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D Hernandez-Verdun

Publications and source records attributed to D Hernandez-Verdun.

At least 73 records · Page 4Linked to original sources
PubMed1986

Sorting of micronuclei from PtK1 cells.

We report here a procedure allowing to select micronuclei corresponding to defined individualized chromosomes in conditions which preserve their synthetic activity. The mammalian PtK1 cells, which possess six chromosome pairs, were micronucleated by colchicine. DNA of the micronucleated cells was labeled by the Hoechst 33342 fluorochrome under vital conditions. The micronuclei were isolated by a gentle procedure and their fluorescence was analysed by flow cytometry. The flow-cytometry parameters were determined for the analysis of non-fixed subdiploid fractions. We obtained five distinct peaks of fluorescence which have been sorted. The sorted micronuclei are different in each peak exhibiting different fluorescence intensity. Peak 3 contains the micronuclei with nucleoli and chromocenters that correspond to the X chromosome in this cell line.

Animals↗
PubMed1985

Silver staining of the nucleolar organizer regions (NORs) on Lowicryl and cryo-ultrathin sections.

Nucleolar organizer region (NOR) silver staining was applied to sections of fixed material. A positive reaction on cryo-ultrathin sections was found as well as on semithin and ultrathin Lowicryl sections. Repeatable staining that was easy to control was obtained by a one-step procedure after aldehyde-Carnoy fixation. Fixation of the material by formaldehyde and glutaraldehyde alone in cacodylate buffer also maintained reaction selectivity when ammonium chloride was used after fixation. Enzymatic digestion by pronase, RNase A, DNase I, or micrococcal nuclease was applied to ultrathin Lowicryl sections. Pronase digestion removed the silver-stained proteins, whereas digestion by the nucleases did not. A routine procedure is proposed for easy NOR silver staining of sections that preserves a good tissue ultrastructure and is also compatible with cytochemical and immunological investigations.

Acrylic Resins↗
PubMed1985

Procedure for isolating micronuclei from rat kangaroo cultured cells containing individualized chromosomes.

Micronuclei are small interphase nuclei containing part of the genome; the DNA content of the smallest micronuclei is equivalent to one chromosome. For analysis by biochemical method and by cytofluorometry of interphase micronuclei containing a single chromosome, several isolation and purification procedures were tested and checked by fluorescent microscopy using the DNA dye Hoechst 33 342 and electron microscopy. Micronucleation of rat kangaroo epithelial cells was induced by colchicine treatment for three days. Micronuclei were isolated in a low ionic strength buffer containing collagenase, with concomitant mechanical shocks. Eighty % of the micronuclei were released after 3 to 7 min, with minimum nuclear breakage. Subsequent filtration through several polycarbonate filters 12, 8 and 5 micron in diameter enabled purification of the smallest micronuclei without aggregates or debris. Micronuclear morphology was well preserved, as shown by electron microscope observations. Therefore, we established the optimal conditions allowing gentle mass isolation of individual micronuclei of cultured PtK1 cells, compatible with flow cytometry analysis.

Animals↗
PubMed1984

Visualization of chromatin distribution in living PTO cells by Hoechst 33342 fluorescent staining.

Chromatin distribution was visualized in living cells with the selective DNA fluorochrome Hoechst 33342. This dye was shown to be non-toxic on the rat kangaroo PTO cell line by measuring the labelled cell growth rate. The aim of this work was firstly to visualize chromatin distribution without fixation or dehydration and secondly to demonstrate that quantitative determination of DNA content was possible under these non-toxic labelling conditions. During interphase, condensed, decondensed and thin network chromatin configurations were visualized. In nucleolar regions the fluorochrome revealed well-defined chromocentres. During mitosis, fluorescent chromosome banding was observed in vital conditions and chromocentres on fixed chromosomes. Chromatin segregation was visualized after micronucleation, which induced chromosomal set distribution in individual micronuclei. By this means, we demonstrated that the chromocentres observed in interphase nuclei were part of nuclear organizer region (NOR)-bearing chromosomes. This vital staining of chromatin was shown to be compatible with the quantitative determination of DNA content, both in living PTO cells and in isolated nuclei.

Anaphase↗
PubMed1984

Relationship between the Ag-NOR proteins and ribosomal chromatin in situ during drug-induced RNA synthesis inhibition.

In human TG tumor cells, the role of silver-NOR proteins was investigated by examining their relationship with the chromatin structure during inhibition of RNA synthesis by actionomycin-D treatment. This induced segregation of the nucleoli into four distinct zones and weakened the silver reaction. The fibrillar components were found to constitute the site of silver-stained proteins segregation. Feulgen-like osmium-ammine staining revealed that the DNA disappeared from the segregated nucleoli except for a network of nonnucleosomal filaments. When Ag-NOR protein detection was combined with chromatin visualization, we found constant overlapping of the silver reaction sites with the nonnucleosomal DNA filaments. Our results indicate that certain Ag-NOR proteins are not directly linked to active rRNA synthesis, but might rather affect the structure of ribosomal genes.

Cell Line↗
PubMed1984

Effects of the vital DNA probe fluorochrome Hoechst 33342 on PtK cell chromatin.

We investigated the effects of DNA labelling by fluorochrome Hoechst 33342, on chromatin distribution and transcription during a short-term non-toxic staining procedure which did not prevent the cell growth. By electron microscope examination we found chromatin condensation and nucleolar fragmentation just after the staining. Both effects appeared to be reversible 24h after staining; 48h afterwards, the morphological pattern was similar to that of control cells. The rate of RNA transcription was reduced at the end of staining, increased at 24h, and was comparable to the control at 48h. We also observed an effect on the cell cycle when the staining pasted 6h. Consequently, 24h after Hoechst vital staining, the effects on chromatin vanished whereas the cells remained fluorescent.

Animals↗
PubMed1983

Structural organization of chromatin in nucleolar organizer regions of nucleoli with a nucleolonema-like and compact ribonucleoprotein distribution.

We have studied the relationship between the structural organization of intranucleolar chromatin and fibrillar nucleolar structures, fibrillar centers, and RNP fibrillar component, which are the interphase counterpart of metaphase nucleolar organizer regions (NORs), in regenerating rat hepatocytes and in a human tumor cell line (TG cells). These two cell types were characterized by a nucleolonema-like and compact nucleolar RNP distribution, respectively. We found that, in sections selectively stained for DNA, the intranucleolar chromatin composed of extended, nonnucleosomal DNA filaments formed roundish agglomerates with a spatial distribution which was superimposable on that of the fibrillar centers and the RNP fibrillar component around them and on sites of the silver reaction in samples selectively stained for Ag-NOR proteins. The agglomerates of extended nonnucleosomal DNA filaments were small and numerous in regenerating hepatocyte nucleoli, in which the RNP components had a nucleolonema-like distribution, whereas they were large and few in TG cell nucleoli, in which the RNP components showed a compact organization. Since the pattern of ribosomal RNA synthesis and processing was similar in the two cell types, a model was proposed in which the difference in size and shape of the agglomerates of extended DNA might be responsible for the different structural organization of the RNP components.

Animals↗
PubMed1983

Visualization of a repeating subunit organization in rat hepatocyte chromatin fixed in situ.

The chromatin structure of rat hepatocytes fixed in situ was investigated in thin sections selectively stained for DNA with the Feulgen-like osmium-ammine reaction. Visualization of the chromatin structure was facilitated by the dispersal that occurred in the chromatin of regenerating rat hepatocytes at 24 h after partial hepatectomy. Our results show that repeating subunits were present both in the clumps and in the extended fibres of chromatin. These subunit particles appeared to be composed of an electron-translucent core delimited by a strongly electron-opaque DNA ring. Depending on the section plane, they displayed a morphology varying from a flat, oval shape to a rough circle. The mean diameter of the repeating subunits was 11 +/- 0.8 nm (S.D.) with a range from 9.5 to 13 nm. The thickness of the DNA ring was about 3 nm. Regarding the association of the subunits in the chromatin fibres, two types of fibres were visualized, 11 nm and 20-25 nm thick, respectively. The thinner fibres were composed of a linear array of adjoining subunits. In the thicker fibres the subunit organization did not clearly form discrete superunit structures.

Animals↗
PubMed1983

Non-nucleosomal configuration of chromatin in nucleolar organizer regions of metaphase chromosomes in situ.

The structure of metaphase chromatin in a human tumor cell line, TG cells, was investigated using thin sections selectively stained for DNA with the Feulgen-like osmium-ammine reaction. The bulk of metaphase chromatin was characterized by the nucleosomal configuration. Some specimens were pretreated by silver staining for selective visualization of acidic proteins of the nucleolar organizer regions. In these specimens, the osmium-amine DNA tracer revealed that the chromatin present at the sites of silver granule localization had a completely extended configuration, and never gave rise to nucleosomal structures.

Cell Line↗
PubMed1982

Visualization of Ag-NOR proteins on nucleolar transcriptional units in molecular spreads.

Application of NOR silver staining to Pleurodeles oocyte nuclei showed selective silver deposits on the fibrillar part of the nucleoli in situ. This staining was adapted to nucleoli spread on grids, by a procedure which allowed the spreading of transcription units from both nucleoli and lampbrush chromosomes on the same grids. This permitted localization of the predominant nucleolar Ag-stained proteins on the nucleolar transcriptional units and not on the lampbrush chromosomes. These proteins were found exclusively on the transcribed part of the nucleolar genes and were not seen in apparently untranscribed spacer regions. The proteins seemed preferentially located on the DNP axis rather than on the RNP fibrils.

Animals↗
PubMed1982

The morphological relationship in electron microscopy between NOR-silver proteins and intranucleolar chromatin.

The relative distribution of NOR proteins and chromatin fibers in the nucleoli was visualized in human cell line. The chromatin was revealed by a Feulgen-like procedure using osmium-ammine as DNA tracer. This selective staining was combined with NOR-silver staining. We provide morphological evidence for constant overlapping of the silver deposit sites with dispersed intranucleolar chromatin fibers. Silver stained proteins were sometimes observed in contact with the chromatin fibers, suggesting that at least some of the Ag-NOR proteins might be closely connected with the dispersed nucleolar DNA.

Cell Line↗
PubMed1980

Ultrastructural localization of Ag-NOR stained proteins in the nucleolus during the cell cycle and in other nucleolar structures.

EM investigation of Ag-AS-NOR staining after short glutaraldehyde prefixation followed by Carnoy fixation maintained good ultrastructural preservation and reactive selectivity. This enables exact localization of silver deposits both in the fibrillar centers of typical or segregated nucleoli during interphase, and in chromosome NORs during mitosis. These results argue in favour of the possibility that fibrillar centers are the interphasic counterpart of chromosome NORs. Special structures such as nucleolar blobs and remnants usually considered to be of nucleolar origin, were also stained. - These findings seem to indicate a relationship between the distribution of the silver-stained proteins, the arrangement of the nucleolar structures and the degree of nucleolar activity resulting from the experimental conditions. These results are of interest at the time when the concept of the nucleolar matrix is gradually emerging.

Animals↗