DNA synthesis progression in 3T3 synchronized fibroblasts: a high resolution approach

Summary In order to investigate at ultrastructural level the mechanism of DNA synthesis progression during the different moments of S-phase a bromodeoxyuridine-anti bromodeoxyuridine (BrdU-anti BrdU) method has been applied to synchronized 3T3 fibroblasts. After 30 min BrdU incorporation, five different labelling patterns can be identified and should be related to early, middle and late S-phase. These patterns are represented mainly by diffuse labelling localized in different nuclear domains and by quite rare cases in which the labelling is limited to isolated clusters of gold particles. After a 5-min pulse with BrdU it is possible to observe isolated clusters of gold particles at each moment of S-phase, which, however, exhibit the same distribution of the five principal labelling patterns observed after 30 min incorporation. In both cases labelling can be detected in the interchromatin regions during early S-phase, at the boundary between interchromatin and heterochromatin during middle S-phase and in the heterochromatin domains during late S-phase. Considering their size, the isolated spots of labelling could be interpreted as single replication units which are subsequently activated throughout the different moments of the S-phase.     

Ultrastructural localization of rRNA in HeLa cells, rat liver cells and Xenopus laevis oocytes by means of the monoclonal antibody--protein a--gold technique

Summary The postembedding localization of rRNA was investigated in ultrathin sections of HeLa cells, rat liver andXenopus laevis oocytes by means of the monoclonal antibody to rRNA and protein A-gold technique. The incidence of gold particles was highest in nucleoli and cytoplasmic areas containing ribosomes. The chromosomes were labelled less than the surrounding cytoplasm in mitotic HeLa cells. In nucleoli of HeLa cells and rat hepatocytes, the labelling of areas containing ribonucleoprotein components was greater than the labelling of fibrillar centres. In segregated nucleoli ofX. laevis oocytes, the labelling of the granular region substantially exceeded that of the fibrillar regions. The incidence of nucleoplasmic gold particles in interphasic HeLa cells was found to be slightly increased in the vicinity of nucleoli. The labelling of clusters of interchromatin granules in rat hepatocytes was not significantly different from that of the rest of the nucleophasmic interchromatin spaces.A part of this study was presented as the poster and abstract at the 8th European Congress on Electron Microscopy 1984 in Budapest.     

Decrease of DNA synthesis in amniotic fluid cells during the middle part of S-phase revealed by differential chromosome staining after incorporation of BrdU

The time sequence of DNA replication in partially synchronized human amniotic fluid cells has been analysed, employing BrdU incorporation techniques. —Regardless of the interval between removal of the methotrexate/uridine block and addition of BrdU during S-phase, the treatment results in an R-type replication pattern. Conversely, replacement of BrdU containing medium by another one with thymidine yields G-type replication patterns. A thymidine pulse during the first 4 h of S-phase results in R-type replication patterns; from 7–10 h after block removal it produces G-type pattern. In between, only faint red staining dots can be found indicating a marked decrease of replicational activity during the middle part of the S-phase.     

Comparison of cell proliferation index in equine and caprine embryos using a modified BrdU incorporation assay

The measurement of cell proliferation and cell viability using 5'bromo-2'deoxy-uridine (BrdU) labelling has been described in several cell types and species. The aim of this study was to adapt this technique to equine embryos and to compare the index of DNA replication (S-phase) between equine and caprine embryos. Seventeen equine embryos were recovered at day 6.5 post-ovulation and 20 caprine embryos were recovered at day 7 after the onset of estrus. Equine embryos were incubated during 1h at 39 degrees C in PBS containing 1mM of BrdU. Embryos were then treated in 0.05% trypsin during 15 min at 39 degrees C to permeabilise the capsule, and then embryos were rinsed in PBS containing 10% of foetal calf serum. After washing, embryos were immediately fixed in 2.5% paraformaldehyde with 0.3M NaOH during 15 min at ambient temperature. The S-phase was detected by immunocytochemistry technique. In caprine embryos, BrdU was visualised by the same technique but without the trypsin treatment. The percentage of cells (+/-S.E.M.) with BrdU incorporated into newly synthesised DNA strands was significantly higher in equine embryos (74+/-1) than in caprine (38+/-2). Our results demonstrated that BrdU incorporation assay can be used in equine embryos. This assay allows the determination of the proliferation index of live cells and could be used as an additional tool for evaluating the viability of embryos. The high percentage of cells incorporating BrdU during 1h of incubation with BrdU suggests that in comparison with the caprine embryos the cellular activity of proliferation is more intense in equine embryos and suggests that the cellular cycle is shorter in equine embryos.     

Immunoelectron microscopy of PCNA as an efficient marker for studying replication times and sites during pollen development

Here we report for the first time the ultrastructural localization of DNA replication sites in the nucleus of plant cells and the timing of replication through the pollen developmental programme by proliferating cell nuclear antigen (PCNA) immunogold labelling. Replication sites were identified by labelling with anti-PCNA antibodies in fibrils of the interchromatin region close to the condensed chromatin, defining a perichromatin subdomain in the interchromatin space where DNA replication takes place. The same nuclear structures are decorated by anti-BrdU (5-bromo-2'-deoxyuridine) immunogold after short pulses of BrdU labelling. Double immunogold labelling for PCNA and DNA show colocalization on these perichromatin structures. PCNA immunoelectron microscopy also allows correlation of replicative activity with the dynamics of chromatin condensation. DNA replication was also monitored at different phases during pollen development by PCNA immunoelectron microscopy, revealing two peaks of DNA synthesis, at the beginning (early tetrad), and the end (late vacuolate), of microspore interphase. High-resolution autoradiography after [3H]thymidine incorporation also showed high replicative activity at the same two periods of microspore interphase. In the bicellular pollen grain, PCNA immunogold labelling revealed that DNA replication in the generative cell starts at an intermediate stage of pollen maturation, whereas the vegetative nucleus does not replicate and is arrested in G1. The use of anti-PCNA antibodies at the ultrastructural level is an easier, faster and more feasible method than the detection of in vivo-incorporated nucleotides, especially in plant systems with long cell cycles. PCNA immunogold labelling is, therefore, proposed as an efficient marker for mapping the sites and timing of replication at the electron microscopy level.     

Dinoflagellate chromosome behaviour during stages of replication.

In most dinoflagellate species, chromosomes are characterized by an almost continuous condensation of the nucleofilaments throughout the cell cycle and the absence of longitudinal differentiation as Q, G, or C banding. Their supercoiled architecture is maintained by divalent cations and structural RNAs. Their chromatin is devoid of histones and nucleosomes and their DNA composition is distinctive: in several species, more than 60% of thymines are replaced by a rare base, hydroxymethyluracil. We report here an immunofluorescence (conventional and confocal laser scanning microscopy, CLSM) and immunogold transmission electron microscopy (TEM) analysis of some stages of the early replication process in Prorocentrum micans dinoflagellate cells, after long pulse incorporation (3, 6 or 9 days) with 50 micrograms/ml bromodeoxyuridine (BrdU) in the presence of 5-fluoro-2'-deoxyuridine (FUdR) and BrdU antibody technique (BAT) detection. The large DNA content (45 pg per nucleus) of P. micans cells is compacted on 100 chromosomes, 10 microns in length. In early S-phase, DNA replication sites are revealed as fluorescent domains organized in clusters, which appear in the periphery of the nucleus unlike other eukaryotes. In late S-phase, the number of labelled clusters increased; helically distributed, they did not appear synchronously in the whole chromosome. Under TEM, spherical domains of equivalent diameter appeared located all along the chromosomes after 6 days BrdU pulse. Replication occurs, but in our experimental conditions, segregation of daughter chromosomes was never observed. The blockade of the cell cycle after BrdU incorporation intervening just before the segregation of daughter chromosomes is discussed.     

多头绒泡菌SC35类蛋白的免疫电镜研究

经抗SC35单克隆抗体标记后,在电子显微镜下观察到多头绒泡菌S、G2、前期、中期和后末期细胞核中存在大量金颗粒,说明多头绒泡菌细胞核含有SC35类蛋白。在G2期和前期时,SC35类蛋白主要分布在细胞核的核仁区域和非核仁区域的染色质间区域;中期和后-末期时,SC35类蛋白主要分布在细胞核内染色体间区域;说明染色质(体)间区域和核仁区域是富含SC35类蛋白的区域。对核仁的进一步观察指出,在核仁中金颗粒主要分布在DFC,FC中的金颗粒很少,说明在核仁中SC35类蛋白主要存在于DFC组分中。     

Phospholipase C digestion induces the removal of nuclear RNA: A cytochemical quantitative study

Summary It has been reported that the incubation of isolated rat liver nuclear matrices with phospholipase C causes the digestion of the matrix-bound phospholipids and the release of most matrix-linked RNAs (Coccoet al., 1980). In this paper, the presence of phospholipids in nuclear substructures and the effects of their removal by phospholipase C digestion have been investigated by means of enzyme—colloidal gold cytochemistry. The nuclear phospholipids appear to be localized in the interchromatin areas and in the nucleolus and are virtually absent in the heterochromatin, when labelled with phospholipase C—colloidal gold. The double labelling test with ribonuclease A and phospholipase C conjugated with gold particles of different diameters shows that the nuclear phospholipids are co-localized with RNA-containing structures. The enzymatic digestion of phospholipids on thin sections of either isolated nuclei or pancreas embedded in LR White resin results in the decrease of the RNase-A colloidal gold labelling of nuclear RNA-containing structures, but not of the rough endoplasmic reticulum. The data confirm the presence of phospholipids in the nucleus in the absence of possible translocation due to isolation procedures and strengthen the hypothesis that they are involved in interactions between nucleic acids and proteins of the nuclear matrix.     

Demonstration of chromosome replication by BrdU antibody technique and electron microscopy

Summary We describe the use of different reporter groups in visualizing replication patterns on metaphase chromosomes after BrdU incorporation by the BrdU antibody technique for the electron microscope. There is an inverse correlation between the density of the label and the size of the reporter particles. This observation alludes to stereo problems interfering with the access of the labeled antibodies into the chromatin. The use of silver enhancement enables easy detection of 1-nm and 5-nm gold particles, which make replication patterns visible in the electron microscope as does the diaminobenzidine/ H₂O₂ reaction. Possible consequences for the demonstration of replication patterns and for nonradioactive DNA in situ hybridization are discussed.     

High-resolution detection of newly synthesized DNA by anti-bromodeoxyuridine antibodies identifies specific chromatin domains

We analyzed the incorporation of bromodeoxyuridine (BrdUrd) into DNA in exponentially growing murine erythroleukemia cells (FLC-745), using fluorescent anti-BrdUrd antibodies with light microscopy and flow cytometry. The fine localization of the DNA replicating sites was investigated at the ultrastructural level by using a second antibody conjugated with colloidal gold. The latter approach, which does not require acidic denaturation of the DNA, enables preservation of good morphology and obtains a better resolution power than that of electron microscopic autoradiography, the percentage of labeled cells obtained with the two techniques being comparable. After short BrdUrd pulses, characteristic distribution of the labeling can be identified in the heterochromatin, in interchromatin domains, or at the boundary between the dispersed and the condensed chromatin. Similar patterns are also observable in the nuclear structures which condense after acid denaturation, suggesting that DNA replication takes place at fixed sites associated with the nuclear matrix.     

Characterization and cell cycle kinetics of hepatocytes during rat liver regeneration: in vivo BrdUrd incorporation analysed by flow cytometry and electron microscopy

The incorporation of bromodeoxyuridine (BrdUrd) into newly synthesized DNA has been analysed during hepatocellular regeneration induced by partial hepatectomy in young rats. The kinetic state of the liver has been studied by flow cytometric analysis of the incorporated BrdUrd, while the fine localization of DNA replication sites through the cell cycle has been investigated at the ultrastructural level by the immunogold technique. Eighteen hours after partial hepatectomy flow cytometry revealed an early S phase distribution which corresponded to a specific staining of the interchromatin domains of the hepatocyte nucleus. Thirty-four hours after hepatectomy, on the other hand, when most cells were in late S, a specific staining of heterochromatin domains was observed. The effect of the BrdUrd technique on nuclear aggregation has also been analysed and discussed. The results demonstrate that specific patterns of DNA replication can be recognized during the cell cycle and that flow cytometry and electron microscopy appear to be complementary in the kinetic study of liver regeneration.     

Analysis of the Length of S-Phase Required to Show Sister Chromatid Differential Staining

Abstract. In vitro studies of BrdU-dependent sister chromatid differential staining typically employ two cycles of BrdU incorporation. Experiments are described which determined the actual fraction of both S-phases that the rat embryonic fibroblasts (Rat-1) cells had to traverse in order to show distinctive differential staining. Following synchronization of cells by a combination of serum deprivation and hydroxyurea blockage, sister chromatid differential staining, labelling index, mitotic index, and per cent DNA replication are determined. Results indicate that only ≤50% of the first S-phase is necessary in order to show distinctive differential staining. the importance of this finding to studies of cellular proliferation using BrdU incorporation is discussed.     

Analysis of the length of S-phase required to show sister chromatid differential staining

In vitro studies of BrdU-dependent sister chromatid differential staining typically employ two cycles of BrdU incorporation. Experiments are described which determined the actual fraction of both S-phases that the rat embryonic fibroblasts (Rat-1) cells had to traverse in order to show distinctive differential staining. Following synchronization of cells by a combination of serum deprivation and hydroxyurea blockage, sister chromatid differential staining, labelling index, mitotic index, and per cent DNA replication are determined. Results indicate that only approximately 50% of the first S-phase is necessary in order to show distinctive differential staining. The importance of this finding to studies of cellular proliferation using BrdU incorporation is discussed.     

Image analysis of the chromatin organization in the nuclear domains of freeze fractured hepatocytes and lymphocytes

The complex organization of the interphase nucleus can be analyzed, by way of thin sectioning and also freeze-fracture. This approach has previously been utilized in association with image analysis to quantitatively describe the organization of isolated rat liver nuclei and nuclear matrices. The main nuclear domains which, in section, present marked differences due to their electron-density, can be identified in replicas with more complex procedures, based on the quantitative evaluation of the number of particles per unit area and mainly by using image analysis. A quantitative analysis of the nuclear substructures has been performed by way of image analysis on in situ nuclei of freeze-fractured cells presenting marked differences in the heterochromatin quantity, such as hepatocytes and lymphocytes. The replicated nuclear particles have been classified according to their diameter and the obtained histograms have been quantitatively evaluated. The nuclear domains, heterochromatin, interchromatin, nucleolus, present characteristic ratios among the three main classes of particles; that is, ribonucleoproteins, solenoid filaments and solenoid fibre aggregates. The typical patterns of the nuclear domains can be further stressed by selecting a single class of particles and by examining its topographic localization. While interchromatin and nucleolar domains present a similar quantitative pattern in hepatocytes and lymphocytes, the heterochromatin of lymphocytes contains a significative higher percentage of solenoid aggregates than that of hepatocytes.     

Regional distribution of Sindbis virus glycoproteins on the plasma membrane of infected baby hamster kidney cells

Sindbis virus-infected baby hamster kidney (BHK) cells were analysed in surface replicas or conventional thin sections after specific immunolabelling with antiviral glycoprotein antibodies in conjunction with colloidal gold-conjugated protein A. Newly synthesized viral glycoproteins were detected, beginning 1 1/2 h after infection, while the virus maturation started 3 h after infection. The glycoproteins appeared to be inserted on the plasma membrane in large spots located mainly in the central area of the cells: no clustering of the labelling was detected. Later, the glycoproteins appeared to arrange linearly in regions in the medial portion of the cells. No labelling was found in the peripheral area or on the cell edges. A drastic change in the surface labelling was detected following the commencement of virus maturation: gold particles were organized mostly in small clusters, each labelling a budding virus. Very few glycoproteins appeared not to be involved in budding figures. The maturation of the virus was clearly regionalized, but during this time it also involved the peripheral area and the cell edges; preferential budding in narrow cellular processes was often observed. It appeared thus that either isolated glycoproteins soon after infection, or clustered glycoproteins at later times, are strictly regionalized on the plasma membrane: however, the early post-infection distribution is clearly different from that seen later during virus maturation. Our experiments support the concept of discrete plasma membrane domains even in cells that do not display distinct specialization of their surface.     

Does ex vivo labelling of proliferating cells in colonic and vaginal mucosa reflect the S-phase fraction in vivo?

The ex vivo labelling of DNA-synthesizing epithelial cells in colonic and vaginal mucosa was compared with in vivo labelling. For this purpose, in vivo S-phase cells were labelled with [3H]thymidine (Tdr) and ex vivo labelling was continued by culturing tissue specimens in bromodeoxyuridine (BrdU). Various methods of tissue culture were employed in order to improve diffusion of medium (and BrdU) in the tissue. BrdU and 3H-TdR labelling were evaluated by immunohistochemistry and autoradiography respectively. Ex vivo labelling resulted in a patchy distribution of labelled cells, which did not correspond with the 3H-TdR labelling pattern obtained in vivo. Under the described conditions ex vivo labelling does not appear to be a reliable for estimation of the proliferative activities in vivo.