Maekawa Y, Yasukawa H, Kawakami B
Tsuruga Enzyme Plant, Toyobo Co., Ltd., Fukui.
J Biochem. 1990 Apr;107(4):645-9. doi: 10.1093/oxfordjournals.jbchem.a123101.
The genes of the BanI restriction-modification system specific for GGPyPuCC were cloned from the chromosomal DNA of Bacillus aneurinolyticus IAM1077, and the coding regions were assigned on the nucleotide sequence on the basis of the N-terminal amino acid sequences and molecular weights of the enzymes. The restriction and modification genes coded for polypeptides with calculated molecular weights of 39,841 and 42,637, respectively. Both the enzymes were coded by the same DNA strand. The restriction gene was located upstream of the methylase gene, separated by 21 bp. The cloned genes were significantly expressed in E. coli cells, so that the respective enzymes could be purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that the catalytically active form of the endonuclease was dimeric and that of the methylase was monomeric. Comparison of the amino acid sequences revealed no significant homology between the endonuclease and methylase, though both enzymes recognize the same target sequence. Sequence comparison with other related enzymes indicated that BanI methylase contains sequences common to cytosine-specific methylases.
从解硫胺素芽孢杆菌IAM1077的染色体DNA中克隆了对GGPyPuCC具有特异性的BanI限制修饰系统的基因，并根据酶的N端氨基酸序列和分子量在核苷酸序列上确定了编码区。限制酶和甲基化酶基因分别编码计算分子量为39,841和42,637的多肽。两种酶均由同一条DNA链编码。限制基因位于甲基化酶基因的上游，相隔21 bp。克隆的基因在大肠杆菌细胞中得到了显著表达，从而可以将各自的酶纯化至均一。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和凝胶过滤分析表明，内切核酸酶的催化活性形式是二聚体，甲基化酶的催化活性形式是单体。氨基酸序列比较显示，尽管两种酶识别相同的靶序列，但内切核酸酶和甲基化酶之间没有明显的同源性。与其他相关酶的序列比较表明，BanI甲基化酶含有胞嘧啶特异性甲基化酶共有的序列。
