Anchorage of the nucleolus in the pore complex-lamina by a DNA-bearing structure masked in situ in rat liver nuclei

We have developed a method by which nuclear shells containing nucleoli can be isolated from membrane-depleted rat liver nuclei. This method involves the removal of the internal chromatin. This chromatin is expelled from the nuclear shell using combinations of low and high ionic strength buffers. The expelled internal part is subsequently digested with DNase I or micrococcal nuclease. Examination by electron microscopy of the nuclear and the nucleolar structures at various steps of the isolation procedure shows that the nucleoli are anchored in the peripheral lamina by a pedicle that is continuous with an intranucleolar network. This network is masked in situ by nucleolar granules. The pedicle and the network which support the nucleolar DNA are composed mainly of non-histone proteins insoluble in 2M NaCl.     

Anchorage of the nucleolus in the pore complex-lamina by a DNA-bearing structure masked in situ in rat liver nuclei

We have developed a method by which nuclear shells containing nucleoli can be isolated from membrane-depleted rat liver nuclei. This method involves the removal of the internal chromatin. This chromatin is expelled from the nuclear shell using combinations of low and high ionic strength buffers. The expelled internal part is subsequently digested with DNase I or micrococcal nuclease. Examination by electron microscopy of the nuclear and the nucleolar structures at various steps of the isolation procedure shows that the nucleoli are anchored in the peripheral lamina by a pedicle that is continuous with an intranucleolar network. This network is masked in situ by nucleolar granules. The pedicle and the network which support the nucleolar DNA are composed mainly of non-histone proteins insoluble in 2M NaCl.     

Evidence for the existence of a nucleolar skeleton attached to the pore complex-lamina in human fibroblasts

This work deals with the types of nuclear skeletal structures obtained from human fibroblast nuclei isolated by different procedures. It is confirmed that, in somatic vertebrate cells, the pore complex-lamina is always observed, whereas the presence of internal nucleolar and extranucleolar residual structures depends upon the method of nuclear isolation used. Furthermore, the results reported here argue for the existence of a nucleolar skeleton different from the nucleolar matrix often observed in different cell types by other investigators. The conditions of nuclear isolation which allow us to visualize this nucleolar skeleton without any other internal residual structures are described. The attachment of the nucleolar skeleton to the lamina suggested by the present data is considered in relation to the in situ position of nucleoli near the nuclear envelope.     

Three-dimensional approaches to the residual structure of histone-depleted HeLa cell nuclei

Histone-depleted nuclei were prepared by high-salt extraction of interphase HeLa cell nuclei. A large amount of the nuclear DNA remained associated with a rapidly sedimenting residual nuclear structure including cytoplasmic (intermediate filament) and nuclear (matrix and lamina) proteins. Electron microscopy allowed detection in the insoluble structure of a residual nuclear envelope, nucleolar residues, and an intranuclear network whose correspondence with components of in situ fixed nuclei is discussed. Using three-dimensional electron microscopy, it is further demonstrated that the salt-insoluble structure remaining after histone depletion in 2 M NaCl is highly ordered. This is of the utmost importance when considering the roles reportedly ascribed to this structure in nuclear functions.     

Isolation of nuclear shells from plant cells

Abstract. Nuclei from Zea mays and Phaseolus vulgaris root meristematic and differentiated cells were treated according to a recently developed simple procedure for isolation of nuclear lamina of Ehrlich Ascite Tumor (EAT) cells. As revealed by electron microscopy, the residual structures obtained represented empty nuclear shells, resembling those previously isolated from animal cells. Moreover, the composition of the residual nuclear structures from plant cells was found to be very similar to that described previously for the nuclear lamina purified by the adopted procedure. As demonstrated by SDS-polyacrylamide gel electrophoresis, the plant nuclear shells contained a small number of proteins in the 65-45 kD range. Two proteins — 62 and 50 kD—were most characteristic for beans, while a 55-kD protein was abundant in maize. When blotted on nitrocellulose paper, some of the proteins of plant nuclear shells were immunoreactive with sera containing antibodies against the proteins of EAT nuclear envelopes. The degree of phosphorylation of the proteins of plant nuclear shells was found to be higher in meristematic than in differentiated maize root cells, correlating with the mitotic activity of the starting material.     

A combined ultrastructural approach to the study of nuclear matrix thermal stabilization.

Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37 degrees C of isolated nuclei. If purified nuclei were heated for 45 min at 37 degrees C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-amine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.     

A combined ultrastructural approach to the study of nuclear matrix thermal stabilization

Summary Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37° C of isolated nuclei. If purified nuclei were heated for 45 min at 37° C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-ammine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.     

Nuclear matrix, hnRNA, and snRNA in friend erythroleukemia nuclei depleted of chromatin by low ionic strength EDTA

A novel and rapid procedure is described for the preparation of chromatin-depleted nuclei (CDN) from Friend erythroleukemia cells under conditions that avoid the use of high salt concentrations. By this procedure isolated nuclei that had previously been incubated with DNase I to partially digest DNA were washed twice in 2 mM EDTA to extract the chromatin. The resulting structures contained 1% of DNA, 65% of total RNA, 60-80% of hnRNA, 74% of snRNA, 29% of protein, and 2% of histones contained in isolated nuclei. Electron microscopy revealed intact, spherical structures similar in diameter to isolated nuclei and consisting of dense networks of fibrils 50-100 A thick surrounded by distinct nuclear laminae. Although no morphological evidence was found for residual nucleoli, C23, a nucleolus-specific phospho-protein, remained centrally localized in CDN, while Sm antigen specific for snRNPs was diffusely localized but absent from central regions. Addition of 2 mM MgCl2 to CDN resulted in the reformation of morphologically distinguishable residual nucleoli. These studies suggest that nucleolar morphology is, in part, dependent upon divalent cations and components unique to the nucleolar matrix and demonstrate that little randomization of nuclear and nucleolar matrix fibrils occurs during CDN isolation in the absence of divalent cations.     

Heat-induced stabilization of the nuclear matrix: A morphological and biochemical analysis in murine erythroleukemia cells

Using mouse erythroleukemia cells we performed a comprehensive morphological and biochemical study of the nuclear matrix obtained after exposure of isolated nuclei to 37 degrees C or from cells heat shocked in vivo at 43 or 45 degrees C. At the ultrastructural level it was possible to see that in the absence of a 37 degrees C incubation of purified nuclei, the final matrix lacked well-defined nucleolar remnants but a peripheral lamina was clearly visible, as well as a sparse fibrogranular network which was located at the periphery of the structures. On the contrary, after a 37 degrees C nuclear incubation, very electron-dense nucleolar remnants were observed along with an abundant meshwork dispersed throughout the interior of the structures. When intact cells were heat shocked in vivo, electron-dense residual nucleoli were present only when isolated nuclei had been exposed to 37 degrees C in vitro, whereas without such an incubation, they were not as easily distinguishable and appeared less electron-dense. In the latter case the inner network was more evenly distributed. After purified nuclei were incubated at 37 degrees C for 45 min, the high salt and DNase I resistant fraction retained about 18% of the nuclear protein whereas if the heating was omitted protein recovery dropped to 6%. An increase in the recovery of intact structures in the matrix fraction was the main reason for the higher protein recovery. Heating nuclei in vitro further increased the amount of nuclear protein present in the matrix fraction even if intact cells had been heat shocked in vivo. No major qualitative differences were seen when the polypeptide pattern of the various types of nuclear matrices was analyzed on one-dimensional polyacrylamide gels and this finding was further supported by Western blot analysis with a monoclonal antibody to lamins A and C. These results show that heating mainly stabilizes the nucleolar remnants of the matrix and to a lesser extent the inner network, but the morphology of the final structures is different depending on whether the stabilization is performed in vivo or in vitro.     

Variation in nucleolar organiser rRNA gene multiplicity in wheat and rye

In hexaploid wheat and diploid rye, different varieties have different numbers of ribosomal RNA genes as indicated by rRNA/DNA hybridisation. Wheat has four different chromosomes which carry nucleolar organisers. Analyses of DNA isolated from substitution lines in which each of these nucleolar organiser chromosomes of several varieties has been substituted one at a time into a common genetic background, have indicated that none of the four organiser chromosomes possess an invariant number of ribosomal RNA genes. The ribosomal RNA gene complement of the varieties investigated can be approximately accounted for by the sum of the ribosomal RNA genes on each of the four nucleolar organiser chromosomes.     

Isolation and initial characterization of residual nuclear structures from yeast

Residual nuclear structures have previously been isolated from a wide range of eukaryotic organisms. When nuclei are isolated from Saccharomyces cerevisiae and then treated with 1.95 M NaCl and DNase I, sedimentable residual structures are obtained similar in several respects to structures isolated from organisms previously studied. These yeast residual nuclear structures retain less than 7% of nuclear DNA, less than 17% of nuclear RNA and less than 50% of nuclear proteins. Electron microscopy suggests that these structures are derived from the nuclear interior and are composed of a sparse fibrogranular network. Replicating DNA is preferentially bound to these yeast residual nuclear structures, just as it is to residual nuclear structures from other organisms.     

Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells

We have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single-stranded DNA and pulse-labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited. When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed. Preparations of purified, high-molecular weight, double-stranded DNA contain variable amounts of fast-sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation. These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts. Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average length practically all DNA was released. The last 1-4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3 in metrizamide density gradients and showing less than 4% retention on filters. At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA. It was shown, however, that digestion of nuclear lamina-DNA complex with EcoRI or Hae III led to the formation of DNA-protein aggregates, which banded at 1.35 g/cm3 in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epigenetic disruption of ribosomal RNA genes and nucleolar architecture in DNA methyltransferase 1 (Dnmt1) deficient cells

The nucleolus is the site of ribosome synthesis in the nucleus, whose integrity is essential. Epigenetic mechanisms are thought to regulate the activity of the ribosomal RNA (rRNA) gene copies, which are part of the nucleolus. Here we show that human cells lacking DNA methyltransferase 1 (Dnmt1), but not Dnmt33b, have a loss of DNA methylation and an increase in the acetylation level of lysine 16 histone H4at the rRNA genes. Interestingly, we observed that SirT1, a NAD+-dependent histone deacetylase with a preference for lysine 16 H4, interacts with Dnmt1; and SirT1 recruitment to the rRNA genes is abrogated in Dnmt1 knockout cells. The DNA methylation and chromatin changes at ribosomal DNA observed are associated with a structurally disorganized nucleolus, which is fragmented into small nuclear masses. Prominent nucleolar proteins, such as Fibrillarin and Ki-67, and the rRNA genes are scattered throughout the nucleus in Dnmt1 deficient cells. These findings suggest a role for Dnmt1 as an epigenetic caretaker for the maintenance of nucleolar structure.     

Nuclear matrix: isolation and characterization of a framework structure from rat liver nuclei

A nuclear framework structure termed the nuclear matrix has been isolated and characterized. This matrix forms the major residual structure of isolated nuclei and consists largely of protein with smaller amounts of RNA, DNA, carbohydrate, and phospholipid. The nuclear matrix can be further resolved by combined treatment with DNase and RNase. The remaining nuclear protein structure, after extraction of 90 percent of the nuclear protein, 99.9 percent of the DNA, and 98 percent of the RNA and phospholipid, is termed the nuclear protein matrix. Electron microscopy of this final nuclear protein matrix reveals an interior framework structure composed of residual nucleolar structures associated with a granular and fibrous internal matrix structure. The internal matrix framework is derived from the interchromatinic structures of the nucleus, and is connected to a surrounding residual nuclear envelope layer containing residual nuclear pore complex structures. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the nuclear matrix proteins demonstrates three major polypeptide fractions, P-1, P-2, and P-3, with average molecular weights of approximately 69,000, 66,000 and 62,000, as well as several minor polypeptides which migrate at approximately 50,000 and at higher molecular weights (>100,000). Polypeptides with molecular weights identical to those of P-1, P-2 and P-3 are also components of isolated nuclear envelopes and nucleoli, whereas isolated chromatin contains no detectable matrix polypeptides. This suggests that the major matrix polypeptides are localized in specific structural regions of the nucleus, i.e., nuclear envelope, nucleoli, and interchromatinic structures. The presence of cytochrome oxidase activity in the isolated nuclear matrix indicates that at least some integral proteins of the nuclear membrane are associated with the matrix.