DNA REPLICATION AND GENE EXPRESSION OF CHLORELLA VIRUSES

小球藻病毒的 DNA 复制和基因表达

• 批准号: 2176575
• 负责人: JAMES L VAN ETTEN
• 金额: $ 20.7万
• 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 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176575
• 关键词: Chlorophyta; DNA methylation; DNA repair; DNA replication; DNA virus; Escherichia coli; alternatives to animals in research; bacterial virus; capsid; chimeric proteins; endonuclease; gene expression; glycosylation; glycosyltransferase; host organism interaction; methyltransferase; molecular cloning; molecular genetics; mutant; plant virus; protein sequence; site directed mutagenesis; virus DNA; virus genetics

DESCRIPTION (Adapted from the applicant's abstract): During the past 10 years, the applicant has isolated and partially characterized many large, polyhedral, dsDNA (>300kb) containing, plaque forming viruses which infect certain unicellular, eucaryotic, chlorella-like green algae. These viruses have several unique features, two of which are the focus of this proposal. (i) They encode DNA methyltransferases and DNA site- specific (restriction) endonucleases. Some of these endonucleases recognize and cleave DNA at the same position as bacterial restriction endonucleases, whereas others have novel specificities. The viruses also contain nonfunctional DNA methyltransferase genes; the applicant suspects they contain nonfunctional restriction endonuclease genes as well. (ii) The viruses contain at least three glycoproteins, including he major capsid protein.However, unlike other glycoprotein containing viruses, the chlorella viruses appear to encode putative glycosyltransferases involved in their glycosylation. The objectives of this proposal are: (i) To continue to isolate and characterize virus encoded DNA methyltransferases and DNA restriction endonucleases suspected of recognizing unique DNA sequences and to clone and sequence their genes. (ii) To determine important domains such as the target recognition domain in selected DNA methyltransferases and DNA restriction endonucleases by comparing functional and nonfunctional genes and making fusion proteins and site-directed mutants. (iii) To determine if the high homology between certain chlorella virus and bacterial DNA methyltransferases indicates natural interkingdom gene exchange between bacteria and the chlorella viruses. (iv)To determine how the chlorella viruses maintain a constant G + C content despite having vastly different levels of 5-methylcytosine. (v) To determine by genetic, molecular genetic, immunological, and biochemical methods those genes and gene products encoded by the prototype virus, PBCV-1, which glycosylate the virus major capsid protein.

描述（改编自申请人的摘要）：过去 10 年 多年来，申请人已经分离并部分表征了许多大型、 多面体、含有 dsDNA (>300kb)、形成空斑的病毒 感染某些单细胞、真核、类小球藻绿藻。 这些病毒有几个独特的特征，其中两个是重点 该提案的。 (i) 它们编码 DNA 甲基转移酶和 DNA 位点- 特异性（限制性）核酸内切酶。 其中一些核酸内切酶 在与细菌限制性酶切相同的位置识别并切割 DNA 核酸内切酶，而其他酶则具有新的特异性。 病毒 还含有非功能性 DNA 甲基转移酶基因；申请人 怀疑它们含有非功能性限制性内切酶基因 出色地。 (ii) 病毒至少含有三种糖蛋白，包括 他是主要的衣壳蛋白。然而，与其他含糖蛋白不同 病毒，小球藻病毒似乎编码假定的 糖基转移酶参与其糖基化。 该提案的目标是： (i) 继续隔离和 表征病毒编码的 DNA 甲基转移酶和 DNA 限制 疑似识别独特 DNA 序列并克隆的核酸内切酶 并对他们的基因进行测序。 (ii) 确定重要领域，例如 选定的 DNA 甲基转移酶和 DNA 中的目标识别域 通过比较功能性和非功能性限制性内切酶 基因并制造融合蛋白和定点突变体。 (三) 至 确定某些小球藻病毒与 细菌DNA甲基转移酶表明天然的界间基因 细菌和小球藻病毒之间的交换。 (iv) 确定 小球藻病毒如何保持恒定的 G + C 含量 5-甲基胞嘧啶的水平差异很大。 (v) 确定 遗传学、分子遗传学、免疫学和生化方法 原型病毒PBCV-1编码的基因和基因产物， 糖基化病毒主要衣壳蛋白。