Düsterhöft A, Erdmann D, Kröger M
Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität Giessen, FRG.
Nucleic Acids Res. 1991 Mar 11;19(5):1049-56. doi: 10.1093/nar/19.5.1049.
The restriction-modification system HgiDI from Herpetosiphon giganteus strain Hpa2 has been cloned in E. coli in a two-step procedure. Selection of the methyltransferase (M.HgiDI) gene in vitro was performed using the heterologous restriction endonuclease AhaII, an isoschizomer of Acyl and HgiDI (GRCGYC). Cloning of the complete HgiDI endonuclease (R.HgiDI) gene could only be achieved in recipient cells harbouring a recombinant plasmid, which was expressing the corresponding methyltransferase and could thereby prevent the host from self-destruction of its genetic material. The HgiDI restriction-modification system was sequenced and functionally correlated with two open reading frames of 309 (M) and 359 (R) codons. In homology studies M.HgiDI showed significant similarities to 20 other m5C-methyltransferases and turned out to be the most compact enzyme of this group described so far. Initial attempts for overexpression of M.HgiDI and partial purification of R.HgiDI have been successful.
巨大吸管菌Hpa2菌株的限制修饰系统HgiDI已通过两步法在大肠杆菌中克隆。使用异源限制性内切酶AhaII（Acyl和HgiDI的同裂酶，识别序列为GRCGYC）在体外筛选甲基转移酶（M.HgiDI）基因。完整的HgiDI内切酶（R.HgiDI）基因只能在携带重组质粒的受体细胞中克隆，该重组质粒表达相应的甲基转移酶，从而防止宿主自身遗传物质的破坏。对HgiDI限制修饰系统进行了测序，并将其功能与两个分别含有309个（M）和359个（R）密码子的开放阅读框相关联。在同源性研究中，M.HgiDI与其他20种m5C甲基转移酶表现出显著相似性，并且是迄今为止该组中结构最紧凑的酶。M.HgiDI的过表达和R.HgiDI的部分纯化的初步尝试已取得成功。
