An in vitro reconstitution system for the assessment of chromatin protein fluidity during Xenopus development

Chromatin fluidity, which is one of the indicators of higher-order structures in chromatin, is associated with cell differentiation. However, little is known about the relationships between chromatin fluidity and cell differentiation status in embryonic development. We established an in vitro reconstitution system that uses isolated nuclei and cytoplasmic extracts of Xenopus embryos and a fluorescence recovery after photobleaching assay to measure the fluidities of heterochromatin protein 1 (HP1) and histone H1 during development. The HP1 and H1 fluidities of nuclei isolated from the tailbuds of early tadpole stage (stage 32) embryos in the cytoplasmic extracts of eggs and of late blastula stage (stage 9) embryos were higher than those in the cytoplasmic extracts of mid-neurula stage (stage 15) embryos. The HP1 fluidities of nuclei isolated from animal cap cells of early gastrula stage (stage 10) embryos and from the neural plates of neural stage (stage 20) embryos were higher than those isolated from the tailbuds of stage 32 embryos in egg extracts, whereas the HP1 fluidities of these nuclei were the same in the cytoplasmic extracts of stage 15 embryos. These results suggest that chromatin fluidity is dependent upon both cytoplasmic and nuclear factors and decreases during development.     

THE FINE STRUCTURE AND CHEMICAL COMPOSITION OF NUCLEI DURING SPERMIOGENESIS IN THE HOUSE CRICKET : I. Initial Stages of Differentiation and the Loss of Nonhistone Protein

The early stages of nuclear differentiation in spermatids of the house cricket are described with regard to the fine structural elements and chemical components which occur. Particular attention is given to the loss of nonhistone protein from the nucleus and its relation to chromatin structure. Granular elements about 25 to 80 mµ in diameter, and fibers about 8 mµ in diameter occur in the earliest spermatid nucleus. The fibers are found in diffuse and condensed chromatin while granules are found only in diffuse material. DNA and histone parallel the chromatin fibers in distribution, while nonhistone protein and RNA parallel the granules in distribution. The granules and most of the nonhistone protein are lost, simultaneously, after the early spermatid stage. The protein loss occurs without detectable change in the structure of chromatin fibers. Chromatin fibers first show a structural change in mid spermiogenesis, when they become thicker and very contorted. Unusually thin fibers (about 5 mµ) also appear in mid spermatid nuclei; they are apparently composed of nonhistone protein and free of DNA and histone.     

FINE STRUCTURAL ALTERATIONS OF INTERPHASE NUCLEI OF LYMPHOCYTES STIMULATED TO GROWTH ACTIVITY IN VITRO

This report describes fine structural changes of interphase nuclei of human peripheral blood lymphocytes stimulated to growth by short-term culture with phytohemagglutinin. Chromatin is found highly labile, its changes accompanying the sequential increases of RNA and DNA synthesis which are known to occur in lymphocyte cultures. In "resting" lymphocytes, abundant condensed chromatin appears as a network of large and small aggregates. Early in the response to phytohemagglutinin, small aggregates disappear during increase of diffuse chromatin regions. Small aggregates soon reappear, probably resulting from disaggregation of large masses of condensed chromatin. Loosened and highly dispersed forms then appear prior to the formation of prophase chromosomes. The loosened state is found by radioautography to be most active in DNA synthesis. Small nucleoli of resting lymphocytes have concentric agranular, fibrillar, and granular zones with small amounts of intranucleolar chromatin. Enlarging interphase nucleoli change chiefly (1) by increase in amount of intranucleolar chromatin and alteration of its state of aggregation and (2) by increase in granular components in close association with fibrillar components.     

Ultrastructural organization of heterochromatin within sea urchin sperm nuclei

Chromatin within swollen or lysed isolated sperm nuclei of the sea urchin, Strongylocentrotus purpuratus, was examined by electron microscopy. Spread preparations of lysed sperm nuclei demonstrated dense aggregates of nondispersed material and beaded filaments radiating from these aggregates. These beaded fibers are similar in size and appearance to the “beads-on-a-string” seen as characteristic of chromatin spreads from numerous interphase nuclei. The beads are nucleosomes that have an average diameter of 130 Å. The interconnecting string is 40 Å indiameter and corresponds to the spacer DNA. In thin sections of swollen nuclei the sperm chromatin appears to be composed of 400 Å superbeads that are closely apposed to form 400 Å fibers. As the chromatin disperses, the superbeads are seen to be attached to one another by chromatin fibers of 110 Å diameter. In thin sections, the 400 Å superbeads appear to disperse directly into the 110 Å fibers with no intervening structures. This work demonstrates that the heterochromatin in Strongylocentrotus purpuratus sperm nuclei is composed of nucleosomes that form 100 Å filaments that are compacted into 400 Å superbeads. The superbeads coalesce to give the morphological appearance of 400 Å fibers.     

肌动蛋白存在于蚕豆细胞核和染色体中

以兔抗肌动蛋白抗体为一抗,FTTC偶联的羊抗兔IgG抗体为二抗进行间接免疫荧光实验,观察到蚕豆(Vicia faba L.)根端分生组织中完整的细胞核和染色体均有明亮荧光。用抗肌动蛋白抗体和蛋白A-胶体金进行标记的免疫电镜实验结果表明,金颗粒分布在蚕豆细胞核中,集缩染色质和核仁中金颗粒较多。经DNaseI消化和2 mol/L NaCl处理得到去除DNA和组蛋白的细胞核和染色体。免疫荧光实验结果指出,去除DNA和组蛋白的细胞核和染色体与抗肌动蛋白抗体呈阳性反应。上述结果说明,肌动蛋白不仅存在于完整的蚕豆细胞核和染色体中,而且存在于去除DNA和组蛋白的蚕豆细胞核和染色体中。另外,用抗原肌球蛋白抗体所做的免疫荧光标记结果表明,原肌球蛋白也存在于蚕豆细胞核和染色体中。对高等植物细胞核和染色体以及核骨架和染色体骨架是否含有肌动蛋白等问题进行了讨论。     

Actin is Immunolocalized in the Nuclei and Chromosomes of Vicia faba

Meristematic cells of Vicia faba L. were labeled with rabbit anti-actin antibody and FITC-conjugated goat anti-rabbit lgG antibody and observed with fluorescence microscopy. Both the nuclei and chromosomes sent forth distinctive fluorescence, indicating that actin is present in the nuclei and chromosomes. Sections were reacted with the anti-actin antibody and protein A-colloidal gold and observed with transmission electron microscopy. Gold particles were found over the whole nuclei, and a lot of particles were concentrated in condensed chromatin areas and nucleoli, confirming the observations with the fluorescence microscopy. V. faba nuclei and chromosomes were treated with DNase Ⅰ and 2 mol/L NaC1, and DNA and histone-depleted nuclei and chromosomes were obtained. Indirect immunofluorescence tests showed that the DNA and histone-depleted nuclei and chromosomes reacted positively with the anti-actin antibody. These results demonstrated that actin exists not only in intact nuclei and chromosomes but also in DNA and histone-depleted nuclei and chromosomes of V. faba. In addition, the authors' results indicate that tropomyosin is present in the nuclei and chromosomes of V. faba. Presence of actin in nuclei and chromosomes as well as in DNA and histone-depleted nuclei and chromosomes of higher plants is discussed.     

The study of chromatin and chromosome structure on preparations of interphase nucleus derivatives resulting from nuclear wall removal.III Structural heterogeneity of chromatin and argyrophilic zone of the nucleolus in stretched membrane-free nuclei and chromatin bodies from human peripheral lymphocytes

Viewed by light microscopy, the majority of lymphocytes in smears of human peripheral blood display a deep staining (with any chromatin- or DNA-specific dye) of the nucleus consisting of densely aggregated chromatin in addition to one or several small nucleoli with a dot- or spot-like argyrophilic zone. Amembraneous nuclei and "free chromatin" structures were isolated from intact lymphocytes gently treated with Triton X-100. Surface stretching of both these nuclei and structures, shortly fixed in methanol--glacial acetic acid (3:1), resulted in spatial separation of thin and thick chromatin or argyrophilic fibres, nucleoli, intranuclear bodies, polymorphous aggregations of chromatin or argyrophilic fibres and incidentally observed splitted or beaded thick chromatin fibres and the chromocenter. The light microscopic pattern of chromatin fibres of stretched amembraneous nuclei, isolated from peripheral lymphocytes, well compares with that of deconvolved images of intact lymphocyte nucleus obtained with optical tomography.     

Immunolcocalization of actin in intact and DNA—and histone—depleted nuclei and chromosomes of allium cepa

The presence of actin in eukaryotic nuclei and chromosomes,and especially in higher plant nuclei and chromosomes,has not been well established.We detected actin in meristematic cells of Allium cepa with indirect immunofluorescence technique and observed bright fluorescence in the intact nuclei and chromosomes,indicating that actin is present in the nuclei and chromosomes of the higher plant.We labeld sections of the meristematic cells of A.cepa with immunogold technique,gold parti cles were concentrated in condensed chromatin and nucleoli,confirming the results of the immunofluoresence observations.We traeated the nuclei and chromosomes of A.cepa with DNase I and 2M NaCl and obtained DNA-and histone-depleted nuclei and chromosomes.Indirect immunofluorescence tests showed that the DNA-and histonedepleted nuclei and chromosomes reacted positively with the anti-actin antibodies.These results demonstrate that the anti-actin antibodies.These results demonstrate that actin exists not only in intact nuclei and chromosomes,but also in DNA-and histone-depleted nuclei and chrmosomes of the plant.In addition,our immuno-fluorescence tests indicate that tropomyosin is present in the nuclei and chromosomes of A.cepa.     

Chromatin structural transitions following histone H1 displacement by phosphatidylserine vesicles and low pH treatment. A multiparametric analysis involving flow cytometry, electron microscopy, and nuclease digestion

We describe several morphological and functional modifications in isolated rat liver nuclei incubated in the presence of phosphatidylserine (PS) multilamellar vesicles (MLV). These effects, which occur through the release of histone H1, induce chromatin decondensation, as shown by electron microscopy and nuclease digestion. Flow cytometry was employed to monitor these changes in chromatin structure in isolated nuclei by means of perpendicular light scatter (PLS) and fluorescence signals. Chromatin decondensation induced by PS or by low pH treatment was accompanied by an increase in perpendicular light scatter and by less efficient binding of ethidium bromide. These flow cytometric findings are peculiar to chromatin decondensation induced by displacement of histone H1. Conversely, chromatin decondensation caused by lowering of the divalent ion concentration, without displacement of histone H1, is characterized only by an increase in perpendicular light scatter.     

Covalent modifications of histones during mitosis and meiosis

Higher order chromosome structures are the hallmark of mitotic and meiotic cells. Chromatin condensation and compaction are essential for rapid chromosome congression and accurate chromosome segregation during cell division. The core histones possess tails at their amino-termini. These tails, which extend from the surface of the nucleosomes, are highly dynamic and subject to an extensive array of covalent modifications. Modified histone tails play an important role, not only in the folding of nucleosomal arrays into higher order chromatin structures but also are crucial role in gene regulation. The combination of these distinct covalent modifications of histones constitutes “the histone code” that regulates various cellular processes, including mitotic and meiotic progression.     

四膜虫(Tetrahymena shanghaiensis)大核核仁与去膜大核能诱导非细胞体系核重建

外源DNA或染色质在非洲爪蟾卵提取物中可以诱导细胞核样结构的重建。重建核除不具有核仁样结构外,在其它形态结构上与真核细胞核十分相似。前人的工作表明在重建核中具有核仁前体结构。但可能是由于缺少活性核仁组织者的缘故,这些核仁前体不能相互融合形成新生核仁。那么活性核仁组织者在重建核中是否能发挥其功能呢?为了研究这一问题,我们提取纯化了四膜虫的大核与大核的周边核仁。进一步去除大核的核被膜,并将去除核被膜的大核与大核核仁分别加入非洲爪赡卵非细胞体系中。通过电镜超薄切片观察,我们发现无论是与大核染色质相连的周边核仁还是分离纯化的核仁结构在非洲爪赡卵非细胞体系中都不能保持其原有结构特征,而是发生了典型核重建变化,并且在诱导形成的重建核中也看不到核仁样结构。这些结果说明具有活性的核仁组织者在加入非洲爪蟾卵提取物后既不能继续保持其原有的RNA转录功能也不能诱导新的核仁的出现。     

Influence of histone H1 on chromatin structure

Removal of histone H1 produces a transition in the structure of chromatin fibers as observed by electron microscopy. Chromatin containing all histone proteins appears as fibers with a diameter of about 250 A. The nucleosomes within these fibers are closely packed. If histone H1 is selectively removed with 50-100 mM NaCl in 50 mM sodium phosphate buffer (pH 7.0) in the presence of the ion-exchange resin AG 50 W - X2, chromatin appears as "beads-on-a-string" with the nucleosomes separated from each other by distances of about 150-200 A. If chromatin is treated in the presence of the resin with NaCl at concentrations of 650 mM or more, the structural organization of the chromatin is decreased, yielding fibers of irregular appearance.     

Analysis of Chlamydomonas reinhardtii histones and chromatin

Chromatin spreads made from isolated nuclei of the unicellular green alga Chlamydomonas reinhardtii show the beaded fibers typical of eukaryotic polynucleosomes. Micrococcal nuclease digestions confirmed the presence of nucleosomes with a repeat length of 189 base pairs, essentially the same as typical mammalian cells. Basic nuclear proteins extracted from isolated nuclei or chromatin with 1 M calcium chloride and 0.3 M hydrochloric acid are resolved into seven major components by electrophoresis in the presence of sodium dodecyl sulfate (SDS). These seven components were subjected to qualitative peptide mapping with V8 protease on SDS gels for comparison with the major histone components of calf thymus. Finally, the C. reinhardtii basic nuclear proteins were fractionated by reversed phase high performance liquid chromatography and their amino acid composition determined. From these studies, we conclude that C. reinhardtii has a full complement of the five histones with properties very similar to those of both higher animals and higher plants.     

Studies on the role of histones H1 (f1) and H5 (f2c) in chromatin structure

Chicken erythrocyte and liver nuclei, isolated and fixed in isotonic saline, contained compact chromatin fibers about 200 Å in diameter. Fibers very similar in dimension and appearance are usually visible in thin sections of fixed nuclei in situ and probably represent chromatin organization close to the native state. After suspension of isolated nuclei in a mildly hypotonic buffer, chromatin fibers extended, became reduced in diameter and apparently unraveled in places. Under such conditions, new detail was revealed suggestive of both helical structure and of subunit organization. The fibers extended and contracted reversibly, changing in diameter from 200 Å to about 100 Å when alternately exposed to isotonic saline and to distilled water. The 200 Å fibers were irreversibly lost, however, following extraction of nuclei with high concentrations of NaCl which selectively removed H1 from liver and H1 plus H5 from erythrocyte chromatin. After extraction of more tightly complexed histones, the residual chromatin consisted mainly of fine filaments less than 30 Å thick. These results suggest a model for native chromatin fibers in which sub-unit organization and coiled configuration are combined, and in which histones H1 and H5 play an integral role in the maintenance of structure.     

Reconstitution of compact polynucleosomes and comparison of the functions of histones H1 and H5

Polynucleosomes with a definite length (about 4,500 base pairs) were prepared from chicken erythrocyte nuclei without depleting magnesium ions from the medium. The polynucleosomes in the presence of Mg2+ ions as well as monovalent salts were more compact than those with monovalent salts alone. We minimized the occurrence of nicks in the DNA of nucleosome fiber during the preparation. When histones H1 and H5 were completely removed from polynucleosomes, linker histone-depleted polynucleosomes sedimented slower than the original ones. When isolated histone H1 or H5 was reassembled with linker histone-depleted polynucleosomes, no significant difference was observed among the reconstituted polynucleosomes with histone H1, the reconstituted polynucleosomes with histone H5, and the original polynucleosomes. We concluded that histones H1 and H5 are similar in their effects on higher order structure of polynucleosomes, as far as can be judged from such characteristics as sedimentation velocity, linker histone content, and the patterns of nuclease digestion.     

Relative position of nucleolar chromatin and nucleolar components in ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis> somatic nuclei

According to our computer modeling data obtained earlier, nucleoli in interphase ciliates Didinium nasutum are complex netlike structures, in which the trabeculumor lamella-shaped fibrillar component is located on the periphery, and the granular component in the central part of the nucleolus. Chromatin bodies connected with nucleoli act as the nucleolar organizers in D. nasutum. In the present work, the arrangement of all chromatin bodies, which could correspond to nucleolar organizers by morphological criteria, is studied by means of a 3D-reconstruction. It is shown that all of these chromatin bodies are localized outside the nucleoli, on the fibrillar component’s periphery. Even those chromatin bodies which appeared to be completely surrounded by the fibrillar nucleolar component on single ultrathin sections are actually settled down in nucleolus cavities open to the nucleoplasm. This proves that the RNA processing in D. nasutum nucleoli is directed toward the center of nucleoli, where the granular component is located. The analysis of the nucleolar chromatin distribution made it possible to conclude that different parts of the complex interfase netlike nucleoli of D. nasutum have approximately the same activity.