CHARACTERIZATION AND EXPRESSION OF THE ESCHERICHIA-COLI MRR RESTRICTION SYSTEM
CHARACTERIZATION AND EXPRESSION OF THE ESCHERICHIA-COLI MRR RESTRICTION SYSTEM
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DOI：
10.1128/jb.173.16.5207-5219.1991
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发表时间：
1991-08-01
影响因子：
3.2
通讯作者：
BENNER, JS
中科院分区：
文献类型：
--
作者：
WAITEREES, PA;KEATING, CJ;BENNER, JS
大肠杆菌K-12的mrr基因参与接受被修饰的外源DNA。 在 Mrr+ 大肠杆菌菌株中，携带 HincII、HpaI 和 TaqI R 和 M 基因的质粒的引入受到严格限制。 克隆来自质粒 pBg3 (B. Sain 和 N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980)的 2-kb EcoRI 片段。 所得质粒恢复了大肠杆菌 mrr 菌株的 Mrr 功能。 mrr 基因的边界是通过亚克隆分析确定的，具有功能性 mrr 基因的质粒产生 33.5 kDa 的多肽。 确定了整个片段的核苷酸序列；除了 mrr 之外，它还包括两个开放阅读框，其中之一编码 hsdR 的一部分。 通过Southern印迹分析，发现大肠杆菌RR1和HB101缺少含有mrr的区域。 测试了含有克隆的 mrr 基因的大肠杆菌中各种克隆的甲基化酶的接受情况。 发现含有 AccI、CviRI、HincII、HinfI (HhaII)、HpaI、NlaIII、PstI 和 TaqI N6-腺嘌呤甲基化酶以及 SssI 和 HhaI C5-胞嘧啶甲基化酶的质粒构建体受到限制。 含有 16 种其他腺嘌呤甲基化酶和 12 种胞嘧啶甲基化酶的质粒构建体不受限制。 尚未确定引起限制的简单共有序列。 已过量产生Mrr蛋白，制备了抗体，并检查了mrr在各种条件下的表达。 建议使用大肠杆菌 mrr 菌株来克隆含有 N6-腺嘌呤和 C5-胞嘧啶甲基的 DNA。
The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified. The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E. coli strains that are Mrr+. A 2-kb EcoRI fragment from the plasmid pBg3 (B. Sain and N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980) was cloned. The resulting plasmid restores Mrr function to mrr strains of E. coli. The boundaries of the mrr gene were determined from an analysis of subclones, and plasmids with a functional mrr gene produce a polypeptide of 33.5 kDa. The nucleotide sequence of the entire fragment was determined; in addition to mrr, it includes two open reading frames, one of which encodes part of the hsdR. By using Southern blot analysis, E. coli RR1 and HB101 were found to lack the region containing mrr. The acceptance of various cloned methylases in E. coli containing the cloned mrr gene was tested. Plasmid constructs containing the AccI, CviRI, HincII, HinfI (HhaII), HpaI, NlaIII, PstI, and TaqI N6-adenine methylases and SssI and HhaI C5-cytosine methylases were found to be restricted. Plasmid constructs containing 16 other adenine methylases and 12 cytosine methylases were not restricted. No simple consensus sequence causing restriction has been determined. The Mrr protein has been overproduced, an antibody has been prepared, and the expression of mrr under various conditions has been examined. The use of mrr strains of E. coli is suggested for the cloning of N6-adenine and C5-cytosine methyl-containing DNA.