Ultrastructural localization of antibody in differentiating plasma cells.

Antibody was localized by electron microscopy within differentiating and mature plasma cells of the spleens of hyperimmunized rabbits. Horseradish peroxidase was used as antigen. Intracellular antibody to peroxidase was revealed in glutaraldehyde-fixed tissue by coupling it with its antigen and then revealing the sites of peroxidase activity cytochemically. Antibody first appears in the perinuclear space of hemocytoblasts where it persists through differentiation into immature plasma cells, but it disappears from this site in mature plasma cells. Concomitant with the development of the ergastoplasm, antibody accumulates in many but not all of its cisternae. Antibody is present in the lamellar portion of the Golgi apparatus in all phases of plasmacytic differentiation. Mature plasma cells exhibit two types of antibody distribution, a concentration into large spherical intracisternal granules or an overflowing into all parts of the cytoplasm.

Mechanisms of transcription in nucleoli of amphibian oocytes as visualized by high-resolution autoradiography.

In oocytes of Pleurodeles waltlii, the method of Miller and Beatty has been combined with a method of high-resolution autoradiography especially suitable for the study of isolated molecules. In vitro labeling of RNA by tritiated precursors was carried out with increasing incubation times (1, 4, 15, 24, 48, and 72 h). Silver grains were present over ribonucleoprotein fibrils in amounts sufficient for quantitative analysis of nucleolar DNA transcription. Statistical analysis of the data revealed that: (a) The units of any one nucleolus exhibited a large degree of heterogeneity in their number of grains. (b) There was a parallelism between the increasing grain number and the ribonucleoprotein-fibril lengthening as observed along the transcription unit.

Structural aspects of intranuclear matrix disintegration upon RNase digestion of HeLa cell nuclei.

The organization of the intranuclear elements observed in histone-depleted (2 M NaCl-extracted) HeLa cell nuclei was investigated by means of electron microscopy and two-dimensional gel electrophoresis. This work was mainly aimed at verifying whether or not an intranuclear skeleton or matrix existed, which could explain the stable attachment of RNA to the residual nuclear structure after high-salt extraction, and its three-dimensional organization. We compared the ultrastructure and the polypeptide composition of RNA-containing and RNA-depleted (RNase-treated) nuclear residues, and we visualized intermediate stages of RNase action on the intranuclear material. We showed that this material was made of two types (fibrillar and granular) of salt-resistant RNP components equally sensitive to RNase when the enzyme was used prior to high-salt extraction. At least in our material and under our experimental conditions, no intranuclear matrix could be distinguished from the residual RNP material. Our results further suggest that formation of such a matrix is a path-dependent phenomenon.

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.

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.

Attachment of DNA to nucleolar and nuclear skeletal structures as visualized by Kleinschmidt molecular spreading.

Co-isolated residual nuclear shells and residual nucleoli from membrane-depleted rat liver nuclei were spread according to Kleinschmidt's method. Comparison of the spread residual structures isolated from nuclear shells and spread pore complex-lamina isolated from nuclear envelopes showed that these residual structures are morphologically identical. Furthermore, our nuclear shell isolation procedure allowed visualization of DNA strands bound to a granular component of the lamina. The fragmentation of nuclear shells allowed us to obtain well-spread nucleolar remnants, in which we observed DNA strands anchored on a residual nucleolar network attached to the lamina. The different molecular features revealed by the spreading of residual nucleolar structures suggest that both non-transcribing nucleolar DNA and active ribosomal genes are linked to the nucleolar network. Although the exact nature of this network remains to be defined, the results of the present study strongly suggest that the DNA molecules of the chromosomes bearing ribosomal genes have many sites of attachment to a non-chromatin nucleolar network which can be referred to as a nucleolar skeletal complex.

Localization of a gene: the nucleolar organizer.

Nucleolus differentiates around the nucleolus organizer regions of the chromosomes or NORs. In the interphasic nucleoli the fibrillar centers are now considered as the NORs. The purpose of this editorial is to review the experimental data which allowed such identification. Our current concepts regarding this point result from three lines of evidence: 1) specific localization during nucleologenesis, 2) in situ hybridization with labeled ribosomal RNA or DNA, and 3) specific staining with silver.

Localization by autoradiography of viral proteins in the parenchymal cells of the liver during frog virus 3 induced hepatitis of mice.

Frog virus 3 inoculated into mice induces an acute degenerative hepatitis. This hepatitis is of toxic origin since the virus is unable to multiply at 37 degrees C. The Kupffer cells, which are the target cells for FV3, reveal the presence of viral particles, viral DNA and proteins. Although the hepatocytes present early and drastic nuclear lesions, viral particles were never observed in these cells. Viral proteins however but not DNA, could be found inside parenchymal cells.

Anchorage of the nucleolus in the pore complex-lamina by a DNA-bearing structure masked in situ in rat liver nuclei.

We have developed a method by which nuclear shells containing nucleoli can be isolated from membrane-depleted rat liver nuclei. This method involves the removal of the internal chromatin. This chromatin is expelled from the nuclear shell using combinations of low and high ionic strength buffers. The expelled internal part is subsequently digested with DNase I or micrococcal nuclease. Examination by electron microscopy of the nuclear and the nucleolar structures at various steps of the isolation procedure shows that the nucleoli are anchored in the peripheral lamina by a pedicle that is continuous with an intranucleolar network. This network is masked in situ by nucleolar granules. The pedicle and the network which support the nucleolar DNA are composed mainly of non-histone proteins insoluble in 2M NaCl.

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.

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.

Preservation of chromosome integrity during micronucleation induced by colchicine in PtK1 cells.

Colchicine induces the formation of small nuclei called micronuclei which contain limited parts of the genome. Some of them exhibit a DNA content equivalent to that of a single chromosome. Our purpose was to study the preservation of chromosome integrity during this micronucleation in PtK1 cells. Observation of karyotypes obtained after 3 days of cell cycle restoration revealed that micronucleation did not affect chromosome integrity or the presence of each chromosome pair in the surviving cells. In 'early restoration' cells, all the chromosomes included a centromere and were represented in the karyotype, but at variable rates. Furthermore, flow cytometry analysis of micronucleated cells, intermediate in DNA rate between control PtK1 cells in G1 and those in G2/M phases, led us to consider the possibility of selective replication of some chromosomes during micronucleation. Using antibodies against the kinetochore proteins, we derived the presence of one centromeric region (1-2 spots) in the smallest micronuclei. Therefore, these data (karyotypes, number of chromosomes, DNA content and kinetochore proteins) seem to indicate that micronucleation does not induce chromosome damages or translocations. Micronuclei are a convenient tool for investigation of the role of the different chromosomes in the organization of the interphase nuclei.

Localization of measles virus nucleic acid sequences in infected cells by in situ hybridization.

Vero cells were infected with measles virus and hybridized in situ to a cloned DNA fragment containing specific sequences for measles nucleocapsid protein. The DNA was labelled with tritium by nick-translation. The viral RNA were detected in the cytoplasm 21 hrs after infection. In many cells, the probe hybridized to nuclear structures, and in several mitotic cells, to chromosomes. After 36 hrs of infection, hybridization sites were found both in the center and in many nuclei of all the polykaryons. These results indicate that cellular distribution of viral RNA molecules varies in the course of infection. They further suggest that the nucleus plays a more active role than expected in measles virus transcription and replication.

Evidence showing that the nucleolus-envelope region is located on the nor-bearing chromosomes in Aotus trivirgatus.

We investigated whether the existence of the Nucleolus-Envelope Region observed in dividing animal cells is related to the Nucleolus Organizer Region or to other chromosome segments of the NOR-bearing chromosomes, since NORs are usually adjacent to the chromosome segments attached to the nuclear envelope such as the centromere, telomere or heterochromatin region. We used Aotus Trivirgatus fibroblasts whose karyotype is characterized by a single pair of NORs located on the long arm of the third pair of chromosomes, far from the centromere, the telomere and any obvious heterochromatin region. All the nucleoli were seen to be clearly associated with the nuclear envelope but separated from it by the outermost layer of chromatin. These results further support the hypothesis that the NOR is a site of attachment of the chromatin to the nuclear envelope by means of the Nucleolus-Envelope Region. They show that genetically active chromatin is also attached to the nuclear envelope. A model of the arrangement of chromatin during interphase is proposed which provides a functional interpretation of the currently available data.

Characterization of lamina-bound chromatin in the nuclear shell isolated from HeLa cells.

The structure and the polypeptide composition of the nuclear shell isolated from interphase HeLa cells have been investigated and compared to those of the intranuclear material. The isolated nuclear shell contains chromatin superstructures (28-32 nm thick fibres) made of tightly packed nucleosomes that resist low ionic strength conditions and that are associated with the three nuclear lamins. Chromatin in the nuclear shell exhibits very simple chemical composition. Especially, non-histone proteins are lacking. The results presented here rule out the possibility that the nuclear shell results from contamination of lamina by intranuclear elements. They suggest that the lamins are directly involved in the specific properties and in the organization of chromatin in the nuclear shell.

Rotation of the cell nucleus in living cells: a quantitative analysis.

Nuclear rotation is observed in a variety of cell types. However, few quantitative analyses are reported and the significance of this phenomenon is still unclear. To investigate this type of nuclear movement, we performed a quantitative analysis in mouse L-929 fibroblasts, a cell line chosen since it displays a high nuclear rotational activity. Analyses were performed using time-lapse microcinematography. The relationship between nuclear rotation and other cellular phenomena such as the cell cycle and locomotion were studied. Then, we investigated the rotation in a population of sister cells to study whether it is genetically determined. Finally, we performed a qualitative analysis of nuclear rotation in different cultured cell lines. Results show that nuclear rotations preferentially occur during the phases of the cell cycle which surround mitosis.

Nuclear RNA-associated proteins and their relationship to the nuclear matrix and related structures in HeLa cells.

The ultrastructure and the polypeptide composition of residual nuclear substructures including nuclear matrices, nuclear ghosts, and residual envelopes were investigated by means of electron microscopy and two-dimensional gel electrophoresis. Nuclear matrices were prepared by digesting isolated nuclei with DNase I alone, followed by high-salt extraction in 2 M NaCl. Nuclear ghosts were obtained by high-salt extraction of nuclei previously digested with DNase and RNase in MgCl2-containing buffers. To prepare residual envelopes, nuclei were first digested with RNase in the presence of EDTA, then digested with Mg2+ -activated DNase I, and extracted in 2 M NaCl. The results of this comparative study support the conclusion that the intranuclear matrix is made of two distinct RNA-containing elements. One of these elements appears on ultrathin sections as a thin fibrillar network. It disappears from RNase-digested nuclei, together with numerous basic proteins, whatever the conditions of digestion. Although this element is present in extranucleolar territories, it is a major component of residual nucleoli. The second element appears as coarse-beaded fibers absent from the nucleolar areas. Its preservation in residual nuclear substructures depends on the presence of Mg2+ ions during RNase digestion of nuclei. It is enriched in two minor basic proteins of relative mass 49 000 and 70 000. The involvement of this fibrogranular element in heterogeneous nuclear RNA attachment to the nuclear matrix is discussed.

Electron microscope fluoro-autoradiography: improvement of efficiency.

The fluorographic process for enhancement of the autoradiographic detection of beta-ray emitted by tritium has been successfully adapted to electron microscope autoradiography. An original scintillator plastic film helped to increase the detection yield of electrons up to 80% without alteration of the morphological aspect of the autoradiograms, and without increasing background fog or causing apparent loss of resolution.