THE GENETICS OF ADENOVIRUS DNA REPLICATION

腺病毒 DNA 复制的遗传学

• 批准号: 3071496
• 负责人: DAVID M REKOSH
• 金额: $ 5.4万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-02-01 至 1990-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3071496
• 关键词: Adenoviridae; DNA directed DNA polymerase; Escherichia coli; HeLa cells; bacteriophage lambda; gene expression; genetic manipulation; genetic recombination; immunochemistry; plasmids; radiotracer; synthetic peptide; temperature sensitive mutant; tissue /cell culture; virus genetics; virus replication; yeasts

This work will focus on the proteins involved in the replication of adenovirus DNA. Specifically, DNA encoding these proteins will be moved onto bacterial plasmids which will allow expression of the viral DNA in bacteria. The proteins of interest are the E2b terminal protein, the E2b encoded polymerase and the 72K DBP from region E2a. These proteins will be synthesized as fusion products containing less than 15 amino acids, and in some cases only 1, from the Beta-galactosidase gene of E. coli or the cro gene of lambda. The microbially synthesized products will be purified and tested in vitro for their ability to function in adenovirus replication assays. Subsequently, specific modifications will be made in the primary structure of these proteins by altering the DNA encoding them. The effect of these alterations on the activity and properties of these proteins will then be tested. Conditional lethal mutations in the adenovirus terminal protein as well as additional mutations within the 120k polymerase gene will also be created in intact virus. These mutations will be characterized for their effect on viral DNA replication and on the virus growth cycle. We will also create antisera against these proteins by utilizing synthetic peptides as well as fragments of the proteins synthesized in bacteria as antigens. These sera will be used to study the synthesis and role of the replication proteins in the infected HeLa cell.

这项工作将重点关注参与复制的蛋白质 腺病毒DNA。 具体来说，编码这些蛋白质的 DNA 将被移动 到细菌质粒上，这将允许病毒 DNA 在 细菌。 感兴趣的蛋白质是 E2​​b 末端蛋白质，即 E2b 编码聚合酶和来自 E2a 区域的 72K DBP。 这些蛋白质将被合成为融合产物，其含量少于 来自 β-半乳糖苷酶基因的 15 个氨基酸，在某些情况下只有 1 个 大肠杆菌或 lambda 的 cro 基因。 微生物合成产品 将在体外纯化并测试其功能能力 腺病毒复制测定。 随后将进行具体修改 通过改变 DNA 来形成这些蛋白质的一级结构 对它们进行编码。 这些改变对活动的影响 然后将测试这些蛋白质的特性。 腺病毒末端蛋白的条件致死突变以及 120k 聚合酶基因内还将产生额外的突变 在完整的病毒中。 这些突变将根据其对 病毒DNA复制和病毒生长周期。 我们还将利用合成材料来制备针对这些蛋白质的抗血清 肽以及细菌中合成的蛋白质片段 抗原。 这些血清将用于研究 受感染的 HeLa 细胞中的复制蛋白。