DNA REPLICATION AND GENE EXPRESSION OF CHLORELLA VIRUSES

描述(改编自申请人的摘要)：过去10年 多年来，申请人已经隔离并部分描述了许多大型、 多角体dsDNA(&gt；300kb)，含有空斑形成病毒， 感染某些单细胞、真核、类小球藻的绿藻。 这些病毒有几个独特的特征，其中两个是重点 这项提议。(I)它们编码DNA甲基转移酶和DNA位点- 特定的(限制性)内切酶。其中一些内切酶 在与细菌限制相同的位置识别和切割DNA 核酸内切酶，而其他的则具有新的特异性。病毒 也含有不起作用的DNA甲基转移酶基因；申请人 怀疑它们含有不起作用的限制性内切酶基因 井。(2)病毒至少含有三种糖蛋白，包括 他主要是衣壳蛋白。然而，与其他含有糖蛋白的 病毒，小球藻病毒似乎编码推定的 糖基转移酶参与它们的糖基化。 这项提议的目标是：(1)继续隔离和 病毒编码的DNA甲基转移酶的特性及DNA限制性内切酶 疑似识别独特DNA序列并克隆的核酸内切酶 并对它们的基因进行测序。(二)确定重要领域等 特定DNA甲基转移酶和DNA中的靶识别结构域 比较功能性和非功能性的限制性内切酶 基因，制造融合蛋白和定点突变体。(Iii) 确定某些小球藻病毒和 细菌DNA甲基转移酶提示天然跨王国基因 细菌与小球藻病毒之间的交换。(四)决定 小球藻病毒如何保持恒定的G+C含量 5-甲基胞嘧啶水平差异很大的。(V)以下列方式决定 遗传学、分子遗传学、免疫学和生化方法 由原型病毒PBCV-1编码的基因和基因产物，它 糖基化病毒主要衣壳蛋白。

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.