Showing papers on "Nucleolar chromatin published in 1977"

Lengths and patterns of transcriptional units in the amplified nucleoli of oocytes of Xenopus laevis

Abstract: Transcriptionally active chromatin from peripheral amplified nucleoli of lampbrush-chromosome stage oocytes of Xenopus laevis was dispersed and spread in various solutions of low salt concentrations (including some with additions of detergents) and examined by electron microscopy. Nucleolar material from oocytes of animals with normal (2-nu) and mutant (1-nu) genetical constitution of nucleolus organizers was compared. Histograms showing the distributions of the lengths of matrix units, apparent spacer intercepts, and the total repeating units of the rDNA containing chromatin axes revealed a significant degree of heterogeneity, with indications of subclasses and predominant repeat unit size classes of 3.3 and 3.8 μm length. The correspondence of matrix unit length to the molecular weight of the first stable product of rDNA transcription was studied using gel electrophoresis of labelled pre-rRNA under non-denaturing and denaturing conditions. Evaluations of individual strands of nucleolar chromatin further demonstrated the existence of both (i) strands with obviously homogeneous repeating units and (ii) strands with intra-axial heterogeneity of rDNA subunits. “Preludecomplexes”, i.e. groups of transcriptional complexes in apparent spacer intercepts, were not infrequently noted. The data are compared with the measurements of lengths of repeating units in fragments of rDNA obtained by digestion with EcoRI endonuclease as described by Morrow et al. (1974) and Wellauer et al. (1974, 1976a, b). The results are discussed in relation to problems of variations in the modes of arrangement of the pre-rRNA genes, the state of packing of rDNA during transcription, and possible mechanisms of the amplification process.

Fidelity of ribosomal ribonucleic acid synthesis by nucleoli and nucleolar chromatin.

Abstract: Isolated nucleoli, nucleolar chromatin, and nucleolar DNA were used as templates for DNA synthesis in appropriately supplemented systems in which RNA polymerases other than RNA polymerase I were blocked by alpha-amanitin. With the aid of nucleotide analysis, DNA-RNA hybridization, and homochromatography fingerprinting, it was found that isolated nucleoli and nucleolar chromatin serve primarily as templates for synthesis of rRNA. However, the products formed with purified nucleolar DNA as a template do not contain the specific rRNA oligonucleotides nor are they appreciably hybridized to the rDNA region on cesium chloride gradients. These results indicate that whole nucleoli and nucleolar chromatin contain control mechanisms that restrict readouts by RNA polymerase I of nucleolar DNA to rDNA.

Antigenic proteins of nucleolar chromatin of Novikoff hepatoma ascites cells.

Abstract: Nucleolar chromatin of Novikoff hepatoma ascites cells contains an antigen (no-Ag1) detected with antinucleolar antibodies by the immunodiffusion technique. This antigen was distinguished from the previously reported nuclear chromatin antigen NAg-1 (19) by the findings that tumor nucleolar antibodies which formed immunoprecipitin bands with no-Ag1 did not do so with NAg-1 and that tumor cytosol, which contains NAg-1, formed immunoprecipitin bands with tumor chromatin antibodies but not with antibodies to tumor nucleoli. Tumor nucleolar chromatin contains both NAg-1 and no-Ag1, but only no-Ag1 formed bands with tumor nucleolar antibodies, no-Ag1 is a component of tumor nucleolar chromatin that was not soluble in 0.075 M NaCl-0.025 M EDTA, pH 8, and only slightly soluble in 0.01 M Tris-HCl, pH 8. NO-Ag1 was not found in liver nucleoli. Antibodies to liver nucleoli formed immunoprecipitin bands with liver nucleolar antigens but none were confluent with those formed between tumor nucleolar antibodies and antigens of tumor nucleolar chromatin. Absorption of the tumor nucleolar antibodies with whole tumor cells or whole liver pressate did not alter band formation with no-Ag1. Three antigens in liver nucleoli were not found in tumor nucleoli.

Fidelity of synthesis of preribosomal RNA in isolated nucleoli and nucleolar chromatin

Abstract: Comparisons were made of the T1 ribonuclease digests of 32P-labeled nucleolar 45S RNA of intact Novikoff hepatoma cells and the RNA synthesized in vitro by isolated nucleoli. Approximately 200 oligonucleotide spots were found in the two-dimensional chromatogram of 45S nucleolar RNA labeled in vivo, which includes fragments of 18S and 28S rRNA and nonconserved spacer regions; four spots containing 2'-O-methyl nucleotides were not found in the corresponding pattern of RNA labeled in vitro. This high degree of fidelity was retained in the patterns of spots from the RNA produced with nucleolar chromatin as template. This specific expression of rDNA was lost when the nucleolar chromatin was completely deproteinized. Specific spots found in the control patterns were absent and many nonspecific oligonucleotides were found to be labeled. A similar nonspecific chromatogram pattern was found when nucleolar chromatin was transcribed with RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of Escherichia coli. These results show that specificity of genetic expression in vitro of isolated chromatin of eukaryotic systems is dependent on the chromatin-associated proteins and the type of RNA polymerase present.

Nucleolar chromatin in Chinese hamster ovary cells. Topographical distribution of ribosomal DNA sequences and isolation of ribosomal transcription complexes.

Abstract: A stepwise physico-chemical dissection of nucleoli isolated from Chinese hamster ovary cells has allowed the isolation of actively transcribed regions of chromatin, highly enriched in ribosomal genes. Nucleolar chromatin could be separated into its various topographical components. This separation should improve our knowledge of the structure/function relationships in chromatin as the fine structure of the nucleolus is very sensitive to changes in ribosomal RNA synthesis. The spatial distribution of ribosomal genes among the various chromatin areas of the nucleolus was assayed by hybridization with rRNA. In exponentially growing Chinese hamster ovary cells ribosomal genes are predominantly located in the intranucleolar stretches of nucleolus-associated chromatin. However, ribosomal sequences represent but a very minor part (less than 1 %) of intranucleolar DNA. In an attempt to improve the analysis of the actively transcribed chromatin areas, intranucleolar chromatin was submitted to additional fractionation steps involving, among others, isopycnic separation in metrizamide gradients. In these latter conditions a small subset of intranucleolar chromatin banded with preribosomal ribonucleoprotein. In this subfraction, about one-third of the DNA sequences corresponded to preribosomal RNA matrix units. The yield of recovery of this intranucleolar chromatin subfraction being closely related to the level of transcriptional activity of ribosomal genes, it appears likely that this separating procedure depends on the presence of ribosomal RNA transcription complexes and could, therefore, represent a general procedure for the isolation of actively transcribed areas from chromatin.

Ultrastrual cell cycle-specific nuclear and nucleolar changes of human leukemic lymphoblasts.

Abstract: Summary To identify cell cycle-specific ultrastructural markers in acute lymphoblastic leukemia, we labeled bone marrow lymphoblasts with [ 3 H]thymidine, separated them according to cell size by velocity sedimentation at 1 × g , and then identified the pattern of changes within the nucleus of the cells according to phase of the cell cycle. The smallest and most slowly sedimenting cells (probably in G 0 phase) had prominent, condensed nuclear chromatin and adjacent small nucleoli with segregated substructure and a distinct focus of pars amorpha, which contained pyroantimonate-reactive cation. Slightly larger cells (probably in G 1 phase) contained a more irregular nuclear profile; dispersed nuclear chromatin; and large, round, centrally located nucleoli with increased granular component, dispersed foci of pars amorpha, dense fibrillar component, and decreased nucleolus-associated chromatin. During early S phase, [ 3 H]thymidine diffusely labeled the nucleus; the nucleoli, usually within the nuclear periphery, had increased nucleolus-associated chromatin. In late S-phase cells, [ 3 H]thymidine was incorporated into the peripheral chromatin and the nucleolus, which was irregularly shaped, contained moderate amounts of granular and dense fibrillar component, and was always surrounded and often penetrated by nucleolus-associated chromatin. Large, rapidly sedimenting cells in G 2 phase contained flattened nucleoli with penetrating nucleolar chromatin and various foci of nucleolar constituents, including pars amorpha. These results demonstrate that leukemic lymphoblasts can be identified at each phase of the cell cycle on the basis of their ultrastructural morphology.

Isolation of transcriptionally active chromatin from mammalian nucleoli

Abstract: Nucleoli isolated from HeLa cells are functionally active but contain large amounts of RNA and proteins (RNA/DNA ratio 1:1; protein/DNA ratio 7:1). We have isolated from the nucleolus a DNA-protein complex that has the characteristics of nucleolar chromatin (RNA/DNA ratio less than 0.05:1; protein/DNA ratio 1.7:1). This nucleolar chromatin has most of the transcriptional activity of the intact nucleolus and, as assayed by circular dichroism and dye binding, has largely preserved its structure. The isolation of a transcriptionally active, fragment of chromatin, which constitutes only a small part of the total genome and codes for only one recognizable product, offers several advantages for the study of chromatin structure and function.

Electron‐microscope investigations on the endonuclear DNA‐RNA bodies of the midgut epithelial cells of Bacillus rossius (insecta, cheleutoptera)

Abstract: Fourth instar larvae and adults of the stick insect B. rossius develop endonuclear bodies in about 10% of the columnar epithelial cells of the midgut. E.M. sections show that the bodies start as loose bundles of fibrils, independent from the intranucleolar chromatin. Large bodies retain a similar structure but also contain larger fibers and electron dense granules, sometimes clustered in small groups. In the nuclei containing these bodies, the other components are similar to those found in ordinary nuclei, apart from the frequent reduction in size and structural changes of the nucleoli. In the cytoplasm of both ordinary cells and those containing bodies, the rough endoplasmic reticulum is strikingly developed and towards the gut lumen many secretion vesicles are found. In EDTA treated sections the endonuclear bodies show intermingled bleached and unbleached areas, the electron density of unbleached structures being increased when compared with controls and reaching the same contrast as the fibrillar-granular components of the nucleolus. In DNase treated specimens both the chromatin and the endonuclear bodies lose their contrast; RNase solutions digest the electron dense granules of the endonuclear bodies and the combined action of RNase and DNase further disintegrates the bodies. Ultrastructural and enzyme digestion data therefore indicate a DNA-RNA composition for the endonuclear bodies and support, together with 3H-thymidine uptake, the hypothesis that they are the expression of a somatic gene amplification. Their origin distinct from the nucleolar chromatin further suggests that the DNA of the bodies is not rDNA and a possible link of the bodies with the production of digestive enzymes is indicated.

Observations of a certain arrangement in the nucleolar chromatin after continuous ethidium bromide treatment

Abstract: This work is an ultrastructural and cytochemical study of a structure observed in the nucleolus of Allium cepa meristematic cells after nucleolar disgregation, by a continuous treatment of 12 h with ethidium bromide. The ultrastructural and cytochemical data allow us to consider this structure as the intranucleolar chromatin collapsed by the effect of the drug.