Structural and functional organization of ribosomal genes within the mammalian cell nucleolus.

in Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society (2006), 54(2), 131-45

Data on the in situ structural-functional organization of ribosomal genes in the mammalian cell nucleolus are reviewed here. Major findings on chromatin structure in situ come from investigations carried ... [more ▼]

Data on the in situ structural-functional organization of ribosomal genes in the mammalian cell nucleolus are reviewed here. Major findings on chromatin structure in situ come from investigations carried out using the Feulgen-like osmium ammine reaction as a highly specific electron-opaque DNA tracer. Intranucleolar chromatin shows three different levels of organization: compact clumps, fibers ranging from 11 to 30 nm, and loose agglomerates of extended DNA filaments. Both clumps and fibers of chromatin exhibit a nucleosomal organization that is lacking in the loose agglomerates of extended DNA filaments. In fact, these filaments constantly show a thickness of 2-3 nm, the same as a DNA double-helix molecule. The loose agglomerates of DNA filaments are located in the fibrillar centers, the interphase counterpart of metaphase NORs, therefore being constituted by ribosomal DNA. The extended, non-nucleosomal configuration of this rDNA has been shown to be independent of transcriptional activity and characterizes ribosome genes that are either transcribed or transcriptionally silent. Data reviewed are consistent with a model of control for ribosome gene activity that is not mediated by changes in chromatin structure. The presence of rDNA in mammalian cells always structurally ready for transcription might facilitate a more rapid adjustment of the ribosome production in response to the metabolic needs of the cell. [less ▲]

Evaluation of the sensitivity of the terminal deoxynucleotidyl transferase-immunogold technique on Balbani ring genes.

in Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society (1998), 46(3), 345-51

Recently, we developed the terminal deoxynucleotidyl transferase (TdT)-immunogold technique for in situ detection of DNA molecules. In this study the potential value and the limitations of the method were ... [more ▼]

Recently, we developed the terminal deoxynucleotidyl transferase (TdT)-immunogold technique for in situ detection of DNA molecules. In this study the potential value and the limitations of the method were evaluated using the giant polytene chromosomes from Chironomus tentans salivary glands. Emphasis was put on the Balbiani rings (BRs), specialized chromosomal sites with exceptionally intense synthesis of large mRNA molecules. Immunolabeling was recorded not only over the bands and interbands of the polytene chromosomes but also over the BR structures. In the BRs, gold particles were present over segments of active transcription units, each with a central chromatin axis and a number of growing RNP products attached to the axis. One third of the transversely sectioned transcription units showed labeling in the central parts, i.e., where the unfolded chromatin axis is located, whereas the growing RNP fibers remained unlabeled. The absence of labeling of the RNP fibers is not likely to be due to lack of accessibility, because anti-RNA antibodies readily decorated the RNP fibers. The nuclear sap and cytoplasm displayed no significant label. These results clearly indicate that the TdT-immunogold technique is specific for DNA and detects not only DNA in compacted chromatin but also fully extended DNA. Its ability to efficiently label a single DNA molecule demonstrates the method's very high sensitivity. [less ▲]

Differential distribution of single-stranded DNA, double-stranded DNA, and RNA in adenovirus-induced intranuclear regions of HeLa cells.

in Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society (1995), 43(8), 749-59

We investigated in great detail the fine spatial distribution of nucleic acids within adenovirus-infected HeLa cells by various immunogold labeling procedures. To detect DNA, we used the in situ terminal ... [more ▼]

We investigated in great detail the fine spatial distribution of nucleic acids within adenovirus-infected HeLa cells by various immunogold labeling procedures. To detect DNA, we used the in situ terminal deoxynucleotidyl transferase-immunogold technique. In addition to the expected evident label over the condensed host chromatin and the structures containing viral double- and single-stranded DNA, label was consistently revealed over round fibrillar spots. By contrast, other virus-induced substructures, such as compact rings, crystalloids, clear amorphous inclusions, and electron-dense amorphous inclusions, displayed no significant label. Except for the viral single-stranded DNA accumulation sites, identical labeling pattern was obtained with the in situ nick-translation-immunogold method. We further labeled the sections with anti-RNA antibodies. Label was present not only over the cytoplasm and the intranuclear fibrillogranular network but also quite obviously over the compact rings and interchromatin granule clusters. None was seen over the other nuclear structures of infected cells, notably over the fibrillar spots. We suggest that these fibrillar spots might be involved in the formation of the viral, non-encapsidated, double-stranded DNA storage site. [less ▲]

Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues.

in Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society (1992), 40(3), 411-9

A new method is described for locating DNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing terminal deoxynucleotidyl transferase (TdT) and ... [more ▼]

A new method is described for locating DNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing terminal deoxynucleotidyl transferase (TdT) and various non-isotopic nucleotide analogues. The labeled nucleotides bound to the surface of ultra-thin sections were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern was strongly dependent on the divalent cation used in the TdT medium. The method revealed with great precision the specific DNA-containing structures within Ehrlich tumor cells, even where DNA was present in very low amounts. The method is compatible with all usual fixation and embedding procedures and can be combined with cytochemical methods. The in situ TdT method provides a very useful tool for pinpointing the precise location of DNA within biological material at the ultrastructural level. [less ▲]

In situ nick translation at the electron microscopic level: a tool for studying the location of DNAse I-sensitive regions within the cell.

in Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society (1991), 39(6), 871-4

The in situ nick translation method was adapted to the ultrastructural level, to study the location of DNAse I-sensitive sequences within the cell. Ultra-thin sections of Lowicryl-embedded cells were ... [more ▼]

The in situ nick translation method was adapted to the ultrastructural level, to study the location of DNAse I-sensitive sequences within the cell. Ultra-thin sections of Lowicryl-embedded cells were incubated in a medium containing DNAse I, DNA polymerase I, and all four deoxyribonucleotides, some being biotinylated. The nick-translated sites were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern is closely dependent on the DNAse I concentration in the nick-translation medium. The method reveals with great precision the specific DNAse I-sensitive regions within the nucleus. This technique can be used to discriminate between active and inactive regions of interphase chromatin. [less ▲]

Ultrastructural cytochemistry of the mammalian cell nucleolus.

in Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society (1990), 38(9), 1237-56

In the present review on the organization of the mammalian cell nucleolus, we report and discuss data obtained during the past 10 years by means of cytochemical and immunocytochemical ultrastructural ... [more ▼]

In the present review on the organization of the mammalian cell nucleolus, we report and discuss data obtained during the past 10 years by means of cytochemical and immunocytochemical ultrastructural techniques. Particular emphasis is placed on the following topics: location of the nucleolus organizer regions in interphasic nucleolar components, structure of nucleolar chromatin in situ, and the structure-function relationship of the nucleolar components. The cytochemical and immunocytochemical results are compared and the concordant data are stressed for each topic. [less ▲]

Reticular fibroblasts in peripheral lymphoid organs identified by a monoclonal antibody.

in Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society (1986), 34(7), 883-90

We have produced a panel of monoclonal antibodies directed against nonlymphoid cells in central and peripheral lymphoid organs. In this paper we present the reactivity of one of these antibodies, ER-TR7 ... [more ▼]

We have produced a panel of monoclonal antibodies directed against nonlymphoid cells in central and peripheral lymphoid organs. In this paper we present the reactivity of one of these antibodies, ER-TR7. This antibody detects reticular fibroblasts, which constitute the cellular framework of lymphoid and nonlymphoid organs and their products. In frozen sections of the spleen incubated with this antibody, the red pulp and white pulp are clearly delineated. Furthermore, the major white pulp compartments--the follicles and periarteriolar lymphoid sheath as well as the marginal zone--are recognized by their characteristic labeling patterns. In lymph nodes, the capsule, sinuses, follicles, paracortex, and medullary cords are clearly delineated. In the thymus and bone marrow no such specialized compartments were demonstrated. ER-TR7 reacts with an intracellular component of fibroblasts. Since ER-TR7 does not react with purified laminin, collagen types I-V, fibronectin, heparan sulfate proteoglycan, entactin, or nidogen, it detects a hitherto uncharacterized antigen. The possible role of the ER-TR7 positive reticular fibroblasts in the cellular organization of peripheral lymphoid organs will be discussed. [less ▲]

Fibronectin presence in native collagen fibrils of human fibroblasts: immunoperoxidase and immunoferritin localization.

in Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society (1980), 28(12), 1319-33

Fibronectin is a major constituent of the fibroblast extracellular matrix. Fibronectin binds to collagen, mediates fibroblast adhesion to collagen, and is synthesized and secreted into the medium of ... [more ▼]

Fibronectin is a major constituent of the fibroblast extracellular matrix. Fibronectin binds to collagen, mediates fibroblast adhesion to collagen, and is synthesized and secreted into the medium of cultured fibroblasts. Affinity-purified antibodies to fibronectin and collagen were localized using the peroxidase-antiperoxidase method or with ferritin-coupled secondary antibodies. Using human fibroblasts cultured under routine conditions, fibronectin and procollagen I react in a nonperiodic manner with: 1) approximately 10 nm extracellular fibrils, 2) cell membrane, and 3) membrane-associated vesicles. All fibrils react with both antibodies, suggesting some form of codistribution of fibronectin and collagen in these fibrils. Treatment with ascorbate leads to the development of a larger diameter extracellular fibril, approximately 40 nm in diameter. These large diameter fibrils are clearly collagen fibrils as documented by the procollagen antibody reaction. Importantly, fibronectin is bound to or a constituent of these "native" or cellular made collagen fibrils. Fibronectin and procollagen antibodies localized with the peroxidase-antiperoxidase method have a 70 nm axial repeat of reaction product on ascorbate-treated fibroblasts. Localization of antibodies with ferritin-labeled secondary antibodies is less satisfactory, but supports the basic observations made with the unlabeled antibody enzyme method. This observation rules out any potential criticisms. Although it is more difficult to observe with immunoferritin, there is an indication that antibodies to fibronectin react with an axial periodicity on cellular produced collagen fibrils. [less ▲]