Ultrastructural localization of Ag-NOR proteins and nucleic acids in reticulated nucleoli

Nucleoli with a thread-like fibrillar structure found in several human breast cancer cells were investigated for the localization of Ag-NOR proteins, RNA, DNA and associated nucleoproteins. Various cytochemical techniques were applied: the one-step silver method for Ag-NOR proteins, the Bernhard's staining preferential for RNP, the Feulgen-like osmium-ammine reaction for DNA and the RNase- and DNase-gold complexes for the demonstration of RNA and DNA respectively. Staining for Ag-NOR proteins was restricted to the edge of the threads or cords and to the nodes of the nucleolar network. The Bernhard's EDTA technique gave a positive staining of the entire nucleoli and on small peripheral granules which were linked to the nucleolar cords by fine RNP fibrils. Localization of RNA molecules by the RNase-gold complex showed a similar pattern, dense labeling by gold particles over the nucleoli and at the periphery of the cords. DNA molecules were revealed by the osmium-ammine technique in small nucleoplasmic clumps and at the edge of the nucleolar cords. Similar results were obtained by applying the DNase-gold complex which showed a labeling by gold particles over the dispersed chromatin and at the edge of the nucleolar cords. The consistency of the results obtained speaks in favor of their specificity and seems to demonstrate the simultaneous localization of certain Ag-NOR proteins, DNA and RNA molecules at the edge of the nucleolar cords. This edge of nucleolar cords becomes then the candidate for the site of nucleolar transcriptional activity.