Nucleosomes in metaphase chromosomes.

Previous studies of the structure of metaphase chromosomes have relied heavily on electron micrography and have revealed the existence of a 10-nm unit fiber that is thought to generate the native 23-30-nm fiber by higher order folding. The structural relationship of these metaphase fibers to the interphase fiber remains obscure. Recent studies on the digestion of interphase chromatin have revealed the existence of a regularly repeating subunit of DNA and histone, the nucleosome that generates the appearance of 10-nm beads connected by a short fiber of DNA seen on electron micrographs. It was therefore of interest to probe the structure of the metaphase chromosome for the presence of nucleosomal subunits. To this end metaphase chromosomes were prepared from colchicine-arrested cultures of mouse L-cells and were subjected to digestion with stayphylococcal nuclease. Comparison of the early and limit digestion products of metaphase chromosomes with those obtained from interphase nuclei indicates that although significant morphologic changes occur within the chromatin fiber during mitosis, the basic subunit structure of the chromatin fiber is retained by the mitotic chromosome.     

Immunoelectronmicroscopy of metaphase chromosomes

Summary A method for the preparation of ultrathin sections of metaphase chromosomes is described. This method was applied to human metaphase chromosomes, which were immunocytochemically stained with anti-DNA and anti-ribonucleoprotein antibodies, derived from patients with auto-immune disease. Conventionally prepared metaphase spreads as well as cytocentrifuge preparations of chromosome suspensions were studied. The results indicate that the ultrastructure of chromosomes and the immunoreactivity of chromosomal constituents are influenced by the applied preparation methods. In comparison with whole mount preparations, ultrathin sections of immunostained chromosomes allow higher resolution and more precise localization of immunoreactive sites within the chromosomal structure.     

Immunoelectronmicroscopy of metaphase chromosomes

A method for the preparation of ultrathin sections of metaphase chromosomes is described. This method was applied to human metaphase chromosomes, which were immunocytochemically stained with anti-DNA and anti-ribonucleoprotein antibodies, derived from patients with auto-immune disease. Conventionally prepared metaphase spreads as well as cytocentrifuge preparations of chromosome suspensions were studied. The results indicate that the ultrastructure of chromosomes and the immunoreactivity of chromosomal constituents are influenced by the applied preparation methods. In comparison with whole mount preparations, ultrathin sections of immunostained chromosomes allow higher resolution and more precise localization of immunoreactive sites within the chromosomal structure.     

Identification of isolated metaphase chromosomes. II. A comparison with the recognizability of chromosomes in metaphase plates

The metaphase chromosomes (MC) isolated from the Chinese hamster cells were identified with the aid of differential staining (G-bands). It was shown that differences in the relative recognizability of MC in metaphase plates and after their isolation are determined by changes in composition of isolated MC, rather than by those in staining capacity of MC after their isolation. The frequencies of identified MC are constant and independent upon the type of MC preparations and relation between identified and unidentified MC in certain preparations. At allows to apply the described method for the analysis of chromosome fractionation, using changes in frequencies of identified MC as a criterion of efficiency of the fractionation method. Possible ways of increasing the recognizability level of isolated MC are discussed.     

Dense chromatin plates in metaphase chromosomes

In a previous work we observed multilayered plate-like structures surrounding partially denatured HeLa chromosomes at metaphase ionic conditions. This unexpected finding has led us to carry out an extensive investigation of these structures. Our results show that plates can also be found in metaphase chromosomes from chicken lymphocytes. We have used atomic force microscopy (AFM) to image and investigate the mechanical properties of plates in aqueous solution. Plates are thin (~6.5 nm each layer) but compact and resistant to penetration by the AFM tip: their Young’s modulus is ~0.2 GPa and the stress required for surface penetration is ~0.03 GPa in the presence of Mg²⁺ (5–20 mM). Low-ionic strength conditions produce emanation of chromatin fibers from the edges of uncrosslinked plates. These observations and AFM results obtained applying high forces indicate that the chromatin filament is tightly tethered inside the plates. Images of metal-shadowed plates and cryo-electron microscopy images of frozen-hydrated plates suggest that nucleosomes are tilted with respect to the plate surface to allow an interdigitation between the successive layers and a thickness reduction compatible with the observed plate height. The similarities between denatured plates from chicken chromosomes and aggregates of purified chromatin from chicken erythrocytes suggest that chromatin has intrinsic structural properties leading to plate formation. Scanning electron micrographs and images obtained with the 200-kV transmission microscope show that plates are the dominant component of compact chromatids. We propose that metaphase chromosomes are formed by many stacked plates perpendicular to the chromatid axis.     

Higher order structure in metaphase chromosomes

The morphology of metaphase chromosome-derived chromatin fibers released from cells by non-ionic detergent cell lysis in the presence of divalent cations has been studied by electron microscopy. In these preparations the euchromatic arms appear as a series of loops, 200–300 Å in diameter, which are composed of closely-apposed nucleosome arrays. The higher order fiber in chromosomes derived from detergent-lysed cells appears to be less stable than chromatin fibers obtained by mechanical cell lysis. The fiber breaks down into a series of non-uniform nucleosome aggregates (superbeads) and finally to chromatin in a beads-on-a-string morphology upon incubation at 31° for 20 min. These observations allow us to suggest a relationship between uniform thick fibers, superbead-containing fibers, and beads-on-a-string chromatin within metaphase chromosomes.     

Photographic equidensitometry of metaphase chromosomes

Summary Photographic equidensitometry, which is a procedure to obtain specific lines or symbols for points of equal optical density, has been utilized in the study of chromosome images. Equidensitometric techniques, using a special contour film, permitted three approaches, namely, production of line equidensities (in the form of families, sequences and contour maps), color equidensities (color conversion of line sequences), and screen equidensities (substituting characteristic symbols for densities). All these techniques have proved very useful to analyze images of metaphase chromosomes and occurrence of spontaneous banding patterns, by showing the precise distribution and relative values of the grey gradient. This report demonstrates the potential of photographic equidensitometrical procedures for chromosome studies, which obviates the need to purchase elaborate equipment.     

Scaffold morphology in histone-depleted HeLa metaphase chromosomes

In a study of HeLa metaphase chromosomes depleted of histories with 2 M NaCl and spread with cytochrome c, two new types of images of chromosome scaffolds have been observed in the electron microscope. In the first type, scaffolds are very large and fibrous but still display the shape typical of metaphase chromosomes. The regularity and lack of distortion in these scaffolds, despite their openness and seeming fragility, support the notion that the underlying scaffold structure is an interconnected network of fibers. In the second type, fibrous regions and dense regions are juxtaposed in the same chromosome scaffold. These micrographs suggest that the dense appearance of some previously observed scaffolds may be the result of incomplete adherence to the cytochrome c monolayer, leading to collapse and aggregation during dehydration.     

X-ray diffraction from isolated metaphase chromosomes

Metaphase chromosomes were isolated from Chinese hamster fibroblasts. They were found to give the same characteristic diffraction pattern in the wet state as isolated nucleohistone but when dry did not give the reflections normally associated with dry nucleohistone. No higher structural order than that associated with the nucleohistone supercoil was detected.     

Reactivity of anti nucleoside antibodies with metaphase chromosomes.

It is shown that the binding of anti-nucleoside antibodies to fixed human metaphase chromosomes can be revealed using the immunoperoxidase procedure while under the same conditions no antibody binding is revealed using the immunofluorescence procedure.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

Silver staining of histone-depleted metaphase chromosomes

To investigate a possible relationship between the core-like structures seen in silver-stained chromosomes (prepared by standard cytogenetic methods) and the scaffolds observed in histone-depleted chromosomes, the ability of the scaffold to stain with silver has been examined. Isolated chromosomes were histone-depleted by washing in ammonium acetate or by spreading the chromosomes on an ammonium acetate hypophase. The residual chromosome structures were carbon-platinum shadowed or stained with silver, and then examined by electron microscopy. The results provide clear evidence that the scaffold structure has a high affinity for silver and is therefore similar in its silver-staining potential to the core structure in standard chromosomes. This suggests that the silver core in standard chromosomes may represent the scaffold visualized by histone depletion. The peripherally dispersed DNA radiating from the scaffold also proved to be silver-reactive, and additional experiments demonstrated that purified DNA is capable of binding silver. This result indicates that cytological silver staining is not simply a matter of staining protein, as has previously been thought, but may also involve the staining of chromosomal DNA. In the ammonium acetate-treated and carbon-platinum-shadowed preparations, the scaffold structure was highly variable in its morphology and appeared to be composed of undispersed or incompletely dehistonized chromatin fibers. The silver-stained scaffold reflected this variability. Taken together with other evidence, these findings lead to a questioning of the reality of chromosome core structures.     

The structure of histone-depleted metaphase chromosomes

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.     

Selective digestion of mouse metaphase chromosomes

Metaphase chromosomes prepared from colcemid-treated mouse L929 cells by non-ionic detergent lysis exhibit distinct heterochromatic centromere regions and associated kinetochores when viewed by whole mount electron microscopy. Deoxyribonuclease I treatment of these chromosomes results in the preferential digestion of the chromosomal arms leaving the centromeric heterochromatin and kinetochores apparently intact. Enrichment in centromere material after DNase I digestion was quantitated by examining the increase in 10,000xg pellets of the 1.691 g/cc satellite DNA relative to main band DNA. This satellite species has been localized at the centromeres of mouse chromosomes by in situ hybridization. From our analysis it was determined that DNase I digestion results in a five to six-fold increase in centromeric material. In contrast to the effect of DNase I, micrococcal nuclease was found to be less selective in its action. Digestion with this enzyme solubilized both chromosome arms and centromeres leaving only a small amount of chromatin and intact kinetochores.     

Isolation of specific human metaphase chromosomes

The human karyotype can be subdivided into seven fractions containing specific chromosomes to provide material for recombinant DNA research. The isolated metaphase chromosomes are sorted according to size by velocity zonal centrifugation, and specific chromosome groups are further purified by electrostatic deflection in a flow microfluorometer. Rapid improvements in technology should soon provide preparations of single chromosomes.