Location of DNA within the nucleolus of rat oocytes during the early stages of follicular growth.

We have investigated the DNA distribution within the rat oocyte nucleolus during the early stages of follicular growth by means of the in situ terminal deoxynucleotidyl transferase method. In the fibrillogranular nucleolus, label is visualized on small clumps of peri- and intranucleolar chromatin. Such labeled clumps are frequently observed inside the interstices surrounding the fibrillar centers. Label is also consistently found in the fibrillar centers whereas the dense fibrillar component and the granular component are devoid of gold particles. These results contradict earlier data but conform with other recent immunocytochemical observations, obtained in nucleoli of a variety of somatic cell types, concerning the correlation between structure and function in the nucleolus.     

In situ hybridization at the electron microscope level: an improved method for precise localization of ribosomal DNA and RNA

In situ hybridization using biotinylated rDNA probes and secondary antibody coupled to gold particles was developed on ultrathin sections of Lowicryl-embedded Ehrlich tumor cells for precise localization of ribosomal RNA (rRNA) and ribosomal DNA (rDNA). For the detection of rDNA, an immunocytochemical approach involving an antibody against single-stranded DNA was used in order to determine the more efficient denaturation procedure. Using this technique, rDNA can be visualized in the fibrillar centers of nucleoli, especially in their peripheral regions at the proximity of both the dense fibrils and the nucleolar interstices as well as within the latter. rDNA was occasionally detected in some clumps of dense nucleolus-associated chromatin. Besides the presence of rRNA in the ribosome-rich cytoplasmic areas and in the dense fibrillar component and the granular component of the nucleolus, rRNA was also found in the fibrillar center areas close to the boundary region to the dense fibrillar component. These results are discussed in the light of the present knowledge on the functional organization of the nucleolus.     

蚕豆细胞核仁和核质中剪接因子SC35的定位研究

SC35 is a non-snRNP spliceosome component purified from mammalian cells by Fu and Maniatis in 1990. In vitro splicing assays showed that SC35 plays a key role in splicing site selection and ATP-dependent pre-spliceosome assembly. In the mammalian nucleus, SC35 has been localized to distinct and dynamic nuclear domains: immunofluorescence observations revealed the presence of SC35 in speckles distributed in various regions throughout the nucleoplasm, which, as identified with immunoelectron microscopy, correspond to the interchromatin granules (IGs) and perichromatin fibrils (PFs). However, there has been no report regarding the presence and distribution pattern of SC35 in higher plant nuclei. Engage in such studies will surely contribute to our understanding of RNA processing and the spatial organization or structure basis of this process in higher plant. In this article, we studied the distribution pattern of SC35 in the nucleus of the root meristematic cells of Vicia faba by immunoelectron microscopy. After immunolabeling with anti-SC35 mAb and protein A-colloidal gold, IGs and PFs in the nucleoplasm and dense fibrillar component (DFC) of the nucleolus were heavily labeled with gold particles, while only a few of the gold particles were found in fibrillar centers (FC) and nucleolar vacuoles (NV) of the nucleolus and the central domains of the condensed chromatin. Densities of gold particles in the areas of DFC and the area of IGs plus PFs were 65.89/microns 2 and 36.28/microns 2 respectively, much higher than that of the central domain of condensed chromatin and that of FC plus NV, which were only 5.90/microns 2 and 6.26/microns 2 respectively. This indicates that DFC of the nucleolus and the area of IGs plus PFs of the nucleoplasm are enriched with SC35 or SC35-like protein. The distribution pattern of SC35 or SC35-like protein in the nucleoplasm of Vicia faba is similar to that of the mammalian nuclei. To the authors' knowledge, it is a new finding that SC35 or SC35-like protein exists in the nucleolus.     

Study of RNA distribution in the nucleolar components of Ehrlich cell using RNase-gold method

The RNA distribution in Ehrlich tumour cell nucleoli has been investigated using RNase-gold method. This technique has been applied to sections of cells prepared under various fixation and embedding conditions. As expected, the specificity and intensity of labelling by gold particles have varied according to the experimental conditions used. Interestingly, however, it has been noted that the localization of gold particles does also vary and in particular within the fibrillar centre. This observation underlines the interest of assaying the RNase-gold complex under various conditions. The gold particles were particularly concentrated over the granular component and to a lesser extent, in the dense fibrillar component. In the latter constituent, it has been noted that the gold markers were preferentially localized at the edge of the dense fibrils. Surprisingly, a few gold particles have also been detected in the fibrillar centres. The weak labelling has persisted even after pepsin or DNase extraction but has completely disappeared after RNase extraction. Further, an inhibition of rRNA synthesis by a treatment with actinomycin D has not produced a significant decrease of the number of gold particles present in the fibrillar centre. These results suggest that fibrillar centres contain a small amount of RNA which would not correspond to pre-rRNA.     

Study of RNA distribution in the nucleolar components of Ehrlich cell using RNase-gold method

Summary The RNA distribution in Ehrlich tumour cell nucleoli has been investigated using RNase-gold method. This technique has been applied to sections of cells prepared under various fixation and embedding conditions. As expected, the specificity and intensity of labelling by gold particles have varied according to the experimental conditions used. Interestingly, however, it has been noted that the localization of gold particles does also vary and in particular within the fibrillar centre. This observation underlines the interest of assyying the RNase-gold complex under various conditions.The gold particles were particularly concentrated over the granular component and to a lesser extent, in the dense fibrillar component. In the latter constitutent, it has been noted that the gold markers were preferentially localized at the edge of the dense fibrils. Surprisingly, a few gold particles have also been detected in the fibrillar centres. The weak labelling has persisted even after pepsin or DNase extraction but has completely disappeared after RNase extraction. Further, an inhibition of rRNA synthesis by a treatment with actinomycin D has not produced a significant decrease of the number of gold particles present in the fibrillar centre. These results suggest that fibrillar centres contain a small amount of RNA which would not correspond to pre-rRNA.     

Cytochemical distinction of various nucleolar components in insect cells.

The fine structure of the insect Sf9 cell nucleolus has been investigated by means of different cytochemical and immunocytochemical techniques at the electron microscope level. Apart from a few perinucleolar condensed chromatin clumps, the insect cell nucleolus comprises two compartments. The first of these consists of a roundish compact zone formed of fibrillar material. The other is composed of fibrillar and granular structures organized into a network separated by interstitial spaces. But, unlike mammalian cell nucleoli, any fibrillar center has been observed in the Sf9 cell nucleolus, even after actinomycin D treatment. We also show that the compact fibrillar zone of Sf9 cell nucleoli contains silver-stainable material and DNA. In actinomycin D-treated cells, a preferential contact of this compact fibrillar zone with condensed chromatin has been visualized. Finally, silver-stainable material has been found to persist throughout the whole mitosis. These results suggest that the compact fibrillar zone at the insect Sf9 cell nucleolus should, at least partly, correspond to the fibrillar center of mammalian cell nucleoli.     

New data concerning the functional organization of the mammalian cell nucleolus: detection of RNA and rRNA by in situ molecular immunocytochemistry.

We have investigated the fine spatial distribution of RNA and rRNA within the Ehrlich tumor cell nucleolus by in situ hybridization with a biotin-labeled probe and by two new strategies, the polyadenylate nucleotidyl transferase-immunogold technique and immuno-labeling with anti-RNA antibodies. Besides the presence, as expected, of RNA and rRNA in the granular component and the dense fibrillar component, we show, for the first time, significant label over all the fibrillar centers of the nucleoli. When RNA and DNA were detected simultaneously on the same sections, only the fibrillar centers were positive for both. These results throw light on the controversial subject of the precise location of transcribing rRNA genes within the nucleolus. The fibrillar centers, and not the dense fibrillar component, should thus be the site of rRNA synthesis.     

Distribution of sugar-binding sites within interphase nuclei and mitotic chromosomes of a human cell line

Using gold labelled neoglycoproteins containing either alpha-D-glucose, N-acetyl-beta-D-glucosamine, alpha-D-mannose, 6-phospho-alpha-D-mannose, and alpha-L-fucose (BSA), we investigated their intranuclear binding sites in the TG human cell line. Although gold-labelled BSA did not give any noticeable labelling, the presence of 1% free BSA in the medium containing the gold labelled neoglycoproteins was revealed to be a key factor of the labelling. During interphase in the presence of free BSA most of the labelling was detected in the nucleoplasm. The border of the condensed chromatin, known to be the site of hnRNA synthesis as well as the interchromatin areas enriched in RNPs were labelled. Condensed chromatin also contained binding-sites. The nucleolus was seen to present low labelling in comparison with the labelling observed over the nucleoplasm. These nucleolar binding sites were located both in the dense fibrillar and granular components. No labelling could be detected over the fibrillar centers which are very conspicuous in this cell line. During mitosis sugar-binding sites were observed over the chromosomes. Data reported here show for the first time that lectin-like proteins and chromatin components are colocalized both during interphase and mitosis. In addition, within the nucleolus the presence of sugar-binding proteins was seen to be restricted to the dense fibrillar and granular components.     

Immunoelectron Microscopy of RNA Combined with Nucleic Acid Cytochemistry in Plant Nucleoli

The immunoelectron microscopy detection of RNA using anti-RNA monoclonal antibodies has been performed for the first time over different plant cells. The use of the methylation-acetylation (MA) method permits clear distinction among the nuclear and nucleolar compartments and can be combined with the immunogold approach. Cytochemical methods for nucleic acids were performed together with the immunoassays, providing additional data about the different composition of the various nucleolar components. Anti-RNA antibodies highly labeled the ribosome-rich areas of the cytoplasm and the nucleolus. The interchromatin region also is labeled. The labeling was intense in the granular component, lower in the dense fibrillar component, and very scarce in the fibrillar centers. The MA method made possible the statistical evaluation of the labeling density in the various nuclear compartments by permitting the clear assignment of the particles to precise nuclear structures.     

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。     

Ultrastructural Distribution of DNA within Plant Meristematic Cell Nucleoli during Activation and the Subsequent Inactivation by a Cold Stress

We have investigated the precise location of DNA within the meristematic cell nucleolus ofZea maysroot cells andPisum sativumcotyledonary buds, in the course of their activation and induced inactivation following a subsequent treatment at low temperature. For this purpose, we combined the acetylation method, providing an excellent distinction between the various nucleolar components, with thein situterminal deoxynucleotidyl transferase-immunogold technique, a highly sensitive method for detecting DNA at the ultrastructural level. In addition to the presence of DNA in the condensed chromatin associated with the nucleolus, we demonstrated that a significant label was detected in the nucleolus of quiescent cells in both plant models. Evident labels were also found in the dense fibrillar component of actived nucleoli. Whereas in inactivated nucleoli no significant label was observed within the dense fibrillar component, an intense label was seen over the large heterogeneous fibrillar centres only during inactivation. The granular component was never significantly labelled. These results appear to indicate that the DNA present in the dense fibrillar component of activated nucleoli withdraws from this structure during its inactivation and becomes incorporated in the large fibrillar centres. These observations suggest that in plant cells inactivation of rRNA genes is clearly accompanied by changes in the conformation of ribosomal chromatin.     

Fine structure of nucleoli in micronucleated cells

The correlation between the number of nucleolus organizing regions (NOR) on metaphase chromosomes and the number of nucleoli was studied in normal and micronucleate cells. Many micronuclei, but not all, were able to form complete nucleoli with fibrillar and granular RNP components and fibrillar centers. Micronuclei which failed to form complete nucleoli often contained multiple electron-dense bodies of fibrillar material. These structures, which were much smaller than nucleoli, reacted with nucleolus-specific antibodies and the Ag-As method in the same way as complete nucleoli, but lacked fibrillar centers and granular RNP components. The data suggest that these nucleolus-like ‘blobs’ contain nucleolar material which, following mitosis, has been enclosed in micronuclei which do not contain nucleolus organizing chromosomes. No evidence was found for the activation of latent NORs not expressed in mononucleate cells.     

Intranucleolar visualization of nucleic acids and acidic proteins in inhibited and reactivated pea cotyledonary buds

Summary Nuclease-colloidal gold complexes and silver staining were used to visualize intranucleolar nucleic acids and argyrophilic proteins of the nucleolar organizers in bud cotyledonary cells ofPisum sativum. In the G_0–1 inhibited bud, a few RNA molecules were detected in the fibrillar component and in the unique fibrillar centre, close to the boundary with the fibrillar component of the nucleolus. DNA was present in the fibrillar component, in the fibrillar centre and in a few fibres crossing the perinucleolar halo. The acidic proteins were localized at the periphery of the fibrillar component but they were also present in the unique fibrillar centre. In the reactivated bud, RNA was particularly concentrated in the granular component and along fibres crossing the perinucleolar halo; a few RNA molecules were also detected at the boundary between the small fibrillar centres and the fibrillar component. DNA was localized in the same nucleolar component as in the inhibited bud, but it was distributed between several fibrillar centres. Acidic proteins coated these DNA loci. In the inhibited and reactivated bud connections between nucleolar DNA containing structures were displayed. The data are discussed in relation to the present knowledge of the functional architecture of the nucleolus.Abbreviations DNA deoxyribonucleic acid - DNase deoxyribonuclease - G_0–1 phase G₁ phase of the cell cycle indefinitely prolonged - PEG polyethylene glycol - RNA ribonucleic acid - RNase ribonuclease - S and G₂ phases synthetic and postsynthetic phases of the cell cycle - SPB saline phosphate buffer