STRUCTURE AND FUNCTION OF THE ADV GENOME

这个项目的目的是研究基因组的结构和功能。 阿留申病细小病毒(ADV)。进一步的研究包括 3株ADV基因组片段的详细物理图谱。 这些克隆是通过复制形式的分子克隆获得的 从受感染细胞培养的Hirt上清液中制备。结果是 表明这三种病毒非常相似，但谨慎 可以在基因组编码病毒的部分检测到差异 结构基因。另一种策略被用来研究来自 不能在细胞培养中生长的病毒。单链病毒DNA是 从感染了这两种病毒的水貂的器官中提纯的病毒制备而成 犹他州I ADV或Pullman ADV。这种DNA在2000年被转化为双链分子 体外，用限制性内切酶切割并克隆。分子克隆是 在大肠杆菌中表达病毒抗原的衍生产品。现在，这将成为可能 比较不同毒株的基因组结构和功能 关注细胞培养适应可能产生的选择因素。 对这些分子克隆的详细分析目前正在进行中。我们有 还构建了ADV-G的全长分子克隆，由 完整复制形式的尾巴。初步分析表明， 这种重组质粒中的DNA在转染时会产生病毒抗原 进入允许ADV复制的细胞的培养中。 最后，ADV基因组的转录也在研究中， 到目前为止的研究结果表明，adv至少编码三种mrna。 大小从2-4kb不等。其中之一，4.0kb的RNA可以被检测到 在可检测到的DNA复制开始之前。这一mRNA可能代表 一些人认为非病毒粒子蛋白在调节病毒方面起作用。 DNA复制。

The purpose of this project is the study of genome structure and function of the Aleutian disease parvovirus (ADV). Further studies have included detailed physical mapping of genomic segments from there strains of ADV. These clones were derived by molecular cloning of replicative forms prepared from Hirt supernatants of infected cell cultures. The results indicated that the three viruses were very similar, but that discreet differences could be detected in the portion of the genome coding for viral structural genes. An alternate strategy was employed to study DNA from viruses that do not grow in cell culture. Single stranded virion DNA was prepared from virus purified from the organs of mink infected with either Utah I ADV or Pullman ADV. This DNA was converted to duplex molecules in vitro, cleaved with restriction enzymes and cloned. Molecular clones were derived that expressed viral antigens in E. coli. It will now be possible to compare the genomic structure and function of ADV strains without concern for possible selection factors exerted by cell culture adaptation. Detailed analysis of these molecular clones is currently underway. We have also constructed a full length molecular clone of ADV-G, developed by tailing of intact replicative form. Preliminary analysis indicates that DNA from this recombinant plasmid produces viral antigen when transfected into cultures of cells permissive for ADV replication. Finally, transcription of the ADV genome is also under study and the results to date suggest that ADV encodes at least three mRNA species ranging in size from 2-4 kb. One of these, the 4.0 kb RNA, can be detected prior to the onset of detectable DNA replication. This mRNA may represent the nonvirion protein thought by some to have a role in modulating viral DNA replication.