Antiserum to a multisubunit
DNA-dependent
RNA polymerase from
vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus
RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of
RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of
mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable
polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early
mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early
mRNA that hybridized to vaccinia virus
DNA. The locations of the subunit genes were determined initially by hybridization of
RNA to a series of overlapping 40-kilobase-pair
DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that
RNA polymerase subunit genes are transcribed early in
infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A.