Blumenthal R M, Gregory S A, Cooperider J S
J Bacteriol. 1985 Nov;164(2):501-9. doi: 10.1128/jb.164.2.501-509.1985.
A 4.84-kilobase-pair plasmid was isolated from Proteus vulgaris (ATCC 13315) and cloned into the plasmid vector pBR322. Plasmid pBR322 contains substrate sites for the restriction endonucleases PvuI and PvuII. The recombinant plasmids were resistant to in vitro cleavage by PvuII but not PvuI endonuclease and were found to cause production of PvuII endonuclease or methylase activity or both in Escherichia coli HB101. The approximate endonuclease and methylase gene boundaries were determined through subcloning, Bal 31 resection, insertional inactivation, DNA-dependent translation, and partial DNA sequencing. The two genes are adjacent and appear to be divergently transcribed. Most E. coli strains tested were poorly transformed by the recombinant plasmids, and this was shown by subcloning and insertional inactivation to be due to the PvuII methylase gene. At a low frequency, stable methylase-producing transformants of a methylase-sensitive strain were obtained, and efficiently transformed cell mutants were isolated from them.
从普通变形杆菌（ATCC 13315）中分离出一个4.84千碱基对的质粒，并将其克隆到质粒载体pBR322中。质粒pBR322含有限制性内切酶PvuI和PvuII的底物位点。重组质粒对PvuII的体外切割具有抗性，但对PvuI内切酶没有抗性，并且发现它们能在大肠杆菌HB101中引起PvuII内切酶或甲基化酶活性或两者的产生。通过亚克隆、Bal 31切除、插入失活、依赖DNA的翻译和部分DNA测序确定了内切酶和甲基化酶基因的大致边界。这两个基因相邻，似乎是反向转录的。大多数测试的大肠杆菌菌株很难被重组质粒转化，通过亚克隆和插入失活表明这是由于PvuII甲基化酶基因所致。在低频情况下，获得了甲基化酶敏感菌株的稳定产甲基化酶转化体，并从中分离出了高效转化的细胞突变体。
