Cloning of an origin of DNA replication of Xenopus laevis.
Cloning of an origin of DNA replication of Xenopus laevis.
复制标题
非洲爪蟾 DNA 复制起点的克隆。
DOI：
10.1073/pnas.77.9.5292
复制
发表时间：
1980
影响因子：
11.1
通讯作者：
Taylor,JH
中科院分区：
文献类型：
--
作者：
Watanabe,S;Taylor,JH
将非洲爪蟾（Xenopus laevis）的DNA片段克隆到大肠杆菌质粒pACYC 189的EcoRI位点，并测试当注射到非洲爪蟾（Xenopus laevis）未受精卵中时重组质粒启动和完成复制的能力。光滑。在测量了许多重组质粒的每个卵的[3 H]-胸苷掺入后，选择分别含有青蛙DNA的一个小片段（550个碱基对）和几个DNA酶的pSW 14和pSW 9进行更广泛的分析。尽管pSW 14中的片段尺寸小，但它在2小时内掺入的标记胸苷至少是pSW 9或单独载体的3倍。在pSW 14中的DNA合成被证明是复制而不是修复合成，基于当碘脱氧尿苷用于标记时产物的浮力密度偏移。为了确定pSW 14的复制数，采用了一种新的方法。因为pSW 14是在EcoRI位点插入爪蟾片段的载体的头对头二聚体，所以该质粒具有三个可甲基化位点--两个包围爪蟾片段，一个与该片段相对。通过共转化E.将pSW 14和含EcoRI甲基化酶基因的pBR 322分别与大肠杆菌连接，将超螺旋的pSW 14甲基化，并用[3 H]胸苷注射到鸡蛋中。随后通过测量随时间推移对EcoRI内切酶的敏感性，通过保守复制使修饰的EcoRI位点消失。结果表明，从受精卵中回收的标记超螺旋DNA中，约有50%对EcoRI酶切敏感，表明在受精卵中，大部分掺入[3 H]胸苷的DNA在4小时内复制了两次。光滑。我们的结论是，pSW 14有一个功能性的起源在非洲爪蟾的DNA片段。
DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Escherichia coli plasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the [3H]-thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (550 base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of the segment in pSW14, it incorporates in 2 hr at least 3 times as much labeled thymidine as either pSW9 or the vector alone. The DNA synthesis in pSW14 was shown to be replication rather than repair synthesis, based on a buoyant density shift of the product when iododeoxyuridine was used for labeling. To determine the number of replications of pSW14, a novel method was employed. Because pSW14 is a head-to-head dimer of the vector with the Xenopus fragment inserted at an EcoRI site, the plasmid has three methylatable sites--two bracketing the Xenopus fragment and one opposite the fragment. By cotransformation of E. coli with pSW14 and pBR322 containing the EcoRI methylase gene, supercoiled pSW14 was methylated and injected into eggs with [3H]thymidine. Disappearance of modified EcoRI sites by semiconservative replication was followed by measuring the sensitivity to EcoRI endonuclease over time. The results showed that about 50% of the labeled, supercoiled DNA recovered from eggs after 4 hr was sensitive to EcoRI digestion, which indicates that most of the DNA that incorporated [3H]thymidine had replicated twice during the 4 hr in the unfertilized eggs of X. laevis. We conclude that pSW14 has a functional origin in the Xenopus DNA segment.