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.

• Klíčová slova
• DNA staining, NADs, Nucleolus, Replication, Transcription activity, rDNA,
• MeSH
• buněčné jadérko genetika   metabolismus   MeSH
• lidé MeSH
• ribozomální DNA genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• ribozomální DNA MeSH

Nuclear actin and nuclear myosin I (NMI) are important players in transcription of ribosomal genes. Transcription of rDNA takes place in highly organized intranuclear compartment, the nucleolus. In this study, we characterized the localization of these two proteins within the nucleolus of HeLa cells with high structural resolution by means of electron microscopy and gold-immunolabeling. We demonstrate that both actin and NMI are localized in specific compartments within the nucleolus, and the distribution of NMI is transcription-dependent. Moreover, a pool of NMI is present in the foci containing nascent rRNA transcripts. Actin, in turn, is present both in transcriptionally active and inactive regions of the nucleolus and colocalizes with RNA polymerase I and UBF. Our data support the involvement of actin and NMI in rDNA transcription and point out to other functions of these proteins in the nucleolus, such as rRNA maturation and maintenance of nucleolar architecture.

• MeSH
• aktiny metabolismus   MeSH
• buněčné jadérko metabolismus   MeSH
• genetická transkripce fyziologie   MeSH
• HeLa buňky MeSH
• imunohistochemie MeSH
• lidé MeSH
• myosin typu I metabolismus   MeSH
• ribozomální DNA metabolismus   MeSH
• RNA ribozomální metabolismus   MeSH
• RNA-polymerasa I metabolismus   MeSH
• transkripční iniciační komplex Pol1 - proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• myosin typu I MeSH
• ribozomální DNA MeSH
• RNA ribozomální MeSH
• RNA-polymerasa I MeSH
• transcription factor UBF MeSH Prohlížeč
• transkripční iniciační komplex Pol1 - proteiny MeSH

Ribosomal genes are organized in clusters termed Nucleolus Organizer Regions (NORs). Essential components of the RNA polymerase I transcription machinery, including Upstream Binding Factor (UBF), can be detected on some NORs during mitosis; these NORs, termed competent, are believed to be transcriptionally active during interphase. In cultured mammalian cycling cells, the number of competent NORs, and their distribution among the different chromosomes, does not vary significantly in the sequential cell cycles. In this work we investigate whether this stable state is achieved by equal distribution of competent NORs during cell division. To answer this question we first studied the state of NORs in telophase HeLa and LEP cells. In both cell lines we found a small but significant difference between the emerging daughter cells in the number of UBF-loaded NORs. To reveal the cause of this difference, we followed the fate of individual NOR using HeLa derived cell line stably expressing UBF-GFP. We demonstrated that some NORs in metaphase are "asymmetrical", i.e. they lack the signal of competence on one of the sister chromatids. Regular presence of such NORs can account for the difference in the number of competent NORs obtained by the daughter cells emerging in mitosis.

• MeSH
• buněčné linie MeSH
• chromatidy MeSH
• genetická transkripce MeSH
• lidé MeSH
• mitóza * MeSH
• organizátor jadérka * MeSH
• transkripční iniciační komplex Pol1 - proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• transcription factor UBF MeSH Prohlížeč
• transkripční iniciační komplex Pol1 - proteiny MeSH

In human cells ribosomal genes are organized as clusters, NORs, situated on the short arms of acrocentric chromosomes. It was found that essential components of the RNA polymerase I transcription machinery, including UBF, can be detected on some NORs, termed "competent" NORs, during mitosis. The competent NORs are believed to be transcriptionally active during interphase. However, since individual NORs were not observed in the cell nucleus, their interphase status remains unclear. To address this problem, we detected the competent NORs by two commonly used methods, UBF immunofluorescence and silver staining, and combined them with FISH for visualization of rDNA and/or specific chromosomes. We found that the numbers of competent NORs on specific chromosomes were largely conserved in the subsequent cell cycles, with certain NOR-bearing homologues displaying a very stable pattern of competence. Importantly, those and only those NORs that were loaded with UBF incorporated bromo-uridine in metaphase after stimulation with roscovitine and in telophase, suggesting that competent and only competent NORs contain ribosomal genes transcriptionally active during interphase. Applying premature chromosome condensation with calyculin A, we visualized individual NORs in interphase cells, and found the same pattern of competence as observed in the mitotic chromosomes.

• MeSH
• barvení stříbrem MeSH
• buněčný cyklus * účinky léků   MeSH
• genetická transkripce * účinky léků   MeSH
• HeLa buňky MeSH
• hybridizace in situ fluorescenční MeSH
• interfáze účinky léků   MeSH
• karyotypizace MeSH
• lidé MeSH
• lidské chromozomy genetika   MeSH
• metafáze účinky léků   MeSH
• organizátor jadérka účinky léků   genetika   MeSH
• puriny farmakologie   MeSH
• roskovitin MeSH
• telofáze účinky léků   MeSH
• transkripční iniciační komplex Pol1 - proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• puriny MeSH
• roskovitin MeSH
• transcription factor UBF MeSH Prohlížeč
• transkripční iniciační komplex Pol1 - proteiny MeSH

As previous studies suggested, nuclear myosin I (NMI) and actin have important roles in DNA transcription. In this study, we characterized the dynamics of these two proteins during transcriptional activation in phytohemagglutinin (PHA) stimulated human lymphocytes. The stimulation led to strong up-regulation of NMI both on the mRNA and protein level, while actin was relatively stably expressed. The intranuclear distribution of actin and NMI was evaluated using immunogold labeling. In nucleoli of resting cells, actin was localized predominantly to fibrillar centers (FCs), while NMI was located mainly to the dense fibrillar component (DFC). Upon stimulation, FCs remained the main site of actin localization, however, an accumulation of both actin and NMI in the DFC and in the granular component was observed. In the nucleoplasm of resting lymphocytes, both actin and NMI were localized mostly in condensed chromatin. Following stimulation, the majority of both proteins shifted towards the decondensed chromatin. In transcriptionally active cells, both actin and NMI colocalized with nucleoplasmic transcription sites. These results demonstrate that actin and NMI are compartmentalized in the nuclei where they can dynamically translocate depending on transcriptional activity of the cells.

• MeSH
• aktiny metabolismus   MeSH
• buněčné jadérko účinky léků   metabolismus   ultrastruktura   MeSH
• buněčné jádro účinky léků   metabolismus   ultrastruktura   MeSH
• fytohemaglutininy farmakologie   MeSH
• genetická transkripce genetika   MeSH
• lidé MeSH
• lymfocyty účinky léků   metabolismus   ultrastruktura   MeSH
• messenger RNA metabolismus   MeSH
• myosin typu I metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• transmisní elektronová mikroskopie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• fytohemaglutininy MeSH
• messenger RNA MeSH
• myosin typu I MeSH