编码Eco29kI限制酶和甲基化酶的质粒携带基因的克隆与序列分析。

Cloning and sequence analysis of the plasmid-borne genes encoding the Eco29kI restriction and modification enzymes.

作者信息

Zakharova M V, Beletskaya I V, Kravetz A N, Pertzev A V, Mayorov S G, Shlyapnikov M G, Solonin A S

机构信息

Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.

出版信息

Gene. 1998 Feb 27;208(2):177-82. doi: 10.1016/s0378-1119(97)00637-9.

DOI:10.1016/s0378-1119(97)00637-9

PMID:9524260

Abstract

The Eco29kI restriction-modification system (RMS2) has been found to be localized on the plasmid pECO29 occurring naturally in the Escherichia coli strain 29k (Pertzev, A.V., Ruban, N.M., Zakharova, M.V., Beletskaya, I.V., Petrov, S.I., Kravetz, A.N., Solonin, A.S., 1992. Eco29kI, a novel plasmid encoded restriction endonuclease from Escherichia coli. Nucleic Acids Res. 20, 1991). The genes coding for this RMS2, a SacII isoschizomer recognizing the sequence CCGCGG have been cloned in Escherichia coli K802 and sequenced. The DNA sequence predicts the restriction endonuclease (ENase) of 214 amino acids (aa) (24,556 Da) and the DNA-methyltransferase (MTase) of 382 aa (43,007 Da) where the genes are separated by 2 bp and arranged in tandem with eco29kIR preceding eco29kIM. The recombinant plasmid with eco29kIR produces a protein of expected size. MEco29kI contains all the conserved aa sequence motifs characteristic of m5C-MTases. Remarkably, its variable region exhibits a significant similarity to the part of the specific target-recognition domain (TRD) from MBssHII--multispecific m5C-MTase (Schumann, J.J., Walter, J., Willert, J., Wild, C., Koch D., Trautner, T.A., 1996. MBssHII: a multispecific cytosine-C5-DNA-methyltransferase with unusual target recognizing properties. J. Mol. Biol. 257, 949-959), which recognizes five different sites on DNA (HaeII, MluI, Cfr10I, SacII and BssHII), and the comparison of the nt sequences of its variable regions allowed us to determine the putative TRD of MEco29kI.

摘要

已发现Eco29kI限制修饰系统（RMS2）定位于大肠杆菌菌株29k中天然存在的质粒pECO29上（佩尔采夫，A.V.，鲁班，N.M.，扎哈罗娃，M.V.，别列茨卡娅，I.V.，彼得罗夫，S.I.，克拉韦茨，A.N.，索洛宁，A.S.，1992年。Eco29kI，一种来自大肠杆菌的新型质粒编码限制性内切酶。《核酸研究》20，1991）。编码此RMS2的基因，一种识别序列CCGCGG的SacII同裂酶，已在大肠杆菌K802中克隆并测序。DNA序列预测有214个氨基酸（aa）（24556道尔顿）的限制性内切酶（ENase）和382个aa（43007道尔顿）的DNA甲基转移酶（MTase），其中基因由2个碱基对分隔并串联排列，eco29kIR在eco29kIM之前。带有eco29kIR的重组质粒产生预期大小的蛋白质。MEco29kI包含m5C - MTases所有保守的氨基酸序列基序。值得注意的是，其可变区与来自MBssHII（一种多特异性m5C - MTase）的特定靶标识别结构域（TRD）的一部分具有显著相似性（舒曼，J.J.，瓦尔特，J.，维勒特，J.，怀尔德，C.，科赫，D.，特劳特纳，T.A.，1996年。MBssHII：一种具有不寻常靶标识别特性的多特异性胞嘧啶 - C5 - DNA甲基转移酶。《分子生物学杂志》257，949 - 959），它识别DNA上的五个不同位点（HaeII、MluI、Cfr10I、SacII和BssHII），并且对其可变区核苷酸序列进行比较使我们能够确定MEco29kI的推定TRD。