Immunological analyses of selected eukaryotic RNA polymerases II

Abstract

Polyclonal antibodies to native RNA polymerase II of Achlya ambisexualis and Agaricus bisporus were produced in rabbits and in mice. Monoclonal antibodies were produced against the α-amanitin resistant RNA polymerase II of the mushroom A. bisporus. These antibodies were used in comparative cross-reactivity studies with five purified RNA polymerases II (A. bisporus, A. ambisexualis, Saccharomyces cerevisiae, wheat germ, and calf thymus). A method for quantitatively comparing cross-reactivity was developed utilizing an enzyme-linked immunosorbant assay (ELISA). ELIS A comparisons indicated that the two filamentous fungi cross-reacted effectively with one another and depending upon the preparation reacted less effectively with yeast and wheat germ RNA polymerases II. Cross-reactivity measurements were also made by immunoblotting sodium dodecyl sulfate – polyacrylamide separated RNA polymerases II. The mouse anti-A. bisporus RNA polymerase II immunoglobulin G (IgG) and the monoclonal antibody preparations did not react with high molecular subunits of A. bisporus RNA polymerase II. The sera did, however, cross-react with high molecular weight subunits of A. ambisexualis. Similarily, rabbit anti-A. ambisexualis RNA polymerase II IgG reacted only with low molecular weight subunits of A. bisporus RNA polymerase II, but reacted with high molecular weight subunits of A. ambisexualis and wheat germ. Our results indicate differences in the cross-reactivity of native and denatured RNA polymerases II and suggest differences in the tertiary and quaternary organization of the enzymes examined.