Effects of bromodeoxyuridine substitution on metaphase chromosome structures examined by scanning electron microscopy

Chinese hamster chromosomes were differentially substituted with 50 M 5-bromodeoxyuridine (BrdU) to obtain chromosomes with bifilarly and unifilarly substituted (BB-TB) and unifilarly and non-substituted (TB-TT) chromatid constitutions. To avoid the effect of Giemsa staining on the ultrastructure of chromosomes, unstained preparations were exclusively used. When TB-TT chromosomes were prepared with the conventional air-drying method followed by the osmium tetroxide-thiocarbohydrazide (OsO₄-TCH) technique and examined by scanning electron microscopy (SEM), the TB-chromatid appeared somewhat more slender and showed more conspicuous spiral structures, thereby appearing more loosened compared to the TT-chromatid. At higher magnifications, however, 30 nm chromatin fibres which were seen to constitute both chromatids showed no discernible differences in dimension between the TT- and TB-chromatids. On the other hand, TB-TT chromosomes specially prepared for SEM without the process of air-drying appeared in their entirety less extended and no spiral configuration was observed even in the TB-chromatid. The TB-chromatid instead appeared rather less loosened than the TT-chromatid whereas thick fibre-like structures which in turn seemed to consist of 30 nm fibres were more easily discernible in the TT-chromatid compared to the TB. Such seemingly contradictory results obtained from the two different preparatory procedures were tentatively explained on the basis of our multiple coiling model (Taniguchi and Takayama 1986).     

Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 30-nm chromatin structure

How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biology. The nucleosome fibre, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fibre and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, we show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibres rather than 30-nm chromatin fibres. Our comprehensive and quantitative study using cryo-electron microscopy and synchrotron X-ray scattering resolved the long-standing contradictions regarding the existence of 30-nm chromatin structures and detected no regular structure >11 nm. Our finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibres, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures.     

Chromosome organization revealed upon the decondensation of telophase chromosomes inAllium

Chromosomes of root tip cells ofAllium cepa andAllium sativum were studied in early, middle and late telophase to examine the organization of mitotic chromosomes, taking advantage of the naturally occurring chromosome dispersion during the process of decondensation in telophase. Longitudinal and transverse sections of telophase chromosomes viewed under the transmission electron microscope showed that mitotic chromosomes inAllium were composed of helically coiled 400–550 nm chromatin fibres. In some regions of the longitudinal sections, these chromatin fibres were seen to be orientated parallel to one another but formed roughly a right angle to the long axis of the chromosome. In transverse sections, the telophase chromosome appeared to have a hollow centre encircled by the 400–550 nm chromatin fibre which in turn was a hollow tube structure formed by the coiling of a thinner fibre of 170–200 nm. In addition, cross views of chromatin fibres of 170–200 nm and 50–70 nm were also identified in telophase chromosome preparations. These two organizational levels of chromatin fibres also showed a hollow centre. The process of decondensation of telophase chromosomes is described, and some morphological characteristics associated with the activities of chromosome decondensation are analysed. Based on the observations made onAllium chromosomes in this study, various models of chromosome organization are discussed.     

Scanning electron microscopic examination of the secondary constriction of Vero cells

Vero cells (African green monkey kidney in origin) were prepared by the conventional air-drying method and then processed for SEM by a modification of the conductive method based on thiocarbohydrazide-osmium binding [3]. Under SEM, not only metaphase chromosomes but also resting nuclei showed distinct fibres 30 nm in diameter. A few such fibres were found to run across the secondary constriction of the NOR-carrying chromosome.     

红翅皱膝蝗减数分裂染色体的螺旋与轴结构

用低渗处理和苯酚品红染色,在经过卡诺液(甲醇3∶冰醋酸1)固定和未经固定的红翅皱膝蝗减数分裂染色体上都看到了螺旋结构。观察和测量结果表明,每条染色单体都是由430nm左右的染色线螺旋形成的。由染色线到染色体的压缩率为4∶1。低渗处理后固定的材料经过银染,则显示了染色体轴结构。同样,未经低渗处理直接固定的材料银染时也出现了轴结构。银染的轴结构位于每个染色单体的中央,并贯穿整个染色单体。在光镜下,这个轴并不是直径均一的棒状结构,而似乎是由许多大小相近的颗粒相连而成。本文对染色体结构的有关模型、骨架和轴结构的真实性以及轴和螺旋的关系等问题进行了讨论。     

Studies in the Mitosis of Euglena I. On the Chromosome Cycle of Euglena viridis Ehrbg.

The chromosome cycle in the vegetative division of Euglena viridis was investigated. The seeming chromatin granules in the interphase nucleus are in reality thread structures, paired and very loosely twisted. Each component of the paired threads is called a chromatid, and consists of a fine thread of even thickness, the chromonema.
In the prophase, linear contraction and thickening of the chromatids occurs by means of the spiralization of them. In the later prophase, the coiled chromonema splits into two finer strands which show the plectonemic spiral. At the metaphase, the chromosomes are arranged in the form of an equatorial ring, encircling the median portion of the elongated endosome. Nearly all of the chromosomes have a submedian or a sub-terminal and a few of them have a terminal kinetochore. In the early anaphase, separation of the sister chromosomes takes place beginning at the kinetochore. The spindle fibres in the metaphase and anaphase were not observed. The two stranded spiral in the chromosomes is separated into distinct components by the uncoiling in the later telophase, and they are transformed, in the interphase nucleus, into the paired chromatids.     

Structural changes of dinoflagellate chromosomes by pronase and ribonuclease

When cells of the dinoflagellates Prorocentrum micans and Gyrodinium cohnii are exposed to the proteolytic enzyme pronase or alternatively to ribonuclease, the structure of chromosomes is markedly altered. These changes have been observed electron microscopically in thin sections and spreads. Treatment of cells with pronase removed the bulk of nonfibrillar chromosome material completely unmasking fine chromosomal DNA fibres. Pronase had similar effect also on the dense material which is in contact with chromosomes; fibrillar loops protruding from chromosomes were exposed. However, pronase had no effect on the structural integrity of chromosomes. On the contrary, treatment of cells with ribonuclease loosened the package of chromosomal fibres. Thin sections showed that the tight package of longitudinal periodic structures seen in untreated chromosome was relaxed; chromosome extended longitudinally and formed a linear array of balls. When ribonuclease-treated chromosomes were spread, they were substantially more stretched than untreated chromosomes because of uncoiling of two oppositehanded spiral chromatid bundles. The effect of ribonuclease treatment suggests that unknown RNA species have an important role in the maintenance of permanent condensation of dinoflagellate chromosomes. On the other hand, proteins removable by pronase are also present. Most probably they are not linked to the chromosome structure but represent the matrix of nuclear activity.     

Packaging the genome: the structure of mitotic chromosomes

Mitotic chromosomes are essential structures for the faithful transmission of duplicated genomic DNA into two daughter cells during cell division. Although more than 100 years have passed since chromosomes were first observed, it remains unclear how a long string of genomic DNA is packaged into compact mitotic chromosomes. Although the classical view is that human chromosomes consist of radial 30 nm chromatin loops that are somehow tethered centrally by scaffold proteins, called condensins, cryo-electron microscopy observation of frozen hydrated native chromosomes reveals a homogeneous, grainy texture and neither higher-order nor periodic structures including 30 nm chromatin fibres were observed. As a compromise to fill this huge gap, we propose a model in which the radial chromatin loop structures in the classic view are folded irregularly toward the chromosome centre with the increase in intracellular cations during mitosis. Consequently, compact native chromosomes are made up primarily of irregular chromatin networks cross-linked by self-assembled condensins forming the chromosome scaffold.     

草鱼染色体的包装（间期—中期）

以草鱼ＺＣ７９０１细胞株为材料,观察鱼类细胞从间期染色质到中期染色体的包装过程。主要通过（１）分裂期与间期细胞融合,诱导染色体早熟凝集;（２）染色体“伸长”处理;（３）培养细胞的低渗处理;（４）染色质辅展等方法,制作染色体标本,进行扫描和透射电镜观察。观察表明,鱼类染色质的基本结构与哺乳类细胞相同,也是直径约１０ｎｍ的核丝。染色体的色装有两种形式：一种是多级螺旋化形成直径约３００ｎｍ的染色单体,     

Dansyl chloride-stained nucleolar organizers and core-like structures in Chinese hamster metaphase chromosomes

When chromosomes containing both BrdU-substituted and unsubstituted regions were treated with hot NaH₂PO₄ at high or low pH and then stained with dansyl chloride, brightly fluorescent nucleolar organizer regions (NORs) and core-like structures were apparent in the chromosomes. These structures closely parallel the appearance of the same structures in silver-stained chromosomes. Since dansyl chloride is a protein-specific fluorochrome, the distribution of fluorescence suggests that the NORs and central zone of each chromatid contain higher concentrations of protein relative to other chromosome regions. The fluorescent core structures are interpreted to be artefacts of the NaH₂PO₄ pretreatment induced by changes in the concentration of chromatin (including protein) between the chromatin-dense center and more dispersed peripheral region of each chromatid.     

Experimental Visualization of Chromoneme as a Higher Level of Chromatin Compactization in the Mitotic Chromosome

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1–0.2 μm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoneme structure of the mitotic chromosomes in the presence of Ca²⁺. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl₂. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosomes swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca₂₊ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl₂, were treated with a photosensitizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and the general pattern of their packaging in the chromatid was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures. __________ Translated from Ontogenez, Vol. 36, No. 5, 2005, pp. 323–332. Original Russian Text Copyright ? 2005 by Burakov, Tvorogova, Chentsov.     

The ultrastructure of G- and C-banded chromosomes

A method of visualizing chromosome bands by electron microscopy has been used to investigate the fine structural organization of G- and C-banded chromosomes. The following information has been obtained:

1. 1. G-bands, produced by trypsinization, were electron dense regions of highly packed chromatin fibres separated by regions in which the chromatin fibres were much less densely packed (interbands).

2. 2. Several degrees of chromatin dispersion were apparent in trypsinized chromosomes. Such dispersion was not a prerequisite for the initial visualization of G-bands, however the progressive pattern of dispersion indicated that the bands were relatively more resistant to dispersion than the interbands.

3. 3. After fixation and trypsinization, individual chromatin fibres measured 250 Å in diameter and appeared morphologically similar to control chromatin fibres seen by whole mount electron microscopy.

4. 4. In trypsinized chromosome complements, the chromosomes often appeared to be interconnected to one another by chromatin fibres. The evidence indicates that these interchromosomal fibres are artefacts produced by the overlapping of dispersed chromatin fibres.

5. 5. When the same metaphase chromosome was observed by both light and electron microscopy, some of the light microscopic G-bands were represented by two or more ultrastructural bands. The number of bands seen in metaphase chromosomes by electron microscopy appears to approach the increased number of bands generally seen in prometaphase chromosomes by light microscopy.

6. 6. C-banding methods (NaOH treatment or overtrypsinization) resulted in the extraction of variable amounts of chromatin from the non C-band regions of the chromosomes, however the constitutive heterochromatin remained highly condensed and resistant to extraction. This result supports the hypothesis that the mechanism of C-banding involves the selective loss of non C-band chromatin.

     

Metaphase chromosome structure: evidence for a radial loop model.

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.     

Superstructures of wet inactive chromatin and the chromosome surface

Superpacking of chromatin and the surface features of metaphase chromosomes have been studied by SiO replication of wet, unstained, and unfixed specimens in an exceedingly thin (≤ 1 nm) aqueous layer, keeping them wet. Hydrophilic Formvar substrates allow controlled thinning of the aqueous layer covering the wet specimens. Whole mounts of chromatin and chromosomes were prepared by applying a microsurface spreading method to swollen nuclei and mitotic cells at metaphase. The highest level of nucleosome folding of the inactive chromatin in chicken erythrocytes and rat liver nuclei is basically a second-order superhelical organization (width 150–200 nm, pitch distance 50–150 nm) of the elementary nucleosome filament. In unfavorable environments (as determined by ionic agents, fixative, and dehydrating agents) this superstructure collapses into chains of superbeads and beads. Formalin (10%) apparently attacks at discrete sites of chromatin, which are then separated into superbeads. The latter consist of 4–6 nucleosomes and seemingly correspond to successive turns of an original solenoidal coil (width 30–35 nm), which forms the superhelical organization. When this organization is unfolded, eg, in 1–2 mM EDTA, DNAse-sensitive filaments (diameter 1.7 nm) are seen to be wrapped around the nucleosomes. The wet chromosomes in each metaphase spread are held to each other by smooth microtubular fibers, 20–30 nm in diameter. Before they enter into a chromsome, these fibers branch into 9–13 protofilaments, each 5 nm wide. The chromosome surface contains a dense distribution of subunits about 10–25 nm in diameter. This size distribution corresponds to that of nucleosomes and their superbeads. Distinct from this beaded chromosome surface are several smooth, 23–30-nm-diameter fibers, which are longitudinal at the centromere and seem to continue into the chromatid structure. The surface replicas of dried chromosomes do not show these features, which are revealed only in wet chromosomes.     

Silver stained core-like structures in chinese hamster metaphase Chromosomes

Chinese hamster metaphase chromosomes, subjected to prolonged hypotonic pretreatment and subsequently stained with ammoniacal silver, contained a darkly-stained core-like structure in each chromatid, surrounded by a halo of dispersed chromatin which was pale yellow to brown in color. The core was variable in its appearance, ranging from a continuous linear configuration to a spiral structure or a discontinuous, particulate structure. Within the centromeric regions, the cores frequently appeared more intensely stained than elsewhere in the chromosome. The nucleolus organizers also stained darkly and appeared to be attached to the core-like structures. It remains to be determined whether the cores represent a real component of metaphase chromosome structure, or whether they are artifacts resulting from abnormal chromatin aggregation arising at the time of chromosome preparation.     

Extracentromeric connections between sister chromatids demonstrated in human chromosomes induced to condense asymmetrically

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.     

Enhanced Hoechst 33258 fluorescence in plants.

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.     

The integrity of the histone-DNA complex in chromatin fibres is not necessary for the maintenance of the shape of mitotic chromosomes

In this study we addressed the question of whether scaffold structures produced from purified mitotic chromosomes are an artefact of dehistonization, and whether the integrity of the chromatin fibres is necessary for the maintenance of the well-known shape of mitotic chromosomes. Purified mitotic chromosomes from Friend erythroleukemia cells were treated either with increasing NaCl concentrations up to 500 mM, or with 6 M urea in the presence or absence of 10 mM 2-mercaptoethanol. The main criterion for the intactness of the overall chromosome shape as seen by electron microscopy was the characteristic X-or U-like appearance with clearly discernable chromatid axes. Histone H1 is known to be essential for the integrity of chromatin fibres. Its removal in sucrose gradients containing 500 mM NaCl did not lead to loss of the overall chromosome shape. However, treatment of chromosomes in sucrose gradients containing 10 mM 2-mercaptoethanol and 6 M urea led to loss of the structure probably due to dissociation (or denaturation) of shape-determining (scaffolding) components. Under these conditions most of the histones remained bound to the chromosomes, and the fibres in this chromatin material, after removal of excess urea and 2-mercaptoethanol, still showed condensation of the nucleosome filaments into the characteristic fibre structures upon increasing ionic strength. Our observations are compatible with the model that specific non-histone components, independently of histone-DNA interactions, organize or stabilize the structure of metaphase chromosomes.     

Structure of human chromosomes visualized at the electron microscopic level

A newly developed technique allows cytological (light microscope level) chromosome preparations to be examined at the electron microscopic level. Ultrathin (50 nm) sections of highly condensed Hela cell metaphase chromosomes show the characteristic mitotic chromosome morphology. In addition a fibrous network (presumably chromosome fibers) can be seen within them. Fibers appear to be gathered at foci along each chromatid. Treatment of chromosomes with trypsin in a trypsin/G-banding procedure reduces the amount of staining material at the electron microscopic level and results in more prominent foci. Thicker (100 nm) sections of less condensed chromosomes prepared from human lymphocytes display a banding pattern similar to G-banding, even without pretreatment with proteases.     

Structural analysis of heavy ion radiation-induced chromosome aberrations by atomic force microscopy

Heavy ion radiation (high linear energy transfer, LET, radiation) induces various types of chromosome aberration. In this report, we describe a new method employing an atomic force microscope (AFM) for nanometer-level structural analysis of chromosome damage induced by heavy ion irradiation. Metaphase mouse chromosomes with chromatid gap or chromatid breaks induced by heavy ion irradiation were marked under a light microscope. Then the detailed structure of chromosomes of Giemsa-stained or unstained samples was visualized by the AFM. The height data of chromosomes obtained by AFM provided useful information to distinguish chromatid gaps and breaks. A fibrous structure was observed on the unstained chromosome, the average width of which was about 45.8 nm in the image of AFM. These structures were considered to be 30-nm fibers on the chromosome. The structure of the break point regions induced by neon- or carbon-ion irradiation was imaged by AFM. In some cases, the fibrous structure of break points was detected by AFM imaging after carbon ion irradiation. These observations indicated that AFM is a useful tool for analysis of chromosome aberrations induced by heavy ion radiation.