摘要
目的 同时克隆 Eco R 限制性内切酶基因 (eco R R)和 Eco R 甲基化酶基因 (eco R M) ,初步探讨该限制 -修饰系统的协同表达机制。方法 采用外切酶 删除法构建单向缺失亚克隆 ,根据亚克隆的酶活性对上述两种基因进行初步定位 ,经核酸序列测定确定亚克隆的缺失部位 ,再以 S1核酸酶保护分析法测定基因的转录起始点。结果 克隆片段上含完整的 eco R R基因和 eco R M基因 ,eco R R基因有两个转录起始点 ;eco R R基因 3′端 132 bp→ 45 8bp和 eco R M基因 3′端 2 0 2 bp为表达活性所必需 ,一个基因的编码区及旁侧序列的缺失并不影响另一基因的表达 ,仅有 eco R R基因表达的缺失体对 dcm+的宿主有致死作用。结论 eco R M基因的有效表达对系统的维持是至关重要的 ,控制 eco R R基因与 eco R
Objective To clone complete EcoRⅡ restriction endonuclease gene (ecoRⅡR) and methyltransferase (ecoRⅡM) gene into one vector and to analyzing the expression of the whole system. Methods Unidirective deletion subclones constructed with ExoⅢ, ecoRⅡR/M genes were preliminarily located in the cloned fragments according to the enzyme activities of each subclone, exact deletion sites were determined by sequencing, and transcriptional start sites were mapped by S1 mapping. Results The DNA fragment which was cloned into pBluescript SK+ contained the complete ecoRⅡR gene and ecoRⅡM gene, there are two transcriptional start sites in ecoRⅡR gene, 132 bp to 458 bp from 3′ and of ecoRⅡR gene are indispensable to enzyme activities and deletion of 202 bp from 3′ end of ecoRⅡM gene made it lose the capability to resist specific cut of EcoRⅡR enzyme, deletion of coding region and flanking sequence of one gene did not affect the expression of the other gene, the recombinant only containing ecoRⅡR gene appeared to be lethal to dcm+ host.Conclusions ecoRⅡM gene closely linking to ecoRⅡR gene was very important for the existence of the RM system in process of evolution, but the key to control EcoRⅡR enzyme acted later than EcoRⅡM enzyme did not exist in transcriptional level.
出处
《中国医学科学院学报》
CSCD
北大核心
2000年第2期111-114,共4页
Acta Academiae Medicinae Sinicae
基金
国家自然科学基金!(39380009)资助
关键词
EcoRⅡ
限制性内切酶
甲基化酶
缺失突变
EcoRⅡ endonuclease
EcoRⅡ methylase
deletion mutation
gene expression
