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DNA-dependent RNA polymerase I was purified from the mouse myeloma, MOPC 315. The enzyme was solubilized from purified nucleoli and subjected to chromatography on CM-Sephadex, DEAE-Sephadex, and phosphocellulose ion exchange resins and to sedimentation in sucrose gradients. These procedures yielded a chromatographically homogeneous enzyme which was purified almost 10,000-fold with respect to whole cell extracts and which had a specific activity similar to those reported for other homogeneous eukaryotic RNA polymerases. Sucrose gradient sedimentation analysis suggested an enzyme molecular weight of 400,000 to 500,000.

RNA polymerase has been solubilized from whole cell homogenates of the mouse myeloma tumor, MOPC 315. Four forms of the solubilized enzyme have been isolated by ion exchange chromatography and designated I, II, IIIa, and IIIb. The enzymes appear analogous to the Class I, II, and III RNA polymerases defined in other eukaryotic systems. Ionic strength optima, metal ion effects, α-amanitin sensitivities, and relative activities with poly[d(A-T)] and calf thymus DNA templates are different for Class I, II, and III RNA polymerases, but are identical for IIIaand IIIb. For the Class I and Class II enzymes ionic strength optima occur, respectively, at 0.05 and 0.09 mammonium sulfate with either calf thymus DNA or poly[d(A-T)] as template. By contrast, the ionic strength activation profiles for Class III RNA polymerases with calf thymus DNA template are biphasic with polytima at 0.055 and 0.17 mammonium sulfate; but with poly[d(A-T)] the profiles are monophasic with optima at 0.055 m. Although RNA polymerase I is completely resistant to α-amanitin at concentrations up to 400 µg per ml, RNA polymerases II and III can both be inhibited completely by the toxin. However, the sensitivity of RNA polymerase II (50% inhibition at 0.025 µg per ml) is much greater than that of the Class III RNA polymerases (50% inhibition at 20 µg per ml). Furthermore, the poly[d(A-T)] to calf thymus DNA activity ratios are considerably greater for the Class III RNA polymerases than for RNA polymerases I and II. By measuring RNA polymerase activity in DEAE-Sephadex fractions at α-amanitin concentrations just sufficient to inhibit RNA polympolymerase II and in the presence of poly[d(A-T)] RNA polymerase III activity has been detected in all tissues examined.

The amounts of the various forms of RNA polymerase (Ia, Ib, IIa, IIb, and III) have been determined in oocytes, eggs, and embryos of Xenopus laevis. During oogenesis the relative proportions of the enzymes remain nearly constant, although the absolute levels of all forms increase dramatically. As a result, the mature oocyte, as well as the unfertilized egg, contains 4 to 5 orders of magnitude more RNA polymerase activity than is present in an individual somatic cell. Both oocytes and eggs contain unusually high levels of forms III and IIain addition to forms I and IIbwhich predominate in somatic cells. The amounts and relative proportions of the five enzymes in the unfertilized egg remain about the same up to gastrulation of the embryo. During subsequent stages of embryogenesis the total amounts of forms I and IIbincrease about 10-fold while the amounts of forms IIaand III increase only slightly. The rapid increase in cell number along with this selective increase in forms I and IIbduring early development re-establishes the same relative and absolute enzyme levels per cell in the swimming embryo that are present in adult somatic cells. Anucleolate mutant embryos at the early swimming stage have the same amount of each enzyme as do control embryos. Furthermore the relative amounts of the various enzymes in purified nuclei are the same in anucleolate embryos as they are in control embryos. Chromatographic heterogeneity in enzyme I was found at all stages of oocyte maturation and throughout embryogenesis of control and anucleolate mutant embryos.