Isolation of multiple forms of DNA-dependent RNA polymerase from calf-livernuclei.

Abstract

Three species of DNA‐dependent RNA polymerase were separated from calf liver nuclei. The nuclei were obtained from up to a maximum of 1800 g of homogenized tissue by centrifugation at low speed and were successively cleaned by centrifugation in 2 M sucrose and 1 mM MgCl2. The purification procedure consisted of sonication at high ionic strength of the nuclear pellet, chromatin removal and ammonium sulphate precipitation of the solubilized activity. The three species were obtained by chromatography on a DEAE‐Sephadex column and have been designated form A(0) and form A, both insensitive to α‐amanitin, and form B, sensitive to this toxin. They could be distinguished according to their dependence on ionic strength and pH, requirement of metal ions, and template specificity. The first peak of activity is not present in preparations from calf thymus, and its characteristics are different from any other nuclear RNA polymerase activity separated by DEAE‐Sephadex chromatography. Its presence seems then to be correlated to the occurrence of tissue specificity of mammalian RNA polymerase.