Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

Abstract

Abstract-Nucleoli are the morphological expression of the activity of a defined set of chromosomal segments bearing rRNA genes. The topological distribution and composition of the intranucleolar chromatin as well as the definition of nucleolar structures in which enzymes of the rDNA transcription machinery reside have been investigated in mammalian cells by various immunogold labelling approaches at the ultrastructural level. The precise intranucleolar location of rRNA genes has been further specified by electron microscopic in situ hybridization with a non-autoradiographic procedure.Our results indicate that the fibrillar centers are the sole nucleolar structures where rDNA, core histones, RNA polymerase I and DNA to po isomerase I are located together.Taking into account the potential value and limitations of immunoelectron microscopic techniques, we propose that transcription of the rRNA genes takes place within the confines of the fibrillar centers, probably close to the boundary regions to the surrounding dense fibrillar component.

The RNA polymerase I transcription factor UBF and rDNA are located at the same major sites in both interphase and mitotic pig embryonic kidney (PK) cells

“…1995). Thus, UBF was revealed predominantly in the dense fibrillar component (DFC) (Roussel et al, 1993), at the periphery of fibrillar centers (FC) (Zatsepina et al, 1993), or in both the DFC and FC Rodrigo et al, 1992;Raska et al, 1995), as well as rDNA (Thiry et al, 1990;Wachtlcret al, 1992;Jimcncz-Garcia et al, 1993).…”

“…in situ hybridization with specific rRNA or rDNA probes has been developed (Evans et al. 1974;Wachtler et al, 1986;Thiry et al. 1990).…”

Assigning functions to nucleolar structures

“…These include autoradiography of cells pulse-labelled with tritiated uridine, selective DNA staining, in situ hybridization with appropriate rDNA probes, immunolocalization of intranucleolar DNA by using antibodies directed against DNA, and immunogold detection of enzymes involved in rDNA transcription such as RNA polymerase I and DNA topoisomerase I (summarized in Scheer and Benavente 1990;Derenzini et al 1990;Thiry et al 1991). These studies have clearly shown that the fibrillar centers are the sole nucleolar elements where rDNA as well as the transcriptional machinery coexist.…”

A novel histone variant localized in nucleoli of higher plant cells

“…Although several nucleolar proteins are known to be targeted to restricted domains within the nucleolus, there is a discrepancy concerning the spatial distribution of ribosomal DNA (rDNA) in these domains. In nucleoli of animal cells, rDNA is suggested to reside in the DFC (Wachtler et al 1990) or in the FC (Thiry et al 1991). On the other hand, in nucleoli of higher plant cells, it is likely that rDNA is mostly localized to the FC (Martin et al 1989;Shaw 1990, Sato andYano 1994).…”