Liu J, Zhao X, Meng Y, Shen J, Xue Y, Shi S, Cai Y
Department of Genetics, Institute of Laboratory Animal Science, CAMS & PUMC, Beijing 100021.
Chin Med Sci J. 2001 Dec;16(4):200-3.
To clone complete EcoRII restriction endonuclease gene (ecoRIIR) and methyltransferase gene (ecoRIIM) in one vector and to analyze the coordinating expression of this whole R-M system.
Unidirectional deletion subclones were constructed with ExoIII. ecoRIIR/ M genes were preliminarily located in the cloned fragment according to the enzyme activities of subclones. Exact deletion sites were determined by sequencing, and transcriptional start sites were determined by S1 mapping.
The DNA fragment which was cloned into pBluescript SK + contained intact ecoRIIR gene and ecoRIIM gene, and two transcriptional start sites of ecoRIIR gene were determined. 132bp to 458bp from 3'end of ecoRIIR gene are indispensable to enzyme activities and deletion of 202bp from 3' end of ecoRIIM gene made enzyme lose the capability in DNA protection to resist specific cut with EcoRII endonuclease (EcoRII. R). Deletion of the coding and flanking sequences of one gene did not affect the expression of the other gene, and the recombinants only containing ecoRIIR gene appeared to be lethal to dcm+ host.
ecoRIIM gene linking closely to ecoRIIR gene is very important for the existence of the R-M system in process of evolution, but the key to control EcoRII R-M order may not exist in transcriptional level .
在一个载体中克隆完整的EcoRII限制性内切酶基因（ecoRIIR）和甲基转移酶基因（ecoRIIM），并分析该完整R-M系统的协同表达。
用ExoIII构建单向缺失亚克隆。根据亚克隆的酶活性，将ecoRIIR/M基因初步定位在克隆片段中。通过测序确定精确的缺失位点，通过S1作图确定转录起始位点。
克隆到pBluescript SK+中的DNA片段包含完整的ecoRIIR基因和ecoRIIM基因，并确定了ecoRIIR基因的两个转录起始位点。ecoRIIR基因3'端132bp至458bp对酶活性不可或缺，ecoRIIM基因3'端缺失202bp使酶失去对EcoRII内切酶（EcoRII.R）特异性切割的DNA保护能力。一个基因的编码和侧翼序列缺失不影响另一个基因的表达，仅含ecoRIIR基因的重组体对dcm+宿主似乎具有致死性。
与ecoRIIR基因紧密相连的ecoRIIM基因对R-M系统在进化过程中的存在非常重要，但控制EcoRII R-M顺序的关键可能不在转录水平上。
