Walder R Y, Hartley J L, Donelson J E, Walder J A
Proc Natl Acad Sci U S A. 1981 Mar;78(3):1503-7. doi: 10.1073/pnas.78.3.1503.
Here we report the cloning and preliminary characterization of the Pst I restriction-modification system of Providencia stuartii 164. Transformants of Escherichia coli carrying the Pst I gene system inserted into the cloning vector pBR322 were selected on the basis of acquired resistance to bacteriophage lambda infection. Pst I endonuclease was detected in osmotic shock fluid from each of the resistant clones. Plasmid and chromosomal DNA from these clones could not be digested by Pst I, indicating that the gene for the corresponding modification enzyme had also been cloned and was being expressed. The smallest recombinant plasmid encoding both activities, pPst201, contains an insert of approximately 4000 base pairs. In vitro transcription studies indicate that this DNA fragment also contains the endogenous promoter(s) of the system. When pPst201 was introduced into a minicell-producing strain of E. coli, two new proteins, 32,000 and 35,000 daltons, were synthesized. We have assigned these to the Pst I modification (methylase) and restriction enzymes, respectively. The active form of the restriction enzyme is a dimer, as determined by gel filtration. Constructed transformants of P. stuartii 164 that carry the Pst I system inserted into pBR322 produce approximately 10 times more Pst I endonuclease activity than does the native strain.
在此我们报告斯氏普罗威登斯菌164的Pst I限制修饰系统的克隆及初步特性分析。携带插入到克隆载体pBR322中的Pst I基因系统的大肠杆菌转化子，是基于对噬菌体λ感染获得性抗性而筛选出来的。在来自每个抗性克隆的渗透休克液中检测到了Pst I核酸内切酶。这些克隆的质粒和染色体DNA不能被Pst I消化，这表明相应修饰酶的基因也已被克隆并正在表达。编码这两种活性的最小重组质粒pPst201含有一个约4000个碱基对的插入片段。体外转录研究表明，该DNA片段还含有该系统的内源启动子。当将pPst201导入产生微小细胞的大肠杆菌菌株时，合成了两种新蛋白质，分子量分别为32,000和35,000道尔顿。我们分别将它们归为Pst I修饰酶（甲基化酶）和限制酶。通过凝胶过滤确定，限制酶的活性形式是二聚体。构建的携带插入到pBR322中的Pst I系统的斯氏普罗威登斯菌164转化子产生的Pst I核酸内切酶活性比天然菌株高约10倍。
