Smith M D, Longo M, Gerard G F, Chatterjee D K
Life Technologies, Inc., Gaithersburg, MD 20878.
Nucleic Acids Res. 1992 Nov 11;20(21):5743-7. doi: 10.1093/nar/20.21.5743.
The genes encoding the endonuclease and the methylase of the PvuI restriction and modification system were cloned in E.coli and characterized. The genes were adjacent in tandem orientation spanning a distance of 2200 bases. The PvuI endonuclease was a single polypeptide with a calculated molecular weight of 27,950 daltons. The endonuclease was easily detectable when the gene was expressed from its endogenous promotor and present on a low copy plasmid, but expression was considerably enhanced when the endonuclease gene was placed under the control of a strong promoter on a high copy plasmid. The methylase did not completely protect plasmid DNA from R.PvuI digestion until the methylase gene was placed under lac promotor control in a multicopy plasmid. In the absence of the M.PvuI methylase, expression of the R.PvuI endonuclease from the lac promotor on a multicopy plasmid was not lethal to wild type E.coli, but was lethal in a temperature-sensitive ligase mutant at the non-permissive temperature. Moreover, induction of the R.PvuI endonuclease under lambda pL promotor control resulted in complete digestion of the E.coli chromosome by R.PvuI.
编码PvuI限制与修饰系统的核酸内切酶和甲基化酶的基因在大肠杆菌中被克隆并进行了特性分析。这些基因以串联方向相邻排列，跨度为2200个碱基。PvuI核酸内切酶是一种单链多肽，计算分子量为27,950道尔顿。当该基因从其内源启动子表达并存在于低拷贝质粒上时，核酸内切酶很容易被检测到，但当核酸内切酶基因置于高拷贝质粒上的强启动子控制下时，表达会显著增强。直到甲基化酶基因置于多拷贝质粒的lac启动子控制下，甲基化酶才完全保护质粒DNA不被R.PvuI消化。在没有M.PvuI甲基化酶的情况下，多拷贝质粒上来自lac启动子的R.PvuI核酸内切酶的表达对野生型大肠杆菌不具有致死性，但在非允许温度下对温度敏感的连接酶突变体具有致死性。此外，在λ pL启动子控制下诱导R.PvuI核酸内切酶会导致R.PvuI对大肠杆菌染色体的完全消化。
