Howard K A, Card C, Benner J S, Callahan H L, Maunus R, Silber K, Wilson G, Brooks J E
Nucleic Acids Res. 1986 Oct 24;14(20):7939-51. doi: 10.1093/nar/14.20.7939.
DdeI, a Type II restriction-modification system from the gram-negative anaerobic bacterium Desulfovibrio desulfuricans, recognizes the sequence CTNAG. The system has been cloned into E. coli in two steps. First the methylase gene was cloned into pBR322 and a derivative expressing higher levels was constructed. Then the endonuclease gene was located by Southern blot analyses; BamHI fragments large enough to contain the gene were cloned into pACYC184, introduced into a host containing the methylase gene, and screened for endonuclease activity. Both genes are stably maintained in E. coli on separate but compatible plasmids. The DdeI methylase is shown to be a cytosine methylase. DdeI methylase clones decrease in viability as methylation activity increases in E. coli RR1 (our original cloning strain). Therefore the DdeI system has been cloned and maintained in ER1467, a new E. coli cloning strain engineered to accept cytosine methylases. Finally, it has been demonstrated that a very high level of methylation was necessary in the DdeI system for successful introduction of the active endonuclease gene into E. coli.
DdeI是一种来自革兰氏阴性厌氧菌脱硫脱硫弧菌的II型限制修饰系统，识别序列CTNAG。该系统已分两步克隆到大肠杆菌中。首先，将甲基化酶基因克隆到pBR322中，并构建了一个表达水平更高的衍生物。然后通过Southern印迹分析定位内切核酸酶基因；将足够大以包含该基因的BamHI片段克隆到pACYC184中，导入含有甲基化酶基因的宿主中，并筛选内切核酸酶活性。这两个基因在大肠杆菌中通过单独但兼容的质粒稳定维持。已证明DdeI甲基化酶是一种胞嘧啶甲基化酶。在大肠杆菌RR1（我们最初的克隆菌株）中，随着甲基化活性增加，DdeI甲基化酶克隆的活力下降。因此，DdeI系统已在ER1467中克隆并维持，ER1467是一种经过改造以接受胞嘧啶甲基化酶的新型大肠杆菌克隆菌株。最后，已经证明在DdeI系统中，为了将活性内切核酸酶基因成功导入大肠杆菌，需要非常高水平的甲基化。
