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

BACKGROUND: Protein exchange kinetics correlate with the level of chromatin condensation and, in many cases, with the level of transcription. We used fluorescence recovery after photobleaching (FRAP) to analyse the kinetics of 18 proteins and determine the relationships between nuclear arrangement, protein molecular weight, global transcription level, and recovery kinetics. In particular, we studied heterochromatin-specific heterochromatin protein 1β (HP1β) B lymphoma Mo-MLV insertion region 1 (BMI1), and telomeric-repeat binding factor 1 (TRF1) proteins, and nucleolus-related proteins, upstream binding factor (UBF) and RNA polymerase I large subunit (RPA194). We considered whether the trajectories and kinetics of particular proteins change in response to histone hyperacetylation by histone deacetylase (HDAC) inhibitors or after suppression of transcription by actinomycin D. RESULTS: We show that protein dynamics are influenced by many factors and events, including nuclear pattern and transcription activity. A slower recovery after photobleaching was found when proteins, such as HP1β, BMI1, TRF1, and others accumulated at specific foci. In identical cells, proteins that were evenly dispersed throughout the nucleoplasm recovered more rapidly. Distinct trajectories for HP1β, BMI1, and TRF1 were observed after hyperacetylation or suppression of transcription. The relationship between protein trajectory and transcription level was confirmed for telomeric protein TRF1, but not for HP1β or BMI1 proteins. Moreover, heterogeneity of foci movement was especially observed when we made distinctions between centrally and peripherally positioned foci. CONCLUSION: Based on our results, we propose that protein kinetics are likely influenced by several factors, including chromatin condensation, differentiation, local protein density, protein binding efficiency, and nuclear pattern. These factors and events likely cooperate to dictate the mobility of particular proteins.

• Publication type
• Journal Article 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

BACKGROUND: The evolution and biology of rDNA have interested biologists for many years, in part, because of two intriguing processes: (1) nucleolar dominance and (2) sequence homogenization. We review patterns of evolution in rDNA in the angiosperm genus Nicotiana to determine consequences of allopolyploidy on these processes. SCOPE: Allopolyploid species of Nicotiana are ideal for studying rDNA evolution because phylogenetic reconstruction of DNA sequences has revealed patterns of species divergence and their parents. From these studies we also know that polyploids formed over widely different timeframes (thousands to millions of years), enabling comparative and temporal studies of rDNA structure, activity and chromosomal distribution. In addition studies on synthetic polyploids enable the consequences of de novo polyploidy on rDNA activity to be determined. CONCLUSIONS: We propose that rDNA epigenetic expression patterns established even in F(1) hybrids have a material influence on the likely patterns of divergence of rDNA. It is the active rDNA units that are vulnerable to homogenization, which probably acts to reduce mutational load across the active array. Those rDNA units that are epigenetically silenced may be less vulnerable to sequence homogenization. Selection cannot act on these silenced genes, and they are likely to accumulate mutations and eventually be eliminated from the genome. It is likely that whole silenced arrays will be deleted in polyploids of 1 million years of age and older.

• MeSH
• DNA, Plant genetics   MeSH
• Epigenesis, Genetic * MeSH
• Phylogeny MeSH
• Evolution, Molecular * MeSH
• Polyploidy MeSH
• DNA, Ribosomal genetics   MeSH
• Nicotiana genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA, Ribosomal MeSH