Efficient Replication of DNA Injected into Xenopus Eggs

我们正在研究控制非洲爪蛙卵中DNA复制启动的分子机制。我们的方法利用了本实验室建立的新技术和发现，其中包括：1)开发一种技术，允许在不激活卵子的情况下将材料注入卵子；2)发现注射的1型牛乳头瘤病毒(BPV1)DNA只有在鸡蛋被激活后才会复制，但在激活之前注射使复制更有效；3)观察到缺乏BPV1复制起点的载体在复制效率上没有表现出同样的提高。这些结果提供了第一个明确的迹象，表明非洲爪哇卵通过使用已知的真核来源启动了DNA复制。为进一步研究鸡蛋-质粒间的相互作用，建议进行以下实验：a)确定DNA/鸡蛋的最适量，从而同步启动第一轮最大分子复制；b)证实双链DNA在激活的鸡蛋中以半保守机制复制；C)确定RNA转录在启动DNA复制中的作用。D)确定缺乏复制起始点的质粒会抑制含有功能起始点的质粒点的有效复制。沿着这些思路进行的实验将有助于测试我们的工作模型，该模型假设未激活鸡蛋细胞质中的蛋白质因素与含有DNA序列的来源相互作用。这些实验的成功完成将使人们能够更深入地了解在活体条件下忠实地启动和复制真核DNA所需的机制和细胞因素。

We are investigating the molecular mechanisms controlling initiation of DNA replication in eggs of the frog Xenopus laevis. Our approach makes use of new techniques and findings established in this laboratory, these include: 1) Development of a technique which permits injection of materials into eggs without activating them; 2) Discovery that injected Type 1 Bovine Papilloma Virus (BPV1) DNA is only replicated after eggs are activated, but replication is made more efficient by injection prior to activation; 3) Observation that vectors which lack the BPV1 origin of replication do not display the same improvement in replication efficiency. These results provide the first clear indication that Xenopus eggs initiate DNA replication via use of a known eukaryotic origin. Experiments aimed at further characterizing the egg-plasmid interactions are proposed: A) Determination of the optimal amount of DNA/egg which yields synchronous initiation of 1st round replication of the maximum number of molecules: B) Confirmation that double stranded DNA is replicated in activated eggs via a semiconservative mechanism; C) Determination, of the role of RNA transcription in the initiation of DNA replication. D) Determination of whether plasmids which lack origins of replication inhibit the efficient replication of those which contain functional origins. Experiments along these lines will help to test our working model which postulates that the protein factors in the cytoplasm of the unactivated egg interact with origin containing DNA sequences. The successful completion of these experiments will allow greater insight into the mechanisms and cellular factors necessary for the faithful initiation and replication of eukaryotic DNA in in vivo conditions.