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.
