The specificity of the anti-RNA polymerase I antibodies raised in rabbits was further confirmed by microinjection of purified IgG into the nuclei of quiescent Swiss 3T3 cells that were subsequently stimulated with serum (in collaboration with Dr. Renato Baserga). The microinjected immune IgG-inhibited nucleolar RNA synthesis (as measured by decrease in the number of grains derived from [?-3?H] uridine) decreased by 50 to 70% which persisted for at least 17 hrs. Preimmune IgG had no effect on the nucleolar RNA synthesis. Under these conditions, nucleoplasmic RNA synthesis was not altered. A 70% decrease in the content of nucleolar RNA was also observed following microinjection of antibodies directed against RNA polymerase I. Previous studies in this laboratory have shown that highly purified RNA polymerase I from rat hepatoma contains protein kinase activity which can phosphorylate and consequently activate the polymerase. In every respect, this kinase resembles nuclear protein kinase NII which consists of two subunits of Mr = 42,000 and 25,000. The possibility that two of the RNA polymerase I subunits of Mr = 42,000 and 25,000 might correspond to the protein kinase NII has been raised. Subsequent studies have shown that sera from patients with certain rheumatic autoimmune diseases contains antibodies to RNA polymerase I and that each patient exhibits a distinct spectrum of antibodies to spoecific subunits of the enzyme. Sera containing antibodies to subunits of Mr = 42,000and 25,000 always had antibodies to protein kinase NII, which suggests a biological association between the two enzymes. To determine whether protein kinase NII plays a role in the accurate transcription of rDNA, transcription of cloned rat rDNA containing the promoter and the initiation site was investigated using a partially purified whole cell extract. Fraction eluting at 175 mM (NH?4?)?2?SO?4? from DEAE-Sephadex column was resolved into two fractions using heparin-sepharose column; one eluting with 0.2 M NH?4?Cl (A) and the other eluting with 1 M NH?4?Cl (B) which contains RNA polymerase I activity. Either fraction A or B by itself did not support accurate transcription, but reconstitution of the two fractions yielded the anticipated transcript. More recent studies have shown that under certain conditions a transcriptionally active fraction containing RNA polymerase I and protein kinase NII can be obtained upon heparin sepharose chromatography. We are now in a position to dissociate this complex and determine the exact role of the protein kinase in rDNA transcription. (G)
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