Efficient Replication of DNA Injected into Xenopus Eggs

注射到非洲爪蟾卵中的 DNA 的高效复制

• 批准号: 8812766
• 负责人: Lawrence Wangh
• 金额: $ 31.6万
• 依托单位: Brandeis University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-02-01 至 1992-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8812766&HistoricalAwards=false
• 关键词: Efficient; Replication; DNA; Injected; into

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.

我们正在研究控制 非洲爪蟾卵中DNA复制的起始 光滑。 我们的方法利用新技术， 本实验室的研究结果包括：1） 开发了一种技术， 材料进入鸡蛋，而不激活它们; 2）发现 注射1型牛乳头状瘤病毒（BPV 1）DNA， 只有在卵子被激活后才能复制， 通过在活化之前注射使其更有效; 3） 观察到缺乏BPV 1起源的载体 复制没有显示相同的改进， 复制效率 这些结果提供了第一 明确表明爪蟾卵启动DNA复制 通过使用已知的真核生物来源。 实验旨在 进一步表征卵-质粒相互作用的是 建议：A）确定DNA/卵的最佳量 其产生第一轮复制的同步启动 最大分子数：B）确认 双链DNA在激活的卵子中通过一种 C.确定保护机制的作用 RNA转录是DNA复制的起始。 D）、 确定是否存在缺乏来源的质粒 复制抑制了那些 包含功能性起源。 沿着这些路线的实验 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 更深入地了解机制和细胞因素 对于忠实地发起和复制所必需的 在体内条件下的真核DNA。