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...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展开更多
EcoR Ⅱ was the first restriction endonuclease ever found requiring the cooperative interaction with the least two DNA sites for digestion activity.To study the specific activity, Eco R Ⅱ was purified from hyperexpre...EcoR Ⅱ was the first restriction endonuclease ever found requiring the cooperative interaction with the least two DNA sites for digestion activity.To study the specific activity, Eco R Ⅱ was purified from hyperexpression engineering bacteria in which the specific expression products increased to about 20% of total cellular protein.By using chromatography on DEAE cellulose column,phosphocellulose column and FPLC of Resource Q,the enzyme was purified from soluble protein fraction.The inclusion bodies were solved and renatured,and the enzyme was purified from this part of protein with higher specific activity by using FPLC of Resource Q.Detection showed that the enzyme was purified to homogeneity and was free of detectable contamination by other DNase(exo and endo).展开更多
文摘目的 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
文摘EcoR Ⅱ was the first restriction endonuclease ever found requiring the cooperative interaction with the least two DNA sites for digestion activity.To study the specific activity, Eco R Ⅱ was purified from hyperexpression engineering bacteria in which the specific expression products increased to about 20% of total cellular protein.By using chromatography on DEAE cellulose column,phosphocellulose column and FPLC of Resource Q,the enzyme was purified from soluble protein fraction.The inclusion bodies were solved and renatured,and the enzyme was purified from this part of protein with higher specific activity by using FPLC of Resource Q.Detection showed that the enzyme was purified to homogeneity and was free of detectable contamination by other DNase(exo and endo).
