Kan T N, Li L, Chandrasegaran S
Department of Environmental Health Sciences, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.
Gene. 1992 Nov 2;121(1):1-7. doi: 10.1016/0378-1119(92)90155-i.
We have cloned and sequenced the cvibIM gene from Chlorella virus NC-1A by selecting for the modification phenotype. The modification gene was cloned on a 7-kb BamHI fragment inserted into the BamHI site of the pUC13 plasmid. The cvibIM gene was localized at the 3' end of this fragment. Sequencing of this region revealed a large open reading frame that codes for methyltransferase (MTase; symbol M.) (predicting 260 amino acids). M.CviBI (GANTC) aa sequence is homologous to M.Dam(GATC), M.DpnII(GATC), and M.T4 (GATC), and not so to M.HinfI(GANTC), M.HhaII (GANTC), and M.DpnA(GATC). We also describe the use of the polymerase chain reaction technique to alter transcriptional and translational signals surrounding this gene so as to achieve overexpression in Escherichia coli. This construct yields M.CviBI at 2-3% of the total cellular protein. The MTase was purified by phosphocellulose, DEAE, and gel filtration chromatography. Its size by SDS-PAGE is approx. 28 kDa, in good agreement with that predicted from the nucleotide sequence.
我们通过选择修饰表型，从小球藻病毒NC-1A中克隆并测序了cvibIM基因。该修饰基因克隆在一个插入到pUC13质粒BamHI位点的7 kb BamHI片段上。cvibIM基因位于该片段的3'端。对该区域进行测序揭示了一个编码甲基转移酶（MTase；符号M.）的大开放阅读框（预测有260个氨基酸）。M.CviBI（GANTC）的氨基酸序列与M.Dam（GATC）、M.DpnII（GATC）和M.T4（GATC）同源，而与M.HinfI（GANTC）、M.HhaII（GANTC）和M.DpnA（GATC）不同源。我们还描述了使用聚合酶链反应技术改变该基因周围的转录和翻译信号，以便在大肠杆菌中实现过表达。该构建体产生的M.CviBI占总细胞蛋白的2 - 3%。甲基转移酶通过磷酸纤维素、DEAE和凝胶过滤色谱法进行纯化。通过SDS-PAGE测定其大小约为28 kDa，与从核苷酸序列预测的结果相符。
