Immunocytogenetics: localization of transcriptionally active rRNA genes in nucleoli and nucleolus organizer regions by use of human autoantibodies to RNA polymerase I

Abstract

Cytological staining with silver nitrate (AgN0₃) has proved useful for the localization of nucleoli in interphase nuclei, as well as of nucleolus organizer regions (NORs) in metaphase chromosomes. The affinity of interphase nucleoli and chromosomal NORs to silver is a direct measure of the ongoing transcriptional activity of the rRNA genes or their activity during the preceding interphase, respectively. Correspondingly, human autoantibodies directed against chromatin-associated RNA polymerase I (RPI) should also be of value in the investigation of transcribed rRNA genes. Indirect immunofluorescence using the anti-RPI antibody as a probe has been employed successfully to visualize the chromosomal distribution of NORs in various mammalian species, as well as in human tumor cells. Immunofluorescence staining even permits the identification of heteromorphisms and small aberrations of the chromosomal NORs. The fluorescent intensity of interphase nucleoli is correlated with the different stages of nucleolar activation. In male gametogenesis, RPI-positive granules are present during meiotic prophase up to pachytene, as well as during the early and middle spermatid stages.

A possible mechanism for the inhibition of ribosomal RNA gene transcription during mitosis.

“…In previous immunocytochemical studies it has been shown that pol I molecules remain associated with the chromosomol NORs as cells progress through mitosis despite littie or no RNA being synthesized during this phase of the cell cycle (Scheer and Rose, 1984;Reimer et al, 1987a;Haaf et al, 1988). We entertained the idea that the polymerases transiently stall during mitosis, perhaps in the form of "frozen" transcriptional units, and are reactivated toward the end of mitosis (Scheer and Rose, 1984).…”

“…On spread metaphase plates of PtK2 cells, the pol I-dependent immunofluorescence has been shown to correspond to the secondary constriction of the X chromosome (Scheer and Rose, 1984). The same antibodies also labeled the NORs of other mammalian species with the difference that the multiple fluorescence dots appeared smaller and less intense (data not shown; see also Haaf et al, 1988).…”

“…It is remarkable that in spite of the mitotic repression of the rRNA genes, major components of the pol I-dependent transcription machinery remain stably associated with the NORs and are probably never completely disengaged from the rDNA template. Among them are pol I (Scheer and Rose, 1984;Haaf et al, 1988), the transcription initiation factor UBF (Chan et al, 1991;Rendon et al, 1992;Roussel et al, 1993;Zatsepina et al, 1993), and DNA topoisomerase I, an enzyme which plays an important role in transcriptional processes (Guldner et al, 1986; for involvement of topoisomerase I in rDNA transcription see Zhang et al, 1988). Though quiescent in RNA synthesis, the NOR-bound pol I molecules seem to be present as engaged transcriptional complexes since they can be reactivated in vitro by Sarkosyl or heparin, i.e., agents that remove histones and other proteins from DNA while blocking new initiation events (Gariglio et al, 1974;Matsui et al, 1979;Matsui and Sandberg, 1985).…”

Extractions reveal specific argentophilic proteins in rat and bull sperm heads

“…The silver-stainability of acidic nonhistone proteins of the nucleolus during interphase and in the NORs of metaphase chromosomes is a direct measure of the transcriptional activity of the ribosomal RNA genes (Hubbell, 1985;Haaf et al, 1988) and has also been used for determining malignant transformations of intratubular germ cells (Delahunt et al, 1990;Loftus et al, 1990). Postmeiotically, silver-stainable nucleolar substance has been shown to abolish after prophase pachytene, reappear in the first stages of spermiogenesis, but to disappear once more during the elongation phase (Hofgartner et al, 1979;Ironside and 1979; Schmid et al, 1982, 1983, Mirre and Knibiehler, 1985Lunghi et al, 1987;Dadoune et al, 1991).…”

An Experimental Approach to the Study of AG-NOR Proteins.

“…Protein C23, also called nucleolin, is another abundant nucleolar phosphoprotein, with a molecular mass of around 100 kDa, which seems to be involved in the early stages of ribosome biogenesis and to be associated with rDNA-containing structures (19,44,48). The interest in Ag-NOR proteins started with the finding that their amount is directly related to the degree of transcriptional activity, its detection being used as a marker of nucleolar activity and cell proliferation (10,12,15,23,30,34,35,38,40).…”