STRUCTURE-FUNCTION OF GENES FOR VACCINIA ENCODED ENZYMES

牛痘病毒是一种大的DNA病毒，生长在真核细胞的细胞质中， 细胞和编码所需的RNA和DNA的酶的整个补充 合成. 编译了一些补充方法来定位 正是编码RNA合成关键酶的基因 还有一些是DNA合成所必需的 这些将包括所有的基因 依赖DNA的RNA聚合酶、mRNA加帽酶、多聚 聚合酶、DNA聚合酶和DNA连接酶。 特定的基因将 插入质粒PMR 100中以制备融合蛋白， β-半乳糖苷酶，以产生针对每种酶的特异性抗体， 产生无意义突变体。 这些含有无义突变的基因将被 通过标记拯救再引入牛痘病毒并分离突变病毒 在抑制型L细胞系上 插入逆转录病毒载体的牛痘基因 将用于衍生合成单个牛痘的L细胞系 多肽。 这些细胞系将用于选择ts突变体， 编码所表达多肽的基因。 变异的牛痘基因 通过标志物拯救引入回病毒中，并且后代病毒在 在表达正常编码的L细胞上的非允许温度 多肽。 对L细胞的空斑扩大试验将表明TS 子代病毒中的突变体和TS病变的位置通过以下方法证实： 在表达正常多肽的L细胞系上形成空斑。 废话 突变体将定义编码病毒所必需的酶亚单位的基因， 生长并将它们的催化和结合结构域定位在多肽上。 Ts突变体在RNA合成中将定义mRNA合成的步骤， mRNA结构变化的影响（即帽的丢失，甲基化的缺乏 在帽中，缺乏Poly A）对mRNA丰度、稳定性和翻译的影响。 Ts DNA合成中的突变体将允许研究DNA复制， 病毒基因表达的相关变化，特别是定位， 这是两类时间上不同的晚期基因的表征。

Vaccinia is a large DNA virus that grows in the cytoplasm of eukaryotic cells and encodes the entire complement of enzymes needed for RNA and DNA synthesis. A number of complementary methods are compiled to locate precisely the genes encoding most of the enzymes vital for RNA synthesis and some required for DNA synthesis. These will include all the genes for the subunits of the DNA dependent RNA polymerase, mRNA capping enzyme, poly a polymerase, DNA polymerase and DNA ligase. Specific genes will be inserted into the plasmid PMR100 to both prepare fusion proteins with B-galatosidase to make specific antibodies against each enzyme and to derive nonsense mutants. These genes containing nonsense mutations will be reintroduced into vaccinia virus by marker rescue and mutant virus isolated on a suppressor L cell line. Vaccinia genes inserted in retrovirus vectors will be used to derive L cell lines that synthesize individual vaccinia polypeptides. These cell lines will be used to select ts mutants in the gene encoding the expressed polypeptide. Mutated vaccinia genes are introduced back into virus by marker rescue, and the progeny virus grown at non permissive temperatures on the L cell expressing the normal encoded polypeptides. Plaque enlargement assays on L cells will indicate the ts mutants in the progeny virus and the location of the ts lesion confirmed by plaquing on the L cell line expressing the normal polypeptide. Nonsense mutants will define genes encoding enzyme subunits essential for virus growth and locate their catalytic and binding domains on the polypeptide. Ts mutants in RNA synthesis will define, the steps in mRNA synthesis, the effects of changes in mRNA structure (i.e. loss of cap, lack of methylation in cap, lack of Poly A) on mRNA abundance, stability and translation. Ts mutants in DNA synthesis will allow the study of DNA replication, and associated changes in viral gene expression, in particular the mapping and characterization of the two temporally distinct classes of late genes.