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1.
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The topological features of isolated Chinese hamster ovary metaphase chromosomes were studied with high resolution scanning electron microscopy (SEM) using the techniques of direct current sputtering for the deposition of metal on the specimens. Metaphase chromosome surfaces consist of numerous compact microconvules of an average diameter of 520 ± 78 Å when corrected for the thickness of the gold-palladium coating (80 ± 2 Å). These microconvules contain several orders of supercoiling. The superhelical structures were detected also in water-spread preparations. Most of the isolated chromosomes had membrane-like structures attached at the distal portions of the chromatids forming a terminal “plate”. Limited tryptic digests of such isolated chromosomes resulted in considerable stretching of the chromatids and revealed a series of interchromatidal fibers with diameters of 203 ± 38 Å (corrected for gold coating). Treatment of these chromosomes with EDTA revealed a longitudinal array of fibers within the chromatids. The diameters of these fibers decreased as the concentration of EDTA was increased. The technique of direct current sputtering for the preparation of chromosomes for scanning microscopy is satisfactory for detailed topological ultrastructural studies in the 70 Å range.  相似文献   

3.
Whole-mounted polytene chromosomes were isolated from nuclei by microdissection in 60% acetic acid and analyzed by electron microscopy. Elementary chromosome fibers in the interchromomeric regions and individual chromomeres can be distinguished in polytene chromosomes at low levels of polyteny (26–27 chromatids). Elementary fibers in the interbands are oriented parallel to the axis of the polytene chromosome. Their number roughly corresponds to the expected level of polyteny. These fibers have an irregular beaded structure, 100–300 Å in diameter, and there is no apparent lateral association between them in the interchromomeric regions. Most bands, in contrast, form continuous structures crossing the entire width of the chromosome. Polytene chromosomes isolated in 2% or 10% acetic acid can be reversibly dispersed in a solution for chromatin spreading. The spread chromosomes consist of long uniform deoxyribonucleoprotein (DNP) fibers with a nucleosome structure. This supports the notion that continuous DNA molecules extend through the entire length of a polytene chromosome and that the nucleosome structure exists both in bands and interbands. Analysis of the band shape and of the fibrillar pattern in the interbands emphasizes that the polytene chromosome assumes a ribbonlike structure from which the more complex three-dimensional structure of the polytene chromosome at higher levels of polyteny develops.  相似文献   

4.
When chromatin from a diverse range of eukaryotes is prepared for the electron microscope by aldehyde fixation followed by centrifugation onto the grid, the fibers are composed of interconnected spherical particles, about 70 Å in diameter. Evidence is presented which suggests that the particles are an in vivo structural repeating unit of deoxyribonucleoprotein, and not a preparation-induced artefact.  相似文献   

5.
In vitro conditions have been determined for obtaining ordered helical ribbons of small ribosomal subunits from Escherichia coli. These ribbons, suitable for study by three-dimensional reconstruction, are the first ordered arrays of ribosomes or ribosomal subunits to be produced in vitro.Although small ribosomal subunits remain in solution for extended periods (up to 6 months) during this procedure, their structural integrity, as assessed by acrylamide/agarose gel electrophoresis, by sucrose gradients, and by electron microscopy, is not significantly altered.Electron micrographs of ribbons of small subunits diffract to 60 Å resolution. Optical diffraction patterns suggest that adjacent subunits within helical ribbons are related by a 2-fold screw parallel to the long axis of the ribbon and the helical repeat distance measured from electron micrographs is 220 Å.  相似文献   

6.
Metaphase chromosome structure: evidence for a radial loop model.   总被引:45,自引:0,他引:45  
M P Marsden  U K Laemmli 《Cell》1979,17(4):849-858
Electron micrographs of thin sections of metaphase chromosomes isolated from HeLa cells provide new insight into the higher-order arrangement of the nucleoprotein fiber. Micrographs obtained from chromosomes swollen by chelation of the divalent cation are particularly revealing. Under these conditions, chromosomes swell in width by a factor of about 4 and the basic, thick nucleoprotein fiber (200–300 Å) relaxes to the thin fiber (100 Å), which is probably a linear array of nucleosomes. Cross sections show a central area from which the fibers emerge in a radial fashion, often forming loops which are 3–4 μm long. Chromosomes fixed in the presence of 1 mM MgCl2 are more compact, having an average chromatid diameter of about 1 μm, and consist of the thick (200–300 Å) fiber. Radial loops of about 0.6 μm can be observed frequently in these chromosomes, although the loops are more difficult to visualize due to the compactness of the structure and the material contaminating the periphery. Chromosomes isolated with the help of hexylene glycol are extremely compact (diameter about 0.6 μm) but quite free of cytoplasmic material. They consist of a 500 Å fiber that forms rather regular projections at the periphery. These projections appear to be loops of the thick fiber (200–300 Å), possibly shortened by twisting into a short supercoil. The chromatin loops observed in the intact chromosomes are thought to be structurally related to the DNA loops observed previously in the histone-depleted chromosomes (Paulson and Laemmli, 1977). In this paper, we discuss a model in which the nucleoprotein fiber is folded into loops which are arranged in the chromatid in radial fashion, in such a way that their bases become the central axis of the chromatid.  相似文献   

7.
It has been possible to identify the chromosomes in electron micrographs of a prophase neuroblast nucleus inDrosophila melanogaster by using stereophotographs of stacked transparencies made from serial sections. Depth was determined by increasing or decreasing the stereo angle. Identification was facilitated by the use of a stock carrying chromosomal rearrangements. In this stock only chromosome 2 is metacentric. Compound chromosomes [symbolized C(3L)RM and C(3R)RM] were formed from the left and from the right arms of chromosome 3, and the X chromosome was intercalated in an inverted position between the two arms of the Y (YSX·YL).—Three-dimensional aspects of the ultrastructure of these chromosomes were observed as follows. The compacted centric regions are composed of fibers coiled in irregular gyres. In the extended regions, the fibers subdivide and appear uncoiled except for regions of compaction—“chromomeres”—which are seen in both homologues. Each homologue appears to be composed of four fibers of 130 Å diameter, a total of eight for the prophase chromosome. Since the larger neuroblast cells are 8C in the G2 phase, it would seem that the 130 Å fiber is equivalent to the cytological chromatid.  相似文献   

8.
The structure of histone-depleted metaphase chromosomes   总被引:1,自引:0,他引:1  
We have previously shown that histone-depleted metaphase chromosomes can be isolated by treating purified HeLa chromosomes with dextran sulfate and heparin (Adolph, Cheng and Laemmli, 1977a). The chromosomes form fast-sedimenting complexes which are held together by a few nonhistone proteins.In this paper, we have studied the histone-depleted chromosomes in the electron microscope. Our results show that: the histone-depleted chromosomes consist of a scaffold or core, which has the shape characteristic of a metaphase chromosome, surrounded by a halo of DNA; the halo consists of many loops of DNA, each anchored in the scaffold at its base; most of the DNA exists in loops at least 10–30 μm long (30–90 kilobases).We also show that the same results can be obtained when the histones are removed from the chromosomes with 2 M NaCl instead of dextran sulfate. Moreover, the histone-depleted chromosomes are extraordinarily stable in 2 M NaCI, providing further evidence that they are held together by nonhistone proteins.These results suggest a scaffolding model for metaphase chromosome structure in which a backbone of nonhistone proteins is responsible for the basic shape of metaphase chromosomes, and the scaffold organizes the DNA into loops along its length.  相似文献   

9.
Giemsa-stained chromosomes as prepared for light microscopy, and including G-banded, C-banded, and FPG-stained chromosomes, were examined by scanning electron microscopy. Although suitable for light microscopy, these chromosomes were too flat for a close examination of their fine structure by scanning electron microscopy. The surface of Giemsa-positive regions was rough and bright, whereas that of unstained or poorly stained regions was smoother and less bright. Giemsa-staining, therefore, seems to produce the bulkiness of the chromosomes. On topographical examination by scanning electron microscopy, the transparent chromosomes as observed with the light microscope proved to be footprints. Stereographical examinations of surface-spread chromosomes showed that minimally stretched chromosomes were composed of a mass of nodular and twisted looping fibers with an average diameter of about 300 Å. The substructure of these chromosome fibers was not determined. The kinetochore region was discernible as a constriction in the mass of the chromosome fibers, and was distinguishable from gaps by the presence of several chromosome fibers parallel to the axis of the chromatid. The organization of the chromosome fibers, however, was disordered rather than regular.  相似文献   

10.
Metaphase chromosomes are extracted from Indian muntjac cultured fibroblasts either through the use of microneedles or by the application of a droplet of silicone oil onto the cell surface. Interconnecting fibers among the chromosomes allow the entire diploid complement to be extracted from the cell. The seven muntjac chromosomes are brought to the surface of a glass coverslip for analysis. Each chromosome can be identified on the basis of morphology, and particular chromosomes or chromosome parts can be isolated. Many of the fibers which interconnect the chromosomes may be attributed to adhesions formed between the sticky chromosome surfaces during extraction. However, when interchromosomal contacts are avoided during extraction, the chromosomes are found to be arranged radially with the centromeres near the center and interconnected by fibers. This arrangement is similar to that seen inside muntjac cells at metaphase. Scanning electron microscopy reveals the chromosome surfaces to consist of looping fibers, except for regions near the centromeres and the secondary constrictions. Chromosome fibers at these sites are organized into parallel bundles. Chromosome interconnections are strands composed of multiple fibers which seem to be continuous with chromosome fibers.  相似文献   

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On the basis of recent results a unified view of different aspects of the higher levels in the organization of chromatin in chromosomes is presented. Basic to these forms of organization is the arrangement of DNA in the complex with nucleosomes and recent studies suggest that at least some species of satellite DNA may maintain a fixed DNA sequence relationship to the phasing of nucleosomes. Special proteins such as the high-mobility group (HMG) proteins or other non-histone proteins could serve specific functions in the recognition of satellite DNA sequences.In the presence of histone H1 the 110 Å nucleosome fiber formed from the basic string of nucleosomes can be further condensed into a thicker 250–300 Å fiber formed by a solenoidal coiling of the 110 Å fiber with about 6–8 nucleosomes per turn and the available evidence suggests that these structures are found in mitotic chromosomes as well as other forms of inactive chromatin. A further level of coiling of the 250–300 Å solenoid has been suggested by our recent studies of disintegrated mitotic chromosomes consisting of a thin-walled tube with an outer diameter of 4000 Å referred to as the unit fiber. This structure would account for a factor of 1400 × contraction of DNA in mitotic chromosomes which in their intact state are only 5-fold more contracted. The recently described “scaffold” proteins could be responsible for this final coiling of the unit fibers in intact chromosomes.Meiotic chromosomes are generally less contracted than mitotic chromosomes. An extreme example of this are lampbrush chromosomes that apart from the axial segments which might contain some structural proteins appear to consist of naked DNA arranged in lateral loops. In the later stages of meiosis more condensed structures arise as exemplified by the synaptonemal complex during the pachytene stage in many organisms. The characteristic features of this structure are interpreted to suggest that the structure consists of lateral components containing two parallel 110 Å nucleosome fibers each representing the axial segments of two sister chromatids. From these paired regions loops protrude laterally in a manner which closely resembles the less condensed lampbrush chromosomes. The implication of this structure in the process of crossingover is discussed.Less is known about the organization of chromatin in interphase nuclei, but structures analogous to the loop-like structures in meiotic chromosomes are suggested on the basis of the isolation of supercoiled DNA loops constrained by RNA-DNA and protein-DNA interactions. The position of these loops is suggested to be fixed by specific repeated DNA sequences that could be associated with specific tenacious non-histone or HMG proteins.  相似文献   

14.
Xu  Peng  Mahamid  Julia  Dombrowski  Marco  Baumeister  Wolfgang  Olins  Ada L.  Olins  Donald E. 《Chromosoma》2021,130(2-3):91-102

“Interphase epichromatin” describes the surface of chromatin located adjacent to the interphase nuclear envelope. It was discovered in 2011 using a bivalent anti-nucleosome antibody (mAb PL2-6), now known to be directed against the nucleosome acidic patch. The molecular structure of interphase epichromatin is unknown, but is thought to be heterochromatic with a high density of “exposed” acidic patches. In the 1960s, transmission electron microscopy of fixed, dehydrated, sectioned, and stained inactive chromatin revealed “unit threads,” frequently organized into parallel arrays at the nuclear envelope, which were interpreted as regular helices with ~ 30-nm center-to-center distance. Also observed in certain cell types, the nuclear envelope forms a “sandwich” around a layer of closely packed unit threads (ELCS, envelope-limited chromatin sheets). Discovery of the nucleosome in 1974 led to revised helical models of chromatin. But these models became very controversial and the existence of in situ 30-nm chromatin fibers has been challenged. Development of cryo-electron microscopy (Cryo-EM) gave hope that in situ chromatin fibers, devoid of artifacts, could be structurally defined. Combining a contrast-enhancing phase plate and cryo-electron tomography (Cryo-ET), it is now possible to visualize chromatin in a “close-to-native” situation. ELCS are particularly interesting to study by Cryo-ET. The chromatin sheet appears to have two layers of ~ 30-nm chromatin fibers arranged in a criss-crossed pattern. The chromatin in ELCS is continuous with adjacent interphase epichromatin. It appears that hydrated ~ 30-nm chromatin fibers are quite rare in most cells, possibly confined to interphase epichromatin at the nuclear envelope.

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15.
Basic proteins were differentially extracted from the purified heads of ejaculated human sperm by successive treatment with (1) 1% Triton X-100, 1% mercaptoethanol (ME); (2) 8 M urea, 1% ME; (3) 8 M urea, 1% ME, 0.2 M NaCl; and (4) 8 M urea, 1% ME, 0.6 M NaCl. Nonhistones were extracted in the first, second, and third treatments; histones, in the second and third; and most of the protamines, in the fourth, together with DNA. Corresponding electro microscopic studies of the sperm pellets after each treatment suggest that the nucleoprotamines are discrete portions of chromatin which are organized in the form of long thick cords and oval bodies of about 400–550 Å in diameter interlinked with very thin strands of fibers about 20–50 Å thick, which could represent the naked DNA depleted of its constituent histones.  相似文献   

16.
When mouse L-cells were treated with a combination of 5-bromodeoxyuridine (BrdUrd) and Hoechst 33258, the metaphase chromosomes revealed undercondensation of the chromatin fibers in the sister centromeres. The application of the osmium-thiocarbohydrazide technique to the air-dried chromosome preparations made it possible to elucidate the ultrastructure of the undercondensed centromeric region at the level of the 30 nm chromatin fiber. Scanning electron microscopy revealed that the undercondensed region consisted of a coiled fiber with a diameter of about 400 nm, and a gyre diameter of approximately 600 nm. The coiled fiber was composed of the 30 nm chromatin fiber loops. These findings indicate that a continuous coiled structure, which is the final higher order structure of the condensed chromatin fiber, exists throughout the entire length of the mouse L-cell metaphase chromosome.  相似文献   

17.
We present a new model of the three-dimensional structure of chromosomes. With DNA and protein staining it could be shown by high-resolution scanning electron microscopy that metaphase chromosomes are mainly composed of DNA packed in "chromomeres" (coiled solenoides) and a dynamic matrix formed of parallel protein fibers. In the centromeric region, the chromomeres are less densely packed, giving insight into the matrix fibers. We postulate that chromosome condensation is achieved by the binding of solenoids to matrix fibers which have contact sites to one another and move antiparallel to each other. As condensation progresses, loops of solenoids accumulate to form additional chromomeres, causing chromosomes to become successively shorter and thicker as more chromomeres are formed. For sterical reasons, a tension vertical to the axial direction forces the chromatids apart. The model can simply explain the enormous variety of chromosome morphology in plant and animal systems by varying only a few cytological parameters. Primary and secondary constrictions and deletions are defined as regions devoid of chromomeres. Even in the highly condensed metaphase, all genes would be easily accessible.  相似文献   

18.
The structural organization of the nuclear matrix of pericentromeric heterochromatin blocks (chromocenters) was examined in cultured murine fibroblasts. After 2 M NaCl extraction without DNase I treatment, chromocenters became extremely swollen and could not be recognized with conventional electron microscopy. Using immunogolding with anti-topoisomerase IIα antibodies, we demonstrated that residual chromocenters were divided into numerous discrete aggregates. After 2 M NaCl extraction with DNase I treatment, the residual chromocenters looked as the dense meshwork of thin fibers and, therefore, were easily distinguished from the rest of nuclear matrix. Extraction with dextran sulfate and heparin resulted in chromocenter decondensation. Chromatin complexes with rosette organization (central core from which numerous DNA fibers radiated) were seen. Most likely, the appearance of these rosettes was a consequence of incomplete chromatin extraction. Thus, the nuclear matrix of pericentromeric chromosome regions in cultured murine fibroblasts is morphologically distinguished from the rest of the nuclear matrix.  相似文献   

19.
Structure of the mammalian kinetochore   总被引:27,自引:0,他引:27  
The structure of the mammalian trilaminar kinetocnore was investigated using stereo electron microscopy of chromosomes in hypotonie solutions which unraveled the chromosome but maintained microtubules. Mouse and Chinese hamster ovary cells were arrested in Colcemid and allowed to reform microtubules after Colcemid was removed. Recovered cells were then swelled, lysed or spread in hypotonic solutions which contained D2O to preserve microtubules. The chromosomes were observed in thin and thick sections and as whole mounts using high voltage electron microscopy. Bundles of microtubules were seen directly attached to chromatin, indicating that the kinetochore outer layer represents a differential arrangement of chromatin, continuous with the body of the chromosome. In cells fixed without pretreatment, the outer layer could be seen to be composed of hairpin loops of chromatin stacked together to form a solid layer. The hypotonically-induced unraveling of the outer layer was found to be reversible, and the typical 300 nm thick disk reformed when cells were returned to isotonic solutions. Short microtubules, newly nucleated after Colcemid removal, were found not to be attached to the kinetochore outer layer, but were situated in the fibrous corona on the external surface of the outer layer. This was verified by observations of thick sections in stereo which made it possible to identify microtubule ends within the section. Thus, kinetochore microtubules are nucleated within the fibrous corona, and subsequently become attached to the outer layer. We dedicate this paper to Wolfgang Beermann on the occasion of his 60th birthday in appreciation of many years of friendship and his pioneering contributions in the field of chromosome biology  相似文献   

20.
Summary Extracentromeric chromatin fibers were proposed to hold sister chromatids together in mitotic chromosomes examined by electron microscopy, but their existence in living cells has not been demonstrated yet. We have performed an in vitro BrdU-H33258 treatment which induced a differential rate of condensation to each sister chromatid, thus producing asymmetrically condensing chromosomes. The fast condensing chromatid pulled the slower sister one, both bending in parallel. Bent chromatids appeared reciprocally connected by loops of chromatin fibers, suggesting they were the links which permitted the physical interplay between the differently condensing chromatids. When sister chromatid exchanges (SCE) intercalated a fast-condensing fragment in the slow-condensing chromatid or vice versa, the chromosome inverted its curvature at the SCE-point.  相似文献   

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