Features of structural organization of mouse centrometric heterochromatin, detected during differential decondensation of chromosomes

The centromeric heterochromatin (CH) of mouse metaphase and interphase chromosomes has been shown to be practically devoid of the chromonemal and chromomeric levels of DNP organization. CH decondensation into DNP-fibrils caused by decreasing Ca2+-ions concentration is accomplished without formation of chromonemata and chromomeres. The peripheral regions of CH, immediately contacting the inner surface of kinetochores, display the highest stability towards the factors inducing the artificial decondensation.     

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.     

The dynamics of the structural reconstruction of mitotic chromosomes after their artificial decondensation in vitro

The treatment of isolated metaphase chromosomes with 5 mM Tris buffer caused their decondensation into DNP fibers 10 nm in diameter. The following increase in CaCl2 concentration induced the transition of nucleosomic DNP fibers into DNP fibers 20 nM and 40-50 nM in diameter, and the recovery of the whole chromosomes. However, in the similar conditions, the typical chromosomes (threads about 100 nm thick), chromomeres and G-bands were not reconstructed. According to these data, we assume that DNP threads 40-50 nm in diameter may be artificial (i.e. "pseudochromonemes"). The treatment of isolated chromosomes with 0.35 and 0.6 M NaCl prevents from formation of nucleomeric and pseudochromomeric fibers, although bodies of chromosomes can be recovered after the removal of HMG and H1 proteins. These observations point to a high stability of chromosomal fasteners providing the structural integrity of mitotic chromosomes.     

Isolation and structural organization of human mitotic chromosomes

New methods are presented for the bulk isolation of metaphase chromosomes from HeLa cells, and an electron microscopic study of thin sections of these chromosomes is presented. The techniques for chromosome isolation were developed to utilize solution conditions that are as mild as possible, so that further biochemical and structural studies can be directly related to the in situ state of chromosomes. — Electron micrographs of thin sections of isolated HeLa metaphase chromosomes reveal the general organization of the nucleosome-containing fibers. Chromosomes in isolation buffer show a dense, relatively uniform distribution of material across the chromatids. Swollen chromosomes reveal the primary mode of organization of the fibers to be a radial distribution from the central axes of the chromatids. A significant proportion of the fibers could also be oriented longitudinally.     

The structural organization of mouse metaphase chromosomes

The binding of highly purified anti-nucleoside antibodies to mouse (Mus musculus) metaphase chromosomes was studied by an immunofluorescence technique. The chromosomal DNA was denatured by one of two selective denaturation procedures because these antibodies reacted with single stranded but not native DNA. After ultraviolet irradiation (UV), which produced single stranded regions primarily in AT rich DNA, the binding of antiadenosine (anti-A) produced a pattern of fluorescent bands similar to that produced by quinacrine (Q-bands). Additional foci of bright fluorescence were observed at the centrometric (C-band) regions, which are known to contain AT rich satellite DNA. After photooxidation, which produced single stranded regions in GC rich DNA, the binding of anti-A produced a fluorescent banding pattern similar to the R-banding pattern seen after thermal denaturation and staining with coriphosphine O. After photooxidation, R-band patterns were also obtained with anti-cytidine (anti-C) and anti-5-methylcytidine (anti-M). After either UV irradiation or photooxidation, anti-M, but not anti-C, showed intense binding to the C-band regions of mouse chromosomes. — These findings led to the following conclusions: (1) Antibody banding patterns reflect the presence of a class of AT rich, GC poor DNA in chromosome regions which show bright quinacrine fluorescence and in the regions that contain the AT rich satellite DNA. (2) The alternate, quinacrine dull regions contain a relatively GC rich class of DNA which appears to be more highly methylated than the AT rich DNA in the Q-bright bands, but not the AT rich satellite DNA in the Q-dull C-bands. (3) 5-Methylcytosine residues occur in a sequence of mouse satellite DNA that contains both adjacent pyrimidines and guanine residues. The basic repeating unit of mouse satellite DNA is known to contain the sequence 5-GAAAAATGA-3 (Biro et al., 1975). Therefore, assuming the antibodies used could detect single bases in denatured DNA, the methylated sequence in mouse satellite DNA      

Visualization of R-bands in human metaphase chromosomes by the restriction endonuclease MseI

Human metaphase chromosomes were treated with the restriction endonuclease MseI, which cuts DNA at TTAA sequences. This enzyme preferentially cuts and extracts DNA from G-bands and thus is the first restriction endonuclease allowing direct R-band visualization. Specific patterns ranging from R+C-like to C-like banding can be induced, depending on the concentration of the enzyme. At intermediate concentrations, only a subset of R-bands are produced, corresponding to GC-rich bands that are especially resistant to heat denaturation (so-called T-bands). These results suggest that compositional differences between chromosomal regions determine the different rates of cleavage by MseI, not only between R- and G-bands but also among different R-bands.     

High resolution R-bands produced in equine chromosomes after incorporation of bromodeoxyuridine

Cell synchronization was used to obtain an adequate percentage of very long chromosomes in equine mitotic spreads. Reported here is our variation, adapted to horse chromosomes, of a method using excess thymidine followed by bromodeoxyuridine incorporation. This technique routinely yields excellent quality cells, predominantly in prometaphase and prophase. Among other differences with the standard technique, this method does not use Colcemid, which, in addition to inhibiting spindle fiber formation, also increases chromosome contraction resulting in thicker and thus fewer bands. Consequently, horse prometaphase chromosomes, which have incorporated BrdU in the late-S-phase, are very long and display a large number of R-bands after the fluorescence-photolysis-Giemsa method. This technique should definitely be useful for the analysis of structural anomalies and the standardization of equine R-bands.     

Reversible differential decondensation of unfixed Chinese hamster chromosomes induced by change in calcium ion concentration of the medium

Differential decondensation of isolated unfixed Chinese hamster metaphase chromosomes was obtained by decreasing the calcium ion concentration in the surrounding medium. A banded appearance of the swollen chromosomes could be observed either directly by phase contrast microscopy or after glutaraldehyde fixation and staining. There was a gradual transition from homogeneously dense to banded and finally to extensively decondensed chromosomes. The patterns induced at different stages were similar to those observed on fixed chromosomes after standard banding procedures (i.e., G-, C-, C_d–, Ag-NOR-staining). Chromosome decondensation could be reversed by the addition of calcium ions to the medium. Ca⁺⁺-dependent reversible differential chromosome decondensation was not observed if the chromosomes were previously treated with 0.35 M NaCl. Chromosome regions which had incorporated BrdU into their DNA were more resistant to a decrease in calcium ion concentration than BrdU non-substituted regions.     

The effect of SH-SS transition in the structural organization of mitotic chromosomes

Summary Human lymphoid cell cultures were treated with 2,2-dithiodipyridine, a thiol reacting agent which produces SS bridges inside the living cells. After 1 to 4h treatment chromosome preparations were made. The chromosomes acquired a peculiar segmentation along the chromatids. The results suggest that the alteration might arise from a direct reaction of the agent with special chromosomal proteins. The resistance of treated chromosomes to alkali-heat treatment used in C-banding technique was different from that of untreated chromosomes. This prefixational procedure seems to be advantageous in analysing both the chromosome organization and the banding mechanism.     

Comparative studies on the structural organization of membrane-depleted nuclei and metaphase chromosomes

Interphase membrane-depleted nuclei and metaphase chromosomes were prepared in parallel with a nonionic detergent lysis procedure at low ionic strength. By flow microfluorometry we showed for the first time that cell lysates contain all stages of the cell cycle in the same proportions as the starting cell population. Morphologically intact membrane-depleted nuclei and metaphase chromosomes were isolated as non-aggregated structures on sucrose gradients. When analysed in the electron microscope, membrane-depleted nuclei that had been treated with 2M NaCl appeared as residual structures containing the pore complex-lamina layer attached to a halo of DNA filaments. In contrast, no distinct high salt-resistant structure was found with metaphase chromosomes. They formed a highly fragile network which disintegrated easily into small complexes connected with DNA filaments. High salt-resistant DNA-protein complexes were purified by Metrizamide density gradient centrifugation. The main difference in the protein composition of interphase and metaphase residual complexes was the presence in interphase of a protein triplet in the 60–75 kilodalton molecular weight range and its absence in metaphase. This protein triplet most likely corresponds to the lamins A, B, and C of the nuclear lamina. The combined results suggest that the main difference in the structural organization of interphase nuclei and metaphase chromosomes is the presence or absence of the pore complex-lamina layer.     

A physical model for the condensation and decondensation of eukaryotic chromosomes

During the eukaryotic cell cycle, chromatin undergoes several conformational changes, which are believed to play key roles in gene expression regulation during interphase, and in genome replication and division during mitosis. In this paper, we propose a scenario for chromatin structural reorganization during mitosis, which bridges all the different scales involved in chromatin architecture, from nucleosomes to chromatin loops. We build a model for chromatin, based on available data, taking into account both physical and topological constraints DNA has to deal with. Our results suggest that the mitotic chromosome condensation/decondensation process is induced by a structural change at the level of the nucleosome itself.     

Ultrastructure of the mitotic chromosomes in pig embryonic kidney cells during their reversible artificial decondensation in vivo

Using methods of in vivo observation and ultrathin sectioning, it is shown that chromosomes of metaphase PE cells, previously treated with diluted Henk's solutions (70, 30 and 15%), undergo some structural transitions resulting in the formation of micronuclei. At the early stages of hypotonic treatment chromosomes are seen considerably swollen and losing the higher levels of organization, including the chromonema and chromomeres. The chromosomal bodies are formed by DNP fibers 10-25 nm in diameter making loops radiating from the central part of the chromatids. Chromosomes are capable of recondensing from this state by consecutive reconstitution of G-bands, chromomeres and the chromonema. The subsequent secondary decondensation of chromosomes is analogous to telophase decondensation at the normal mitosis, but it results in the formation of a great number of small nuclei (micronuclei). The chromatin structure in micronuclei as well as their ability to synthesize RNA and to replicate DNA show these effects to be reversible. It has been suggested that the loop organization of DNP may be essential for sustaining the structural integrity of the mitotic chromosome.     

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.

     

Differential decondensation of mitotic chromosomes during the hypotonic treatment of cells as a possible cause of G-banding

Chinese hamster metaphase chromosomes were studied after treating the cells with a hypotonic 0.075 M KCl solution, routinely used for differential staining of chromosomes. After the incubation of cells in KCl for 5 seconds-40 minutes and fixation with glutaraldehyde, the chromosomes displayed DNP fibrils about 20 nm in diameter. These fibrils were unevenly packed along the chromosomal arms and formed sites of differential density. In sister chromatids, the even density sites were located symmetrically. The use of serial ultrathin sections of marker chromosomes (e.g., chromosomes with a telomeric disposition of nucleolar organizing regions) made it possible to establish a good correlation between the sites with the light packing of DNP-fibrils and G-bands, identified in the same chromosomes by the standard Giemsa-staining technique. Fixation of the KCl-incubated cells with the methanol: glacial acetic acid (3:1) solution did not change the DNP packing heterogeneity. The chromosomal banding state is reversible, for with the transfer of cells from KCl to the normal cultural medium the chromosomes become homogeneous in length. Thus, the data obtained allow to propose that the capacity of chromosomes for differential staining may be based on an uneven sensitivity of G- and R-bands to the decondensing effect of hypotonic treatment.     

Cathepsin L inhibitor I blocks mitotic chromosomes decondensation during cleavage cell cycles of sea urchin embryos

We have previously reported that sperm histones (SpH) degradation after fertilization is catalyzed by a cystein-protease (SpH-protease). Its inhibition blocks the degradation of SpH in vivo and also aborts sea urchin development at the initial embryonic cell cycles. It remains unknown if this effect is a consequence of the persistence of SpH on zygotic chromatin, or if this protease is involved per-se in the progression of the embryonic cell cycles. To discriminate among these two options we have inhibited this protease at a time when male chromatin remodeling was completed and the embryos were engaged in the second cell cycle of the cleavage divisions. The role of this enzyme in cell cycle was initially analyzed by immuno-inhibiting its SpH degrading activity in one of the two blastomeres after the initial cleavage division, while the other blastomere was used as a control. We found that in the blastomere injected with the anti-SpH-protease antibodies the cytokinesis was arrested, the chromatin failed to decondense after mitosis and BrdU incorporation into DNA was blocked. Since the N-terminal sequence and the SpH protease was homologous to the cathepsin L (Cat L) family of proteases, we subsequently investigated if the deleterious effect of the inhibition of this protease is related to its Cat L activity. In this context we analyzed the effect of Cat L inhibitor I (Z-Phe-Phe-CH(2)F) on embryonic development. We found that the addition of 100 uM of this inhibitor to the embryos harvested at the time of the initial cleavage division (80 min p.i.) mimics perfectly the effects of the immuno-inhibition of this enzyme obtained by microinjecting the anti-SpH-protease antibodies. Taken together these results indicate that the activity of this protease is required for embryonic cell cycle progression. Interestingly, we observed that when this protease was inhibited the chromatin decondensation after mitosis was abolished indicating that the inhibition of this enzyme affects chromosomes decondensation after mitosis.     

Reversible hypercondensation and decondensation of mitotic chromosomes studied using combined chemical-micromechanical techniques

We show that the chromatin in mitotic chromosomes can be drastically overcompacted or unfolded by temporary shifts in ion concentrations. By locally 'microspraying' reactants from micron-size pipettes, while simultaneously monitoring the size of and tension in single chromosomes, we are able to quantitatively study the dynamics of these reactions. The tension in a chromosome is monitored through observation and calibration of bending of the glass pipettes used to manipulate the chromosomes. For concentrations > 500 mM of NaCl and > 200 mM of MgCl2, we find that the initially applied tensions of approximately 500 pN relax to zero and that mitotic chromatin temporarily disperses in agreement with previous work (Maniotis et al. [1997] J. Cell. Biochem. 65:114-130). This unfolding occurs in about 1 s, and is reversible once the charge density is returned to physiological levels, if the exposure is not longer than approximately 1 min. Low concentrations of NaCl (< 30 mM) also induces a decrease in tension and increase in size. We observe this swelling to be isotropic in experiments on chromosomes under zero tension, a behavior inconsistent with the existence of a well-defined central chromosome 'scaffold'. By contrast 10 mM of divalent cations (MgCl2 and CaCl2) induces an extremely rapid and reversible increase in tension and a reduction in the size of mitotic chromosomes. Hexaminecobalt trichloride (trivalent cation) has the same effect as MgCl2 and CaCl2, except the magnitude of force increase and size change are much larger. Hexaminecobalt trichloride reduces mitotic chromosomes to 65% of their original volume, indicating that at least 1/3 of their apparent volume is aqueous solution. These results indicate that chromatin inside mitotic chromatids has a large amount of conformational freedom allowing dynamic unfolding and refolding and that charge interactions play a central role in maintaining mitotic chromosome structure.     

Centromere organization in chromosomes of the mouse

The ultrastructure of the centromere region of chromosomes from mouse L929 cells treated with agents that affect centromere condensation have been examined using light, transmission electron, and scanning electron microscopic techniques. Micrographs of expanded centromeres from treated chromosomes illustrate that both the biarmed chromosomes that were generated by Robertsonian fusion during the past history of the strain and the functional centromere of the multicentromeric marker chromosomes display a prominent gap. This gap probably represents the original site of association of the acrocentric chromosomes and is also the site of the kinetochore. Despite the multicentromeric nature of the marker chromosome a single pair of kinetochores were found only at the central heterochromatic region. The functional implications of these structural findings are discussed.     

Analysis of high-resolution R-bands, obtained by heat-denaturation and Giemsa staining, on human prophase chromosomes

RHG-bands (heat-denatured Giemsa R-bands) of human prophase chromosomes were analyzed at high resolution, and the banding patterns at prophase and metaphase are presented. The bands were compared with those of the International Standard Cytogenetic Nomenclature idiograms and of the G-band idiograms proposed by J. J. Yunis. The number, size, and position of the RHG-bands correspond rather well with their equivalent G-negative bands, but some differences were noted in the zones of preferential stretching, the juxtacentromeric regions, and the telomeres. Variations in the centromere index and the banding pattern in heterochromatin were also discussed.