Chromosomal constitution of nucleolus-associated chromatin in man

Summary Use of specific stains permits analysis of the frequency of nucleolus-associated heterochromatin in chromosomes 1 and 9 from human fibroblasts. In 81% of interphase nuclei the heterochromatic segment of both No. 1 chromosomes is associated with the nucleolus, while in 19% only one heterochromatic segment shows such an association with the other occupying a random position in the nucleoplasm. The nucleolar association of chromosome 9 heterochromatin is less constant: in 42.3% of the nuclei both segments are associated with the nucleolus, in 39% of the nuclei only one heterochromatic segment presents such an association, and in 18.7% neither of the two heterochromatic segments is in nucleolar association. In 6% of the cells, one or two chromosome 9 heterochromatic segments are in contact with the nuclear membrane.In situ hybridization using tritium-labeled 28S and 18S RNA shows that in the interphase nucleus the acrocentric short arms, carriers of ribosomal cistrons, are associated with the nucleolus.These observations demonstrate the complexity of the nucleolus-associated chromatin which, in addition to segments of chromosomes 1, 9, 13, 14, 15, 21 and 22, may include the Y chromosome. They also confirm that the nucleolus constitutes one of the orientation points determining the relative localization of chromosomes in the interphase nucleus.     

Chromosome-like bodies of dysentery ameba Entamoeba histolytica

Intact and surface stretched amembraneous nuclei of Entamoeba histolytica (Rhizopoda, Lobosea, Entamoebidae) trophozoites were studied by light and electron microscopy. A moderately dense karyosome about 1.5 microns in diameter, localized in the central part of the interphase nucleus, contains the bulk of nuclear DNA. Within the karyosome, beaded and ribbon-like chromatin bodies surrounding a loose fibrillar core are commonly recognized. The peripheral domain of both interphase and several mitotic nuclei is filled with a heterogeneous material similar in its ultrastructure to the nucleolar substance. A wide fibrogranular domain lies between this unusual nucleolus and the karyosome. Rosette-like intranuclear inclusions 0.2-0.4 micron in diameter are often seen in both the fibrogranular and nucleolar domains. At the prophase-metaphase, nearly 50 linear chromosome-like bodies are detected as being in close association with several large beaded and ribbon-like chromatin bodies. At the anaphase-telophase, the chromatin bodies per surface-stretched daughter nucleus of live entamoebae, and in each amembraneous daughter nuclear preparation number nearly 14 and 6, respectively. Besides, in each amembraneous DAPI-stained nucleus a set of 50 or so linear chromosome-like bodies are clearly identified. We infer that the nucleus of E. histolytica contains more than 50 linear chromosomes which at different stages of the cell cycle can unite into several beaded and ribbon-like associations. These form a single moderately dense chromatin karyosome in the central part of the interphase nucleus.     

Nuclear matrix proteins (with molecular masses of 38 and 50 kDa) are transported by chromosomes in mitosis

Using the immunofluorescence method, sera M-68 and K-43 from patients with autoimmune diseases were shown to stain interphase nuclei and the periphery of mitotic chromosomes of pig embryo kidney cells. Western blotting revealed a polypeptide with a molecular mass of 50 kDa in M-68 serum and polypeptide with a molecular mass 38 kDa in K-43 serum. In the nuclear protein matrix, the antibodies to protein with a molecular mass of 38 kDa stained only the nucleolar periphery, while the antibodies to protein with a molecular mass of 50 kDa stained not only the nucleolar periphery, but also all interphase nuclei. It was shown that, among all components of the nuclear protein matrix (lamina, internuclear network, residual nucleoli), only the nucleolar periphery contained the 38-kDa protein, while the 50-kDa protein was part of the residual nucleolar periphery and participated in the formation of a nuclear-protein network. Both proteins in interphase cell in situ were located in nuclei, but one of them with a molecular mass of 50 kDa was in the form of small, clearly outlined granules, while the other protein (38 kDa) was in the form of small, bright granules on a background of a diffusely stained nucleus. Both proteins also were revealed as a continuous rim around the nucleolar periphery. During all mitotic stages, the 50-kDa protein was seen over the whole chromosomal periphery as a sheath, while the 38-kDa protein formed individual fragments and granules around them. After the decondensation of the nucleus and chromosomes induced by hypotonic treatment, both antibodies stained interphase nuclei diffusely, whereas, in mitotic cells, they stained the surfaces of swollen chromosomes. Polypeptide with a molecular mass of 50 kDa maintained a strong connection with the periphery of the chromosome in the norm during decondensation induced by hypotonic treatment and during subsequent recondensation in isotonic medium, while, during recondensation, protein with a molecular mass of 38 kDa partially lost contact with the chromosome and, at the same time, appeared in the form of granules in the cytoplasm. The obtained data allow one to conclude that nuclear matrix proteins can be transferred with peripheral chromosomal material; similar to the main nucleolar proteins (fibrillarin, B-23, nucleolin, et al.) and some non-nucleolar components of the nuclear protein matrix, they can also have connections of different stabilities with chromosomal periphery.     

Human Y-chromatin : I. Dispersion and condensation

The fluorescent Y-body was observed to manifest striking variations in morphology in interphase lymphocyte and fibroblast nuclei. These variations appeared to be largely independent of the specific fixation or hypotonic treatment. The Y-chromatin body ranged from a highly condensed mass to a constellation of smaller discrete particles. Dispersion of Y-chromatin was not seen in mitotically inactive cells from buccal mucosa and hair root sheaths. Comparison of uncultured and cultured lymphocytes confirmed a higher proportion of dispersed Y-bodies in cultured cells, particularly those with a relatively large mean nuclear diameter. In serially cultured lymphocytes, the frequency of Y-body dispersion showed a direct relationship to the time in culture. Mean nuclear diameter also increased directly with time in culture. In cultured fibroblasts dispersion of the Y-body increased during the logarithmic phase of growth and declined during the post-logarithmic phase.     

Actin Is Localized in the Nucleolar Skeleton of Physarum polycephalum

Nucleoli were isolated from the interphase nuclei of Physarum polycephalum Schw. 'lhe nucleolar skeleton was obtained after DNA and most of the nucleolar proteins were extracted with DNase I 0.25 mol/L ( NH4)2SO4 and 2 mol/L NaC1. The nucleolar skeleton appeared as a fibrous network structure composed of fibres about 10 to 30 nm in diameter when observed under the electron microscope. SDS-PAGE analyses revealed about 20 polypeptides in the nucleolar skeleton, including a 43 kD pelypeptide which is equivalent to actin in molecular weight, lmmunofiuorescence observations upon slide preparations of the nueleolar skeleton labeled with anti-actin antibody showed that the nucleolar skeleton emanated bright fluorescence, indicating the existence of thc antigen, lmmunodotting assays further localized actin in the protein preparations of the nucleolar skeleton. Results of immuno-electron microscopy, with anti-actin antibody and protein A-gold as probes, indicated that gold particles were distributed all over the nucleolus of the interphase nucleus.     

多头绒泡菌核仁骨架的研究

从多头绒泡菌（Ｐｈｙｓａｒｕｍ　ｐｏｌｙｃｅｐｈａｌｕｍ　Ｓｃｈｗ）间期细胞核中分离出核仁,用ＤＮａｓｅ　Ｉ、０．２５ｍｏｌ／Ｌ（ＮＨ４）２ＳＯ４和２ｍｏｌ／ＬＮａＣｌ去除核仁ＤＮＡ和大部分蛋白质,得到核仁骨架。核仁骨架是直径１０３０ｎｍ的纤维组成的网络结构,含有约２０种多肽,其中包括与肌动蛋白电泳迁移率相当的４３ｋＤ左右的多肽。免疫荧光检测结果表明,核仁骨架能与肌动蛋白抗体结合而发出明亮的荧光。免疫斑点印迹结果进一步证实,核仁骨架的蛋白质成分中存在肌动蛋白。免疫电镜结果指出,代表肌动蛋白的金颗粒分布在整个核仁中。     

ELECTRON MICROSCOPIC STUDIES OF MITOSIS IN AMEBAE : I. Amoeba proteus

Individual organisms of Amoeba proteus have been fixed in buffered osmium tetroxide in either 0.9 per cent NaCl or 0.01 per cent CaCl₂, sectioned, and studied in the electron microscope in interphase and in several stages of mitosis. The helices typical of interphase nuclei do not coexist with condensed chromatin and thus either represent a DNA configuration unique to interphase or are not DNA at all. The membranes of the complex nuclear envelope are present in all stages observed but are discontinuous in metaphase. The inner, thick, honeycomb layer of the nuclear envelope disappears during prophase, reappearing after telophase when nuclear reconstruction is in progress. Nucleoli decrease in size and number during prophase and re-form during telophase in association with the chromatin network. In the early reconstruction nucleus, the nucleolar material forms into thin, sheet-like configurations which are closely associated with small amounts of chromatin and are closely applied to the inner, partially formed layer of the nuclear envelope. It is proposed that nucleolar material is implicated in the formation of the inner layer of the envelope and that there is a configuration of nucleolar material peculiar to this time. The plasmalemma is partially denuded of its fringe-like material during division.     

The interphase distribution of satellite DNA-containing heterochromatin in mouse nuclei

The distribution of sites capable of binding mouse satellite-complementary RNA in the cytological hybridization reaction has been examined in mouse liver and testis interphase nuclei. The approach taken has been to combine hybridization with semi-thin sectioning and autoradiography in order to obtain a clear picture of the relationship of satellite DNA-containing structures to the rest of the interphase nucleus. In liver nuclei, hybridization occurs primarily with blocks of heterochromatin associated with the nuclear envelope. The most prominent of these, in terms both of size and intensity of hybridization, is the nucleolar stalk and the rest of the nucleolus-associated heterochromatin. The nucleolar body itself is not labeled, nor is much of the peripheral condensed chromatin ; in fact, a polarized distribution of satellite DNA is evident. In Sertoli and spematid nuclei, satellite DNA is found in a small number of large heterochromatin blocks with which the nucleolus is associated; some of this material bears a relationship to the nuclear envelope in these cells also.     

Mitosis in the Hemoflagellate Trypanosoma cyclops*

SYNOPSIS. The ultrastructure of interphase and mitotic nuclei of the epimastigote form of Trypanosoma cyclops Weinman is described. In the interphase nucleus the nucleolus is located centrally while at the periphery of the nucleus condensed chromatin is in contact with the nuclear envelope. The nucleolus fragments at the onset of mitosis, but granular material of presumptive nucleolar origin is often recognizable in the mitotic nucleus. Peripheral chromatin is in contact with the nuclear envelope throughout mitosis, and it seems reasonable to assume that the nuclear envelope is involved in its segregation to the daughter nuclei. Spindle microtubules extend between the poles of the dividing nucleus and terminate close to the nuclear envelope. The basal body and kinetoplast divide before the onset of mitosis and do not appear to have any morphologic involvement in that process. Spindle pole bodies, kinetochores, and chromosomal microtubules have not been observed.     

An autoimmune antibody from scleroderma patients recognizes a component of the plant cell nucleolus

Summary An auto-antibody from human serum of patients with the autoimmune disease scleroderma was used to localize the nucleolus in meristematic cells of onion and soybean roots using indirect immunofluorescence microscopy. Similar lots of antiserum recognized a single 34 kD, nucleolar protein, fibrillarin, in a variety of animal cells (Ochs, et al. 1984, 1985). In both plants, antibody linked fluorescence is associated with the one to several nucleoli present in the interphase nucleus. The fluorescence becomes diffuse around condensing prophase chromosomes and becomes more diffused at metaphase with slightly more intense fluorescence surrounding the chromosomes. At anaphase-telophase the fluorescence is localized in dense areas within the chromosomes, presumably representing prenucleolar bodies which will form the interphase nucleoli of the daughter nuclei. This antiserum provides a new, valuable tool for the study of the nucleolus and the highly conversed nucleolar antigen(s) that it recognizes.     

Ultrastructural,cytochemical and immunocytochemical investigation of the interphase nucleus in the unicellular green alga Chlamydomonas reinhardtii

Summary— The ultrastructural organization of the interphase nucleus of the green alga Chlamydomonas reinhardtii was investigated and found to be largely dependent on the fixation conditions. In specimens stained with bismuth, densely contrasted granules ranging from 25 to 45 nm in diameter were localized throughout the interchromatin space and often formed clusters. These granules were labeled by RNase A-gold complexes and may represent the counterparts of animal and higher plant cll interchromatin granules. Within the nucleolus the Ag-NOR and pyroantimonate stains and, to a lesser extent, the bismuth stain reacted with the nucleolar dense fibrillar component (DFC). When cells were subjected to a heat shock at 42°C, the nucleolar DFC was found to progressively separate from the nucleolus and, after 3 h, appeared as a continuous meandering thread about 0.1 μm in width. Within the nucleolus, labeling on conventional preparations occurred as small clusters with antibodies to H3 histones or to DNA whereas RNase A-gold complexes labeled most of it including fibrillar centers. Improved ultrastructural preservation in cryofixed, cryosubstituted specimens gently fixed in glutaraldehyde permitted to localize nucleolar DNA predominantly at the outer edge of fibrillar centers and to a lesser extent within the neighbouring DFC. Our results indicate that the structure and composition of Chlamydomonas interphase nuclei are comparable, despite particularities, to those of animal and higher plant nuclei.     

The nucleoli matrix proteins with molecular masses 40 and 27 kDa are transported as peripheral chromosomal material

Using immunofluoresence method, sera M-311 and K-30 obtained from patients with autoimmune disease were shown to stain interphase nuclei and the periphery of chromosomes. Western blotting revealed a polypeptide with mol. mass 27 kDa in serum K-30. Both proteins were localized in the karyoplasm. One of them (27 kDa) has a diffuse form and contains small granules, while the other (40 kDa) is in the form of small clearly outlined granules. Both proteins are also revealed around the nucleolar periphery, making a continental ring, while the main part of the nucleolus remains unstained. During pro- and metaphase, these proteins were associated with the chromosomal periphery: 27 kDa protein formed separate groups, and 40 kDa protein was seen over the whole chromosomal periphery. After nuclear and chromosomal decondensation, induced by hypotonic treatment (15% of culture medium solution), both antibodies stain diffusively interphase nuclei, but in mitotic cells they stained the surface of the swollen chromosomes. After chromatin recondensation in isotonic medium these proteins were localized similarly as in normal cells. Thus, both proteins maintained their association with the periphery of chromosomes. To reveal the nuclear protein matrix, cells were treated with 2M NaCl, DNAase and RNAase A. After this procedure, the antibodies stained only the nucleolar periphery, and no fluorescence in the karyoplasm was seen. It shows that of all the components of the nuclear protein matrix (lamina, internuclear network, residual nucleoli) only 27 and 40 kDa proteins are contained in the nucleolar rim. The data allow to suggest that the nucleolar matrix proteins may be transported to new cell nuclei as part of the peripheral chromosomal material likely as other nucleolar (fibrillarin, B-23, and others) or some non-nuclear components of the nuclear protein matrix are transported.     

Interphase chromosome arrangement in Arabidopsis thaliana is similar in differentiated and meristematic tissues and shows a transient mirror symmetry after nuclear division

Whole-mount fluorescence in situ hybridization (FISH) was applied to Arabidopsis thaliana seedlings to determine the three-dimensional (3D) interphase chromosome territory (CT) arrangement and heterochromatin location within the positional context of entire tissues or in particular cell types of morphologically well-preserved seedlings. The interphase chromosome arrangement was found to be similar between all inspected meristematic and differentiated root and shoot cells, indicating a lack of a gross reorganization during differentiation. The predominantly random CT arrangement (except for a more frequent association of the homologous chromosomes bearing a nucleolus organizer) and the peripheric location of centromeric heterochromatin were as previously observed for flow-sorted nuclei, but centromeres tend to fuse more often in nonendoreduplicating cells and NORs in differentiated cells. After mitosis, sister nuclei revealed a symmetric arrangement of homologous CTs waning with the progress of the cell cycle or in the course of differentiation. Thus, the interphase chromosome arrangement in A. thaliana nuclei seems to be constrained mainly by morphological features such as nuclear shape, presence or absence of a nucleolus organizer on chromosomes, nucleolar volume, and/or endopolyploidy level.     

BEHAVIOR OF NUCLEOLI AND CONTRACTING NUCLEOLAR VACUOLES IN TOBACCO CELLS GROWING IN MICROCULTURE

The ability to observe for extended periods of time individual tobacco cells growing in microculture has made it possible to describe the behavior of their nucleoli and contracting nucleolar vacuoles. Nucleoli typically disappeared in prophase and reappeared in telophase. If several nucleoli were present in telophase they generally fused to form only one or two during interphase. In one instance a nucleolus was seen to separate into two nucleoli prior to disappearance in late prophase. In aging and senescent cells the number of nucleoli or bodies similar to normal nucleoli often increased, and occasionally fragmentation of nucleoli was noted prior to death of cells. Budding of solid material from the nucleolus was also observed. The amount of nucleolar material decreased rapidly prior to death of tobacco cells. Nucleolar vacuoles were found to be a general and consistent component of tobacco cells in microculture. Nucleolar vacuoles typically formed and contracted repeatedly in interphase nuclei and apparently released a fluid material into the nucleus. Associated with the contraction of the nucleolar vacuoles was a corresponding decrease in diameter of the nucleolus. Nucleolar vacuoles were observed to occur in about 70% of the actively growing cells examined, whereas they were present in only 33% of the senescent or weakened cells. These data indicate a relationship between nucleolar vacuoles and the morphogenic status of the cells. Since it has been shown by others that the nucleolus is an active site of RNA metabolism, it is suggested that the contracting nucleolar vacuoles may be involved in the controlled release of a soluble product associated with RNA metabolism.     

Nucleolar DNA organization in the interphase nuclei of mouse fibroblasts.

The organization of nucleolar DNA in interphase nuclei of somatic cells was studied at the ultrastructural level using oxidized DAB as a nucleic acid stain. Some finely filamentous networks of DNA-containing structures were observed within the nucleolar fibrillar component. They originated from the perinucleolar shell of condensed chromatin and from a chromatinic area with a honeycomb like structure. The latter was significantly associated with nucleoli and is believed to be a part of the nucleolar organizer region.     

Nucleolus degradation and growth induced by uv-microbeam irradiation of interphase cells grown in culture

In contrast to total cell irradiation, local UV-microbeam irradiation can stimulate a significant diminution in the irradiated mature nucleoli in interphase mammalian cells in culture. This diminution is accompanied by the concomitant expansion of the unirradiated nucleoli within the same nucleus, and the total nucleolar volume per nucleus does not change appreciably. It is suggested that these nucleolar volume changes are the result of the dispersion, migration, and redistribution of the nucleolar material between competitive nucleolar organizer regions of the interphase nucleus.     

Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.     

Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells

The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.     

Ultrastructural features of mitosis inLeishmania adleri

Summary The interphase nucleus ofLeishmania adleri has clumps of chromatin associated with the nuclear envelope and a large centrally located nucleolus. Prior to mitosis the basal bodies replicate at the cell anterior. Subsequently, dense plaques appear in the equatorial region of the nucleus at the time of spindle development. Microtubules appear in the nucleus adjacent to the nuclear envelope and embedded in the matrix of the plaques. A central spindle composed of a single bundle of microtubules develops and spans the nucleus. Plaques and nucleolar components laterally associate with the spindle and migrate towards the poles. The central spindle elongates to three to four times its original length separating the forming daughter nuclei and producing an interzonal spindle. A remnant of the interzonal spindle remains attached to each of the daughter nuclei until late into cytokinesis. The kinetoplast does not divide until after the completion of mitosis.