GENETIC AND BIOCHEMICAL ANALYSIS OF AD5 REGION 2

这项提案中描述的工作的目标是获得一个 理解控制机制在调节中的作用 用腺病毒5型(Ad5)早期区域2A进行真核基因表达 作为模型系统。早期区域2A的基因产物为529个氨基酸 与病毒有关的酸性单链DNA结合蛋白(DBP) DNA复制、病毒的宿主范围、病毒组装、 早期区域4(E4)的转录与早期mRNAs的稳定性 区域1A和1B。 拟议工作的直接目标是在 参与DNA结合和DNA复制的DBP结构域。三 到目前为止，已经确定了区域，并且有可能其他区域 的蛋白质也参与其中。每个确定的区域都将是 进行饱和诱变以确定其贡献 不同氨基酸对DBP在DNA结合和DNA中的作用 复制。突变的DBP将被检测它们的能力 参与腺病毒DNA的起始和延伸反应 体外复制。 DBP突变体将被用来检测蛋白质在控制 E4启动子转录及E1a和E1a基因的稳定性 E1B。RNA的稳态水平将通过Northern印迹法测量 核测量mRNAs的合成和衰变速率 连续贴标签技术和连续贴标签技术。 DBP在自动监管中的可能作用将通过审查 蛋白质对同源和异源mRNAs的亲和力，以及它的 对这些RNA的体外翻译的影响。RNAs将被合成 在体外，与E2A和其他mRNAs的不同区域结合 滤膜结合试验及DBP对体外培养细胞的影响 检查了这些mRNAs的翻译。 DBP和腺病毒DNA聚合酶之间的联系已经被证实 被认为在复制的渐进性质中很重要，但 这种联系从未被证明过。我们将使用合成材料 与DBP区域相对应的多肽来确定这一潜力 关联可能被破坏，如果是这样，DBP的哪些区域 牵涉其中。 这些实验的成功结果将为我们深入了解 真核基因表达调控的分子机制。

The objective of the work described in this proposal is to gain an understanding of the control mechanisms operative in the regulation of eukaryotic gene expression, using adenovirus type 5 (Ad5) early region 2A as the model system. The gene product of early region 2A is a 529 amino acid single-stranded DNA binding protein (DBP) that is involved in viral DNA replication, the host range of the virus, virus assembly, the transcription of early region 4 (E4) and the stability of mRNAs from early regions 1A and 1B. The immediate goal of the proposed work is to precisely define within the DBP domains that are involved in DNA binding and DNA replication. Three regions have been identified to date, and it is possible that other regions of the protein are also involved. Each region that is identified will be subjected to saturation mutagenesis to define the contribution of individual amino acids to the function of the DBP in DNA binding and DNA replication. The mutant DBPs will be assayed for their ability to participate in the initiation and elongation reactions of adenovirus DNA replication in vitro. DBP mutants will be used to examine the role of the protein in controlling transcription from the E4 promoter and the stability of mRNAs from E1A and E1B. Steady-state levels of RNAs will be measured by Northern blot analysis, and rates of synthesis and decay of mRNAs measured by nuclear run-on and continuous labeling techniques, respectively. A possible role for the DBP in autoregulation will be explored by examining the affinity of the protein for homologous and heterologous mRNAs, and its effect on the in vitro translation of these RNAs. RNAs will be synthesized in vitro, binding to various regions of E2A and other mRNAs measured by filter binding assays, and the effect of the DBP on the in vitro translation of these mRNAs examined. An association between the DBP and the adenovirus DNA polymerase has been postulated to be important in the precessive nature of replication, but such an association has never been demonstrated. We will use synthetic peptides corresponding to regions of the DBP to determine if this potential association can be disrupted, and if so, which regions of the DBP are involved. The successful outcome of these experiments will provide insight into the molecular mechanisms by which eukaryotic gene expression is regulated.