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.     

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.     

TEM cytochemical study of the localization of phospholipids in interphase chromatin in rat hepatocytes

Summary The electron microscopy cytochemical detection of phospholipids in well-defined areas in the interphase nuclei of hepatocytes has been obtained by the acid haematein test, modified for electron microscopy and by the phospholipase A2-colloidal gold method. The specificity of both methods were controlled by enzymatic digestion with phospholipase. The main intra-nuclear localization of phospholipids is at the border between the condensed and dispersed chromatin, where non-ribosomal RNA is also revealed by RNase-gold labelling. Phospholipids are detected, too, over the clusters of interchromatin granules and in the fibrillar component of the nucleolus.     

Localization of Nopp140 within mammalian cells during interphase and mitosis

We investigated distribution of the nucleolar phosphoprotein Nopp140 within mammalian cells, using immunofluorescence confocal microscopy and immunoelectron microscopy. During interphase, three-dimensional image reconstructions of confocal sections revealed that nucleolar labelling appeared as several tiny spheres organized in necklaces. Moreover, after an immunogold labelling procedure, gold particles were detected not only over the dense fibrillar component but also over the fibrillar centres of nucleoli in untreated and actinomycin D-treated cells. Labelling was also consistently present in Cajal bodies. After pulse-chase experiments with BrUTP, colocalization was more prominent after a 10- to 15-min chase than after a 5-min chase. During mitosis, confocal analysis indicated that Nopp140 organization was lost. The protein dispersed between and around the chromosomes in prophase. From prometaphase to telophase, it was also detected in numerous cytoplasmic nucleolus-derived foci. During telophase, it reappeared in the reforming nucleoli of daughter nuclei. This strongly suggests that Nopp140 could be a component implicated in the early steps of pre-rRNA processing.     

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.     

Nuclear architecture and DNA location in two VAM fungi

Summary The ultrastructural organization of nuclei in Glomus versiforme and G. mosseae hyphae was examined by sampling at different times during the fungus life cycle. In young and active hyphae, the nucleus is chromocentric with dispersed chromatin and a well-developed nucleolus. On aging, and particularly during arbuscule collapse, the chromatin condenses to give a pyk-notic nucleus with blebs in the envelope and conspicuous spaces between this and the nucleoplasm. DNA location by immunogold or enzyme-gold techniques resulted in uniform labelling over the chromatin. Nuclear degeneration is mirrored by decreased gold labelling and may thus be regarded as an important parameter for evaluation of fungus viability and the ephemeral life of the arbuscule.     

Relative position of nucleolar chromatin and nucleolar components in ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis> somatic nuclei

According to our computer modeling data obtained earlier, nucleoli in interphase ciliates Didinium nasutum are complex netlike structures, in which the trabeculumor lamella-shaped fibrillar component is located on the periphery, and the granular component in the central part of the nucleolus. Chromatin bodies connected with nucleoli act as the nucleolar organizers in D. nasutum. In the present work, the arrangement of all chromatin bodies, which could correspond to nucleolar organizers by morphological criteria, is studied by means of a 3D-reconstruction. It is shown that all of these chromatin bodies are localized outside the nucleoli, on the fibrillar component’s periphery. Even those chromatin bodies which appeared to be completely surrounded by the fibrillar nucleolar component on single ultrathin sections are actually settled down in nucleolus cavities open to the nucleoplasm. This proves that the RNA processing in D. nasutum nucleoli is directed toward the center of nucleoli, where the granular component is located. The analysis of the nucleolar chromatin distribution made it possible to conclude that different parts of the complex interfase netlike nucleoli of D. nasutum have approximately the same activity.     

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.     

The 3-dimensional organization of the nucleoli of benign and malignant tumor cells from different human organs]

The method of ultrathin serial sections was used to perform a comparative ultrastructural and 3-dimensional analysis of nucleoli for the following variants of human tumours: benign (fibroadenoma) and malignant (infiltrating ductal carcinoma) tumours of one organ (mammary gland); malignant tumours of epidermal genesis in different organs (squamous cell carcinomas of skin, larynx, lung, gullet, uterus); two forms of malignant tumours (squamous cell and small cell carcinomas) of one organ (lung). The spatial models of nucleoli in these tumour cells are given. The specific signs in architecture of tumour nucleoli was found. Nucleoli of fibroadenomas have well pronounced 1-4 fibrillar centres forming a united system with a lacunar component and intranucleolar chromatin. Unlike benign tumour cells, nucleoli of infiltrating ductal carcinomas are characterized by large, prominent nucleoli containing giant, multiform fibrillar centres with a complicated surface, a well developed granular component and an unusually organized lacunar system. In squamous cell carcinomas of various localization, active, hypertrophied nucleoli with pseudonucleolonemal organization were found. The small cell carcinoma of lung differs from the squamous cell cancer of the same organ by dense, fibrillar nucleoli with a small amount of granular component located on the periphery of the nucleolar body. Nucleolar type reflecting the functional state of malignization process may serve as an additional diagnostic criterion for tumour identification.     

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.     

Spatial redistribution of ribosomal chromatin in the fibrillar centres of human circulating lymphocytes after stimulation of transcription

We have studied the distributional changes of the completely extended ribosomal chromatin present in the fibrillar centres of resting human lymphocytes after phytohemagglutinin (PHA) treatment. In thin sections of resting lymphocytes selectively stained for DNA, the extended non-nucleosomal chromatin was located in a solitary, large agglomerate which corresponds to the solitary, large fibrillar centre observed in uranium-lead-stained sections. At 20 h after PHA stimulation the ribosomal chromatin agglomerate appeared to be fragmented into smaller agglomerates which correspond to numerous fibrillar centres surrounded by a thick rim of dense fibrillar component. The mean area of ribosomal chromatin agglomerates from resting lymphocytes was found to be 0.772 mu 2 + 0.125 SD, whereas in stimulated lymphocytes it was found to be 0.184 mu 2 + 0.052 SD. At 20 h after PHA treatment ribosomal RNA (rRNA) synthesis was 8-fold greater than the control value, whereas DNA synthesis had not started. These results indicate that ribosomal chromatin of resting lymphocyte fibrillar centres contains transcribable sequences, temporally not expressed.