CLONING OF A RESTRICTION-MODIFICATION SYSTEM FROM PROTEUS-VULGARIS AND ITS USE IN ANALYZING A METHYLASE-SENSITIVE PHENOTYPE IN ESCHERICHIA-COLI
CLONING OF A RESTRICTION-MODIFICATION SYSTEM FROM PROTEUS-VULGARIS AND ITS USE IN ANALYZING A METHYLASE-SENSITIVE PHENOTYPE IN ESCHERICHIA-COLI
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DOI：
10.1128/jb.164.2.501-509.1985
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发表时间：
1985-01-01
影响因子：
3.2
通讯作者：
COOPERIDER, JS
中科院分区：
文献类型：
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作者：
BLUMENTHAL, RM;GREGORY, SA;COOPERIDER, JS
从普通变形杆菌（ATCC 13315）中分离4.84-内切酶对质粒，并克隆到质粒载体pBR 322中。质粒pBR 322含有限制性内切核酸酶PvuI和PvuII的底物位点。重组质粒在体外裂解PvuII，但不是PvuI内切酶，并发现导致生产的PvuII内切酶或甲基化酶活性或两者在大肠杆菌HB 101。通过亚克隆、Bal 31切除、插入失活、DNA依赖性翻译和部分DNA测序来确定近端核酸内切酶和甲基化酶基因的边界。这两个基因是相邻的，似乎是不同的转录。大多数E.大肠杆菌菌株被重组质粒转化的效果很差，亚克隆和插入失活表明这是由于PvuII甲基化酶基因所致。在低频率下，获得了甲基化酶敏感菌株的稳定的甲基化酶产生转化体，并从它们中分离出有效转化的细胞突变体。
A 4.84-kilobase-pair plasmid was isolated from Proteus vulgaris (ATCC 13315) and cloned into the plasmid vector pBR322. Plasmid pBR322 contains substrate sites for the restriction endonucleases PvuI and PvuII. The recombinant plasmids were resistant to in vitro cleavage by PvuII but not PvuI endonuclease and were found to cause production of PvuII endonuclease or methylase activity or both in Escherichia coli HB101. The aproximate endonuclease and methylase gene boundaries were determined through subcloning, Bal 31 resection, insertional inactivation, DNA-dependent translation, and partial DNA sequencing. The two genes are adjacent and appear to be divergently transcribed. Most E. coli strains tested were poorly transformed by the recombinant plasmids, and this was shown by subcloning and insertional inactivation to be due to the PvuII methylase gene. At a low frequency, stable methylase-producing transformants of a methylase-sensitive strain were obtained, and efficiently transformed cell mutants were isolated from them.