Showing papers on "Nucleolus published in 1978"

Chapter 27. Transcription of RNA in Isolated Nuclei

Abstract: Publisher Summary This chapter describes a system that fulfills many of the requirements for the study of gene control in vitro. The system makes possible the study of the primary transcription event separate from later maturation and transport events in RNA synthesis. While the results in the chapter are described for myeloma cells, lines derived from the MPC-11 tumor, the preliminary results with chick embryos, and those of others with HeLa cells and other myeloma cells suggest that these methods may be generally applicable. There are many examples of systems of isolated nuclei where the RNA product is small. Moreover, the isolated nuclei synthesize a very small product, but the nuclear subfraction they isolate (nucleoli) synthesizes very large RNA. This chapter illustrates that crude homogenates and crude nuclei have minimal RNA synthetic activity but activity becomes evident on further purification. Thus, in each system careful evaluation of conditions for optimal activity is required and they may well vary extensively.

Structure-activity relationships of anthracyclines relative to effects on macromolecular syntheses.

Abstract: The anthracyclines studied may be divided into two classes on the basis of their effects on DNA and RNA syntheses. Class I anthracyclines-adriamycin, carminomycin, and pyrromycin-inhibit DNA, whole cell RNA, and nucleolar RNA syntheses at approximately comparable concentrations. Class II anthracyclines-aclacinomycin, marcellomycin, and musettamycin-inhibit whole cellular RNA synthesis at 6-7-fold lower concentrations than those required to inhibit DNA synthesis, and nucleolar RNA synthesis at 170-1250-fold lower concentrations than necessary to inhibit DNA synthesis. Structure-activity relationship studies suggest that the presence of di- or trisaccharides confers nucleolar RNA synthesis-inhibitory specificity on anthracyclines. None of the anthracyclines studied had demonstrable effects on processing of preribosomal RNA.

Patterns of rna synthesis in early meiotic prophase oocytes from fetal mouse ovaries.

Abstract: In a study of the early meiotic prophase stages of mouse oogenesis from d12 of gestation to 10d post-partum the patterns of RNA synthesis during these stages of oogenesis using H3-uridine incorporation as visualized by light microscope autoradiography are reported. We find that chromosomal RNA synthesis occurs in all stages except early to mid-pachytene, the time of maximum chromosome condensation. Diplotene and dictyate nuclei are the most heavily labelled stages. Nucleolar labelling ceases before leptotene and reappears in late pachytene or early diplotene, even though nucleoli can be identified in all stages except early to mid-pachytene.

The Ultrastructure of Immature Sertoli Cells. Maturation-Like Changes During Culture and the Maintenance of Mitotic Potentiality

Abstract: Cell aggregates dissociated from 10-day-old rat testes form epithelial colonies in culture. These epithelial cells have been identified as supporting cells (immature Sertoli cells) by the close similarity between the nuclear and cytoplasmic structures of the cultured cells and those of supporting cells in vivo. The cultured supporting cells undergo maturation-like changes in serum-enriched media containing follicle stimulating hormone (FSH). These changes include a change in the pattern of chromatin condensation, the development of large nucleoli and nuclear infoldings, a progressive predominance of the smooth over the rough endoplasmic reticulum and the appearance of luminal (apical) differentiations. The differentiations of the luminal surface consist of finger-like cytoplasmic extensions, a regional accumulation of microfilaments and microtubules and the junctional complexes between neighboring cells near the lumen. In the presence of FSH there is a low but sustained mitotic activity in the population of cultured supporting cells. This FSH-dependent mitotic activity was observed from Day 4-15 in culture. It is suggested that while several structural differentiations of the supporting cells develop under these conditions in Vitro, the luminal region of these cells does not differentiate in the same way as the apical region of Sertoli cells in vivo.

Large scale isolation of nuclei and nucleoli from vitellogenic oocytes of Xenopus laevis.

Abstract: A new method is described which allows the purification of large quantities of nuclei (germinal vesicles) from Xenopus laevis vitellogenic oocytes of different developmental stages. From the isolated nuclei, purified nucleoli were obtained. The isolated germinal vesicles are transcriptionally active showing endogenous RNA polymerase activity as well as a high level of activity with an exogenously added template.

Ultrastructure and activity of the nucleolar organizer in the mouse oocyte during meiotic prophase

Abstract: The mouse oocyte is the site of nucleolar synthesis during pachytene. The chromosomes containing a nucleolar organizer are attached to the nuclear envelope by their paracentromeric heterochromatin, either alone or by taking part in the formation of a chromocentre. The nucleolus appears at the junction of the paracentromeric heterochromatin with the euchromatic portion of the bivalent. In this zone, 5.0-nm-diameter fibres, thinner than those of the rest of the chromosome (10.0 nm), extend from the lateral element of the synaptonemal complex up to the nucleolar fibrillar centre in which they penetrate. At the onset of its synthesis, the nucleolus only contains the fibrillar centre and an electron-dense fibrillar component in continuity with the latter. Growth of the nucleolus often takes place in the form of a strand whose proximal end, in contact with the fibrillar centre, is formed by preribosomal fibrils and whose distal end is at first fibrillo-granular then granular. Following brief incorporation of tritiated uridine, nucleolar labelling is active in oogonia. No ribosomal RNA-synthetic activity is revealed during leptotene and zygotene. Incorporation resumes at mid-pachytene, with labelling located over the electron-dense fibrillar component adjacent to the fibrillar centre. These observations suggest that the rDNA is located in both the fibrillar centre and its associated electron-dense fibrillar component and that the rDNA transcription occurs in the latter.

Quantitative analysis of rat liver nucleolar and nucleoplasmic ribosomal ribonucleic acids.

Abstract: rRNA from detergent-purified nuclei was fractionated quantitatively, by two independent methods, into nucleolar and nucleoplasmic RNA fractions. The two RNA fractions were analysed by urea/agar-gel electrophoresis and the amount of pre-rRNA (precursor of rRNA) and rRNA components was determined. The rRNA constitutes 35% of total nuclear RNA, of which two-thirds are in nucleolar RNA and one-third in nucleoplasmic RNA. The identified pre-rRNA components (45 S, 41 S, 39 S, 36 S, 32 S and 21 S) are confined to the nucleolus and constitute about 70% of its rRNA. The remaining 30% are represented by 28 S and 18 S rRNA, in a molar ratio of 1.4. The bulk of rRNA in nucleoplasmic RNA is represented by 28 S and 18 S rRNA in a molar ratio close to 1.0. Part of the mature rRNA species in nucleoplasmic RNA originate from ribosomes attached to the outer nuclear membrane, which resist detergent treatment. The absolute amount of nuclear pre-rRNA and rRNA components was evaluated. The amount of 32 S and 21 S pre-rRNA (2.9 x 10(4) and 2.5 x 10(4) molecules per nucleus respectively) is 2-3-fold higher than that of 45 S, 41 S and 36 S pre-rRNA.

Differences in nucleolar antigens of rat liver and Novikoff hepatoma ascites cells.

Abstract: Antisera to nucleoli of Novikoff hepatoma ascites and normal rat liver cells were produced in rabbits by injection of whole, isolated nucleoli. These antisera have been used to compare the nucleolar antigens that were partially fractionated by differential solubilization from nucleoli. Fourteen antigens were detected by these antisera; ten of these antigens were detected by both antisera. Ouchterlony double diffusion analysis of soluble extracts from normal rat liver and Novikoff hepatoma ascites nucleoli and fetal rat liver nuclei provided evidence for antigens found only in liver extracts, only in tumor extracts, or only in tumor and fetal extracts. Antisera preabsorbed to remove antibodies to common antigens of liver and tumor provided confirmatory evidence for one nucleolar antigen in liver that was not found in tumor or fetal rat liver, one antigen in tumor that was not found in adult or fetal rat liver, and three antigens in both tumor and fetal rat liver that were not found in adult rat liver. In addition, the antitumor nucleolar antiserum preabsorbed with liver nuclear extracts still produced positive nucleolar fluorescence in Novikoff hepatoma ascites cells but not in liver cells. Conversely, anti-liver nucleolar antiserum preabsorbed with tumor nucleolar extracts did not produce detectable tumor nucleolar fluorescence but did produce positive fluorescence in liver nucleoli.

Isolation and purification of nucleoli and nucleolar chromatin from mammalian cells.

Abstract: Publisher Summary This chapter describes the methods for isolation of nucleoli from various cell types and for purification of chromatin from isolated nucleoli. The nucleolus is known to be the site of ribosomal RNA (rRNA) synthesis and ribosome assembly. Association of histones and nonhistone proteins with the nucleolar DNA provides an opportunity to study the regulatory function of these proteins in a DNA-protein complex, designated as chromatin that is actively synthesizing one species of RNA. The chapter focuses on the intactness of the macromolecular structure of the nucleolus to retain in vivo function in isolated nucleoli. Isolation of nucleoli consists of three steps: (1) isolation of nuclei from whole homogenate, (2) disruption of nuclei, and (3) purification of nucleoli from the disintegrated nuclear material. Combined with advanced procedures for purification of eukaryotic RNA polymerases from various sources and with the improved techniques of analysis of histone and nonhistone proteins, isolated nucleoli and nucleolar chromatin constitute a good system for the study of gene regulation in eukaryotic cells.

Comparative ultrastructural studies of nucleoli of tumor cells treated with adriamycin and the newer anthracyclines, carminomycin and marcellomycin.

Abstract: This study was designed to determine the effects of several antitimor anthracyclines, including Adriamycin and its analogs, carminomycin and marcellomycin, on the ultrastructure of nucleoli of Novikoff hepatoma cells. Adriamycin and carminomycin, which are structurally related, induce nucleolar segregation following the formation of conspicuous fibrillar centers. Marcellomycin did not induce formation of nucleolar fibrillar centers. Instead, numerous microspherules formed following treatment with marcellomycin; later complete nucleolar segregation developed. The microspherules were observed to be in various stages of extrusion from the nucleolar body. This microspherule "migration" appeared to be both time and drug concentration dependent. These results show that the rate and extent of nucleolar ultrastructural aberration may be related to structural differences of the various anthracyclines.

Comparison of silver staining of nucleolus organizer regions in human lymphocytes and fibroblasts.

Abstract: Ag-staining of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q-banding in repeated lymphocyte and skin fibroblast cultures from three different individuals. A similar pattern of Ag-stainability of NORs was found in the two tissues in each individual. Small differences concerning, in each case, only one of the acrocentric chromosomes were found between repeated lymphocyte cultures, as well as between lymphocyte and fibroblast cultures of the same individual without indication of any prevalence of one tissue type in a certain direction. The possibility that these differences are caused by different stages of NOR activation is discussed.

Processing and migration of ribosomal ribonculeic acids in the nucleolus and nucleoplasm of rat liver nuclei

Abstract: Kinetic studies on the labelling in vivo with [14C]orotate of rat liver nucleolar and nucleoplasmic pre-rRNA (precursor of rRNA) and rRNA, isolated from detergent-purified nuclei, were carried out The mathematical methods used for the computer analysis of specific-radioactivity curves are described Evaluation of the experimental data permitted the selection of the most probable models for the processing of pre-rRNA and the nucleo-cytoplasmic transfer of rRNA It was shown that considerable flexibility exists in the sequence of endonuclease attacks at critical sites of 45 and 41 S pre-rRNA chains, resulting in the simultaneous occurrence of several processing pathways However, the phosphodiester bonds involved in the formation of mature 28 and 18 S rRNA appear to be protected until the generation of their immediate pre-rRNA The turnover rates and half-lives of all pre-rRNA and rRNA pools were determined The turnover rate of 45 S pre-rRNA corresponds to the formation of 1100 ribosomes/min per nucleus The model for the nucleolus-nucleoplasm-cytoplasm migration of rRNA includes a 'nucleoplasm' compartment in which the small ribosomal subparticle is in rapid equilibrium with the respective cytoplasmic pool At equimolar amounts of nuclear 28 and 18 S rRNA this model explains the faster appearance of labelled small ribosomal subparticles in the cytoplasm simultaneous with a lower labelling of nuclear 18 S rRNA as compared with 28 S rRNA

Characterization of proteins from nucleolar preribosomes of mouse leukemia cells by two-dimensional polyacrylamide gel electrophoresis.

Abstract: Proteins of isolated 80-S and 60-S nucleolar preribosomal particles were characterized by means of two-dimensional polyacrylamide gel electrophoresis, in the lymphocytic mouse leukemia cells L5178Y. Their identification and metabolic relationships with ribosomal subunit proteins were investigated using co-electrophoresis of unlabeled polysomal proteins with labeled proteins of either nucleolar preribosomes or ribosomal subunits. The large and small ribosomal subunits contain 40 and 31 proteins, respectively. The nucleolar 80-S preribosomes were analysed after 2 and 5 h of incubation with tritiated valine and leucine and were shown to contain about 55 proteins. Most of them were identical to cytoplasmic ribosomal subunit proteins. The nucleolar 60-S preribosomes contain all the proteins which are common to 80-S preribosomes and large ribosomal subunits, and one additional protein (L10). The ribosomal proteins which were absent from nucleolar particles were found to be labeled in the cytoplasmic ribosomes after the same incubation period. Thus, in addition to the association of the bulk of ribosomal proteins with 45-S RNA within the 80-S preribosomes, results indicate that a group of ribosomal proteins and particularly from the small subunits, become associated at later stages of the maturation process of mammalian ribosomes. It was further shown that a set of 10 proteins, different from ribosomal polypeptides, were present in nucleolar preribosomal particles. Several of them were associated with polyribosomes in the cytoplasm, whereas the others were unique to the nucleolus.

The organization, composition and matrix of hepatocyte nuclei exposed to alpha-amanitin.

Abstract: Alterations in the structure and molecular composition of avian hepatocyte nuclei were compared following administration in vivo of lethal and sub-lethal doses of α-amanitin. This toxin interferes with extranucleolar transcription by direct inhibition of RNA polymerase II activity. The resultant effects include: extensive condensation of chromatin, displacement of nucleoplasmic contents and fragmentation of nucleoli. Changes in nuclear morphology were quantitated by stereometry and related to variations in RNA and residual, non-histone proteins (NHP). Gross alterations in nuclear structure and depletion of RNA and NHP levels were of similar magnitude with both doses of amanitin. The effects were fully reversible, however, with a minimal dose but terminal with a lethal dose. DNA and histone protein levels remained unchanged at all stages.

Localization of simian adenovirus 7 (SA 7) transcription and replication in lytic infection. An ultracytochemical and autoradiographical study

Abstract: We have studied SA7 (simian adenovirus 7) lytic infection at the ultrastruct level. The use of cytochemical techniques which specifically stain DNA or preferentially stain ribonucleoproteins permitted the analysis of the structure of the virus-induced nuclear inclusions, and revealed presumed virus DNA before the appearance of other nuclear alterations. Correlation of these findings with high resolution autoradiography enabled the functions of virus DNA replication and transcription to be ascribed to a defined nuclear inclusion. We demonstrate that the nucleolus remains distinct from the inclusion body, contrary to the situation in other adenovirus-infected cells. The functional role of host cell chromatin and of the nucleolus are discussed.

Morphology of transcriptional units at different states of activity

Abstract: The morphology of two forms of transcriptionally active chromatin, the nucleoli and the loops of lampbrush chromosomes, has been examined after fixation in situ or after isolation and dispersion of the material in media of low ionic strengths, using a variety of electron microscopic preparation techniques (e.g. spread preparations with positive or negative staining or without any staining at all, with bright and dark field illumination, with autoradiography, after pretreatment of the chromatin with specific detergents such as Sarkosyl NL-30; transmission and scanning transmission electron microscopy of ultrathin sections). Nucleolar chromatin and chromosomes from oocytes of various amphibia and insects as well as from green algae of the family of the Dasycladaceae were studied in particular detail. The morphology of transcriptional units that are densely packed with lateral ribonucleoprotein fibrils, indicative of great transcriptional activity, was compared with that of chromatin of reduced lateral fibril density, including stages of drug-induced inhibition. The micrographs showed that under conditions which preserve the nucleosomal organization in condensed chromatin studied in parallel, nucleosomes are not recognized in transcriptionally active chromatin. This holds for the transcribed regions as well as for apparently untranscribed (i.e. fibril-free) regions interspersed between (‘spacer’) and/or adjacent to transcribed genes and for the fibril-free regions within transcriptional units of reduced fibril density. In addition, comparison of lengths of repeating units of isolated rDNA with those observed in spread nucleolar chromatin indicated that this DNA is not foreshortened and packed into nucleosomal structures. Granular particles which were observed, at irregular frequencies and in variable patterns, in some spacer regions, did not result in a proportional shortening of the spacer axis, and were found to be resistant to detergent treatment effective in removing most of the chromatin associated proteins including histones. Thus, these particles behave like RNA polymerases rather than nucleosomes. It is suggested that structural changes from nucleosomal packing to an extended form of DNA are involved in the transcriptional activation of chromatin.

Transcriptional properties of nucleoli isolated from Tetrahymena

Abstract: Nucleoli can be isolated from Tetrahymena in a yield of 30-60%. The isolated nucleoli contain rDNA (at least 90% pure) and have a protein to DNA ratio of 30:1. The endogenous RNA-polymerase activity of the r-chromatin has the following properties: (i) The in vitro transcript has a maximal size identical to the in vivo 35S rRNA precursor, demonstrating correct termination on the gene, (ii) 79% of the in vitro transcript is complementary to cDNA of 17S and 25S rRNA which is close to the theoretical maximum for the 35S rRNA precursor, (iii) the elongation rate of the endogenous RNA-polymerase molecules is 9-12 nucleotides/sec, (iv) an average of 4-16 active RNA polymerases are associated with each rDNA molecule depending upon the preparation.

Nucleolar size in parallel with ribosomal RNA synthesis at diapause termination in the eggs of Bombyx mori

Abstract: The eggs of Bombyx mori, both in diapause and nondiapause, were subjected to cytological examination of nucleoli and measurement of RNA precursor incorporation (2 hours) into ribosomal RNA. In diapause eggs, the nucleoli were very small and the rate of ribosomal RNA synthesis was the lowest of the samples tested. Most cells in diapause possessed nuclei with one nucleolus. In contrast, the eggs activated from diapause by long chilling attained the largest size of nucleoli and the highest rate of ribosomal RNA synthesis. A significant proportion of the cell nuclei still had only one nucleolus at this stage. Three days after activation, the eggs exhibited intermediate levels in both the size of nucleoli and the rate of ribosomal RNA sythesis. At this stage, about half of the egg cell nuclei had two nucleoli.

Chromosome location of the ribosomal genes in Triturus vulgaris meridionalis (Amphibia Urodela). III. Inheritance of the chromosomal sites for 18S + 28S ribosomal RNA.

Abstract: In Triturus vulgaris meridionalis, the 18S + 28S rDNA sequences have been shown to be located in a number of additional chromosomal sites besides the nucleolus organizing region. The additional ribosomal sites have been found to vary as to their number and chromosomal location in different individuals of the species.—The data presented in this study concern the chromosomal distribution of the ribosomal sequences as analyzed by in situ hybridization technique in two individuals as well as in their offspring. The evidence obtained by this analysis indicates quite clearly that all 18S + 28S rRNA sites present in each individual genome are inherited according to simple mendelian principles.

RNA synthesis by nuclei and nucleoli from ischemic liver cells.

Abstract: Nuclei and nucleoli were isolated from rat livers subjected to an interruption of the blood supply for periods of different duration, as well as after restoration of the blood supply. They were assayed for RNA synthesis under conditions of diverse ionic strengths, and in the presence of an exogenous template, such as poly d (A-T), and actinomycin to inactivate the endogenous template; alpha-amanitin was made used of to distinguish polymerase I and polymerase II dependent RNA synthesis. Nuclei and nucleoli from ischemic livers showed a severe impairment of RNA synthesis, which is likely to be due to decreased initiation frequency of the engaged polymerases, while free polymerases were essentially unchanged. Both form I and II polymerase were equally involved. After restoration of the blood supply RNA synthesis recovered with an overshooting well above normal levels of activity, lasting for at least 24 hours. Increased RNA synthesis was not followed by thymidine incorporation into DNA.