scholarly journals Ultrastructural distribution of DNA and RNA within the nucleolus of human Sertoli cells as seen by molecular immunocytochemistry

1993 ◽  
Vol 105 (1) ◽  
pp. 33-39 ◽  
Author(s):  
M. Thiry
Keyword(s):  
Sertoli Cells ◽  
Ultrastructural Level ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dense Fibrillar Component ◽  
Granular Component ◽  
Dna And Rna ◽  
Nucleotidyl Transferase ◽  
Immunocytochemical Techniques ◽  
Fibrillar Component

The precise distribution of DNA and RNA within the human Sertoli cell nucleolus has been investigated, at the ultrastructural level, by cytochemical and molecular immunocytochemical techniques. In Sertoli cells, the nucleolar components show a typical spatial distribution. The fibrillar centres are not surrounded by a layer of dense fibrillar component, but come in contact only with strands of dense fibrillar component. These fibrillar parts of strands are the extensions of granular strands connected to a large granular mass. These strands delimit numerous nucleolar interstices in which chromatin fibres are clearly obvious. Using the in situ terminal deoxynucleotidyl transferase/immunogold procedure for detecting DNA, we find evident label exclusively over the chromatin fibres enclosed in the nucleolar interstices and over the fibrillar centres, and no significant label over the dense fibrillar component and granular component of the nucleolus. Furthermore, using the polyadenylate nucleotidyl transferase/immunogold procedure for detecting RNA, we show that label is deposited not only over the granular component and dense fibrillar component, as expected, but also quite obviously over the fibrillar centres. No label is seen over the interstices containing chromatin.

Ultrastructural association of the chromatin-containing lacunar spaces with the granular component of the interphase nucleolus in Allium cepa

1981 ◽  
Vol 59 (11) ◽  
pp. 2073-2076
Author(s):  
L. A. Chouinard
Keyword(s):  
Granular Material ◽  
Allium Cepa ◽  
Ultrastructural Level ◽  
Dense Fibrillar Component ◽  
Active Chromatin ◽  
Granular Component ◽  
Meristematic Cells ◽  
Partial Association ◽  
Root Meristematic Cells ◽  
Fibrillar Component

At the ultrastructural level, the interphase nucleolus in root meristematic cells of Allium cepa is characterized by the presence of chromatin-containing lacunar spaces associated with the dense-fibrillar component of the nucleolar mass. The present observations reveal that a number of these chromatin-containing lacunar spaces also exhibit partial association with the dense-granular component of the nucleolus. Under the electron microscope, such lacunar spaces are indeed seen to be enclosed or walled off, on one side, by dense-fibrillar material, and on the other side, by dense-granular material continuous with and indistinguishable from the dense-granular component of the nucleolus. The relevant observational evidence would be consistent with the view that loops of transcriptionally active chromatin, emanating from the nucleolar organizing region, project radially into either only the dense-fibrillar or both the dense-fibrillar and the dense-granular material bordering the lacunar spaces in question.

A study on nucleolar DNA: isolation of DNA from fibrillar components and ultrastructural localization of different DNA probes

1993 ◽  
Vol 104 (4) ◽  
pp. 1199-1205 ◽  
Author(s):  
P. Hozak ◽  
C. Schofer ◽  
J. Sylvester ◽  
F. Wachtler
Keyword(s):  
Ribosomal Rna ◽  
Sertoli Cells ◽  
Ultrastructural Localization ◽  
Ribosomal Rna Genes ◽  
Border Region ◽  
Dense Fibrillar Component ◽  
Mitotic Chromosomes ◽  
Electron Microscopic ◽  
Fibrillar Component ◽  
Rna Genes

The nature and localization of DNA contained in the fibrillar centres and the dense fibrillar component (the fibrillar complex) in the nucleoli, was studied in human LEP cells, Sertoli cells, spermatogonia A and in mitotic chromosomes of stimulated lymphocytes. A novel procedure for isolating the intact fibrillar complex from LEP cells was used; the complex contains DNA that hybridizes to secondary constrictions of mitotic chromosomes and to 28 S rDNA sequences, on Southern blots. Electron microscopic DNA-DNA in situ hybridization was performed, with (a) a probe prepared from DNA extracted from the fibrillar complex of LEP cells, (b) a probe for human total genomic DNA, and (c) a probe for the transcribed part of human rDNA. On the basis of the results obtained we conclude that the ribosomal RNA genes in human Sertoli cells and spermatogonia A are predominantly associated with the dense fibrillar component, including the border region between fibrillar centres and the dense fibrillar component. The ribosomal RNA genes are the main, if not exclusive, DNA type present in the fibrillar complex in the studied cell types.

scholarly journals Immunodetection of RNA on ultra-thin sections incubated with polyadenylate nucleotidyl transferase.

1993 ◽  
Vol 41 (5) ◽  
pp. 657-665 ◽  
Author(s):  
M Thiry
Keyword(s):  
Divalent Cations ◽  
Ultrastructural Level ◽  
Ehrlich Tumor ◽  
Precise Location ◽  
Great Precision ◽  
Thin Sections ◽  
Nucleotidyl Transferase ◽  
Ehrlich Tumor Cells ◽  
Labeling Pattern

A new method is described for locating RNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing polyadenylate nucleotidyl transferase (PnT) and biotinylated ATP. The labeled nucleotides bound to RNA at the surface of the ultra-thin sections were than visualized by an indirect immunogold labeling technique. The resulting labeling pattern was dependent on the presence of divalent cations in the PnT medium. The method revealed with great precision the specific RNA-containing structures within Ehrlich tumor cells. The method is applicable to Epon sections. However, the labeling intensity varies according to the fixation used. Best results were obtained on acetylated cell sections. The method can be combined with EDTA regressive staining. The in situ PnT method provides a very useful tool for pinpointing the precise location of RNA within biological material at the ultrastructural level.

Distinguishing the sites of pre-rRNA synthesis and accumulation in Ehrlich tumor cell nucleoli

1991 ◽  
Vol 99 (4) ◽  
pp. 759-767
Author(s):  
M. Thiry ◽  
G. Goessens
Keyword(s):  
Tumor Cell ◽  
Condensed Chromatin ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Dense Fibrillar Component ◽  
Ehrlich Tumor ◽  
Granular Component ◽  
Fibrillar Component

The precise location of transcribing rRNA genes within Ehrlich tumor cell nucleoli has been investigated using two approaches: high-resolution autoradiography of cells pulse-labelled with tritiated uridine, varying the exposure time, and in situ-in vitro transcription coupled with an immunogold labelling procedure. When autoradiographic preparations are exposed for a short time, silver grains are found associated almost exclusively with interphasic cell nucleoli. Labelling of extranucleolar areas requires longer exposure. Within the nucleolus, the first sites to be revealed are in the dense fibrillar component. Prolonging exposure increases labelling over the dense fibrillar component, with label becoming more and more apparent over the fibrillar centers. Under these conditions, however, labelling does not extend into the granular component, and no background is observed. Initiation of transcription on ultrathin cell sections occurs preferentially at the borders of condensed chromatin blocks and in their close vicinity. The condensed chromatin areas themselves remain unlabelled. Inside most nucleoli, gold-particle clusters are mainly detected in the fibrillar centers, especially at their periphery, whereas the dense fibrillar component and the granular component remain devoid of label. These results, together with previous observations made on the same cell type, clearly indicate that the fibrillar centers are the sites of rRNA gene transcription in Ehrlich tumor cell nucleoli, while the dense fibrillar component is the site of pre-rRNA accumulation.

Revealing the unseen: the organizer region of the nucleolus

2001 ◽  
Vol 114 (17) ◽  
pp. 3199-3205 ◽  
Author(s):  
Marco Biggiogera ◽  
Manuela Malatesta ◽  
Sousan Abolhassani-Dadras ◽  
François Amalric ◽  
Lawrence I. Rothblum ◽  
...  
Keyword(s):  
Inner Core ◽  
Organizer Region ◽  
Exact Distribution ◽  
Dense Fibrillar Component ◽  
Fibrillar Center ◽  
Energy Filtering ◽  
Dna And Rna ◽  
Highly Sensitive ◽  
Fibrillar Component ◽  
First Time

We carried out a high-resolution ultrastructural analysis of the nucleolus in mouse P815 cells by combining specific DNA and RNA staining, anti-fibrillarin immunolabeling, contrast enhancement by energy filtering TEM and phosphorus mapping by ESI to visualize nucleic acids. We demonstrated that specifically contrasted DNA, fibrillarin and phosphorus overlap within the nucleolar dense fibrillar component. Moreover, we describe a ‘DNA cloud’ consisting of an inner core of DNA fibers (fibrillar center) and a periphery made of extremely thin fibrils overlapping the anti-fibrillarin immunolabeling (dense fibrillar component). This highly sensitive approach has allowed us to demonstrate, for the first time, the exact distribution of DNA within the decondensed interphase counterpart of the NOR, which includes both the fibrillar center and the dense fibrillar component.

scholarly journals Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus.

1989 ◽  
Vol 37 (9) ◽  
pp. 1371-1374 ◽  
Author(s):  
M Biggiogera ◽  
S Fakan ◽  
S H Kaufmann ◽  
A Black ◽  
J H Shaper ◽  
...  
Keyword(s):  
Hela Cell ◽  
Immunoelectron Microscopy ◽  
Silver Staining ◽  
Dense Fibrillar Component ◽  
Specific Antibodies ◽  
Granular Component ◽  
Protein B23 ◽  
Reported Data ◽  
Fibrillar Component

The intranucleolar distribution of phosphoproteins B23 and C23 was visualized simultaneously by post-embedding immunoelectron microscopy in HeLa cell nucleoli, using specific antibodies. The data show that proteins B23 and C23 co-localize to the same nucleolar compartments, i.e., the dense fibrillar component and the granular component. Neither of the two antibodies is significantly associated with the fibrillar centers in these cells, although the fibrillar centers appear positive after silver staining. These findings suggest that other unidentified components must be responsible for the silver staining observed in the fibrillar centers of interphase nucleoli. The results are discussed in the light of previously reported data obtained by preembedding immunolabeling techniques and by silver staining, which both suggested a localization of protein C23 inside the fibrillar centers.

3-D organization of ribosomal transcription units after DRB inhibition of RNA polymerase II transcription

1999 ◽  
Vol 112 (13) ◽  
pp. 2145-2154 ◽  
Author(s):  
S.L. Panse ◽  
C. Masson ◽  
L. Heliot ◽  
J.M. Chassery ◽  
H.R. Junera ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase Ii ◽  
Single Gene ◽  
Functional Domain ◽  
Dense Fibrillar Component ◽  
Rdna Transcription ◽  
Active Transcription ◽  
The Individual ◽  
Fibrillar Component

In each bead of the nucleolar necklace, using adenosine analog DRB-treated PtK1 cells, we investigated the three components of rDNA transcription, i.e. the gene, transcription factor UBF and transcripts. In situ hybridization revealed the unraveling and 3-D dispersion of most of the rDNA coding sequences within the nucleus. The signals were small, of similar intensity and tandemly organized in the necklace. This observation is compatible with the fact that they might correspond to single gene units. Active transcription was visualized in these units, demonstrating that they were active functional units. Transcript labeling was not similar for each unit, contrary to UBF labeling. UBF and rRNA transcripts were only partially colocalized, as demonstrated by 3-D image analysis and quantification. As visualized by electron microscopy, the necklace was composed of a small fibrillar center partially surrounded by a dense fibrillar component. The 3-D arrangement of this individual unit in the necklace, investigated both by confocal and electron microscopy in the same cells, showed that the individual beads were linked by a dense fibrillar component. The reversibility of this organization after removal of DRB indicated that the beads in the necklace are certainly the elementary functional domain of the nucleolus. In addition, these results lead us to suggest that the organization of a functional domain, presumably corresponding to a single gene, can be studied by in situ approaches.

Cytochemical features common to nucleoli and cytoplasmic nucleoloids of Olea europaea meiocytes: detection of rRNA by in situ hybridization

1994 ◽  
Vol 107 (2) ◽  
pp. 621-629 ◽  
Author(s):  
J.D. Alche ◽  
M.C. Fernandez ◽  
M.I. Rodriguez-Garcia
Keyword(s):  
In Situ Hybridization ◽  
Olea Europaea ◽  
Electron Microscopic ◽  
Phosphorylated Proteins ◽  
Immunocytochemical Techniques ◽  
Fibrillar Component ◽  
Immunohistochemical Techniques ◽  
Microscopic Techniques ◽  
Olea Europaéa

We used light and electron microscopic techniques to study the composition of cytoplasmic nucleoloids during meiotic division in Olea europaea. Nucleoloids were found in two clearly distinguishable morphological varieties: one similar in morphology to the nucleolus, and composed mainly of dense fibrillar component, and another surrounded by many ribosome-like particles. Cytochemical and immunocytochemical techniques showed similar reactivities in nucleoloids and the nucleolus: both are ribonucleoproteic in nature, and possess argyrophillic, argentaffinic and highly phosphorylated proteins. Immunohistochemical techniques failed to detect DNA in either structure. In situ hybridization to a 18 S rRNA probe demonstrated the presence of ribosomal transcripts in both the nucleolus and nucleoloids. These similarities in morphology and composition may reflect similar functionalities.

scholarly journals Building an efficient factory

2002 ◽  
Vol 157 (5) ◽  
pp. 739-741 ◽  
Author(s):  
Sui Huang
Keyword(s):  
Rrna Synthesis ◽  
Dense Fibrillar Component ◽  
Rdna Transcription ◽  
Granular Component ◽  
Fibrillar Center ◽  
Pol I ◽  
Fibrillar Component ◽  
Polymerase I

The subnucleolar structure that is involved in rDNA transcription has been controversial. A report by Koberna et al. (2002)(this issue, page 743) adds significant weight toward the idea that dense fibrillar components (DFCs)**Abbreviations used in this paper: DFC, dense fibrillar component; FC, fibrillar center; GC, granular component; Pol I, polymerase I. and fibrillar center (FC)/DFC borders are the sites of pre-rRNA synthesis.

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