Walder R Y, Langtimm C J, Chatterjee R, Walder J A
J Biol Chem. 1983 Jan 25;258(2):1235-41.
The gene for the MspI modification enzyme from Moraxella was cloned in Escherichia coli using the plasmid vector pBR322. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by MspI. Both chromosomal and plasmid DNA were modified in the selected clones. None of the clones obtained produced the cognate restriction enzyme which suggests that in this system the genes for the restriction enzyme and methylase are not closely linked. Crude cell extracts prepared from the recombinant strains, but not the host (E. coli HB101), contain an S-adenosylmethionine-dependent methyltransferase specific for the MspI recognition site, CCGG. Production of the enzyme is 3-4-fold greater in the transformants than in the original Moraxella strain. 5-Methylcytosine was identified as the product of the reaction chromatographically. The outer cytosine of the recognition sequence, *CCGG, was shown to be the site of methylation by DNA-sequencing methods. This modification blocks cleavage by both MspI and its isoschizomer HpaII. HpaII, but not MspI, is able to cleave the unmethylated strand of a hemimethylated substrate. The relevance of these results to the use of MspI and HpaII to analyze patterns of methylation in genomic DNA is discussed.
利用质粒载体pBR322，在大肠杆菌中克隆了莫拉克斯氏菌（Moraxella）的MspI甲基化酶基因。携带该基因的转化子的筛选基于编码该酶的修饰质粒对MspI切割的抗性。在所选克隆中，染色体DNA和质粒DNA均被修饰。所获得的克隆均未产生同源限制酶，这表明在该系统中，限制酶基因和甲基化酶基因没有紧密连锁。从重组菌株而非宿主（大肠杆菌HB101）制备的粗细胞提取物含有一种对MspI识别位点CCGG具有特异性的依赖S-腺苷甲硫氨酸的甲基转移酶。该酶在转化子中的产量比原始莫拉克斯氏菌菌株高3至4倍。通过色谱法鉴定出反应产物为5-甲基胞嘧啶。通过DNA测序方法表明，识别序列*CCGG的外侧胞嘧啶是甲基化位点。这种修饰会阻断MspI及其同裂酶HpaII的切割。HpaII能够切割半甲基化底物的未甲基化链，而MspI则不能。文中讨论了这些结果与使用MspI和HpaII分析基因组DNA甲基化模式的相关性。
