Showing papers on "Nucleolus published in 1988"

Reproducible compartmentalization of individual chromosome domains in human CNS cells revealed by in situ hybridization and three-dimensional reconstruction.

Abstract: Specific chromosome domains in interphase nuclei of neurons and glia were studied by three-dimensional (3-D) reconstruction of serial optical sections from in situ hybridized human CNS tissue. Overall patterns of centromere organization, delineated with alphoid repeats, were comparable to those seen in mouse, and are clearly conserved in mammalian evolution. Cloned probes from other individual chromosome domains were used to define interphase organization more precisely. Homologous chromosomes were spatially separated in nuclei. In large neurons, probes specific for 9q12, or 1q12 showed that at least one homolog was always compartmentalized together with centromeres on the nucleolus, while the second signal either abutted the nucleolus or was on the nuclear membrane. A telomeric Yq12 sequence also localized together with perinucleolar centromeres in a completely non-Rabl orientation. In astrocytes, these three chromosome regions were on the membrane and not necessarily associated with nucleoli. Therefore there are different patterns of interphase chromosome organization in functionally distinct cell types. In contrast to the above domains, a 1p36.3 telomeric sequence embedded in a large Alu-rich and early replicating chromosome region, was always found in an interior euchromatic nuclear compartment in both neurons and glial cells. In double hybridizations with 1q12 and 1p36.3 probes, 1p arms were clearly separated in all cells, and arms projected radially into the interior nucleoplasm with non-Rabl orientations. There was no absolute or rigid position for each 1p arm with respect to each other or to the major dendrite, indicating that individual chromosome arms may be dynamically positioned even in highly differentiated cell types. We suggest that centromeric and other highly repeated non-transcribed sequence domains may act as general organizing centers for cell type specific interphase patterns that are conserved in mammalian evolution. Such centers would allow selected groups of chromosome arms to extend into (and contract from) an interior, presumably transcriptionally active, nuclear compartment.

Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein.

Abstract: We have produced monoclonal antibodies against purified nuclei from the yeast Saccharomyces cerevisiae and have characterized three different antibodies that recognize a protein with an apparent molecular weight of 38,000, termed p38. Subcellular fractionation shows that virtually all of p38 occurs in the nuclear fraction. High concentrations of salt (1 M) or urea (6 M) effectively solubilize p38 from a nuclear envelope fraction prepared by digestion of nuclei with DNase. Indirect immunofluorescence demonstrates a crescent shaped distribution of p38 at the inner periphery of the nucleus, with p38 extending between dividing pairs of cells during (closed) mitosis. Postembedding immunogold electron microscopy shows decoration of the densely stained "crescent" region of the yeast nucleus, confirming the localization of p38 to the nucleolus. One of the monoclonals, D77, cross reacts on immunoblots with a single protein of molecular weight 37,000 from purified rat liver nuclei. Indirect immunofluorescence localizes this protein to the nucleolus, and shows that it is dispersed throughout the cell during mitosis. The yeast and rat liver nucleolar proteins behave similarly when electrophoresed in two dimensions, and appear to have basic pI values. Analysis of immunological cross-reactivity using D77, and antibodies specific for nucleolar proteins from other sources, suggests that the rat liver protein is fibrillarin, and demonstrates that p38 shares epitopes with fibrillarin, as well as with other vertebrate nucleolar proteins.

Characterization of the thermotolerant cell. II. Effects on the intracellular distribution of heat-shock protein 70, intermediate filaments, and small nuclear ribonucleoprotein complexes.

Abstract: Here we further characterize a number of properties inherent to the thermotolerant cell. In the preceding paper, we showed that the acquisition of the thermotolerant state (by a prior induction of the heat-shock proteins) renders cells translationally tolerant to a subsequent severe heat-shock treatment and thereby results in faster kinetics of both the synthesis and subsequent repression of the stress proteins. Because of the apparent integral role of the 70-kD stress proteins in the acquisition of tolerance, we compared the intracellular distribution of these proteins in both tolerant and nontolerant cells before and after a severe 45 degrees C/30-min shock. In both HeLa and rat embryo fibroblasts, the synthesis and migration of the major stress-induced 72-kD protein into the nucleolus and its subsequent exit was markedly faster in the tolerant cells as compared with the nontolerant cells. Migration of preexisting 72-kD into the nucleolus was shown to be dependent upon heat-shock treatment and independent of active heat-shock protein synthesis. Using both microinjection and immunological techniques, we observed that the constitutive and abundant 73-kD stress protein similarly showed a redistribution from the cytoplasm and nucleus into the nucleolus as a function of heat-shock treatment. We show also that other lesions that occur in cells after heat shock can be prevented or at least minimized if the cells are first made tolerant. Specifically, the heat-induced collapse of the intermediate filament cytoskeleton did not occur in cells rendered thermotolerant. Similarly, the disruption of intranuclear staining patterns of the small nuclear ribonucleoprotein complexes after heat-shock treatment was less apparent in tolerant cells exposed to a subsequent heat-shock treatment.

Evolutionary diversity of reverse (R) fluorescent chromosome bands in vertebrates

Abstract: Mitotic chromosomes, interphase cell nuclei, and male meiosis of 41 species representing all vertebrate classes were analyzed with distamycin A/mithramycin counterstaining. The purpose of the study was to recognize differences and common characteristics in the reverse (R) fluorescent banding patterns in the chromosomes of vertebrate species at various stages of evolution. In contrast to the warm-blooded mammals and birds, the euchromatic segments in the chromosomes of most reptiles, amphibians, and fishes contain no multiple fluorescent R-bands. This is thought to be due to the absence of the long homogeneous regions (isochores) in the DNA of the cold-blooded vertebrates. Distamycin A/mithramycin banding specifically reveals the GC-rich constitutive heterochromatin in all vertebrates. In most of the vertebrate chromosomes examined, the heterochromatic regions have opposite staining properties with mithramycin and quinacrine. Mithramycin labels the nucleolus organizer regions very brightly in the karyotypes of fishes, amphibians, reptiles and birds, but not of mammals. The lack of mithramycin fluorescence at the nucleolus organizer regions of mammals is attributed to the relatively low level of redundancy of the GC-rich ribosomal DNA in their genomes. Studies on the various meiotic stages of the cold-blooded vertebrates show that the mithramycin labeling of the nucleolus organizers is independent of their state of activity. This can be confirmed by mithramycin fluorescence at the nucleoli of actinomycintreated cells.

A Drosophila rRNA gene located in euchromatin is active in transcription and nucleolus formation.

Abstract: P-element transformants of a single rRNA gene (rDNA) were used to investigate the relationship between the organization of the nucleolus organizer (NO) and rDNA function in Drosophila melanogaster. In situ hybridization to rRNA in polytene nuclei of salivary glands demonstrated that an rRNA gene can be transcribed at a high rate when inserted into chromosomal sites other than the NO. Structures that resemble morphologically the endogenous nucleoli ('mininucleoli') were associated with four different euchromatic sites of rDNA insertion. Molecular analyses revealed that these mininucleoli contained both rRNA and an antigen specific to nucleoli. Phenotypes resulting from rDNA deficiencies were rescued partially by the presence of the transformed rDNA, indicating that the transcripts and mininucleoli associated with the rDNA insertion sites were functional. Thus, two conserved features of rDNA organization in eukaryotes, namely tandem repetition and heterochromatic localization, are not required for rRNA gene function. We conclude that 'nucleolar organizing activity' is an intrinsic property of the rDNA or its RNA products.

A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells.

Abstract: A novel 5S RNA-protein (RNP) complex in human and mouse cells has been analyzed using patient autoantibodies. The RNP is small (approximately 7S) and contains most of the nonribosome-associated 5S RNA molecules in HeLa cells. The 5S RNA in the particle is matured at its 3' end, consistent with the results of in vivo pulse-chase experiments which indicate that this RNP represents a later step in 5S biogenesis than a previously described 5S*/La protein complex. The protein moiety of the 5S RNP has been identified as ribosomal protein L5, which is known to be released from ribosomes in a complex with 5S after various treatments of the 60S subunit. Indirect immunofluorescence indicates that the L5/5S complex is concentrated in the nucleolus. L5 may therefore play a role in delivering 5S rRNA to the nucleolus for assembly into ribosomes.

Nucleolus organizer regions and nucleoli in preattachment bovine embryos

Abstract: Embryos (1-cell to elongated blastocyst stage) were recovered from superovulated heifers at surgery (Days 2-4; oestrus = Day 0), after slaughter (Day 4), or by transcervical flushing (Days 6, 7 and 14). The 175 embryos were cultured for 4, 8, 24 or 48 h, fixed on slides and sequentially stained with Giemsa and silver nitrate. Twenty-three 2-cell to blastocyst-stage embryos were fixed, embedded and examined by transmission electron microscopy. Argentophilic nucleolus organizer regions (Ag-NORs), indicative of transcriptionally active rRNA genes, were observed in embryos in which short- or long-term culture began at or after the late 8-cell stage. The nucleoli of embryonic cells also showed increased affinity for silver from the 8-cell stage onward. Differences in the number of Ag-NORs observed after the 8-cell stage reached statistical significance only when Day-5 and Day-7 embryos cultured for 4 h were compared. Ultrastructurally, the nucleoli were seen to develop from small, dense, fibrillar masses at the 2-cell stage, to ring-shaped structures (signifying a low level of activity) at the 8-cell stage. At the 16-cell stage the nucleoli became reticulated, suggesting an increase in activity, and by the morula and blastocyst stages they were characteristic of fully active nucleoli. It is concluded that a significant transcriptional activity of the rRNA genes in the embryos of cattle begins around the 8-cell stage.

Remodeling of mouse thymocyte nuclei depends on the time of their transfer into activated, homologous oocytes

Abstract: The potential of parthenogenetically activated mouse oocytes to remodel somatic cell nuclei was studied by ultrastructural means using oocyte-thymocyte hybrids. Complete nuclear remodeling, initiated by nuclear envelope breakdown and chromosome condensation (which is followed by formation of pronucleus-like nucleus) is possible only during a short time gap between metaphase II and telophase of meiotic division. Maturation-promoting factor activity is high during this period. The thymocyte nucleus can follow the sequence of morphological changes only in concert with the development of the native nucleus and only after exposure of the chromatin to the ooplasm. If hybridization is effected with pronucleate oocytes, the thymocyte nucleus retains its interphase character but shows particular modifications in nucleolar morphology (identical to changes observed during reactivation of the nucleolus in stimulated lymphocyte) and in the activity of the nuclear envelope (blebbing). Thus the nucleus not exposed to maturation-promoting factor activity may be influenced by a 'programme' specific for oocyte (blebbing) and by a programme inherent in the introduced somatic cell nucleus.

Identification and Characterization of a Human Proliferation-associated Nucleolar Antigen with a Molecular Weight of 120,000 Expressed in Early G1 Phase

Abstract: Tumor nucleoli were treated with polyclonal antisera to normal human tissue nucleoli to block some determinants common to tumor and normal tissue nucleoli. Immunization of mice with these immune complexes resulted in the development of a monoclonal antibody (FB2) to a novel Mr 120,000 nucleolar proliferation-associated antigen. By indirect immunofluorescence, antibody FB2 produced bright nucleolar staining in a variety of malignant tumors, including cancers of the breast, liver, gastrointestinal tract, genitourinary tract, blood, lymph system, lung, and brain. Although specific nucleolar immunofluorescence was not detectable in most normal tissues, it was detectable in some proliferating nonmalignant tissues including spermatogonia of the testes, ductal regions of hypertrophied prostates, and phytohemagglutinin-stimulated lymphocytes. The Mr 120,000 antigen was not detectable in 48-h serum-deprived HeLa cells but was readily detectable (within 30 min) following serum refeeding. The Mr 120,000 antigen was not detected in retinoic acid-treated HL-60 cells following morphological differentiation but was detectable in 48-h phytohemagglutinin-treated lymphocytes. These studies suggest that the Mr 120,000 antigen is a proliferation-associated antigen which plays a role in the early G1 phase of the cell cycle.

Observations on the number, size and localization of nucleoli in hyperplastic and neoplastic prostatic disease.

Abstract: Specimens of prostate obtained for histopathological and cytological examination by urethral resection and 'punch' biopsies showing carcinoma, atypical and typical hyperplasia were re-examined with regard to the number, size and localization of nucleoli. With increasing degrees of malignancy the number and size of the nucleoli increased and the position of the nucleolus shifted to the periphery of the nucleus. In the cells in typical and slightly atypical prostatic hyperplasia there were few nucleoli which were localized to the centre of the nucleus. In moderately atypical hyperplasia there were intermediate locations, while in severe atypical hyperplasia and 'borderline' malignant cases there was a distinct shift towards the periphery of the nucleus as well as an increase in the number of nucleoli in each nucleus. It is suggested that careful analysis of the number and localization of nucleoli can be helpful in the analysis of the differential diagnosis between carcinoma and atypical hyperplasia of the prostate, which can be difficult in histopathological and cytopathological material.

Intranucleolar localization of human proliferating cell nucleolar antigen p120.

Abstract: The human proliferation-associated nucleolar antigen p120 was localized to substructures within HeLa cell nucleoli by immunofluorescence and immunoelectron microscopy of cells whose nucleoli were segregated by drug treatment or extracted with nucleases. By indirect immunofluorescence, protein p120 was localized diffusely throughout all interphase nucleoli. However, high resolution immunoelectron microscopy demonstrated that protein p120 staining delineated a network of 20–30-nm diameter beaded fibrils distributed throughout the nucleolus. This distribution was unique compared to that of the nucleolar proteins p145, RNA polymerase I, or B23 which were examined simultaneously. Drug-induced segregation of nucleoli by actinomycin D or dichlorobenzimidazole riboside, followed by immunoelectron microscopy, indicated that protein p120 was concentrated at the periphery of the granular region in segregated nucleoli. In situ nuclease digestion of cells with DNase I and/or RNase A did not release p120 from the nucleolus. Instead, p120 immunoreactivity was retained within phase-dense residual nucleoli. These results provide evidence that protein p120 is associated with, and in fact delineates, a network of fibrils which is retained in the nucleolar residue fraction of proliferating cells.

Spatial Relationship between the Nucleolus and the Nuclear Envelope: Structural Aspects and Functional Significance

Abstract: Publisher Summary This chapter reviews the structural aspects and functional significance of spatial relationship between the nucleolus and the nuclear envelope. The descriptive studies of the position of the nucleolus and chromosomes provide interesting data about the relationship between the spatial organization of the nucleus and some of its activities. The position of the nucleolus in the nucleus shows that the nucleoli are located close to or are in contact with the nuclear envelope in dividing somatic animal and plant cells, as well as in gametes. So far, the only cells in which the nucleolus occupies a central position are highly differentiated and do not divide after differentiation. The position of the nucleolus is a consequence of the chromosomal organization characterizing the different cell types. Thus, in plant cells and animal gametes in the early stages of meiosis, the telomeres of the nucleolus organizing regions (NOR)-bearing chromosomes form a small knob of condensed chromatin attached to the nuclear envelope. The present development of three-dimensional computer-assisted analysis and the display of nuclear structures in the field of cell biology will lead to a better understanding of both the architecture and functions of the cell nucleus along with its relationships with the cytoplasm.

Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells

Abstract: After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtK2) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed. These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent “nucleolar segregation”. The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase.

A major 62-kD intranuclear matrix polypeptide is a component of metaphase chromosomes.

Abstract: We have isolated and partially characterized a major intranuclear matrix polypeptide from rat liver. This polypeptide, which is reversibly stabilized into the intranuclear matrix under conditions which promote intermolecular disulfide bond formation, has a Mr of 62,000 and pI of 6.8-7.2 as determined by two-dimensional IEF/SDS-PAGE. A chicken polyclonal antiserum was raised against the polypeptide purified from two-dimensional polyacrylamide gels. Affinity-purified anti-62-kD IgG was prepared and used to immunolocalize this polypeptide in rat liver tissue hepatocytes. In interphase hepatocytes the 62-kD antigen is localized in small, discrete patches within the nucleus consistent with the distribution of chromatin. The staining is most prominent at the nuclear periphery and somewhat less dense in the nuclear interior. Nucleoli and cytoplasm are devoid of staining. During mitosis the 62-kD antigen localizes to the condensed chromosomes with no apparent staining of cytoplasmic areas. The chromosomal staining during mitosis is uniform with no suggestion of the patching seen in interphase nuclei. Fractionation and immunoblotting studies using rat hepatoma tissue culture cells blocked in metaphase with colcemid confirm the chromosomal localization of this 62-kD intranuclear protein during mitosis. The 62-kD polypeptide fractionates completely with metaphase chromosome scaffolds generated by sequential treatment of isolated chromosomes with DNAse I and 1.6 M NaCl, suggesting that this major 62-kD intranuclear protein may be involved in maintaining metaphase chromosomal architecture.

Ultrastructural cytochemistry of the nucleolus in rat oocytes at the end of the folliculogenesis.

Abstract: Various ultrastructural changes occur during follicular growth in the rat oocyte nucleolus. The nucleolus, which has a reticulated fibrillogranular structure at the primordial and primary follicle stages, becomes entirely compact and is made up of a conspicuous and homogeneous mass at the antral follicle stage. In order to define the nature and the functions of this homogeneous mass, cytochemical methods allowing detection of nucleic acids, proteins and lipids were performed at the light microscopic and ultrastructural levels. The results obtained suggest that this nucleolar mass is probably composed of acid proteins which are not silver stained. This proteinaceous mass could be a special kind of nucleolar secretion providing material for meiotic resumption in the oocyte. Cytochemical researches now in progress should supply new information concerning the exact nature and the role of the nucleolar compact mass, which is the essential nucleolar component at the antral follicle stage and which really plays a role in the nucleolus in the first stages of embryogenesis.

RNA in the nucleus of a motile plant spermatozoid: characterization by enzyme-gold cytochemistry and in situ hybridization.

Abstract: To investigate the decrease of transcription and to determine the localization of RNA molecules during spermiogenesis in the fern Scolopendrium, we have used the enzyme-gold electron microscope method. During cell differentiation, the labeling decreases over the cytoplasm and increases over the nucleus. In the latter, the nucleolus disappears at the beginning of differentiation and the gold particles that were at first situated over the dispersed chromatin are then located over the condensed chromatin. In mature gametes, gold particles linked to RNA are abundant over the dense and homogeneous nucleus and sparse over the cytoplasm. By using in situ hybridization of RNA-poly(A) tails with radioactive poly(U) probes, we have shown that these RNA molecules are mRNA; they are located in the condensed chromatin of the mature sperm nucleus.

A nuclear protein associated with cell divisions in plants.

Abstract: A nuclear protein, present in carrot meristems and rapidly proliferating cultured cells of carrot (Daucus carota L.) has been identified by the use of a monoclonal antibody (MAb 21D7). By combining the techniques of two-dimensional polyacrylamide gel analysis and blotting separated proteins onto nitrocellulose sheets, it was shown that the antibody detected a single polypeptide of apparent molecular mass (Mr) of 45000 and an isoelectric focusing point (pI) of 6.7. This protein was found by subcellular fractionation and immunofluorescence to be highly concentrated in the nucleoli of somatic and zygotic embryos of a wide range of plants. It was not detectable in logarthmically growing cells ofEscherichia coli, yeast, embryos ofDrosophila melanogaster or cultured C3H mouse cells. These data indicate that this protein is a highly conserved non-histone protein associated with nuclei of rapidly dividing plant cells.