Tanaka T, Weisblum B
J Bacteriol. 1975 Jan;121(1):354-62. doi: 10.1128/jb.121.1.354-362.1975.
A composite plasmid has been constructed in vitro from colicin E1 factor (mass of 4.2 megadaltons [Md]) and nontransmissible resistance factor RSF 1010 (mass, 5.5. Md) deoxyribonucleic acids (DNAs) by the sequential action of Escherichia coli endonuclease (RI (Eco RI) and T4 phage DNA ligase on the covalently closed circular forms of the constituents. The composite plasmid was selected and amplified in vivo by sequential transformation of E. coli C600 with the ligated mixture and selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-cesium chloride solution. Treatment of the composite plasmid with Eco RI yielded two fragments with mobilities corresponding to the linear forms of the parental plasmids, whereas Serratia marscesens endonuclease R (SmaR), which introduces a single scission in the colicin E1 factor but not in RSF 1010, convErted the composite plasmid to a single linear molecule (mass, 9.7 Md). Sequential degradation of colicin E1 factor with Sma R and Eco RI produced two fragments with masses of 3.5 and 0.7 Md; sequential degradation of RSF 1010 produced only one fragment (due to the cleavage with Eco RI), and sequential degradation of the composite plasmid produced the expected three fragments--an RSF 1010 Eco RI linear and the two expected products from the colicin E1 factor moiety. The composite plasmid conferred on the host cell resistance to streptomycin, sulfonamides, and colicin E1, but colicin E1 itself was not synthesized. In contrast, colicin E1 was synthesized by cells containing simultaneously both colicin E1 factor and RSF 1010 as separate entities. In the presence of chloramphenicol, the composite plasmid continued to replicate for 6 h. whereas replication of RSF 1010 and chromosomal DNA stopped within 2 h. Continued replication in the presence of chloramphenicol suggests that the replicator of the colicin E1 factor is functional in the composite plasmid.
通过大肠杆菌内切酶（RI（Eco RI））和T4噬菌体DNA连接酶对组成成分的共价闭合环状形式依次作用，已在体外从大肠杆菌素E1因子（质量为4.2兆道尔顿[Md]）和非传递性抗性因子RSF 1010（质量为5.5 Md）的脱氧核糖核酸（DNA）构建了一种复合质粒。通过用连接混合物依次转化大肠杆菌C600并在含有链霉素加大肠杆菌素E1的培养基中选择转化体，然后在氯霉素存在下进行扩增，并通过在溴化乙锭 - 氯化铯溶液中进行染料浮力密度梯度离心来纯化提取的质粒，从而在体内选择并扩增该复合质粒。用Eco RI处理复合质粒产生了两个片段，其迁移率与亲本质粒的线性形式相对应，而粘质沙雷氏菌内切酶R（SmaR）在大肠杆菌素E1因子中引入了一个单一的切口，但在RSF 1010中没有，它将复合质粒转化为一个单一的线性分子（质量为9.7 Md）。用Sma R和Eco RI依次降解大肠杆菌素E1因子产生了两个质量分别为3.5和0.7 Md的片段；RSF 1010的依次降解仅产生一个片段（由于用Eco RI切割），复合质粒的依次降解产生了预期的三个片段——一个RSF 1010 Eco RI线性片段和来自大肠杆菌素E1因子部分的两个预期产物。该复合质粒赋予宿主细胞对链霉素、磺胺类药物和大肠杆菌素E1的抗性，但不合成大肠杆菌素E1本身。相比之下，同时含有大肠杆菌素E1因子和RSF 1010作为独立实体的细胞合成了大肠杆菌素E1。在氯霉素存在下，复合质粒继续复制6小时，而RSF 1010和染色体DNA的复制在2小时内停止。在氯霉素存在下的持续复制表明大肠杆菌素E1因子的复制子在复合质粒中起作用。
