Nucleoli are formed on the basis of ribosomal DNA (rDNA) clusters called Nucleolus Organizer Regions (NORs). Each NOR contains multiple genes coding for RNAs of the ribosomal particles. The prominent components of the nucleolar ultrastructure, fibrillar centers (FC) and dense fibrillar components (DFC), together compose FC/DFC units. These units are centers of rDNA transcription by RNA polymerase I (pol I), as well as the early processing events, in which an essential role belongs to fibrillarin. Each FC/DFC unit probably corresponds to a single transcriptionally active gene. In this work, we transfected human-derived cells with GFP-RPA43 (subunit of pol I) and RFP-fibrillarin. Following changes of the fluorescent signals in individual FC/DFC units, we found two kinds of kinetics: 1) the rapid fluctuations with periods of 2-3 min, when the pol I and fibrillarin signals oscillated in anti-phase manner, and the intensities of pol I in the neighboring FC/DFC units did not correlate. 2) fluctuations with periods of 10 to 60 min, in which pol I and fibrillarin signals measured in the same unit did not correlate, but pol I signals in the units belonging to different nucleoli were synchronized. Our data indicate that a complex pulsing activity of transcription as well as early processing is common for ribosomal genes.

• Keywords
• fibrillarin, fluctuation, pol I, rDNA, transcription pulsing,
• MeSH
• Cell Nucleolus chemistry   enzymology   MeSH
• Chromosomal Proteins, Non-Histone chemistry   metabolism   MeSH
• DNA-Directed RNA Polymerases chemistry   metabolism   MeSH
• HeLa Cells MeSH
• Immunohistochemistry MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromosomal Proteins, Non-Histone MeSH
• DNA-Directed RNA Polymerases MeSH
• fibrillarin MeSH Browser

The essential structural components of the nucleoli, Fibrillar Centers (FC) and Dense Fibrillar Components (DFC), together compose FC/DFC units, loci of rDNA transcription and early RNA processing. In the present study we followed cell cycle related changes of these units in 2 human sarcoma derived cell lines with stable expression of RFP-PCNA (the sliding clamp protein) and GFP-RPA43 (a subunit of RNA polymerase I, pol I) or GFP-fibrillarin. Correlative light and electron microscopy analysis showed that the pol I and fibrillarin positive nucleolar beads correspond to individual FC/DFC units. In vivo observations showed that at early S phase, when transcriptionally active ribosomal genes were replicated, the number of the units in each cell increased by 60-80%. During that period the units transiently lost pol I, but not fibrillarin. Then, until the end of interphase, number of the units did not change, and their duplication was completed only after the cell division, by mid G1 phase. This peculiar mode of reproduction suggests that a considerable subset of ribosomal genes remain transcriptionally silent from mid S phase to mitosis, but become again active in the postmitotic daughter cells.

• Keywords
• FC/DFC units, cell cycle, nucleolus, rDNA, replication,
• MeSH
• Cell Nucleolus metabolism   MeSH
• HeLa Cells MeSH
• Humans MeSH
• S Phase MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Nucleoli are formed on the basis of ribosomal genes coding for RNAs of ribosomal particles, but also include a great variety of other DNA regions. In this article, we discuss the characteristics of ribosomal DNA: the structure of the rDNA locus, complex organization and functions of the intergenic spacer, multiplicity of gene copies in one cell, selective silencing of genes and whole gene clusters, relation to components of nucleolar ultrastructure, specific problems associated with replication. We also review current data on the role of non-ribosomal DNA in the organization and function of nucleoli. Finally, we discuss probable causes preventing efficient visualization of DNA in nucleoli.

• Keywords
• DNA staining, NADs, Nucleolus, Replication, Transcription activity, rDNA,
• MeSH
• Cell Nucleolus genetics   metabolism   MeSH
• Humans MeSH
• DNA, Ribosomal genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• DNA, Ribosomal MeSH

Polycomb group (PcG) proteins of the Polycomb repressive complex 1 (PRC1) are found to be diffusely distributed in nuclei of cells from various species. However they can also be localized in intensely fluorescent foci, whether imaged using GFP fusions to proteins of PRC1 complex, or by conventional immunofluorescence microscopy. Such foci are termed PcG bodies, and are believed to be situated in the nuclear intechromatin compartment. However, an ultrastructural description of the PcG body has not been reported to date. To establish the ultrastructure of PcG bodies in human U-2 OS cells stably expressing recombinant polycomb BMI1-GFP protein, we used correlative light-electron microscopy (CLEM) implemented with high-pressure freezing, cryosubstitution and on-section labeling of BMI1 protein with immunogold. This approach allowed us to clearly identify fluorescent PcG bodies, not as distinct nuclear bodies, but as nuclear domains enriched in separated heterochromatin fascicles. Importantly, high-pressure freezing and cryosubstitution allowed for a high and clear-cut immunogold BMI1 labeling of heterochromatin structures throughout the nucleus. The density of immunogold labeled BMI1 in the heterochromatin fascicles corresponding to fluorescent "PcG bodies" did not differ from the density of labeling of heterochromatin fascicles outside of the "PcG bodies". Accordingly, an appearance of the fluorescent "PcG bodies" seems to reflect a local accumulation of the labeled heterochromatin structures in the investigated cells. The results of this study should allow expansion of the knowledge about the biological relevance of the "PcG bodies" in human cells.

• Keywords
• BMI1 protein, PcG body, Polycomb group proteins, correlative light-electron microscopy, heterochromatin, high-pressure freezing, immunogold labeling,
• MeSH
• Microscopy, Electron * MeSH
• Heterochromatin metabolism   MeSH
• Immunohistochemistry MeSH
• Cryopreservation MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Polycomb-Group Proteins MeSH
• Repressor Proteins chemistry   metabolism   MeSH
• Light * MeSH
• Pressure MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Heterochromatin MeSH
• Polycomb-Group Proteins MeSH
• Repressor Proteins MeSH

The nucleolus is a nuclear compartment that plays an important role in ribosome biogenesis. Some structural features and epigenetic patterns are shared between nucleolar and non-nucleolar compartments. For example, the location of transcriptionally active mRNA on extended chromatin loop species is similar to that observed for transcriptionally active ribosomal DNA (rDNA) genes on so-called Christmas tree branches. Similarly, nucleolus organizer region-bearing chromosomes located a distance from the nucleolus extend chromatin fibers into the nucleolar compartment. Specific epigenetic events, such as histone acetylation and methylation and DNA methylation, also regulate transcription of both rRNA- and mRNA-encoding loci. Here, we review the epigenetic mechanisms and structural features that regulate transcription of ribosomal and mRNA genes. We focus on similarities in epigenetic and structural regulation of chromatin in nucleoli and the surrounding non-nucleolar region and discuss the role of proteins, such as heterochromatin protein 1, fibrillarin, nucleolin, and upstream binding factor, in rRNA synthesis and processing.

• MeSH
• Cell Nucleolus genetics   metabolism   ultrastructure   MeSH
• Chromatin genetics   ultrastructure   MeSH
• Epigenesis, Genetic * MeSH
• Transcription, Genetic MeSH
• Genes, rRNA MeSH
• Histones metabolism   MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• DNA, Ribosomal genetics   MeSH
• Ribosomes genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Comparative Study MeSH
• Names of Substances
• Chromatin MeSH
• Histones MeSH
• RNA, Messenger MeSH
• DNA, Ribosomal MeSH

Pontin is a multifunctional protein having roles in various cellular processes including regulation of gene expression. Here, we addressed Pontin intracellular localization using two different monoclonal antibodies directed against different Pontin epitopes. For the first time, Pontin was directly visualized in nucleoli where it co-localizes with Upstream Binding Factor and RNA polymerase I. Nucleolar localization of Pontin was confirmed by its detection in nucleolar extracts and by electron microscopy, which revealed Pontin accumulation specifically in the nucleolar fibrillar centers. Pontin localization in the nucleolus was dynamic and Pontin accumulated in large nucleolar dots mainly during S-phase. Pontin concentration in the large nucleolar dots correlated with reduced transcriptional activity of nucleoli. In addition, Pontin was found to associate with RNA polymerase I and to interact in a complex with c-Myc with rDNA sequences indicating that Pontin is involved in the c-Myc-dependent regulation of rRNA synthesis.

• MeSH
• ATPases Associated with Diverse Cellular Activities MeSH
• Cell Nucleolus enzymology   ultrastructure   MeSH
• DNA Helicases metabolism   MeSH
• Transcription, Genetic MeSH
• HeLa Cells MeSH
• Humans MeSH
• RNA, Ribosomal biosynthesis   MeSH
• RNA Polymerase I metabolism   MeSH
• Pol1 Transcription Initiation Complex Proteins metabolism   MeSH
• Microscopy, Electron, Transmission MeSH
• Carrier Proteins metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATPases Associated with Diverse Cellular Activities MeSH
• DNA Helicases MeSH
• RNA, Ribosomal MeSH
• RNA Polymerase I MeSH
• RUVBL1 protein, human MeSH Browser
• transcription factor UBF MeSH Browser
• Pol1 Transcription Initiation Complex Proteins MeSH
• Carrier Proteins MeSH

T he organization of transcriptionally active ribosomal genes in animal cell nucleoli is investigated in this study in order to address the long-standing controversy with regard to the intranucleolar localization of these genes. Detailed analyses of HeLa cell nucleoli include direct localization of ribosomal genes by in situ hybridization and their indirect localization via nascent ribosomal transcript mappings. On the light microscopy (LM) level, ribosomal genes map in 10-40 fluorescence foci per nucleus, and transcription activity is associated with most foci. We demonstrate that each nucleolar focus observed by LM corresponds, on the EM level, to an individual fibrillar center (FC) and surrounding dense fibrillar components (DFCs). The EM data identify the DFC as the nucleolar subcompartment in which rRNA synthesis takes place, consistent with detection of rDNA within the DFC. The highly sensitive method for mapping nascent transcripts in permeabilized cells on ultrastructural level provides intense and unambiguous clustered immunogold signal over the DFC, whereas very little to no label is detected over the FC. This signal is strongly indicative of nascent "Christmas trees" of rRNA associated with individual rDNA genes, sampled on the surface of thin sections. Stereological analysis of the clustered transcription signal further suggests that these Christmas trees may be contorted in space and exhibit a DNA compaction ratio on the order of 4-5.5.

• MeSH
• Bromouracil analogs & derivatives   MeSH
• Cell Nucleolus genetics   ultrastructure   MeSH
• Microscopy, Electron MeSH
• Transcription, Genetic physiology   MeSH
• Genes, rRNA genetics   MeSH
• HeLa Cells MeSH
• In Situ Hybridization MeSH
• Humans MeSH
• Permeability MeSH
• Uridine analogs & derivatives   pharmacokinetics   MeSH
• Gold MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5-bromouridine MeSH Browser
• Bromouracil MeSH
• Uridine MeSH
• Gold MeSH

Under electron microscope, the matrix of sectioned mitochondria exhibits ribosomes and an oval, electron-transparent zone which is devoid of ribosomes and is named chondriolite. Fine fibers or clumps of an electron-dense material appeared in this zone after several fixation and contrasting steps and were identified with mitochondrial DNA by cytologists. To verify this assumption, we labeled DNA by a monoclonal antibody and a secondary antibody coupled to immunogold. The label was observed in the nucleus and in the chondriolite zone of sectioned mitochondria. Because the ultrastructure of chondriolites resembles that of nucleoids of prokaryotes, we suggest the term mitochondrial nucleoid for the zone of mitochondrial matrix devoid of ribosomes and containing DNA.

• MeSH
• Aspartate Carbamoyltransferase analysis   MeSH
• DNA, Fungal analysis   MeSH
• Microscopy, Immunoelectron methods   MeSH
• Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) analysis   MeSH
• DNA, Mitochondrial analysis   MeSH
• Mitochondria chemistry   ultrastructure   MeSH
• Multienzyme Complexes analysis   MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• Saccharomyces cerevisiae chemistry   genetics   ultrastructure   MeSH
• Submitochondrial Particles chemistry   ultrastructure   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Aspartate Carbamoyltransferase MeSH
• DNA, Fungal MeSH
• Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) MeSH
• DNA, Mitochondrial MeSH
• Multienzyme Complexes MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• URA2 protein, S cerevisiae MeSH Browser