Hiraga S, Sudo T, Yoshida M, Kubota H, Ueyama H
Proc Natl Acad Sci U S A. 1982 Jun;79(12):3697-701. doi: 10.1073/pnas.79.12.3697.
Recombinant plasmids carrying a segment of Xenopus laevis chromosomal DNA were constructed with plasmid pBR322 as the vector. A recombinant plasmid pXY65 carrying a 3.2-kilobase BamHI segment of the chromosome of X. laevis has been found to contain a repetitive sequence dispersed throughout the X. laevis chromosomes. This plasmid initiated replication in vitro when the supercoiled circular molecules were incubated in a replication system. The other recombinant plasmids tested and the pBR322 vector were not replicated. Electron microscopic analysis of the replicative intermediates showed that the replication was initiated at a specific site in the 3.2-kilobase BamHI segment of pXY65 and that the replication usually proceeded bidirectionally. Analysis of the reaction products by centrifugation in alkaline sucrose gradients indicated that short pieces were synthesized in the in vitro replication system. DNA synthesis was inhibited in vitro by the addition of aphidicolin and by omission of dNTPs. These results indicate that the X. laevis segment cloned in pXY65 contains a site capable of initiating replication in vitro.
以质粒pBR322作为载体构建了携带非洲爪蟾染色体DNA片段的重组质粒。已发现携带非洲爪蟾染色体3.2千碱基BamHI片段的重组质粒pXY65含有分散在整个非洲爪蟾染色体中的重复序列。当超螺旋环状分子在复制系统中孵育时，该质粒在体外起始复制。所测试的其他重组质粒和pBR322载体未进行复制。对复制中间体的电子显微镜分析表明，复制在pXY65的3.2千碱基BamHI片段中的一个特定位点起始，并且复制通常双向进行。通过在碱性蔗糖梯度中离心分析反应产物表明，在体外复制系统中合成了短片段。通过添加阿非科林和省略脱氧核苷三磷酸在体外抑制了DNA合成。这些结果表明，克隆在pXY65中的非洲爪蟾片段含有一个能够在体外起始复制的位点。
