DNA REPLICATION AND GENE EXPRESSION OF CHLORELLA VIRUSES

描述（改编自申请人摘要）：在过去10年中 多年来，申请人已经分离和部分表征了许多大的， 多角体，含dsDNA（> 300 kb），噬斑形成病毒， 感染某些单细胞、真核、类似绿藻的绿色藻类。 这些病毒有几个独特的特点，其中两个是重点 这一提议。 (i)它们编码DNA甲基转移酶和DNA位点- 特异性（限制性）内切核酸酶。 这些核酸内切酶中的一些 在与细菌限制性内切酶相同的位置识别和切割DNA 核酸内切酶，而其他具有新的特异性。 的病毒 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， 好. (ii)这些病毒含有至少三种糖蛋白，包括 主要衣壳蛋白。然而，与其他含有 病毒，该病毒似乎编码推定的 参与其糖基化的糖基转移酶。 这项建议的目标是：㈠继续孤立和 表征病毒编码的DNA甲基转移酶和DNA限制 怀疑识别独特DNA序列的核酸内切酶， 并对它们的基因进行排序。 (ii)为了确定重要的领域， 所选DNA甲基转移酶和DNA中的靶识别结构域 通过比较功能性和非功能性限制性内切核酸酶 基因，制造融合蛋白和定点突变体。 (iii)到 确定是否在某些大肠杆菌病毒和 细菌DNA甲基转移酶指示天然界间基因 细菌和病毒之间的交换。 ㈣确定 如何在一个恒定的G + C含量， 它们的5-甲基胞嘧啶水平相差很大。 (v)通过以下方式确定 遗传学、分子遗传学、免疫学和生物化学方法， 由原型病毒PBCV-1编码的基因和基因产物， 使病毒主要衣壳蛋白糖基化。

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.