DNA REPLICATION & GENE EXPRESSION OF CHLORELLA VIRUSES

DNA复制

• 批准号: 3281271
• 负责人: JAMES L VAN ETTEN
• 金额: $ 16.21万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-28 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3281271
• 关键词: Chlorophyta; DNA directed DNA polymerase; DNA directed RNA polymerase; DNA methylation; DNA virus; autoradiography; bacteriophage lambda; endonuclease; enzyme mechanism; eukaryote; gene expression; genetic library; genetic manipulation; genetic mapping; genetic promoter element; genetic regulation; genome; high performance liquid chromatography; methyltransferase; molecular cloning; mutant; nucleic acid probes; nucleic acid sequence; plant genetics; plant virus; plaque assay; plasmids; protein sequence; protoplast /spheroplast; virus DNA; virus RNA; virus genetics; virus infection mechanism; virus protein; virus replication; virus virus interaction

We have isolated and partially characterized 30 large polyhedral, dsDNA containing (greater than 300 kbp), plaque forming viruses which infect a unicellular, eukaryotic, Chlorella-like green alga. The plaque assay, the ability to synchronously infect the host, the short life cycle, and the ability of the viruses to undergo homologous recombination make them excellent model systems for studying gene regulation and expression in a photosynthetic eukaryote. These are the first plant-virus systems amenable to standard bacteriophage technology. At least some of these viruses, whose genomes contain various levels of methylated bases (0.1 to 47% 5mC and 0 to 37% 6mA), encode for DNA modification and restriction systems. The virus infected algae are a new source of type II DNA restriction endonucleases and the first source from a nonprokaryotic system. Our objectives are to: (i) clone and characterize some of the methyltransferase and restriction endonuclease genes. Sequencing these genes will provide information not only on the promoters and other regulatory elements of early virus genes but also allow us to compare them to genes for bacterial enzymes which recognize identical base sequences. (ii) Characterize viruses containing mutations in their modification and restriction systems; comparing the mutant viruses with their parents may reveal the biological function of these systems. (iii) Determine the structure of virus DNA replicative intermediates and the intracellular site of virus DNA replication and DNA methylation. (iv) Examine progeny viruses form Chlorella simultaneously infected with two viruses possessing different modification and restriction systems to determine if only one or both viruses replicate in the same cell. (v) Isolate and characterize virus DNA and RNA polymerases from different virus-host combinations and compare the ability of the enzymes to function with different methylated DNA templates.

我们已经分离并部分表征了 30 个大型多面体， 含有双链DNA（大于300 kbp）、噬斑形成病毒 它感染单细胞、真核、类似小球藻的绿藻。 噬菌斑测定，同步感染宿主的能力， 病毒的生命周期短，生存能力强 同源重组使它们成为优秀的模型系统 用于研究光合作用中的基因调控和表达 真核生物。 这些是第一个适合的植物病毒系统 标准噬菌体技术。 至少其中一些 病毒，其基因组含有不同水平的甲基化碱基 （0.1 至 47% 5mC 和 0 至 37% 6mA），编码 DNA 修改和限制系统。 病毒感染藻类 是 II 型 DNA 限制性内切酶的新来源 首先来自非原核系统。 我们的目标是：（i）克隆并表征一些 甲基转移酶和限制性内切酶基因。 对这些基因进行测序不仅可以提供有关 早期病毒基因的启动子和其他调控元件，但是 还允许我们将它们与细菌酶的基因进行比较 识别相同的碱基序列。 (ii) 表征 修饰和限制中含有突变的病毒 系统；将突变病毒与其亲本进行比较可能 揭示这些系统的生物学功能。 (三) 确定 病毒DNA复制中间体的结构和 病毒DNA复制和DNA甲基化的细胞内位点。 (iv) 同时检查小球藻的子代病毒 感染了两种具有不同变异的病毒 限制系统来确定是否只有一种或两种病毒 在同一个细胞中复制。 (v) 病毒 DNA 的分离和表征 和来自不同病毒-宿主组合的RNA聚合酶和 比较不同酶的作用能力 甲基化 DNA 模板。