DNA REPLICATION & GENE EXPRESSION OF CHLORELLA VIRUSES

我们已经分离并部分表征了30个大型多面体， DsDNA(大于300kbp)，空斑形成病毒 感染单细胞、真核、类小球藻的绿藻。 空斑分析，同步感染宿主的能力， 短的生命周期，以及病毒经历 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。这些都是第一批适合于 标准噬菌体技术。至少其中的一部分 病毒，其基因组包含不同水平的甲基化碱基 (0.1到47%5 mC和0到37%6 mA)，DNA编码 修改和限制制度。病毒感染了藻类 是II型DNA限制性内切酶的新来源， 第一个来源是非原核系统。 我们的目标是：(I)克隆和鉴定一些 甲基转移酶和限制性内切酶基因。 对这些基因进行测序将不仅提供关于 早期病毒基因的启动子和其他调控元件，但 也让我们可以将它们与细菌酶的基因进行比较 它们识别相同的碱基序列。(Ii)描述 病毒在其修饰和限制中含有突变 系统；将突变病毒与它们的双亲进行比较可能 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功能 病毒DNA复制和DNA甲基化的细胞内位置。 (四)同步检测小球藻后代病毒 感染了两种具有不同修饰的病毒和 限制系统，以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的分离和鉴定 和来自不同病毒-宿主组合的RNA聚合酶和 比较酶在不同环境中的作用能力 甲基化的DNA模板。

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.