Showing papers on "Nucleolus published in 1981"

A nuclear antigen associated with cell proliferation and blast transformation.

Abstract: A nuclear antigen associated with cell proliferation (proliferating cell nuclear antigen-PCNA) and blast transformation is recognized by autoantibodies in the sera of some patients with systemic lupus erythematosus This autoantibody is a precipitating antibody and also reacts in immunofluorescence, staining the nucleoplasm of proliferating and blast-transformed cells The autoantibody was used as a reagent to determine the distribution of PCNA in a synchronized continuous B lymphoid cell line (WiL-2) and in mitogen-induced blast-transformed lymphocytes In WiL-2 cells, PCNA was detected as speckled nucleoplasmic staining in G1, S, and G2 phases of the cell cycle In addition, during late G1 and early S phases, PCNA was also detected in the nucleolus During mitogen-induced blast transformation of lymphocytes, PCNA was noticed in the nucleolus before the initiation of DNA synthesis and later became nucleoplasmic with disappearance of nucleolar staining These studies demonstrate that the relationship of PCNA to proliferation and blast transformation may be associated with events related to DNA synthesis in these cells

The life history of cells in renewing systems.

Abstract: Cell populations may be classified into three groups: static, which do not divide in the adult (e.g., neurons), expanding, which may divide but at a decreasing rate with age (e.g., kidney cells), and renewing, which undergo active division throughout life (e.g., blood cells, intestinal and seminiferous epithelium). The first two groups consist of nonrenewing populations whose cells usually survive as long as the body itself. In contrast, the cells of renewing populations have a short life, of the order of 2 to 3 days in the intestinal epithelium of rodents and 7 to 8 weeks in their seminiferous epithelium. In the present work, the life of a nonrenewing cell—the proximal convoluted tubule cell of kidney—is compared to that of the renewing cells of small intestine and testis. The proximal convoluted tubule cell arises during embryonic development, when it has few organelles, many free ribosomes, a pale nucleus with diffuse chromatin, and a large open-network nucleolus. As the cell differentiates, organelles accumulate while free ribosomes decrease in number, chromatin masses appear in the nucleus, and the nucleolus becomes rather dense. With old age, some of the cells show features interpreted as senescence, particularly a small dense nucleolus. The short-lived columnar cells of small intestine in adult rats arise from stem cells present in the base of the crypts. These cells have features similar to those of the embryonic proximal tubule cell, particularly many free ribosomes and a large open-network nucleolus. The stem cells give rise to cells which ascend crypts and villi. Shortly after the cells reach the villus, they acquire a full set of organelles, suggesting maturity, while free ribosomes decrease in number and the nucleolus undergoes condensation. Meanwhile radioautographic tests indicate that the production by the cells of glycoproteins identified as intestinal enzymes reaches a high level. However, when cells arrive in the villus tip region, their activity decreases, as shown by reduced enzyme production, while the nucleolus appears atrophic. The cells are then lost to the lumen. The cells of the seminiferous epithelium also arise in the adult from stem cells with embryonic features. In the course of a complex differentiation, the nucleolus becomes atrophic and disappears. Soon thereafter the fragile spermatozoon stage is reached. Thus, the renewing cells of the adult show in rapid succession some of the features occurring slowly during the life of nonrenewing cells: origin from embryonic-like elements, maturation, and senescence indicated by a fragile state before life's end.

Ultrastructural localization of nuclei acids by the use of enzyme-gold complexes.

Abstract: A cytochemical technique for the ultrastructural localization of substrates using enzyme-gold complexes is reported. RNase A and DNase I have been labeled with gold particles. The RNase-gold and dNase-gold complexes obtained were applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Different cellular compartments were labeled by these enzyme-gold complexes. Using the RNase-gold complex the rough endoplasmic reticulum appeared decorated with gold particles. The gold marker was also present over the nucleus, especially over the nucleolus; mitochondria were weakly labeled. Using the DNase-gold complex, gold particles were concentrated over the euchromatin of the nucleus and the mitochondria. The heterochromatin and the nucleolus showed a less intense labeling. For both enzyme-gold complexes, the Golgi area, the secretory granules and the extracellular space appeared free of label. In those control conditions where the substrates were added to the enzyme-gold complexes a major reduction...

Immunocytochemical localization of S-100 protein in the brain of adult rat. An ultrastructural study.

Abstract: The cellular and subcellular distribution of the nervous system-specific S-100 protein has been investigated in the brain of adult rat at the ultrastructural level by the pre-embedding unlabelled antibody PAP method. The protein is found in both fibrous and protoplasmic astrocytes and in the ependymal cells. The neurons, the oligodendrocytes as well as the microglial cells are lacking S-100. The labelled cells show a reaction product diffusely distributed in the cytoplasmic matrix and on specialized membranes, namely plasma membranes, outer mitochondrial membranes and membranes of the endoplasmic reticulum and Golgi apparatus. The astrocytic filaments and the axonemes of the ependymal cilia exhibit a strong immunoreactivity. The reaction product is also present in the nucleoplasm of the astrocytes and ependymal cells but it is absent from the nucleolus and nuclear envelope. This immunocytochemical data on tissue with satisfactory ultrastructural preservation, provides new information on the localization of the S-100 protein, and could contribute to the understanding of the biological role of the protein.

Distribution of kinetochore (centromere) antigen in mammalian cell nuclei.

Abstract: Antigens associated with mammalian centromeres were localized at the high and electron microscopic levels using the peroxidase-labeled antibody method. The antibody used was of a type naturally occurring in the sera of patients with scleroderma. At the light microscopic level, it reacts specifically with the centromere regions of chromosomes in a variety of mammalian species and strains in discrete foci in interphase nuclei. We find that the number of foci approximates the number of chromosomes present in the various cell types. At the ultrastructural level, the antigenic foci are confirmed to lie in the kinetochore regions of each chromosome. In interphase nuclei, the antigenic foci were usually associated either with the inner surfaces of the nuclear envelope or with the nucleoli. These observations indicate that the centromere regions of the chromosomes in interphase are not randomly distributed within the nucleus but are usually fixed either to the inner surface of the nuclear envelope or to nucleoli.

Human nucleolus organizers on nonhomologous chromosomes can share the same ribosomal gene variants.

Abstract: The distributions of three human ribosomal gene polymorphisms among individual chromosomes containing nucleolus organizers were analyzed by using mouse--human hybrid cells. Different nucleolus organizers can contain the same variant, suggesting the occurrence of genetic exchanges among ribosomal gene clusters on nonhomologous chromosomes. Such exchanges appear to occur less frequently in mice. This difference is discussed in terms of the nucleolar organization and chromosomal location of ribosomal gene clusters in humans and mice.

Ultrastructural organization, sites of transcription and distribution of fibrillar centres in the nucleolus of the mouse oocyte.

Abstract: The emergence of newly formed nucleoli and their development have been studied in mouse oocytes from pachytene to diplotene stages. At mid-pachytene, the nucleolus first appears as a fibrillar centre surrounded by a layer of electron-dense fibrils and penetrated by chromatin fibres emanating from the secondary constriction region of the nucleolar bivalent. Since this bivalent contains 2 paired nucleolar organizers, 2 nucleoli are formed in a symmetrical fashion. At advanced pachytene, the nucleoli are extended by strands of fibrillar component which become fibrillogranular distally. The 2 nucleoli fuse together at late pachytene. At diplotene, the nucleolus becomes large and reticulated. The development of the nucleolonema coincides with the appearance of numerous secondary fibrillar centres. Three-dimensional reconstruction of the reticulated nucleolus shows that the number of fibrillar centres largely exceeds that of nucleolar organizers. Radioautography after [3H]uridine incorporation demonstrates that during the first step of nucleologenesis the labelling is limited to the layer of electron-dense fibrils surrounding the fibrillar centre. Study of the time course of tritiated uridine incorporation from pachytene to diplotene shows that the labelling extends with the extending strands of fibrillar component. In the fully developed nucleolus, all fibrillar strands are labelled and contain, therefore, actively transcribed rDNA. These observations suggest that the rDNA, which is initially compacted in the primary fibrillar centre at the onset of nucleogenesis, progressively unravels and becomes distributed throughout the fibrillar parts of the nucleolonema. The lack of labelling of the secondary fibrillar centres suggests that they are zones of inactivity of the ribosomal genes where the rDNA remains locally compacted. A model of the ultrastructural organization of the nucleolus is proposed based on our observations.

A nucleolar skeleton of protein filaments demonstrated in amplified nucleoli of Xenopus laevis.

Abstract: The amplified, extrachromosomal nucleoli of Xenopus oocytes contain a meshwork of approximately 4-nm-thick filaments, which are densely coiled into higher-order fibrils of diameter 30-40 nm and are resistant to treatment with high- and low-salt concentrations, nucleases (DNase I, pancreatic RNase, micrococcal nuclease), sulfhydryl agents, and various nonionic detergents. This filamentous "skeleton" has been prepared from manually isolated nuclear contents and nucleoli as well as from nucleoli isolated by fluorescence-activated particle sorting. The nucleolar skeletons are observed in light and electron microscopy and are characterized by ravels of filaments that are especially densely packed in the nucleolar cortex. DNA as well as RNA are not constituents of this structure, and precursors to ribosomal RNAs are completely removed from the extraction-resistant filaments by treatment with high-salt buffer or RNase. Fractions of isolated nucleolar skeletons show specific enrichment of an acidic major protein of 145,000 mol wt and an apparent pI value of approximately 6.15, accompanied in some preparations by various amounts of minor proteins. The demonstration of this skeletal structure in "free" extrachromosomal nucleoli excludes the problem of contaminations by nonnucleolar material such as perinucleolar heterochromatin normally encountered in studies of nucleoli from somatic cells. It is suggested that this insoluble protein filament complex forms a skeleton specific to the nucleolus proper that is different from other extraction-resistant components of the nucleus such as matrix and lamina and is involved in the spatial organization of the nucleolar chromatin and its transcriptional products.

Protein A24 lyase activity in nucleoli of thioacetamide-treated rat liver releases histone 2A and ubiquitin from conjugated protein A24.

Abstract: Isolated liver nucleoli from rats treated for 3 days with thioacetamide contained an enzyme activity which specifically degraded conjugate protein A24. Two-dimensional polyacrylamide gel electrophoresis indicated that the amount of protein A24 in chromatin decreased during incubation at 37 degrees C for 60 min with these nucleoli. Concomitantly, a marked increase was found in the content of free ubiquitin, the nonhistone component of protein A24. Incubation of 3H-labeled protein A24 with the thioacetamide-treated liver nucleoli resulted in the linear release of 3H-labeled histone 2A and 3H-free ubiquitin in the presence of phenylmethanesulfonyl fluoride (PMSF) for 2 h. Pretreatment of the nucleoli with trypsin or by heating at 80 degrees C for 10 min inhibited their ability to cleave protein A24. Protein A24 lyase catalyzes the reaction: protein A24 leads to histone 2A plus ubiquitin.

Polyamine-activated protein kinase reaction from nuclei and nucleoli of Physarum polycephalum which phosphorylates a unique Mr 70 000 nonhistone protein.

Abstract: Methods are described for the detection and purification of a protein kinase from nuclei and nucleoli of Physarum polycephalum which catalyzed transfer of phosphate from [gamma-32P]ATP to a unique nonhistone protein of Mr 70 000 in a reaction that was polyamine dependent. Enzymatic phosphorylation of the nonhistone protein by the purified protein kinase was stimulated greatly, at times more than 60-fold, by the polyamines spermidine and spermine. This unique polyamine-dependent reaction was localized on the rDNA minichromosome of the nucleolus. The polyamine-dependent protein kinase, which was first partially purified with the acidic nonhistone protein fraction from isolated nucleoli, was resolved from at least six other protein kinases by phosphocellulose chromatography into a catalytic component of Mr 26 000 and a complex comprised of the catalytic component associated with a phosphate acceptor protein of Mr 70 000. The complex also catalyzed polyamine-dependent phosphorylation of the endogenous Mr 70 000 component. The resolved catalytic component catalyzed polyamine-dependent phosphorylation of a dephosphorylated Mr 70 000 nonhistone protein that had been independently isolated from nucleoli and previously demonstrated to have properties concordant with a specific regulatory role in rRNA gene transcription [Keuhn, G. D., Affolter, H. U., Atmar, V. J., Seebeck, T., Gubler, U., & Braun, R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2541-2545]. These studies indicate one way that the polyamines may regulate rRNA gene transcription through the mediation of a highly specific nonhistone protein kinase.

Localization of nucleoli in Drosophila melanogaster polytene chromosomes

Abstract: The majority of D. melanogaster salivary gland nuclei contains many nucleoli which vary in size and number. All nucleoli hybridize in situ with a cloned Drosophila DNA fragment containing 26 S ribosomal gene. Autoradiographic analysis of preparations after pulse H3-uridine or H3-thymidine labelling of the salivary gland indicates an intensive transcription and replication of DNA within nucleoli. The nucleoli are bound to different sites of polytene chromosomes by chromatin fibers similar to strands of ectopic pairing and they are most often bound to regions which may be defined as intercalary heterochromatin.

Thioacetamide-Induced Hepatocarcinoma in Rat

Abstract: Out of 56 thioacetamide (TAA)-fed rats hepatocellular carcinoma were found in four instances after 300, 360, 450 and 495 days in the present experiment. Of the four tumours, two showed lung metastasis. The size of TAA-treated hepatocyte nucleoli increased greatly after several weeks of feeding. Two enzymes, succinate dehydrogenase and glucose-6-phosphatase were found in both cytoplasma and nucleoli of hepatocytes of which nucleolar localizations are quite new. In hepatocellular carcinoma, malignant cells lost glucose-6-phosphatase activity completely in cytoplasm and nucleoli though succinate dehydrogenase activity was present in these organelles.

In vitro RNA synthesis in oocyte nuclei of the newt Notophthalmus

Abstract: An incubation medium is described which supports RNA synthesis in isolated oocyte nuclei of the newt Notophthalmus, and which permits subsequent autoradiographic examination of the lampbrush chromosomes and nucleoli. By using different concentrations of α-amanitin we distinguish RNA synthesis due to RNA polymerases I, II and III. All RNA synthesis on loops is inhibited by 0.5 μ/ml of α-amanitin and is therefore due to polymerase II. Polymerase III is responsible for RNA synthesis at a small number of discrete sites in condensed chromatm. These include the centromere bars of three of four chromosomes, which probably represent 5S RNA synthesis, as well as 15–20 lesser sites scattered elsewhere. Polymerase I activity is confined to the nucleoli.

Nuclease-Sensitive Regions on the Extrachromosomal r-Chromatin from Tetrahymena pyriformis

Abstract: The extrachromosomal DNA coding for the ribosomal RNA precursor in Tetrahymena contains a transcribed region with a size of 6 × 103 base pairs plus non-transcribed central and distal spacers. In the present study the chromatin structure of the transcribed region and the terminal spacer have been compared. Micrococcal nuclease and DNase I were used to investigate the nucleosomal and the higher order structures. The specific DNA fragments were visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets and hybridization with specific 32P-labelled RNA probes. Investigations of the cleavage patterns demonstrate the presence of a defined nucleosomal structure in the non-transcribed region, while there is no indication of a nucleosomal pattern in the transcribed region. Specific regions on the r-chromatin are hypersensitive to DNase I. The first cleavage occurs in the non-transcribed central spacer region, while the second cleavage takes place in a region near the 3′ end. The hypersensitivity of the central part of r-chromatin is also found by autodigestion in isolated nucleoli.

Nucleolar silver stained granules in rat Yoshida sarcoma cells after RNA synthesis inhibition.

Abstract: The number of nucleolar silver stained granules representing nucleolus organizer regions in interphase nuclei was studied in Yoshida ascitic sarcoma cells without and after the inhibition of the nucleolar RNA synthesis with actinomycin D or cyclophosphamide. The results demonstrated that the number of nucleolar silver stained granules decreased after the inhibition of the nucleolar RNA synthesis with these drugs disregarding the mode of their action. In addition, the decrease of nucleolar silver stained granules in number produced by actinomycin D was dose dependent. Similarly, the number of nucleoli without silver stained granules increased depending on the dose of the administered actinomycin D.

Differential conjugation of polyamines to calf nuclear and nucleolar proteins.

Abstract: Nuclei and nucleoli isolated from calf liver contain acid-precipitable putrescine, spermidine and spermine conjugates. The polyamines are released upon peptide bond hydrolysis. All of the nuclear putrescine conjugate and a major portion of the polyamine conjugates are localized within the nucleolus. Nuclei and nucleoli also contain, in proportions consistent with the nucleolar/nuclear protein ratio, the putative conjugating enzyme, transglutaminase, as well as amine acceptor substrates to which radiolabeled putrescine can be conjugated by endogenous enzyme. Extraction of the isolated organelles with saline solutions of increasing ionic strength showed a differential distribution of the polyamine derivatives: all the covalently linked putrescine was associated with the less soluble components of the chromatin residue, while the spermidine and spermine conjugates were associated with several salt-extractable protein fractions as well as tightly bound to the chromatin pellet. Mono-gamma-glutamyl putrescine was detected after proteolytic digestion of the 600 mM NaCl fraction, further suggesting the enzymatic action of transglutaminase(s) in the conjugation process.

The behaviour of silver-positive structures during meiotic prophase of male mice

Abstract: Air dried preparations and sections of mouse seminiferous tubules were stained with an ammoniacal silver solution to study the behaviour of silver positive structures in meiotic prophase I nuclei. The presence of RNA was investigated using specific staining techniques and RNase digestion. Pachytene nuclei showed silver precipitation at the paracentromeric region of two to six autosomal bivalents. In spermatogonia at least the chromosome nos. 12, 15, 16, 17 and 18 proved to have transcriptionally active nucleolus organizer regions. From late pachytene the Ag-positive structures migrate towards the sex vesicle. In prediffuse diplotene, when the silver spots reached the sex vesicle, a vacuole-like body appeared near the sex vesicle. At the same time significant amounts of RNA accumulate near to the sex vesicle. Finally, in diffuse diplotene a tripartite structure could be observed, composed of (a) a horseshoe-shaped structure adjacent to the sex vesicle, which contains a great deal of RNA, (b) a vacuole-like body, being enclosed by the horseshoe and (c) an Ag-positive mass, migrated from the nucleolus organizer regions. It is probable that the tripartite structure, or at least a part of it, is a large nucleolus. The significance of the structure is discussed.