STRUCTURE-FUNCTION OF GENES FOR VACCINIA ENCODED ENZYMES

痘苗病毒是一种生长在真核生物细胞质中的大型dna病毒。 细胞，编码RNA和DNA所需的全部酶 综合。编译了许多补充方法来定位 确切地说，编码对RNA合成至关重要的大多数酶的基因 还有一些是DNA合成所必需的。这些将包括所有的基因 DNA依赖的RNA聚合酶、mRNA封闭酶、多聚酶的亚基 A聚合酶、DNA聚合酶和DNA连接酶。特定的基因将是 将其插入到pMR100中以制备融合蛋白 B-半乳糖苷酶，以产生针对每种酶的特异性抗体，并 衍生出无意义的变种。这些含有无义突变的基因将是 标记救助法将痘苗病毒重新导入痘苗病毒并分离到突变病毒 在一株抑制细胞株L上。插入逆转录病毒载体的牛痘病毒基因 将用于衍生合成单个牛痘病毒的L细胞系 多肽。这些细胞系将被用来选择ts突变体。 编码表达多肽的基因。突变的牛痘基因 通过标记救援法重新引入病毒，后代病毒在 表达正常编码的L细胞上的非允许温度 多肽。L细胞上的空斑扩大分析将表明TS 子代病毒中的突变体和ts损伤的位置 表达正常多肽的L细胞系上的空斑形成。废话 突变体将定义编码病毒必需的酶亚基的基因 生长并在多肽上定位它们的催化和结合区。 RNA合成中的TS突变体将定义，RNA合成中的步骤， 信使核糖核酸结构变化的影响(即帽子缺失、甲基化缺失 在CAP中，缺少Poly A)对mRNA的丰度、稳定性和翻译都有影响。TS DNA合成中的突变体将允许研究DNA复制，以及 病毒基因表达的相关变化，特别是作图和 两类时间上不同的晚期基因的特征。

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.