Immunolocalization of a subnucleolar nucleoprotein complex containing RNA polymerase 1 in ascites hepatoma cells...

We have isolated a discrete subnucleolar macromolecular nucleoprotein complex by direct treatment of Novikoff ascites hepatoma nucleoli by MspI restriction digestion. Using a monoclonal antibody made against the subnucleolar nucleoprotein complex that was shown to inhibit RNA polymerase (pol) 1 activity in vitro, we localized an Mr approximately 55,000 protein subunit which was demonstrated previously by an enzyme-linked immunosorbent assay and Western blotting to share epitopes with the RNA pol 1 moiety of the subnucleolar complex. By indirect immunofluorescence the distribution of the Mr approximately 55,000 component of the subnucleolar nucleoprotein complex was examined at various phases of the cell cycle. At prophase, it was localized in large (approximately 1.5 microns in diameter) ball-like structures associated with the nuclear periphery and nuclear peripheral chromatin, suggesting that these structures might be related to preribosomal elements. After chromatin condensation and the pairing of daughter chromosomes, the large ball-like spheres increased in size and were associated with propidium iodide staining at one side of the nucleus; whereas throughout and especially at the opposite side of the nucleus, smaller, round, punctate structures of approximately 0.5 micron in diameter were visibly labeled that were not associated with propidium iodide staining. At later stages of the cell cycle, these small round structures were again associated with propidium iodide staining, suggesting that they may be related to prenucleolar and/or preribosomal elements which would likely contain the appropriate nucleic acid in association with RNA pol 1 and cofactors of RNA pol 1.