Efficient Replication of DNA Injected into Xenopus Eggs

我们正在研究控制非洲爪蟾卵DNA复制起始的分子机制。我们的方法利用了本实验室建立的新技术和发现，包括：1)开发了一种技术，可以在不激活卵子的情况下将材料注入卵子；2)发现注射1型牛乳头瘤病毒（BPV1） DNA只有在卵子被激活后才能复制，但在激活前注射能使复制更有效；3)观察到缺乏BPV1复制源的载体在复制效率上没有同样的提高。这些结果提供了第一个明确的迹象，即非洲爪蟾卵通过使用已知的真核起源启动DNA复制。本文提出了进一步表征卵-质粒相互作用的实验：A)确定同步启动第一轮复制的最大分子数的最佳DNA/卵量；B)确认双链DNA在活化卵中通过半保存机制复制；C)确定RNA转录在DNA复制起始中的作用。D)确定缺乏复制起始点的质粒是否会抑制含有功能起始点的质粒的有效复制。沿着这些路线进行的实验将有助于测试我们的工作模型，该模型假设未激活卵子细胞质中的蛋白质因子与含有DNA序列的起源相互作用。这些实验的成功完成将使我们更深入地了解真核生物DNA在体内条件下忠实起始和复制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.