Ultrastructure of the centrometric regions of mouse chromosomes in metaphase chromosomes and interphase nuclei]

The chromatin ultrastructure was studied in the centromeric region of mitotic chromosomes and in interphase nuclei of mouse cells after differential staining on C-band. A new method is suggested to study centromeric region of chromosomes treated by the Giemsa banding technique. Fibers of chromosomes appeared to be packed denser in the centromeric regions of mitotic chromosomes than in arms. The disposition of chromatin fibers in the centromeric chromocentres of interphase nuclei is the same as in the centromeric regions of mitotic chromosomes.     

Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro

Higher-order chromatin structural domains approximately 130 nm in width are observed as prominent components of both Drosophila melanogaster and human mitotic chromosomes using buffer conditions which preserve chromosome morphology as determined by light microscopic comparison with chromosomes within living cells. Spatially discrete chromatin structural domains of similar size also exist as prominent components within interphase nuclei prepared under equivalent conditions. Examination of chromosomes during the anaphase-telophase transition suggests that chromosomes decondense largely through the progressive straightening or uncoiling of these large-scale chromatin domains. A quantitative analysis of the size distribution of these higher-order domains in telophase nuclei indicated a mean width of 126±36 nm. Three-dimensional views using stereopairs of chromosomes and interphase nuclei from 0.5 m thick sections suggest that these large-scale chromatin domains consist of 30 nm fibers packed by tight folding into larger, linear, fiber-like elements. Reduction in vitro of either polyamine or divalent cation concentrations within two different buffer systems results in a loss of these large-scale domains, with no higher-order chromatin organization evident above the 20–30 nm fiber. Under these conditions the DNA distribution within mitotic chromosomes and interphase nuclei appears significantly diffuse relative to the appearance by light microscopy within living cells, or, by electron microscopy, within cells fixed directly without permeabilization in buffer. These results suggest that these large-scale chromatin structural domains are fundamental elements of chromosome architecture in vivo.     

Distribution of the rDNA and three classes of highly repetitive DNA in the chromatin of interphase nuclei of Arabidopsis thaliana

The distribution of the ribosomal RNA (rRNA) genes and three classes of highly repetitive DNA in the chromatin of interphase nuclei of Arabidopsis thaliana was studied for the first time through non-isotopic in situ hybridization and luminescence digital imaging microscopy. Each of the three classes of highly repetitive DNA exhibited a characteristic hybridization pattern, and one class was seen to be primarily localized on two chromocentres, which would allow it to distinguish a particular chromosome. The rDNA was consistently localized on the two largest chromocentres and on one or two smaller chromocentres. A limited number of nuclei exhibited more than four labelled chromocentres, indicative of either polypoidy or differential amplification of the rDNA. In nuclei where the nucleolus could be clearly observed, the nucleolar associated chromocentres (NACs) were seen to be labelled by the ribosomal DNA (rDNA) probe.by W. Hennig     

The state of the chromosomes in the interphase nucleus

In the living interphase nucleus no chromosomal structures are visible. Yet in the injured cell and after treatment with most histological fixatives chromatin structures become apparent. Under certain conditions this appearance of structure in the living interphase nucleus is reversible. We have found that this change in the interphase nucleus is the result of a change in the state of the chromosomes. In the living nucleus the chromosomes are in a greatly extended state, filling the entire nucleus. Upon injury the chromosomes condense and therefore become visible. At the same time the nuclear volume decreases. This behavior of the chromosomes is connected with their content of desoxyribonucleic acid (DNA). This view is based on the following observations: (a) Distribution of DNA in the Nucleus.-(1) The living interphase nucleus of uninjured cells absorbs diffusely at 2537 A. No chromosomal structures are visible in ultraviolet photographs unless they are also distinct in ordinary light. If the chromosomes are made to condense they become visible and the absorption at 2537 A is now localized in these structures. (2) After fixation with formalin and osmic acid interphase nuclei stain diffusely with Feulgen. These fixatives preserve the extended state of the chromosomes. (3) If nuclei are teased out in non-electrolytes (sucrose, glycerin) the chromosomes are extended. Such nuclei stain homogeneously with methyl green. On adding salts the chromosomes condense and the methyl green is now restricted to the visible structures. (b) Extension and Condensation of Isolated Chromosomes.-When chromosomes isolated from interphase nuclei of calf thymus are suspended in sucrose, their volume is four to five times larger than in saline, but they retain their characteristic shapes. Chromosomes from which DNA and histone have been removed do not show this reversible extension and condensation, neither do lampbrush chromosomes of frog oocytes which contain very little DNA. During mitosis a partial condensation of the DNA occurs in prophase, so that the mitotic chromosomes now occupy a much smaller volume of the nucleus. At telophase the chromosomes swell again to fill the entire nucleus.     

Dynamic inorganic ion-protein interactions in structural organization of DNA of living cell nuclei.

Staining polarization optical techniques showed differences in the structural organization of DNA of chromatin in interphase nuclei and in mitotic chromosomes. The DNA was non-birefringent in intact interphase cell nuclei, but birefringent in chromosomes and in isolated nuclei incubated in a physiological electrolyte solution. The birefringence of DNA appears to be related to an unfolding of DNA filaments induced by free cations and to the oriented binding of dye molecules to DNA phosphates. We propose that the actual concentration of free cations inside the living cell nuclei is regulated by a dynamic interaction between nuclear proteins and ions.     

大麦细胞核及染色体肌球蛋白免疫细胞化学定位

对去除DNA、组蛋白和大部分非组蛋白的大麦(Hordeum vulgare)细胞核和染色体间接免疫荧光标记实验结果表明:抗肌球蛋白抗体的荧光标记弥散分布在整个细胞核和染色体上;进一步应用免疫胶体金技术分析肌球蛋白在细胞核和染色体的分布情况,发现在染色体中散布着大量的胶体金颗粒;间期细胞核中胶体金颗粒主要分布在核仁和染色质中。上述实验结果表明:肌球蛋白是细胞核及染色体非组蛋白组成成分。本文还对肌球蛋白在细胞核和染色体中的分布规律进行了讨论。     

Analysis of Y chromosome heterochromatin in Rumex thyrsiflorus

The Y chromosome heterochromatin in Rumex thyrsiflorus has been analyzed. In natural populations the Y chromosome shows a higher morphological variability than the X chromosome. The total duration of replication of Y chromosomes is about 2 hrs longer than that of euchromatin. Autoradiography with tritiated thymidine showed that chromocentres formed by Y chromosomes in interphase nuclei retain their heterochromatic form during DNA replication. — Y chromosome heterochromatin in interphase nuclei is stained pink, while the rest of the nucleus stains green after fast green-eosin staining for histones. — During the premeiotic stage of PMC development Y chromosomes are no longer visible as compact bodies and become more fuzzy in appearance. A diffuse state of Y coincides with intense RNA synthesis. Therefore genetic activity of Y chromosomes or their parts during premeiotic stage of microsporogenesis is postulated.     

DNA-fluorescence of mammalian intra-nucleolar chromatin detected by confocal laser scanning microscopy (CLSM)

The complex spatial DNA distribution in the mammalian interphase nucleus was investigated in Feulgen stained thick sections through mouse trophoblast giant nuclei after Lowicryl embedding. DNA-fluorescence was visualized using confocal laser scanning microscopy. Our results show that the spatial arrangement of major interphase chromatin areas can be precisely documented, including the distribution of small intra-nucleolar chromatin zones.     

Mitotic chromatin condensation in vitro using somatic cell extracts and nuclei with variable levels of endogenous topoisomerase II

We report the development of a new method for producing mitotic extracts from tissue culture cells. These extracts reproducibly promote the condensation of chromatin in vitro when incubated with purified interphase nuclei. This condensation reaction is not species specific, since nuclei from chicken, human, and hamster cell lines all undergo chromatin condensation upon incubation with the extract. We have used this extract to investigate the role of DNA topoisomerase II (topo II) in the chromosome condensation process. Chromatin condensation does not require the presence of soluble topo II in the mitotic extract. However, the extent of formation of discrete chromosome-like structures correlates with the level of endogenous topo II present in the interphase nuclei. Our results further suggest that chromatin condensation in this extract may involve two processes: chromatin compaction and resolution into discrete chromosomes.     

Transition from chromatin bodies to linear chromosomes in nuclei of murine PreB cells synchronized in S phase

Chromatin structures and individual interphase chromosomes escaping nuclei of reversibly permeabilized cells were analyzed in a cell cycle-dependent manner. Cells were synchronized by counterflow centrifugal elutriation. Individual interphase chromosomes became visible as distinct fibrous chromatin bodies from mid-S-phase, turning to elongated chromosomes by the end of S phase. Major interphase chromosomal forms include (1) mid-S-phase chromatin bodies at 3.0 C-value, (2) elongated chromatin bodies later in mid-S-phase (3.25 C-value), (3) chromatin bodies with head and leg portions later in S phase (3.5 C-value), (4) supercoiled ribbons later in S phase seen as twisted prechromosomes (3.7 C-value), and (5) end-S-phase elongated, bent prechromosomal structures (3.9 C-value). The first karyotype analysis of the earliest forms of chromosomes referred to as chromatin bodies was performed.     

Rumex acetosa Y chromosomes: constitutive or facultative heterochromatin?

Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw.) Steam roots. It was shown that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.     

Features of interactions of p68 anchorosomal protein with the nuclear envelope in the cell cycle

Chromatin associated with the nuclear envelope appears in the interphase nuclei as a layer of anchorosomes, granules 20-25 nm in diameter. The fraction of chromatin directly associated with the nuclear envelope is resistant to decondensation, shows a low level of DNA methylation, and contains specific acid-soluble proteins. However, mechanisms underlying the interaction of chromatin with the nuclear envelope are not fully understood. Specifically, it is not known whether anchorosomes are permanent structures or if they undergo reversible disassembly during mitosis, when contacts between chromatin and the nuclear envelope are destroyed. We obtained immune serum recognizing a 68 kDa protein from the nuclear envelopes fraction and studied the localization of this protein in interphase and mitotic cells. We show that this protein present in the NE/anchorosomal fraction does not remain bound with chromosomes during mitosis. It dissociates from chromosomes at the beginning of the prophase and then can be identified again at the periphery of the newly forming nuclei in the telophase.     

Condensation of interphase chromatin in nuclei of synchronized chinese hamster ovary (CHO-K1) cells

Reversibly permeabilized cells have been used to visualize interphase chromatin structures in the presence and absence of biotinylated nucleotides. By reversing permeabilization, it was possible to confirm the existence of a flexible chromatin folding pattern through a series of transient geometric forms such as supercoiled, circular forms, chromatin bodies, thin and thick fibers, and elongated chromosomes. Our results show that the incorporation of biotin-11-dUTP interferes with chromatin condensation, leading to the accumulation of decondensed chromatin structures. Chromatin condensation without nucleotide incorporation was also studied in cell populations synchronized by centrifugal elutriation. After reversal of permeabilization, nuclei were isolated and chromatin structures were visualized after DAPI staining by fluorescent microscopy. Decondensed veil-like structures were observed in the early S phase (at an average C-value of 2.21), supercoiled chromatin later in the early S (2, 55 C), fibrous structures in the early mid S phase (2, 76 C), ribboned structures in the mid-S phase (2, 98 C), continuous chromatin strings later in the mid-S phase (3,28), elongated prechromosomes in the late S-phase (3, 72 C), precondensed chromosomes at the end and after the S phase (3, 99 C). Fluorescent microscopy revealed that neither interphase nor metaphase chromosomes are separate entities but form a linear array arranged in a semicircle. Linear arrangement was confirmed by computer image analysis.     

The ultrastructure and properties of centromeric heterochromatin in cultured bovine trachea cells]

The fine organization of the centromere structural heterochromatin (CSH) in a cell culture of calf trachea (TR) was studied by the methods of light and electron microscopy after fixation in native conditions and after treatments with water Henk's solutions and solutions of Ca2+ of different concentrations. In interphase nuclei the CSH forms compact blocks--the chromocentres, which are connected with the nuclear envelope or the nucleolus. The diameter of the main class of DNP fibers in the CSH, chromosomal arms and chromocentres after fixation of control cells and after treatment with 50% Henk's solution is 20-25 nm. 10-15 nm DNP fibers are largely found in the contacts with kinetochores. After 20% Henk's solution treatment 10-15 nm fibers predominate in the CSH and chromosomal arms. A chromonema--a 100 nm chromatin fiber--is detected in the CSH after treatments with different concentrations of Ca2+ solutions, as well after fixation in native conditions. The peculiarity of structural organization and properties of the calf CSH at interphase and mitosis as compared with analogous regions in mouse chromosomes are suggested to be connected with the composition of its DNA and proteins.     

Interphase chromosome arrangement in Anopheles atroparvus

The arrangement of chromosomes in interphase nuclei of Anopheles atroparvus has been inferred from an analysis of: 1. The early stages of mitosis as seen following Quinacrine staining, and 2. The reversible effects on the chromatin pattern obtained following the treatment of living cells with various NaCl solutions, and the following conclusions have been reached: (a) The chromatin is connected to the nuclear membrane, (b) Homologous chromosomes show close side-by-side somatic pairing, (c) The long arms of the sex chromosomes form a fluorescent peripheral body, (d) The autosomes are strongly reflexed at the centromeres, (e) The autosomal centromeric regions are polarized towards the peripheral body, (f) The telomeric regions of all the autosomes are closely apposed.--A ring-shaped pattern of interphase chromatin is constantly and reversibly induced by NaCl 0.15 to 0.18 M solutions.--These relationships indicate a peripheral arrangement of the interphase somatic complement.--The distribution of the chromosomes in polytene nuclei and at the beginning of meiosis resembles that suggested above for somatic interphase cells. This distribution may apply more widely in the Diptera.     

In situ factors affecting stability of the DNA helix in interphase nuclei and metaphase chromosomes

The data from earlier cytochemical studies, in which the metachromatic fluorochrome acridine orange (AO) was used to differentially stain single vs double-stranded DNA, suggested that DNA in situ in intact metaphase chromosomes or in condensed chromatin of G0 cells is more sensitive to denaturation, induced by heat or acid, than DNA in decondensed chromatin of interphase nuclei. Present studies show that, indeed, DNA in permeabilized metaphase cells, in contrast to cells in interphase, when exposed to buffers of low pH (1.5-2.8) becomes digestible with the single-strand-specific S1 or mung bean nucleases. A variety of extraction procedures and enzymatic treatments provided evidence that the presence of histones, HMG proteins, and S-S bonds in chromatin, as well as phosphorylation or poly(ADP)ribosylation of chromatin proteins, can be excluded as a factor responsible for the differential sensitivity of metaphase vs interphase DNA to denaturation. Cell treatment with NaCl at a concentration of 1.2 N and above abolished the difference between interphase and mitotic cells, rendering DNA in mitotic cells less sensitive to denaturation; such treatment also resulted in decondensation of chromatin visible by microscopy. The present data indicate that structural proteins extractable with greater than or equal to 1.2 N NaCl may be involved in anchoring DNA to the nuclear matrix or chromosome scaffold and may be responsible for maintaining a high degree of chromatin compaction in situ, such as that observed in metaphase chromosomes or in G0 cells. Following dissociation of histones, the high spatial density of the charged DNA polymer may induce topological strain on the double helix, thus decreasing its local stability; this can be detected by metachromatic staining of DNA with AO or digestion with single-strand-specific nucleases.     

Localization of tandemly repeated DMA sequences in Arabidopsis thaliana

In-situ hybridization to interphase nuclei and chromosomes of Arabidopsis thaliana (2n= 10) shows that there are four sites of rDNA in a diploid nucleus. The sites are located on chromosomes 2 and 4, and the strength of hybridization indicates that copy number is similar at both pairs of sites. Hybridization to trisomic line 4 revealed five hybridization sites. Silver staining of nucleoli demonstrates that all four loci can be active in diploid interphase nuclei. The tandemly repeated probe pAL1 hybridizes near to the centromeres of all five chromosome pairs. In diploid interphase nuclei, 10 sites of hybridization are detected, while 15 are seen in triploid nuclei. The sites of hybridization co-localize with the centromeric heterochromatin visualized by staining DNA with the fluorochrome DAPI. The results demonstrate that molecular cytogenetics can be applied to A. thaliana and high resolution physical chromosome maps can be generated. Both probes may be useful for interphase cytogenetics, where they enable chromosome number and aneuploidy to be examined in tissues without divisions. The physical localization of these hybridization sites provides a starting point for linking RFLP and physical chromosome maps.     

Chromatin organization during meiotic prophase ofBombyx mori

Chromatin organization during the early stages of male meiotic prophase inBombyx mori was investigated by electron microscopy. The analysis of nuclei prepared by the Miller spreading procedure, suggests that chromatin fibers which are 200–300 Å in diameter undergo an orderly folding coincident with the formation of the synaptonemal complex. In very early stages the chromatin is released in linear arrays typical of interphase chromatin material. With time loops containing 5–25 of B conformation DNA, initially visualized at the periphery of early meiotic prophase nuclei, aggregate into discrete foci. These foci coalesce to form the longitudinal axis of the chromosome in conjunction with the initial appearance of the axial elements of the synaptonemal complex. At pachytene, the loops are evenly distributed along the length of the chromosome and extend radially so that in well spread preparations the chromosome has a brush-like appearance. Throughout this period nascent RNP-fibers were visualized along some of the loops.     

Some observations on the structure, cation content and possible evolutionary status of dinoflagellate chromosomes

SICEE, D. C., 1984. Some observations on the structure, cation content and possible evolutionary status of dinoflagellate chromosomes. Dinoflagellate chromosomes have a well-ordered structure, as observed in living cells, glutaraldehyde/osmium tetroxide-fixed cells, ultrathin cryosections and freeze-etch preparations. It is suggested that the stabilization of this chromatin in the living cell is largely mediated by divalent cations, acting as bridging molecules between the DNA superstructure and the protein matrix. Studies using X-ray micro-analysis and autoradiography have shown that these chromosomes have high levels of bound Ca and transition metals, and that these are associated with both the DNA and surrounding proteins.
The organization and stabilization of chromatin in dinoflagellate chromosomes is quite different from that of the cells of other eukaryotes, but shows some resemblance to the dispersed chromatin of bacteria. The evolution of dinoflagellate chromosomes from a prokaryote-like ancestral genome is attributed to two main factors–the retention of a primitive cationic non-histone stabilization system, and a pronounced evolutionary trend towards high DNA values. On this theory, dinoflagellate chromosomes are phylogenetically distinct from all other eukaryote chromosomes, and provide a separate evolutionary route for the attainment of high DNA levels and increased cell size.