Purification of DNA‐Dependent RNA Polymerases A and B from Ovaries of Xenopus laevis

A method was developed for preparing RNA polymerases A and B from ovaries of adult female Xenopus laevis, avoiding sonication procedures at high ionic strength. The technique involves homogenisation in low ionic strength glycerol buffers, precipitation of contaminants with streptomycin sulphate and concentration of enzymes by ammonium sulphate precipitation; this is followed by resuspension of the protein pellets, centrifugation through glycerol density gradients and chromatography on DEAE‐cellulose and phosphocellulose. A and B enzymes, separated at the DEAE‐cellulose step, were purified 90‐fold and 200‐fold respectively by these procedures and were free of ribonuclease and deoxyribonuclease contamination. The two enzymes exhibited properties known to be characteristic of the major eukaryotic RNA polymerases in their ionic strength optima, cation and template requirements and responses to α‐amanitin.