DNA-dependent
RNA polymerase was solubilized from nuclei of Ehrlich
ascites carcinoma (EAC) cells by sonic disruption in the presence of 0.3 M (NH4)2 SO4, and the multiple
RNA polymerases were separated by chromatography on
DEAE-Sephadex A-25. Elution with a nine-step gradient of (NH4)2 SO4 yielded five peaks of activity designated
RNA polymerases Ia, Ib, IIa, IIb, and III, of which IIb was the most prominent. Linear-gradient elusion also yielded five peaks of the same designation, but Ia and Ib, as well as IIa and IIb, were not well separated. IIa and IIb were inhibited completely by 0.1 mug
alpha-amanitin/ml, whereas the other forms were not. EAC
RNA polymerases Ia, Ib, IIa, and IIb possessed Mg2+ ion, Mn2+ ion, and (NH4)2 SO4 optima, molecular weights, and thermal sensitivities similar to those reported for other mammalian
DNA-dependent RNA polymerases. As measured by relative ribonucleoside monophosphate incorporation, with native
calf thymus DNA template, EAC
RNA polymerases Ia and Ib synthesized
ribosomal RNA-like products, whereas forms IIa, IIb, and the parent
enzyme mixture synthesized compounds that were more similar to
DNA. No species specificity was found for
DNA templates, and denatured
DNA was consistently preferred to the native template by
RNA polymerases IIa and IIb; the two kinds of template were about equally efficient for
RNA polymerases Ia and Ib. Although EAC
RNA polymerases Ia, IIa, and IIb were inhibited by
daunomycin, form IIa was preferentially affected. 3',5'-Cyclic
AMP, 3',5'-Cyclic GMP, and
gibberellic acid, implicated as
RNA polymerase regulators in other systems, were generally ineffective. The levels of
nuclear RNA polymerase activities, per mg
DNA, of 3 mouse
ascites tumors (EAC, 6C3HED
lymphosarcoma, and TA3
adenocarcinoma) were compared with those from 3 normal mouse tissues (kidney, liver, and spleen). On the average, the
tumor cell nuclei contained (per mg of
DNA) 8.9, 1.5, 2.7, 20.0, and 3.8 times the activities of
RNA polymerases Ia, Ib, IIa, IIb, and III, respectively, as the normal cells, but the difference was significantly only for IIb.