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Previously, other workers have shown that isolated nucleoli can synthesize RNA in vitro that resembles pre-rRNA in base composition, marker oligonucleotides, and hybridization analysis (4-10). The present study further supports the fidelity of pre-rRNA synthesis by isolated nucleoli and nucleolar chromatin, using the homochromatographic fingerprinting technique, which permits a direct comparison of patterns of virtually all the oligonucleotides obtained by T1 ribonuclease digestion of 45S pre-rRNA.Abbreviation: pre-rRNA, preribosomal RNA. MATERIALS AND METHODSIsolation of Nucleoli and Nucleolar Chromatin. Nucleoli were isolated from 6-day-old Novikoff hepatoma ascites cells (11). They were suspended in 0.05 M Tris-HCI, pH 7.9/50% glycerol/i mM MgCl2/0.1 mM EDTA/1 mM dithiothreitol. RNA Synthesis In Vitro. RNA was synthesized in a standard system (6, 12). Similar conditions were used for assays with nucleolar chromatin template and solubilized polymerase I except that 5 mM MgCl2 was replaced by 2.5 mM MgCl2 and 2 mM MnCI2.When the in vitro transcripts were to be analyzed by ho-

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