Connaughton J F, Vanek P G, Lee-Lin S Q, Chirikjian J G
Department of Biochemistry, Georgetown University, School of Medicine, Washington, D. C. 20007.
Gene Anal Tech. 1988 Nov-Dec;5(6):116-24. doi: 10.1016/0735-0651(88)90011-8.
We wish to report the initial characterization of a recombinant clone containing the BamHI methylase gene. Genomic chromosomal DNA purified from Bacillus amyloliquefaciens was partially cleaved with HindIII, fractionated by size, and cloned into pSP64. Plasmid DNA from this library was challenged with BamHI endonuclease and transformed into Escherichia coli HB101. A recombinant plasmid pBamM6.5 and a subclone pBamM2.5 were shown to contain the BamHI methylase gene based on three independent observations. Both plasmids were found to be resistant to BamHI endonuclease cleavage, and chromosomal DNA isolated from E.coli HB101 cells harboring either of the plasmids pBamM6.5 or pBamM2.5 was resistant to cleavage by BamHI endonuclease. In addition, DNA isolated from lambda phage passaged through E.coli HB101 containing either plasmid was also resistant to BamHI cleavage. Expression of the BamHI methylase gene is dependent on orientation in pSP64. In these clones preliminary evidence indicates that methylase gene expression may be under the direction of the plasmid encoded LacZ promoter.
我们希望报告一个含有BamHI甲基化酶基因的重组克隆的初步特征。从解淀粉芽孢杆菌中纯化的基因组染色体DNA用HindIII进行部分切割，按大小分级分离，然后克隆到pSP64中。用BamHI核酸内切酶对该文库的质粒DNA进行检测，并转化到大肠杆菌HB101中。基于三个独立的观察结果，证明重组质粒pBamM6.5和一个亚克隆pBamM2.5含有BamHI甲基化酶基因。发现这两种质粒都对BamHI核酸内切酶的切割具有抗性，并且从携带质粒pBamM6.5或pBamM2.5之一的大肠杆菌HB101细胞中分离的染色体DNA对BamHI核酸内切酶的切割具有抗性。此外，从通过含有任一质粒的大肠杆菌HB101传代的λ噬菌体中分离的DNA也对BamHI切割具有抗性。BamHI甲基化酶基因的表达取决于其在pSP64中的方向。在这些克隆中，初步证据表明甲基化酶基因的表达可能受质粒编码的LacZ启动子的调控。
