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Computer graphics of SEM images facilitate recognition of chromosome position in isolated human metaphase plates.

There is general agreement that at the time of mitosis chromosomes occupy precise positions and that these positions likely affect subsequent nuclear function in interphase. However, before such ideas can be investigated in human cells, it is necessary to determine first the precise position of each chromosome with regard to its neighbors. It has occurred to us that stereo images, produced by scanning electron microscopy, of isolated metaphase plates could form the basis whereby these positions could be ascertained. In this paper we describe a computer graphic technique that permits us to keep track of individual chromosomes in a metaphase plate and to compare chromosome positions in different metaphase plates. Moreover, the computer graphics provide permanent, easily manipulated, rapid recall of stored chromosome profiles. These advantages are demonstrated by a comparison of the relative position of group A-specific and groups D- and G-specific chromosomes to the full complement of chromosomes in metaphase plates isolated from a nearly triploid human-derived cell (HeLa S3) to a hypo-diploid human fetal lung cell.

Cells, Cultured↗

Cytoskeletal organization of rat oocytes during metaphase II arrest and following abortive activation: a study by confocal laser scanning microscopy.

In metaphase II arrested rat oocytes (M II), microtubules were found in the taper-shaped meiotic spindle and in the cytoplasm as asters and free microtubules. Whereas spindle microtubules were acetylated, those located in the cytoplasm were not. Cytoplasmic microtubules were also labile as assessed by mild cooling. In contrast to mouse oocytes, rat microtubule organizing centers (MTOCs) did not react with MPM-2 antibody by immunofluorescence despite the fact that this antibody reacts with several proteins as shown by immunoblot. However, cytoplasmic MTOCs in M II-arrested rat oocytes could be detected by their nucleating capacity in the presence of taxol, a drug that induced the formation of numerous cytoplasmic asters. In addition, taxol caused a change in the spindle shape and the formation of astral microtubules at the spindle poles. Meiotic spindles (as well as chromosomes devoid of microtubules after nocodazole-treatment) were overlaid by an actin-rich domain. Spontaneous abortive activation led to the extrusion of the second polar body followed by another metaphase arrest--metaphase III; however, normal spindles did not form and dispersed chromosomes surrounded by microtubules were observed. Electron microscopic studies confirmed these observations and revealed that the kinetochores, are located deep within the chromosomes in contrast to mouse kinetochores, and this might be responsible for the absence of a metaphase III spindle in the rat oocyte. Induced activation caused transition to interphase with the formation of a characteristic microtubule network. This study shows that there are several significant differences in the cytoskeletal organization of rat and mouse oocytes.

Animals↗

Localization and identification of galactose/N-acetylgalactosamine and sialic acid-containing proteins in Chinese hamster metaphase chromosomes.

Four lectins were used to recognize galactose/N-acetyl-galactosamine (Gal/GalNAc) and sialic acid residues in proteins of Chinese hamster metaphase chromosomes. In situ binding pattern of a fluorescein isothiocyanate-labelled (Gal/GalNAc)-specific lectin Sophora japonica agglutinin (SJA) showed that chromosomal SJA-binding proteins are primarily localized to the helically coiled substructure of chromatids. Numerous SJA-binding proteins were identified in Western blots of chromosomal proteins, their molecular weights ranging from 26 to 200 kDa. Another Gal/GalNAc-specific lectin, peanut agglutinin (PNA), with a slightly different sugar binding specificity, did not bind to Chinese hamster metaphase chromosomes, and in Western blots only two chromosomal protein bands were faintly stained. The in situ labelling patterns of two sialic acid-specific lectins, Maackia amurensis (MAA) and Sambucus nigra (SNA) agglutinins, both showed that the helically coiled substructure of chromatids is also enriched in sialylated proteins. In Western blot analysis 11 MAA-binding protein bands with molecular weights ranging from 54 to 215 kDa were identified, while SNA only bound to one protein band of 67 kDa. MAA and SNA are specific for alpha(2-->3)- and alpha(2-->6)-linked sialic acid residues, respectively. Thus, it is likely that alpha(2-->3)-linked sialic acid residues are more common in chromosomal proteins than alpha(2-->6)-linked sialic acid residues. These data suggest that Gal/GalNAc and sialic acid-containing glycoproteins exist in metaphase chromosomes and that these proteins may have a role in the formation of higher order metaphase chromosome structures.

Acetylgalactosamine↗

Arrest in metaphase and anatomy of mitotic catastrophe: mild heat shock in two human osteosarcoma cell lines.

The exits from metaphase arrest and anatomy of mitotic catastrophe were studied in two human osteosarcoma cell lines, nontumorigenic HOS TE85 and its chemically transformed strain MNNG-HOS, applying mild genotoxic damage by heat shock at 41.8 degrees C for 24 h. Under these conditions, both cell lines doubled or tripled their mitotic index entering arrest in metaphase. On return to 37 degrees C, the arrest was either released or ended in apoptosis. The transformed strain showed a greater capacity to arrest in metaphase as well as a greater probability of developing the third pathway: to restitute this arrest in polyploid interphase. This, in turn, either entered an 'endocycle' or, following a delay, apoptosis. Thus, arrest in metaphase was a cross-point of the mitotic cycle, apoptosis, and endocycle. Mitotic catastrophe can morphologically manifest combinations of elements of these three processes.

Anaphase↗

The oocyte metaphase arrest.

Usually, oocyte meiosis reinitiation appears as a two step process during which release from the prophase block is followed by a second arrest in metaphase I or II. In this review, we will examine the mechanisms required to maintain the metaphase arrest and stabilize MPF activity at this stage. Then, we will analyse the processes required to exit from the metaphase block. These may drive the cells forward to the metaphase-anaphase transition, as a result of fertilization, activation or protein synthesis inhibition. Instead, inhibiting protein phosphorylation drives the oocyte back to interphase. All these treatments result in derepression of DNA synthesis.

Amphibians↗

Fluorescent in situ hybridization to soybean metaphase chromosomes.

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescent in situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosomes showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescent in situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.

Biotin↗

Natural size classes in DNA from Chinese hamster metaphase chromosomes.

DNA from isolated Chinese hamster ovary (CHO) metaphase chromosomes can be obtained in three different molecular weight classes. The two largest forms have sedimentation coefficients of 80 and 120 S at 7,500 rpm. Based on sedimentation and speed dependence analysis these have molecular weights of 220 million and above 5,000 million, and are thought to be analogs of DNA classes observed in a prior study of human metaphase chromosomes. An extract can be converted to primarily the 80 S form through alkaline pH treatment of metaphase DNA. The third class (45 S DNA) is formed as a result of metaphase chromosome exposure to the nuclease Bal31, and has a mass distribution analogous to the CHO replicon.

Animals↗

The central localization of the small and early replicating chromosomes in human diploid metaphase figures.

Centromere-center distances are analyzed in 700 metaphase plates, which belong to four different samples. The descriptive analysis of the chromosome distribution shows that smaller, earlier replicating, genet-dense chromosomes are preferentially found near the metaphase plate center, surrounded by longer chromosomes which finish their replication rather late during S phase. This general pattern is highly constant in diploid metaphase samples and independent of sex, culture time, and number of individuals used in the sample. There is accumulating evidence that this overall distribution is not the result of technical artifacts. The metaphase plate data are complementary to the concept of an interphase nucleus structure in which late-replicating, genetically less active chromatin is accumulated at the periphery of the nucleus, while other, earlier replicating chromatin is connected with the intranuclear matrix. Although the currently available data should not be overinterpreted, an extension of the "bodyguard" hypothesis, which was suggested for C heterochromatin, provides a functional interpretation for these data: The peripherally localized, late-replicating genetic material protects the centrally localized euchromatin against mutagens, clastogens, and maybe also against viruses.

Adult↗

Transmission of three radiation-induced translations in the Syrian hamster. I. Chromosome studies of male meiosis: pachytene to metaphase II.

Chromosome behaviour during meiosis in male Syrian hamsters heterozygous for one of three translocations was analysed as part of a study of the transmission of these structural changes. Synapsis was studied using preparations of synaptonemal complexes, and chiasmate associations and the results of anaphase I segregation were studied in air-dried preparations of metaphases I and II respectively. The main findings were: (i) that, at least in the two trivalent-forming translocations, there is no simple relationship between either the frequency or the extent of synapsis and chiasma formation between the chromosomes involved in the translocation; (ii) that the presence of a univalent in a substantial proportion of metaphase I cells does not necessarily lead to irregular segregation as judged by analysis of metaphase IIs; and (iii) conversely, that in translocation heterozygotes in which metaphase I contains the chromosomes involved in the translocation as a quadrivalent or as two bivalents, with no univalents or trivalents, unexpected numerical segregation can be found. The observations of meiotic chromosomes behaviour reported here show that it is not always possible to predict the effects of structural change, or to determine the basis of these effects, from an analysis of any stage of meiosis taken in isolation, or from an analysis of an apparently similar change.

Animals↗

Isolation of chromosome clusters from metaphase-arrested HeLa cells.

We have developed a simplified approach for the isolation of metaphase chromosomes from HeLa cells. In this method, all the chromosome from a cell remain together in a bundle which we call a "metaphase chromosome cluster". Cells are arrested to 90-95% in metaphase, collected by centrifugation, extracted with non-ionic detergent in a low ionic strength buffer at neutral pH, and homogenised to strip away the cytoskeleton. The chromosome clusters which are released can then be isolated in a crude state by pelleting or they can be purified away from nearly all the interphase nuclei and cytoplasmic debris by banding in a PercollTM density gradient. -- This procedure has the advantages that it is quick and easy, metaphase chromatin is recovered in high yield, and Ca++ is not needed to stabilise the chromosomes. Although the method does not yield individual chromosomes, it is nevertheless very useful for both structural and biochemical studies of mitotic chromatin. The chromosome clusters also make possible biochemical and structural studies of what holds the different chromosomes together. Such information could be useful in improving chromosome isolation procedures and for understanding suprachromosomal organisation of the nucleus.

Cell Fractionation↗

The radial positions of metaphase chromosomes may be a consequence of the relative strength of their interaction with the spindle and their size.

Microtubule oriented forces acting on chromosomes on spindles in mitosis and meiosis will produce a radial component of force in the plane of the metaphase plate. The strength of this vector will depend on the angle at which the microtubule meets the plate. Radial forces will tend to segregate chromosomes to peripheral or central positions, depending on their size, and also on the strength of the activity of individual centromeres. In prometaphase, forces pushing chromosomes from the poles will tend to force them to the periphery of the metaphase plate, as seen in radial metaphases. Tension towards the poles at late metaphase will pull smaller chromosomes and those with more powerfully active kinetochores towards the centre of the plate. If the two genomes in a hybrid cell have different centromeric activities, their chromosomes will be segregated. Microtubule assembly and disassembly, and motor proteins such as the kinesins and dynein which haul organelles along microtubules, can provide forces in both directions.

Biomechanical Phenomena↗

Relationship between the levels of wheat-rye metaphase I chromosomal pairing and recombination revealed by GISH.

The metaphase I and anaphase I stages of meiosis of wheat x rye hybrids carrying the ph1b mutation were analyzed by genomic in situ hybridization. This technique allows distinction between three different types of wheat-rye associations in metaphase I configurations as well as detection of wheat-rye recombinant chromosomes in anaphase I cells. The frequency of associations between wheat and rye chromosomes greatly exceeded the level of wheat-rye recombination found in the three hybrids examined. Extremely distal associations, which account for about 50% of the total wheat-rye metaphase I chromosomal pairing, can explain such a discrepancy between metaphase I and anaphase I data. It is further discussed whether these associations reflect very distally located chiasmata or nonchiasmatic pairing. The sizes of the segments exchanged in wheat-rye recombinant chromosomes provide cytological evidence that wheat-rye recombination is restricted to the distal chromosomal regions.

Anaphase↗

Structural elements of bulk chromatin within metaphase chromosomes.

We have performed a very extensive electron microscopy investigation of the chromatin structures extruded from partially denatured metaphase chromosomes from HeLa cells under a wide variety of conditions. Denatured chromosomes having fibres as the dominant structural element are obtained in the presence of buffers of very low concentration or after incubation with water. At slightly higher ionic concentrations, metaphase chromosomes become granulated. The most frequently observed granules have a diameter of about 35 nm and show the same structural characteristics as the compact cylindrical chromatin bodies previously found in our laboratory in studies performed using small chromatin fragments. Our results suggest that fibres are formed by the face-to-face association of 35-nm chromatin bodies. We have observed a very compact morphology of chromosomes in solutions containing intracellular concentrations of monovalent cations and the Mg2+ concentration found in metaphase. The most abundant structural elements observed in chromatin extruded from partially denatured compact metaphase chromosomes are multilayered plate-like structures. This is the first time that these planar structures have been reported. The observation of the irregular plates found in some preparations and of the small planar structures seen in aggregates of small chromatin fragments suggests that plates are formed by side-by-side association of compact chromatin bodies.

Acetic Acid↗

Factors influencing ADP-ribosylation differences between chromosomal proteins of interphase and metaphase HeLa cells.

Fundamental differences were previously discovered in the ADP-ribosylation of proteins from metaphase chromosomes and interphase nuclei of HeLa cells. The number of modified nonhistone species was found to be dramatically reduced for metaphase chromosomes. An investigation has therefore been made of factors which could influence, and therefore be responsible for, this change in ADP-ribosylation during the cell cycle. Modified proteins were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing mitotic and interphase samples from permeabilized cells that had been incubated with [32P]NAD. Whole cells showed a difference between interphase and metaphase similar to that for isolated nuclei and chromosomes. Chromosome expansion, disruption of chromosomes or nuclei, DNA nicking, and cellular growth activity significantly changed the incorporation of 32P label. Inhibitors of protein, RNA, and DNA synthesis did not, however, greatly affect ADP-ribosylation. The pattern of labeled species was not altered by the presence of nonradioactive NAD, though the extent of labeling declined. The results were not artifactually due to the procedure used to arrest cells in mitosis. Similar results were found with Novikoff rat hepatoma cells, demonstrating that the difference between metaphase and interphase is not confined to HeLa cells.

Adenosine Diphosphate Ribose↗

Effects of Ca2+ ions on the formation of metaphase chromosomes and sperm pronuclei in cell-free preparations from unactivated Rana pipiens eggs.

Nuclei transplanted into unactivated amphibian eggs are known to condense into metaphase chromosomes whereas those transplanted into activated eggs decondense and enlarge. We have made cell-free cytoplasmic preparations from Rana pipiens eggs which can induce demembranated Xenopus laevis sperm to undergo changes similar to those seen in intact eggs. Sperm chromatin which is incubated for 3 hr in unactivated egg preparations made using a buffer containing 3 mM EGTA is induced to form metaphase chromosomes. However, decondensed interphase nuclei are formed when chromatin is incubated in unactivated egg preparations made without EGTA as well as in activated egg preparations. When Ca2+ ions are added to unactivated egg preparations made with EGTA, the preparations lose the ability to induce metaphase chromosome formation and become capable of decondensing sperm chromatin. Once the ability to decondense chromatin has developed, either in unactivated or activated egg preparations, it cannot be suppressed by the addition of EGTA. However, decondensation of sperm chromatin in activated egg preparations can be suppressed by the addition of unactivated egg preparations made with EGTA. In this case, the incubated sperm chromatin is induced to form metaphase chromosomes. These results may indicate that the chromosome condensation activity of unactivated egg cytoplasm can be sustained in cell-free preparations when Ca2+ ion levels are kept low, but when Ca2+ ion levels increase this activity is lost and replaced by a new activity which can decondense chromatin. Since this change in cytoplasmic activities is comparable to that occurring in the intact egg following fertilization, these results suggest that Ca2+ ions play a crucial role during activation in altering the cytoplasmic activities which control nuclear behavior.

Animals↗

Interactions between metaphase and interphase factors in heterokaryons produced by fusion of mouse oocytes and zygotes.

The cytoplasmic factor responsible for chromosome condensation was introduced into mouse zygotes at different times after fertilization by fusion of the zygotes with metaphase I oocytes. In 72% of heterokaryons obtained after fusion of early zygotes (14-18 hr post-human chorionic gonadotrophin (HCG) with oocytes, the male and female pronuclei of the zygote decondensed. At the same time, the oocyte chromosomes became enclosed in a nuclear envelope and decondensed to an interphase state. However, in the rest of the heterokaryons, the chromatin of the pronuclei condensed to metaphase chromosomes, thus resulting in three sets of chromosomes. Fusion of zygotes that had begun DNA synthesis (20-22 hr post-HCG) with oocytes induced chromosome condensation of the pronuclei in 76% of the cases. In some heterokaryons, however, the oocyte chromosome decondensed to an interphase state similar to the zygote pronuclei. Fusion between late zygotes (27-29 hr post-HCG) with oocytes resulted in chromosome condensation of the pronuclei in all heterokaryons. On the basis of these results, the formation of the pronuclei and their progression toward mitosis in the zygote may be explained by changing levels of a metaphase factor in the cell, or by a balance between interphase and metaphase factors.

Animals↗

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.

Animals↗

In situ factors affecting stability of the DNA helix in interphase nuclei and metaphase chromosomes.

The data from earlier cytochemical studies, in which the metachromatic fluorochrome acridine orange (AO) was used to differentially stain single vs double-stranded DNA, suggested that DNA in situ in intact metaphase chromosomes or in condensed chromatin of G0 cells is more sensitive to denaturation, induced by heat or acid, than DNA in decondensed chromatin of interphase nuclei. Present studies show that, indeed, DNA in permeabilized metaphase cells, in contrast to cells in interphase, when exposed to buffers of low pH (1.5-2.8) becomes digestible with the single-strand-specific S1 or mung bean nucleases. A variety of extraction procedures and enzymatic treatments provided evidence that the presence of histones, HMG proteins, and S-S bonds in chromatin, as well as phosphorylation or poly(ADP)ribosylation of chromatin proteins, can be excluded as a factor responsible for the differential sensitivity of metaphase vs interphase DNA to denaturation. Cell treatment with NaCl at a concentration of 1.2 N and above abolished the difference between interphase and mitotic cells, rendering DNA in mitotic cells less sensitive to denaturation; such treatment also resulted in decondensation of chromatin visible by microscopy. The present data indicate that structural proteins extractable with greater than or equal to 1.2 N NaCl may be involved in anchoring DNA to the nuclear matrix or chromosome scaffold and may be responsible for maintaining a high degree of chromatin compaction in situ, such as that observed in metaphase chromosomes or in G0 cells. Following dissociation of histones, the high spatial density of the charged DNA polymer may induce topological strain on the double helix, thus decreasing its local stability; this can be detected by metachromatic staining of DNA with AO or digestion with single-strand-specific nucleases.

Acridine Orange↗