Characterization of early DNA synthesis in Xenopus eggs after injection of circular plasmid DNA.
Characterization of early DNA synthesis in Xenopus eggs after injection of circular plasmid DNA.
复制标题
注射环状质粒 DNA 后非洲爪蟾卵中早期 DNA 合成的表征。
DOI：
10.1093/nar/17.10.3709
复制
发表时间：
1989
影响因子：
14.9
通讯作者：
Ryoji,M
中科院分区：
文献类型：
--
作者：
Yasui,W;Ryoji,M
已知环质粒DNA在注射到非洲爪哇未受精卵中时会复制。我们对复制之前的早期DNA合成进行了表征。将放射性前体掺入共价闭合环状DNA中，最早在注射后2分钟就可以检测到。从对蜂毒灵的敏感性来看，复制型α聚合酶或δ参与了这一反应。然而，对密度标记产物的分析以及通过限制性内切酶Dpn1酶切检测新合成的未甲基化的链，都表明合成的DNA中的大部分(如果不是全部)以短修复样斑块的形式存在于注射的DNA分子中。它们随机存在于注射的DNA的所有HaeIII片段中，插入到质粒中的5S RNA基因的转录激活不影响分布。只有一小部分注入的DNA被用于这一反应，其中12%的DNA分子被进一步追逐到复制的后代DNA中。这种复制效率与未经早期DNA合成的批量注射DNA的复制效率几乎相同。我们的结论是，尽管复制型DNA聚合酶的使用很普遍，但早期的DNA合成和随后的DNA复制是相互独立的过程。
Circular plasmid DNA is known to replicate when injected into unfertilized eggs ofXenopus laevis. We characterized early DNA synthesis which precedes the replication. Incorporation of a radioactive precursor into covalent closed circular DNA becomes detectable as early as 2 minutes after injection. Judging from the sensitivity to aphidicolin, replicative DNA polymerase α or δ is involved in this reaction. However, analysis of density-labeled product as well as detection of newly synthesized, unmethylated strands by restriction endonuclease Dpnl digestion, both indicated that most if not all, of the synthesized DNA is present as short repair-like patches in the injected DNA molecules. They are present randomly in all the HaeIII fragments of injected DNA, and transcriptional activation of the 5S RNA gene inserted in the plasmid does not affect the distribution. Only a minor fraction of injected DNA is utilized for this reaction, and 12% of such DNA molecules are further chased into replicated progeny DNA. This efficiency of replication is nearly the same as that of bulk injected DNA which has not been subjected to the early DNA synthesis. We concluded that, despite the common use of replicative DNA polymerase, the early DNA synthesis and the subsequent DNA replication are mutually independent processes.