Immunofluorescent staining of human metaphase chromosomes with monoclonal antibody to histone H2B

A mouse monoclonal IgM antibody against the core histone H2B has been shown, by indirect immunofluorescence, to stain metaphase chromosomes from a variety of cultured cell types. Experiments carried out with human HeLa cells showed that the intensity of staining varied along the length of chromosome arms giving in some cases a rudimentary banded staining pattern. Considerable variation in staining intensity was noted between individual chromosomes and between different metaphase spreads. It was noted that chromosomes having a more swollen appearance stained more intensely than those with a more compact structure, which were often unstained. Preincubation of unfixed metaphase chromosomes in buffered salt solutions virtually eliminated the cell to cell and chromosome to chromosome variation in staining, even when no visible effect on chromosome morphology was caused by such treatment. It is concluded that the determinant recognised by antibody HBC-7 is ubiquitous but is inaccessible in some chromosomes or chromosome regions. Digestion of purified chromatin (primarily interphase) with DNAase 1 or micrococcal nuclease resulted in a several-fold increase in the binding of antibody HBC-7 measured by solid-phase radioimmunoassay. This increase was abolished by subsequent treatment with trypsin, which suggests that the antigenic determinant recognised by antibody HBC-7 lies in the trypsin-sensitive N-terminal region of nucleosomal H2B. As the cationic N-terminal regions of the core histones are involved in DNA binding, it is likely that the accessibility of the determinant recognised by antibody HBC-7 is influenced by the relationship between the core histones and their associated DNA.     

红翅皱膝蝗减数分裂染色体轴的形成与联会复合体

本文通过延长低渗处理、压片和硝酸银染色技术,对红翅皱膝蝗减数分裂中期Ⅰ染色体轴的形成过程及其联会复合体(Synaptonemal complex,SC)与染色体轴形成的关系进行了研究。我们的结果表明,中期Ⅰ染色体轴是在晚双线期到终变期的过程中逐渐在染色体中形成的。染色体轴形成的动态行为,一方面暗示了这种结构在染色体集缩和维持中期染色体的形态方面起某种重要作用;另一方面说明了轴是染色体中存在的一种真实结构。同时,本文的结果还指出,SC在早双线期到中双线期就解体了,而中期Ⅰ染色体轴是在晚双线期才开始形成。这两种轴结构之间很明显不是连续的。染色体轴的形成与SC的侧轴无直接的相关性。它们是减数分裂染色体中先后出现的两种不同的轴结构。     

Chinese hamster metaphase chromosomes isolated under physiological conditions. A partial characterization of associated non-histone proteins and protein cores

In a previous report [2] we have described a non-histone protein core which could be isolated from Chinese hamster metaphase chromosomes. This core structure maintained the overall morphology of the metaphase chromosome even after removal of all of the histones, together with many of the non-histone proteins and the bulk of the DNA. As part of our work on the characterization of these core structures, we have developed a novel procedure for the isolation of metaphase chromosomes which avoids the use of high pH buffers and hexylene glycol, as well as eliminating the numerous centrifugation and resuspension steps previously employed. Chromosome cores prepared by 2 M NaCl extraction and DNase I digestion from metaphase chromosomes isolated under these more gentle, quasi-physiological conditions, are shown to contain a relatively simple subset of non-histone proteins. One-dimensional SDS-polyacrylamide gel electrophoresis shows two major groups of polypeptides having molecular weights 48 000-52 000 and 65 000-72 000 D respectively, with similarities in mobilities to the nuclear pore complex-lamina polypeptides and tubulins. However, more detailed analysis by two-dimensional gel electrophoresis and peptide mapping has failed to detect these proteins. A 52 000 D polypeptide component of the core is tentatively identified as the intermediate filament protein vimentin. The in vivo significance of chromosome cores is discussed.     

Detection of post-repair DNA damage and heterogeneity of its distribution in fractions of metaphase chromosomes of various sizes

The distribution of single-strand DNA breaks induced by various N-methyl-N-nitrosourea concentrations in S-phase of the cell cycle has been studied in Djungarian hamster fibroblast culture in the isolated samples of fractions of metaphase chromosomes of varying size. For the first time, a dose-effect relationship between the molecular mass of single-stranded DNA fragments isolated in an alkaline sucrose gradient from small chromosomes and the lack of the effect on DNA in large chromosomes has been established. The phenomenon detected is being discussed in terms of structural heterogeneity of interphase nuclear chromatin, i.e. irregular distribution of eu- and heterochromatin in small and large chromosomes. Another possible explanation is the storage of various damaged chromosome fragments in small chromosomes. The data obtained, apart from their significance for chromatin structure analysis, can serve as an experimental basis for the detection of, otherwise undetectable, postreparative molecular DNA damages.     

STUDIES ON THE ISOLATION OF METAPHASE CHROMOSOMES

A method for the isolation of metaphase chromosomes from mouse L1210 leukemia cells has been developed. Cells, arrested at metaphase with colchicine, were exposed to hypotonic solution and the pH was then adjusted to 5.6 to stabilize the chromosomes. The metaphase figures were subsequently disrupted and the chromosomes isolated by a series of differential centrifugations in sucrose. The isolated chromosomes were well preserved, as judged by morphological criteria. The effect of various enzymes and chemical agents on the isolated chromosomes was studied. Chymotrypsin, trypsin, and deoxyribonuclease caused a marked disintegration of the chromosomes, whereas treatment with pepsin and ribonuclease induced no significant morphological alterations.     

Mitotic and meiotic chromosomes in Somatochlora metallica (Cordulidae,Odonata). The absence of localized centromeres and inverted meiosis

Spermatogonial metaphase chromosomes were examined in two dragonfly species, Somatochlora metallica (Cordulidae) and Aeshna grandis (Aeshnidae), and the behaviour of male meiotic chromosomes was studied in S. metallica. Both in S. metallica and A. grandis the male mitotic metaphase chromosomes from cells treated with colchicine consisted of two equidistantly aligned chromatids, showing no primary constriction. In meiosis the chromosomes of S. metallica males showed telokinetic activity during the first meiotic division, and kinetic activity was restricted in the middle parts of chromosomes during the second division. The kinetic behaviour of the chromosomes both in mitosis and meiosis showed that they were holocentric. One chiasma arises interstitially in each bivalent in S. metallica male meiosis. The chiasmata retain their interstitial position at metaphase I and do not terminalize. At metaphase I bivalents co-orient with homologous telomere regions towards the opposite poles. Thus genuine dyads segregate at the first anaphase. Meiosis in these male dragonflies is thus pre-reductional or conventional, not post-reductional or inverted, as has been previously proposed.     

Self-assembly of thin plates from micrococcal nuclease-digested chromatin of metaphase chromosomes

The three-dimensional organization of the enormously long DNA molecules packaged within metaphase chromosomes has been one of the most elusive problems in structural biology. Chromosomal DNA is associated with histones and different structural models consider that the resulting long chromatin fibers are folded forming loops or more irregular three-dimensional networks. Here, we report that fragments of chromatin fibers obtained from human metaphase chromosomes digested with micrococcal nuclease associate spontaneously forming multilaminar platelike structures. These self-assembled structures are identical to the thin plates found previously in partially denatured chromosomes. Under metaphase ionic conditions, the fragments that are initially folded forming the typical 30-nm chromatin fibers are untwisted and incorporated into growing plates. Large plates can be self-assembled from very short chromatin fragments, indicating that metaphase chromatin has a high tendency to generate plates even when there are many discontinuities in the DNA chain. Self-assembly at 37°C favors the formation of thick plates having many layers. All these results demonstrate conclusively that metaphase chromatin has the intrinsic capacity to self-organize as a multilayered planar structure. A chromosome structure consistent of many stacked layers of planar chromatin avoids random entanglement of DNA, and gives compactness and a high physical consistency to chromatids.     

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.     

Localization and development of kinetochores and a chromatid core during meiosis in grasshoppers

Positive staining of kinetochores and a chromatid core has been achieved using a simplified silver staining method in squash preparations from meiotic chromosomes of grasshoppers. This technique permits the exact localization of kinetochores on the chromosomes whether metacentric, acrocentric or ‘telocentric’. The sister kinetochores can be observed from mid-diplotene stages but they are not differentiated during first meiotic metaphase. However they can be observed again at the onset of anaphase 1. The existence of a positively stained chromatid core in meiotic divisions is also reported. This core appears well defined inside each chromatid from diplotene to the end of the second meiotic division. The visualization of these cores in first meiotic metaphase clearly shows the points at which the chiasmata took place.     

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.     

Calreticulin as a new histone binding protein in mitotic chromosomes

Calreticulin (CRT) is a multifunctional Ca(2+)-binding protein that mainly functions in the endoplasmic reticulum as a molecular chaperone for newly synthesized proteins. Recently we reported the protein composition of human metaphase chromosomes (Uchiyama et al., 2004), which included CRT. Here we describe new characteristics of CRT in vitro as well as its localization on the surface of metaphase chromosomes in vivo. CRT was detected in the chromosomal fraction by Western blotting and its binding partners were identified as core and linker histones by ligand overlay assay. Surface plasmon resonance sensor analyses revealed that CRT is bound to chromatin fibers. Moreover, we found that CRT has both supercoiling activity, which assists core histone assembly into chromatin fibers, and binding ability to histone H2A/H2B dimers and histone H3/H4 tetramers. Unlike the chromosome scaffold proteins, indirect immunofluorescent staining revealed that CRT is located on the surface of metaphase chromosomes. These results suggest that CRT plays a role which involves chromatin dynamics on the surface of mitotic chromosomes.     

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.     

Skeletor, a novel chromosomal protein that redistributes during mitosis provides evidence for the formation of a spindle matrix

A spindle matrix has been proposed to help organize and stabilize the microtubule spindle during mitosis, though molecular evidence corroborating its existence has been elusive. In Drosophila, we have cloned and characterized a novel nuclear protein, skeletor, that we propose is part of a macromolecular complex forming such a spindle matrix. Skeletor antibody staining shows that skeletor is associated with the chromosomes at interphase, but redistributes into a true fusiform spindle structure at prophase, which precedes microtubule spindle formation. During metaphase, the spindle, defined by skeletor antibody labeling, and the microtubule spindles are coaligned. We find that the skeletor-defined spindle maintains its fusiform spindle structure from end to end across the metaphase plate during anaphase when the chromosomes segregate. Consequently, the properties of the skeletor-defined spindle make it an ideal substrate for providing structural support stabilizing microtubules and counterbalancing force production. Furthermore, skeletor metaphase spindles persist in the absence of microtubule spindles, strongly implying that the existence of the skeletor-defined spindle does not require polymerized microtubules. Thus, the identification and characterization of skeletor represents the first direct molecular evidence for the existence of a complete spindle matrix that forms within the nucleus before microtubule spindle formation.     

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.     

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.     

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.     

Chromosome length and DNA loop size during early embryonic development of Xenopus laevis

The looped organization of the eukaryotic genome mediated by a skeletal framework of non-histone proteins is conserved throughout the cell cycle. The radial loop/scaffold model envisages that the higher order architecture of metaphase chromosomes relies on an axial structure around which looped DNA domains are radially arranged through stable attachment sites. In this light we investigated the relationship between the looped organization and overall morphology of chromosomes. In developing Xenopus laevis embryos at gastrulation, the bulk of the loops associated with histone-depleted nuclei exhibit a significant size increase, as visualized by fluorescence microscopy of the fully extended DNA halo surrounding high salt treated, ethidium bromide stained nuclei. This implies a reduction in the number of looped domains anchored to the supporting nucleoskeletal structure. The cytological analysis of metaphase plates from acetic acid fixed whole embryos, carried out in the absence of drugs inducing chromosome condensation, reveals a progressive thickening and shortening of metaphase chromosomes during development. We interpret these findings as a strong indication that the size and number of DNA loops influence the thickness and length of the chromosomes, respectively. The quantitative analysis of chromosome length distributions at different developmental stages suggests that the shortening is timed differently in different embryonic cells.     

Distribution of human chromosomes on the metaphase plate using banding techniques

Summary This study deals with the problem of distribution of the 46 centromeres on the human metaphase plate after treatment with colchicine and hypotonic shock.The location of the centromeres was recorded for 400 metaphases in which the chromosomes were identified by bands.A quantitative analysis of chromosome distribution ascertains the absence of proximities between homologous chromosomes. The already known ones between acrocentric chromosomes are found at a very high level. The nature and intensity of these proximities vary from one sample to another as is the case between heterologues.A three-dimensional graphic technique is proposed to summarize all of the interchromosome proximities.     

A new method for the preparation of metaphase chromosomes for flow analysis

A new method for the preparation of metaphase chromosomes for flow analysis has been evaluated. It has been shown that this method, which involves detergent lysis of metaphase cells and polyamines to stabilize the DNA, yields lower coefficients of variation and background levels in the DNA histograms than is currently obtained by hexylene glycol based methods. A conventional flow cytometer (FACS-II) has been used to resolve the human karyotype into about 14 peaks after ethidium bromide staining and excitation with a relatively low level of illumination (0.4 W at 488 nm). Flow karyotypes have also been obtained from suspension cell lines, in particular from the mouse cell line, Friend 707/B10. The only disadvantage of this method is that the chromosomes are highly condensed and therefore banding studies on sorted chromosomes may not be possible.