EXPRESSION AND REGULATION OF VACCINIA VIRUS LATE GENES

为简单起见，痘苗病毒(VV)基因通常被归类为早期 或较晚的类，取决于它们的表达式是否独立于 依赖于病毒DNA的合成。人们对此知之甚少 控制早期和晚期基因表达切换的机制 VV复制周期中的模式。最近，相当数量的 已经获得了关于结构、核苷酸序列和 一些典型的VV早期遗传基因座的调控。此信息 为后续VV早期基因的鉴定提供依据 监管信号和识别它们的因素。然而到目前为止， 类似的分析还没有对VV晚期基因进行。它是朝着 这一问题是本提案中概述的实验所针对的。 一些VV晚期基因的基因组位置将由两个决定 方法：研究方法。首先，将利用DNA介导的标记救援技术来 绘制温度敏感型或抗药性VV突变体的位置图 在体内表现出有缺陷的晚期表型。第二，VV晚期mRNA 将在无细胞蛋白质合成系统和晚期基因中翻译 根据其酶活性鉴定的产品(病毒粒子 酶)或多肽结构(例如，VP62或VP11)。这些是无细胞的 然后，分析可以与杂交抑制或杂交选择相结合 程序，以映射这些功能。曾经的代表VV迟到了 基因已经定位，最新的重组DNA和分子 生物学技术将被用于研究细胞的结构 转录单位，它们是如何表达和调控的 感染，以及编码多肽的性质和活性。什么时候 与已有的关于VV的信息进行比较和对比 早期基因，这些结果应该会提供一些机制的洞察力 VV用什么来实现其复合体的有序表达 受感染细胞内的发育程序。

For simplicity, vaccinia virus (VV) genes are routinely grouped into early or late classes depending on whether their expression is independent of, or dependent on, viral DNA synthesis. Very little is known concerning the mechanisms which govern the switch between early and late gene expression modes during the VV replicative cycle. Recently a considerable amount of data has been obtained concerning the structure, nucleotide sequence, and regulation of some "typical" early VV genetic loci. This information should provide the basis for the subsequent identification of VV early gene regulatory signals and the factors which recognize them. To date however, similar analyses have not been carried on the VV late genes. It is towards this problem that the experiments outlined in this proposal are directed. The genomic location of a number of VV late genes will be determined by two methods. First, DNA-mediated marker rescue techniques will be employed to map the positions of temperature-sensitive or drug-resistant VV mutants which exhibit a defective late phenotype in vivo. Second, VV late mRNA will be translated in cell-free protein synthesizing systems and late gene products identified on the basis of their enzymatic activities (virion enzymes) or polypeptide structure (e.g., VP62, or VP11). These cell-free assays can then be coupled with hybrid-arrest or hybrid-selection procedures in order to map these functions. Once representative VV late genes have been located, state-of-the-art recombinant DNA and molecular biology techniques will be used to study the structure of the transcriptional units, how they are expressed and regulated during infection, and the nature and activity of the encoded polypeptides. When compared and contrasted to the information already available concerning VV early genes, these results should provide some insight into the mechanisms which VV employs to achieve the ordered expression of it's complex developmental program within the infected cell.