色体区域未发生重复。无论整合的pBR322的方向或位置如何，它都作为染色体的一部分稳定维持。发现克隆在pBR322中的长达17 kb的DNA片段可通过转化轻易转移到枯草芽孢杆菌染色体上。结果表明，使用从已在不同位点获得pBR322序列的菌株中分离的染色体DNA，通过连续转化可将第二个pBR322序列有效地引入染色体的不同区域。同样，也可以引入第三个pBR322序列。通过这种方法，可以在不连锁的位点整合两个或三个pBR322序列，而不会影响枯草芽孢杆菌基因组的整体结构。
