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D Hernandez-Verdun

Publications and source records attributed to D Hernandez-Verdun.

At least 37 records · Page 2Linked to original sources
PubMed1997

When rDNA transcription is arrested during mitosis, UBF is still associated with non-condensed rDNA.

The mechanisms that control inactivation of ribosomal gene (rDNA) transcription during mitosis is still an open question. To investigate this fundamental question, the precise timing of mitotic arrest was established. In PtK1 cells, rDNA transcription was still active in prophase, stopped in prometaphase until early anaphase, and activated in late anaphase. Because rDNA transcription can still occur in prophase and late anaphase chromosomes, the kinetics of rDNA condensation during mitosis was questioned. The conformation of the rDNA was analyzed by electron microscopy from the G2/M transition to late anaphase in the secondary constriction, the chromosome regions where the rDNAs are clustered. Whether at transcribing or non-transcribing stages, non-condensed rDNA was observed in addition to axial condensed rDNA. Thus, the persistence of this non-condensed rDNA during inactive transcription argues in favor of the fact that mitotic inactivation is not the consequence of rDNA condensation. Analysis of the three-dimensional distribution of the rDNA transcription factor, UBF, revealed that it was similar at each stage of mitosis in the secondary constriction. In addition, the colocalization of UBF with non-condensed rDNA was demonstrated. This is the first visual evidence of the association of UBF with non-condensed rDNA. As we previously reported that the rDNA transcription machinery remained assembled during mitosis, the colocalization of rDNA fibers with UBF argues in favor of the association of the transcription machinery with certain rDNA copies even in the absence of transcription. If this hypothesis is correct, it can be assumed that condensation of rDNA as well as dissociation of the transcription machinery from rDNA cannot explain the arrest of rDNA transcription during mitosis. It is proposed that modifications of the transcription machinery occurring in prometaphase could explain the arrest of transcription, while reverse modifications in late anaphase could explain activation.

Animals↗
PubMed1996

Conditions favoring RNA polymerase I transcription in permeabilized cells.

RNA synthesis can be detected in nuclei using modified RNA precursors (Br-UTP) introduced in permeabilized cells. Surprisingly, RNA pol I transcripts are detected only after inhibition of RNA pol II or salt enhancement of RNA pol I activity. By modifying a previously reported protocol, we found that RNA pol I transcripts can be detected selectively or simultaneously with RNA pol II transcripts without any drug treatment. Removing glycerol from the permeabilization and transcription buffers and improving the permeabilization using Triton X-100 revealed RNA pol I transcription in two cell lines (mammalian and Xenopus) and in isolated mouse oocytes. The transcripts were most probably rRNA because they were detected in the nucleoli, digested by RNase, sensitive to actinomycin D, and resistant to alpha-amanitin. We found by microinjection of the Br-UTP precursors in living cells that low ionic strength allows the detection of RNA pol I transcription. Electron microscopy of mouse oocytes showed that the "looseness" of the nucleolar organization is associated with the detection of the RNA pol I transcription; this detection does not necessarily need nucleolar disorganization. The data obtained with both permeabilized cells and microinjections of RNA precursors in the absence of glycerol support the hypothesis that the degree of hydration of the cell plays a role in RNA pol I transcription.

Animals↗
PubMed1996

News and views on the nucleolus in 1996. Report on the 'Colloquium on the nucleolus', a meeting at Paris-Grignon, July 18-20, 1996.

The new data presented by the participants during the 'Colloquium on the Nucleolus' were reviewed and regrouped in different topics concerning the molecular and functional significance of nucleolar structure. The topics included: rRNA transcription and nucleolar organization, the nucleolus in relation to cell proliferation including nucleolus in pathology during the cell cycle and cell division, nucleologenesis, and nucleolar proteins.

Animals↗
PubMed1996

The rDNA transcription machinery is assembled during mitosis in active NORs and absent in inactive NORs.

In cycling cells, the rDNAs are expressed from telophase to the end of G2 phase. The early resumption of rDNA transcription at telophase raises the question of the fate of the rDNA transcription machinery during mitosis. At the beginning of mitosis, rDNA transcription is arrested, and the rDNAs are clustered in specific chromosomal sites, the nucleolar organizer regions (NOR). In human cells, we demonstrate that the rDNA transcription machinery, as defined in vitro, is colocalized in some NORs and absent from others whatever the mitotic phase: RNA polymerase I and the RNA polymerase I transcription factors, upstream binding factor and promoter selectivity factor (as verified for TATA-binding protein and TATA-binding protein-associated factor for RNA polymerase I [110]), were colocalized in the same NORs. The RNA polymerase I complex was localized using two different antibodies recognizing the two largest subunits or only the third largest subunit, respectively. These two antibodies immunoprecipitated the RNA polymerase I complex in interphase cells as well as in mitotic cells. These results clearly indicated that the RNA polymerase I complex remained assembled during mitosis. In addition, RNA polymerase I and the transcription factors varied in the same proportions in the positive NORs, suggesting stoichiometric association of these components. The fact that the rDNA transcription machinery is not equally distributed among NORs most likely reflects the implication of the different NORs during the subsequent interphase. Indeed, we demonstrate that only positive NORs exhibit transcription activity at telophase and that the level of transcription activity is related to the amount of rDNA transcription machinery present in the NOR. We propose that assembly of rDNA transcription machinery preceding mitosis determines expression of the rDNAs at the beginning of the next cell cycle. Consequently, the association of rDNAs with the rDNA transcription machinery defines the "active" NORs and the level of activity at the transition telophase/interphase.

Cell Nucleolus↗
PubMed1996

The Ag-NOR proteins present a crescent-shaped distribution at the secondary constrictions of metaphase PtK1 chromosomes.

The ultrastructural distribution of the proteins responsible for the silver staining of NORs was analyzed on serial thin sections of preembedded silver-stained rat kangaroo PtK1 metaphase chromosomes. Our results show that the Ag-NOR proteins present a crescent-shaped distribution at the secondary constriction of each chromatid. Moreover, in some cases the crescent-shaped structures of both chromatids are not symmetrically arranged but show different orientations. These observations, together with our own and previously reported light microscopy results obtained on silver-stained mammalian chromosomes, lead us to suggest a mechanism for the formation of apparent secondary constrictions at NORs.

Animals↗
PubMed1996

Osmium ammine: review of current applications to visualize DNA in electron microscopy.

This review has been collectively written. The contribution of the authors is mentioned for each part. References have been grouped at the end of the review. The objective of this review is to outline the principle of the method for electron microscopy, to emphasize the major applications and recent developments of this technique for DNA detection and finally to compare this technique with some other methods of DNA detection.

Coloring Agents↗
PubMed1995

Quantitative changes of the two major AgNOR proteins, nucleolin and protein B23, related to stimulation of rDNA transcription.

The relationship between the amount of the two major AgNOR proteins, nucleolin and protein B23, and the rate of ribosomal RNA (rRNA) synthesis was studied in cortisol-treated and regenerating rat hepatocytes after partial hepatectomy. In both experimental models the synthesis of rRNA was greatly stimulated, but only in regenerating hepatocytes was the increased synthesis associated with cells entering the mitotic cycle. Nucleolin and protein B23 were identified on SDS-polyacrylamide gels of nuclear proteins transferred to nitrocellulose and detected (1) by immunoreaction with specific monoclonal antibodies revealed by second antibodies coupled to peroxidase followed by chemiluminescence or (2) by the silver-staining procedure for AgNOR proteins. Nucleolin and protein B23 were then quantified by computerized densitometric analysis of the immunolabeling signals or the silver-stained bands at 105 kDa (nucleolin) and 39 kDa (protein B23). The synthesis of rRNA was measured by evaluating the amount of radioactivity incorporated into pre-rRNA after [3H]orotic acid injection. Densitometric analysis of silver-stained bands and immunolabeling signals showed no change in nucleolin and protein B23 amounts in cortisol-stimulated hepatocytes, whereas a moderate increase was found in regenerating hepatocytes at 12 h after partial hepatectomy. In both cortisol-stimulated and regenerating hepatocytes the synthesis of rRNA was highly increased (2.6-fold and 4.3-fold above the control level, respectively). To ascertain the relationship between quantitative changes in nucleolin and protein B23 and stimulation of rRNA transcriptional activity in regenerating hepatocytes, the quantitative distribution of these proteins was also investigated in the early times of regeneration using silver-stained nitrocellulose-transblotted nuclear proteins. The quantity of protein B23 was unchanged until 12 h after partial hepatectomy, whereas nucleolin appeared to be slightly increased at 9 h (1.15-fold above the control value) after partial hepatectomy. On the other hand, at just 6 h after partial hepatectomy, a significant increase of rRNA synthesis occurred in regenerating rat hepatocytes (1.8-fold above the control value). These data demonstrated that stimulation of rRNA transcriptional activity occurring in rat hepatocytes after cortisol treatment and in the early times after partial hepatectomy was not associated with quantitative changes in the amounts of nucleolin and protein B23.

Animals↗
PubMed1995

Amount variability of total and individual Ag-NOR proteins in cells stimulated to proliferate.

Ribosomal genes are associated with a subset of acidic proteins called Ag-NOR proteins. The amount of nucleolar Ag-NOR proteins varies, depending on nucleolar activity and/or cell proliferation. To understand the linkage between the amount of Ag-NOR proteins, ribosome biogenesis, and cell proliferation, we investigated the variability of Ag-NOR proteins in rRNA-stimulated cells maintained in G1 and in rRNA-stimulated cells entering the mitotic cycle. Rat hepatocytes were stimulated with cortisol for rRNA synthesis (1, 4, and 8 hr) and the cell cycle was induced by hepatectomy in regenerating hepatocytes (3-21 hr). In non-stimulated hepatocytes, nucleolin and protein B23 were the two major Ag-NOR proteins, corresponding to 70% of total Ag-NOR staining. In hepatocytes stimulated for rRNA synthesis in G1, the amount of Ag-NOR proteins was only slightly increased, whereas in cycle-stimulated cells it was increased 3.04-fold. This is the consequence of a differential increase of the major Ag-NOR proteins that appears earlier and is proportionally more important for nucleolin (3.5-fold) than for protein B23 (twofold) and also for the increase of several minor Ag-NOR proteins. We conclude that, in dividing cells, the mean value of the Ag-NOR proteins measured reflects the percentage of cells in the different phases. This could explain why the amount of Ag-NOR proteins can be used as a marker of cell proliferation.

Animals↗
PubMed1995

The three-dimensional organization of ribosomal genes and the architecture of the nucleoli vary with G1, S and G2 phases.

The three-dimensional (3-D) organization of the nucleolus, a defined nuclear territory containing tandem repeats of the ribosomal genes (rDNA), was investigated in PtK1 cells. Identification of the interphase stages was performed in single cells using DNA quantification by cytometry for the G1 and G2 phases while the S phase was identified by immunolabelling of the proliferating cell nuclear antigen (PCNA). The 3-D organization of the rDNA in the nucleolus was analyzed by fluorescence in situ hybridization using confocal microscopy. All the rDNA was located inside the nucleolar structures during all stages and the two rDNA loci were orthogonal. The rDNA was heterogeneously distributed in each nucleolus during G1, S and G2, with alternate sites of clustered genes (spots) and of genes in more extended configurations. The number of spots (4 to 6 in G1) increased during S phase (7 to 12) and their 3-D organization was progressively relaxed from G1 to G2. Double spots in G2 could reflect a similar gene organization of two chromatids. During mid-S phase, PCNA co-localized with some clustered genes (spots), indicating that rDNA replication occurs inside nucleoli and at different sites of the same locus simultaneously. The evaluation of the rDNA transcription units in 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)-treated cells indicated a mean of 16 units per G1 nucleus and 25 units per G2 nucleus. For G1 and G2, the fine 3-D structure of nucleoli was reconstructed using ultrathin serial sections after specific contrast of DNA and RNA, digitization of the serial section images and computer-assisted 3-D architecture. Fibrillar centers (FCs) formed discrete structures (about 10 in G1 and 20 in G2) connected by a network of the dense fibrillar component. The 3-D arrangement of the FCs in G1 and G2 are similar to the rDNA spots. In conclusion, the architecture of the nucleoli during interphase reflects the distribution of the rDNA that is characterized by alternation of clustered and extended genes.

Animals↗
PubMed1994

The chromosome periphery during mitosis.

A complex structure, visible by electron microscopy, surrounds each chromosome during mitosis. The organization of this structure is distinct from that of the chromosomes and the cytoplasm. It forms a perichromosomal layer that can be isolated together with the chromosomes. This layer covers the chromosomes except in centromeric regions. The perichromosomal layer includes nuclear and nucleolar proteins as well as ribonucleoproteins (RNPs). The list of proteins and RNAs identified includes nuclear matrix proteins (perichromin, peripherin), nucleolar proteins (perichro-monucleolin, Ki-67 antigen, B23 protein, fibrillarin, p103, p52), ribosomal proteins (S1) and snRNAs (U3 RNAs). Only limited information is available about how and when the perichromosomal layer is formed. During early prophase, the proteins extend from the nucleoli towards the periphery of the nucleus. Thin cordon-like structures reach the nuclear envelope delimiting areas in which chromosomes condense. At telophase, the proteins are associated with the part of the chromosomes remaining condensed and accumulate in newly formed nucleoli in regions where chromatin is already decondensed. The perichromosomal layer contains several different classes of proteins and RNPs and it has been attributed various roles: (1) in chromosome organization, (2) as a barrier around the chromosomes, (3) involvement in compartmentation of the cells in prophase and telophase and (4) a binding site for chromosomal passenger proteins necessary to the early process of nuclear assembly.

Animals↗
PubMed1994

Identification of Ag-NOR proteins, markers of proliferation related to ribosomal gene activity.

During mitosis, ribosomal genes are associated with a set of silver-stained nucleolar proteins designated Ag-nucleolar organizer region (NOR) proteins. The amount of Ag-NOR protein, estimated during interphase, may be used as marker of cell proliferation with a prognostic value for several human cancers. Our objective was to identify the Ag-NOR proteins in human transformed cell lines at specific phases of the cell cycle and in a hamster cell line that serves as model for active ribosomal transcription. During interphase, the major Ag-NOR proteins in both human and hamster cells were nucleolin and protein B23 and also proteins of 42, 40, and 29 kDa, which accounted for a small amount of the silver stain. The pIs of these proteins were between 4.5 and 5.6. During mitosis, the Ag-NOR proteins were either solubilized in the cytoplasm, distributed around the chromosomes, or associated with the ribosomal genes, i.e., in the NORs. The major Ag-NOR proteins associated with the ribosomal genes were the largest RNA polymerase I subunit, the 135-kDa NOR protein, the UBF transcription factor, and a 50-kDa protein. Less than 5% of the total nucleolin remained associated with ribosomal genes during mitosis. Using the purified RNA polymerase I complex from yeast, we demonstrate that the 190-, 43-, and 34.5-kDa subunits are Ag-NOR proteins in this species. This study demonstrates that the major Ag-NOR proteins in nucleoli during interphase are not the same as those associated with the ribosomal genes during mitosis. We conclude that the prognostic test for human cancer cell proliferation is largely based on the amount of the nucleolar proteins, nucleolin, and protein B23, which are not directly involved in ribosomal gene transcription. In contrast, the evaluation of active NORs in karyotypes during mitosis is based on the presence of some proteins of the ribosomal gene transcription machinery.

Antigens, Nuclear↗
PubMed1994

Fate of specific nucleolar perichromosomal proteins during mitosis: cellular distribution and association with U3 snoRNA.

In mammalian cells, the nucleoli disintegrate during mitosis and some nucleolar proteins disperse at the periphery of all chromosomes forming a novel class of chromosomal passenger proteins. The nucleolar components which participate in the formation of this perichromosomal layer have been investigated to elucidate the role of these perichromosomal proteins in the assembly and disassembly of the nucleoli. i) Electron microscopy immunolabelling reveals that these proteins are predominantly located in the granular component of the nucleoli during interphase. ii) Immunoprecipitation data suggest that they are distributed at the chromosome periphery in association with U3 small nucleolar RNA (snoRNA). In addition, the distribution of U3 snoRNA visualized by in situ hybridization, is similar to that observed for the perichromosomal proteins. iii) In cells which possess a nucleolar remnant during mitosis, U3 snoRNA and perichromosomal proteins were found both in the perichromosomal layer and in the nucleolar remnant. iv) Some of these proteins are conserved from yeast to man such as fibrillarin and a protein of 52 kDa. v) The location of these proteins observed in yeast by confocal microscopy shows that they are not dispersed during mitosis. Their partition between the two daughter cells is performed by scission of nucleolar structures forming a rod during the budding process. Therefore RNP complexes related to the processing steps of ribosome biogenesis in mammalian cells quit the nucleolus in late G2 and associate with the chromosome periphery until late telophase. They associate in the perichromosomal layer in human and PtK1 cells and both in the perichromosomal layer and the nucleolar remnant in CHO cells.

Animals↗
PubMed1994

Quantification of Ag-NOR proteins using Ag-NOR staining on western blots.

Ribosomal genes are associated with a set of silver-stained nucleolar proteins, the Ag-NOR proteins, whose amount is directly related to the duration of the cell cycle. Quantification of Ag-NOR proteins by image analysis is presently used to evaluate the rate of proliferation of cancer cells and nucleolar activity. Our objective was to establish a procedure to quantify independently each major Ag-NOR protein in cell extracts. Computerized densitometry established that the specific silver staining of Ag-NOR proteins (Ag-NOR staining) performed on Western blots makes it possible to quantify Ag-NOR proteins. Using purified Ag-NOR proteins, nucleolin, and protein B23, we observed that the intensity of Ag-NOR staining is proportional to the amount of protein. A linear relationship exists between the intensity of Ag-NOR staining and the amount of nucleolin, in the range of 0.2-1.6 micrograms. Using total nuclear extracts prepared from mammalian cells, the proportionality was maintained for total Ag-NOR-stained proteins or for a particular protein. We also determined the levels of nuclear proteins suitable for quantitative analysis. Individual Ag-NOR proteins can be quantified by computerized densitometry in nuclear extracts after Ag-NOR staining on Western blots. This procedure can be applied to establish the contribution of each Ag-NOR protein in general staining, estimate the variability of each Ag-NOR protein in normal and pathological conditions, and quantify each Ag-NOR protein contained per cell.

Animals↗
PubMed1993

Three-dimensional organization of the ribosomal genes and Ag-NOR proteins during interphase and mitosis in PtK1 cells studied by confocal microscopy.

The three-dimensional (3-D) organization of rDNA-containing chromatin and the set of protein markers of active ribosomal genes, the Ag-NOR proteins, were investigated by confocal laser scanning microscopy (CLSM). The rDNA genes of marsupial cells (PtK1) were mapped using biotinylated DNA probes for 45S rDNA sequences and the Ag-NOR protein distribution was revealed by specific Ag-NOR staining. We used PtK1 cells because each nucleolus possesses only one nucleolar organizer region (NOR). In metaphase chromosomes, nonisotopic in situ hybridization demonstrated the presence of rDNA in the secondary constriction of the X chromosomes with an axial distribution and also lateral expansions. 3-D reconstruction of the Ag-NOR protein signals revealed the presence of these proteins in the secondary constriction where they formed a crescent-shaped structure around the axial chromatin pedicule. The organization of the secondary constriction in PtK1 chromosomes is discussed. During interphase, nonisotopic in situ hybridization in intact cell monolayers and isolated nuclei showed the rDNA genes distributed as intense fluorescent spots linked by weak signals in the inner regions of the nucleoli. We conclude that the rDNA is not homogeneously distributed in the internal regions of the nucleoli. In the same nucleolar regions, the Ag-NOR proteins were revealed as granules linked by thin filaments. These images indicate similar 3-D distributions for rDNA probes and Ag-NOR proteins. The beaded organization of the transcriptional regions in the nucleoli is discussed.

Animals↗
PubMed1993

Localization of the RNA polymerase I transcription factor hUBF during the cell cycle.

Autoantibodies directed against nucleoli that recognized a doublet of 97-94 kDa in HeLa nuclear protein extracts were identified. The two polypeptides bound equal amounts of antibody, and each was recognized by antibodies affinity purified using the other polypeptide. These antigens were localized in the secondary constriction of PtK1 cells, i.e. the nucleolar organizer regions (NORs) where ribosomal genes accumulate. They were observed in human cells in the same sites as the NOR-silver-stained proteins. The molecular mass of the antigens, their characteristics in Western blotting and their localization in nucleoli and NORs during mitosis are consistent with them being RNA polymerase I transcriptional factor, UBF. This identification was confirmed on Western blotted proteins by their identical labelling patterns, using these autoantibodies and an anti-mUBF antibody that had been previously described. We obtained definitive evidence that these autoantibodies recognize UBF by the strong positive labelling of purified hUBF (1 to 4 ng). During interphase, these autoantibodies directed against UBF labelled in a folded filament pattern as small beads that may correspond to individual transcriptional units. In electron microscopy, the antibodies were observed in the dense fibrillar component (DFC) of the nucleoli and at the periphery of the fibrillar centers (FCs). At the end of G2 phase, transcription inactivation was concomitant with the gathering of UBF at mitotic NORs. UBF was not equally distributed between NORs in human cells: some NORs scored negative (2 to 4) and the intensity of labelling of positive NORs (6 to 8) differed. In confocal microscopy, 3-dimensional analysis of mitosis indicated that UBF remained associated with NORs during all mitotic stages and that there was equal partition of UBF between the daughter cells. The relationship between proteins associated with the NORs and ribosomal gene transcription is discussed.

Antibodies, Antinuclear↗
PubMed1992

The ultrastructure of the chromosome periphery in human cell lines. An in situ study using cryomethods in electron microscopy.

We studied the chromosome periphery in human HeLa and TG cells using cryomethods in electron microscopy. A contrasted layer of peripheral chromosomal material (PCM) was visible in cryo-ultrathin sections of mitotic cells. This PCM was composed of closely packed fibrils associated with granules. The PCM did not cover the entire chromosome surface but was found around most of the chromosomes and even between two chromatids. The organization of the PCM was not affected by colchicine treatment of mitotic cells. In cells prepared by quick-freezing, the PCM appeared to be a fibrous material at the chromosome periphery, and was also associated with granules that resembled inter-chromatin granules in size and shape. At higher magnification, direct contacts between the chromosomes and the fibrils of the PCM were observed. The cryotechniques used are known to preserve the native organization of cells. Therefore, the architecture of the perichromosomal region analysed presumably corresponds to that in vivo during mitosis. These observations show that in HeLa and TG cells, a particular structure present at the chromosome periphery in the form of PCM is persistent and ubiquitous. In addition, we showed by immunolabelling that the PCM is the specific site of accumulation of nucleolar antigens during mitosis. These two results, i.e. the identification of specific morphological structures and the compartmentation of proteins, indicate that this layer is a specific region of mitotic cells.

Cell Line↗
PubMed1992

Revealing nucleolar architecture by low ionic strength treatment.

The internal nucleolar architecture of HeLa cells was revealed after a short hypotonic treatment. The response of nucleoli to a gradual reduction in the ionic strength of incubation buffer was assessed by immunofluorescence, confocal microscopy using human autoimmune sera monospecific for antigens present in different nucleolar components, and electron microscopy. The granular component dispersed first, followed by the dense fibrillar component, leaving distinct fibrillar centers remaining. This demonstrates differential sensitivity to low ionic strength treatment in the transcription and the maturation territories of the nucleoli. The changes described develop in only a few minutes and this approach can reveal momentary in situ intranucleolar arrangements. We suggest that the fibrillar centers provide a structural support for RNA-polymerase I complexes and are possibly also attached to a nuclear skeleton. The evidence presented implicates the fibrillar centers as the core elements of nucleoli and that functional nucleoli arise around them.

Antigens↗