Replication of antibiotic resistance plasmid R6K DNA in vitro.
Replication of antibiotic resistance plasmid R6K DNA in vitro.
复制标题
抗生素抗性质粒 R6K DNA 的体外复制。
作者：
M. Inuzuka;D. Helinski
从携带抗生素抗性质粒R6 K的大肠杆菌菌株的细胞制备的可溶性提取物能够进行R6 K DNA复制的完整过程。体外DNA合成依赖于四种脱氧核糖核苷酸和核糖核苷酸三磷酸，对利福平和链霉素敏感，它们是DNA依赖性RNA聚合酶的抑制剂。脱氧核糖核苷酸掺入R6 K DNA也对放线菌素D、新生霉素、阿拉伯呋喃糖基CTP和N-乙基马来酰亚胺敏感。合成的动力学是线性的60至120分钟。复制的进行是保守的和超螺旋的闭环DNA分子的合成。通过碱性蔗糖梯度离心的分析表明，早期R6 K DNA产物含有约18 S大小的DNA片段，对应于在体内观察到的R6 K α复制起点和复制末端之间的长度。加入外源性超螺旋R6 K DNA对体外系统有抑制作用，而加入松弛复合物形式的R6 K DNA在很小程度上刺激R6 K DNA的合成。
A soluble extract prepared from cells of an Escherichia coli strain carrying the antibiotic resistance plasmid R6K is capable of carrying out the complete process of R6K DNA replication. DNA synthesis in vitro is dependent on the four deoxyribo- and ribonucleotide triphosphates and is sensitive to rifampin and streptolydigin, inhibitors of DNA-dependent RNA polymerase. The incorporation of deoxyribonucleotides into R6K DNA also is sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP, and N-ethylmaleimide. Kinetics of synthesis are linear for 60 to 120 min. Replication proceeds semiconservatively and supercoiled closed-circular DNA molecules are synthesized. Analysis by alkaline sucrose gradient centrifugation indicated that the early R6K DNA products contain DNA fragments of approximately 18 S in size, corresponding to the length between the R6K alpha origin of replication and the terminus of replication observed in vivo. Addition of exogenous supercoiled R6K DNA is inhibitory to the in vitro system, whereas the addition of R6K DNA in the form of relaxation complex stimulates R6K DNA synthesis to a small extent.