The diplochromosome of endoreduplicated cells: A new approach to highlight the mechanism of sister chromatid exchange

Chinese hamster lung embryonic cells (CL1) were treated with colchicine in order to induce endoreduplication and subsequently with mitomycin-C (MMC) to induce exchanges within the diplochromosome. The use of chromosomal differential staining through incorporation of 5-bromodeoxyuridine, resulting in only one stained chromatid, has allowed the analysis of all classes of exchanges among the four chromatids of the diplochromosome. Three classes of exchanges may occur: intradiplochromatid exchanges (ICEs) between the two inner chromatids, cousin chromatid exchanges (CCEs) between one inner and one outer chromatid, and sister chromatid exchanges (SCEs) between the two sister chromatids of the diplochromosome. The results show that MMC treatment, in the last cell cycle of endoreduplication, as expected, significantly increases only the frequency of SCEs, whereas the frequency of ICEs and CCEs remains unchanged. This result supports replication models of formation of SCEs. Furthermore the fact that the number of ICEs does not increase means that the molecular mechanism of somatic crossing over is not related to that of SCE formation, or very rarely. The results also indicate a statistically significant lower induction of SCEs in endoreduplicated metaphases as compared with diploid ones both in control and MMC-treated cells. Such a result may be due to structural restrictions within the diplochromosome. Received: 29 December 1995; in revised form; 4 March 1996 / Accepted: 24 March 1996     

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.     

Scaffold-like structures in mouse chromosomes revealed by restriction endonuclease digestion and electron microscopy

A scaffold-like structure is observed under the electron microscope when mouse chromosomes are digested with the restriction endonuclease Hae III. This structure, located in the inner part of chromatids, may correspond to those fragments of chromatin loops anchored to the chromosome scaffold and is obtained when chromosomes are treated either in suspension or attached to grids. The width of the structure is correlated with the extent of digestion in chromosomes treated in suspension. Those treated on grids show this structure whenever chromatids do not collapse. These results agree with the model of chromosome organization based on a non-histone protein scaffold.     

Chromosome reproduction: units of DNA for segregation

Evidence is summarized which indicates that the DNA loop anchoring proteins in chromosomes are effectively heterodimers that stack and are fastened into a bilaterally symmetrical array along the chromonemal axis. The evidence consists primarily of the observations made twenty five to thirty years ago on the pattern of sister chromatid exchanges and the way the DNA chains are sorted in the formation of diplochromosomes in cells that have undergone endoreduplication. The evidence indicates that each chain of DNA in the single duplex, which is assumed to run the length of a chromosome, is anchored to a bilaterally symmetrical axis of heterodimers that sort the two original chains among the four derived chromatids of each diplochromosome in a very precise way. These observations are considered in the context of investigations on the nature of scaffold proteins and the loop anchorage sequences, as well as the advances being made on the nature of DNA binding proteins and the roles of topoisomerase II.     

Configurational changes in chromatids from helical to banded structures

Induction of configurational changes in the helical chromatids of air dried chromosomes was used to explore the mechanism of G-banding. From the water-Giemsa stained metaphase spreads of Chinese hamster cells, chromosomes having clearly helical chromatids were selected and photographed. Then the chromosomes were decolorized, treated with trypsin, and restained with saline-Giemsa (1 x SSC). Such procedures were repeatedly carried out upon the same chromosomes. Subsequent examination of the chromosomes showed that configurational changes from a helical structure to a banded structure had occurred. Some chromosomes revealed a variety of transitional changes between these two configurations. During the repeated G-banding treatments, the distances between bands along the same chromatids changed each time. The results obtained seem to indicate that the G-banding results from locally induced compaction of chromosomal materials along the chromatids.     

Induction of endoreduplication in double minutes of the human neuroblastoma cells and the replication pattern.

Induction of endoreduplication (ERD) using Hoechst 33258 as well as colcemid was carried out in cultured neuroblastoma (NB) line cells. In these endoreduplicated cells, the majority of double minutes (DMs) appeared to take a diplochromosome like configuration to form a cluster consisting of four minute elements, assuming a complex DM. Sister chromatid differential staining (SCD) using 5-bromo-2'-deoxyuridine (BrdUrd) revealed the non-random distribution of the stained chromatids among four chromatids composing each diplochromosome, suggesting the occurrence of so-called "outside replication" of DNA strands during the process of ERD. The same pattern of differential staining was also found in the quadruple minutes of each endoreduplicated DM. Since DMs are acentric, the present results suggest that centromeres do not play any essential role in the formation of diplochromosomes observed in the conventional cytologic preparations and that centromeres are probably not responsible for the phenomenon of the "outside replication" of DNA strands.     

Geregelte Anordnung der Chromatiduntereinheiten in den Diplochromosomen bei der Endoreduplikation

Zusammenfassung An durch -Merkaptoäthanol induzierten Endoreduplikationen in Blutkulturen wurden morphologische und autoradiographische Untersuchungen angestellt. Merkaptoäthanolinduzierte Endoreduplikationen gleichen sowhl in morphologischer als auch funktioneller Hinsicht weitgehend spontan auftretenden Endoreduplikationen. Auf das identische Verhalten der Chromosomen des einzelnen Diplochromosoms in verschiedenen Eigenschaften (Zentromer, sekundäre Konstriktionen, Verdrehungen der Chromatiden) wird besonders hingewiesen. Die Endoreduplikation besteht aus zwei aufeinanderfolgenden DNS-Synthesen ohne Kernteilung dazwischen. Wird in der ersten Synthese H³-Thymidin eingebaut, so finden sich in der späteren Metaphase jeweils die außen gelegenen Chromatiden des Diplochromosoms matkiert. Dieses Verhalten und seine Bedeutung für das Verständnis der Chromosomenreplikation wird diskutiert.

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By means of -merkaptoethanol a distinct increase of endoreduplications in blood cultures of human origin can be produced. Such merkaptoethanol-induced endoreduplications were the object of morphological research and experiments concerning the pattern of DNA-synthesis.Merkaptoethanol-induced endoreduplications are to a high degree similar to spontaneous endoreduplications both in morphology and function. In the chromosomes formed into pairs in endoreduplication (socalled diplochromosomes) morphologically distinguishing marks are always equally developed (e.g. secondary constrictions, division of the centromere). Even twists of the chromatids within a chromosome are to be found in a like manner in the other partner of the diplochromosome.Endoreduplication consists of two successive DNA-replications without mitosis between.If H³-thymidin is incorporated during the first DNA-replication the respective chromatids of the diplochromosome lying at the outside are labelled in metaphase whereas all the chromatids of the diplochromosomes are labelled, if H³-thymidin is incorporated during the second reduplication.The lying outside of the labelled chromatids allows the conclusion that (1) there is a regular distribution of half-chromatids during the DNA-replication. In addition to that the must be (2) a difference concerning the affinity between certain chromatids, which causes the single chromosomes of diplochromosomes to lie in a certain order to each other during the preparation of the chromosomes.A knowledge of these relations may be useful to set up a model of the chromosome.

     

Scanning electron microscopy of mammalian chromosomes from prophase to telophase

Changes in the morphology of human and murine chromosomes during the different stages of mitosis have been examined by scanning electron microscopy. Two important findings have emerged from this study. The first is that prophase chromosomes do not become split into pairs of chromatids until late prophase or early metaphase. This entails two distinct processes of condensation, the earlier one starting as condensations of chromosomes into chromomeres which then fuse to form a cylindrical body. After this cylindrical body has split in two longitudinally, further condensation occurs by mechanisms that probably include coiling of the chromatids as well as other processes. The second finding is that the centromeric heterochromatin does not split in two at the same time as the rest of the chromosome, but remains undivided until anaphase. It is proposed that the function of centromeric heterochromatin is to hold the chromatids together until anaphase, when they are separated by the concerted action of topoisomerase II acting on numerous similar sites provided by the repetitive nature of the satellite DNA in the heterochromatin. A lower limit to the size of blocks of centromeric heterochromatin is placed by the need for adequate mechanical strength to hold the chromatids together, and a higher limit by the necessity for rapid splitting of the heterochromatin at anaphase. Beyond these limits malsegregation will occur, leading to aneuploidy. Because the centromere remains undivided until anaphase, it cannot undergo the later stage of condensation found in the chromosome arms after separation into chromatids, and therefore the centromere remains as a constriction.by U. Scheer     

Immunocytology of chiasmata and chromosomal disjunction at mouse meiosis

Immunocytological and in situ hybridization evidence supports the hypothesis that at meiosis of chiasmate organisms, chromosomal disjunction and reductional segregation of sister centromeres are integrated with synaptonemal complex functions. The Mr 125,000 synaptic protein, Syn1, present between cores of paired homologous chromosomes during pachytene of meiotic prophase, is lost from synaptonemal complexes coordinately with homolog separation at diplotene. Separation is constrained by exchanges between non-sister chromatids, the chiasmata. We show that the Mr 30,000 chromosomal core protein, Cor1, associated with sister chromatid pairs, remains an axial component of post-pachytene chromosomes until metaphase I. We demonstrate that at this time the chromatin loops are still attached to their cores. A reciprocal exchange event between two homologous non-sister chromatids is therefore immobilized by anchorage of sister chromatids to their respective cores. Cores thus contribute to the sister chromatid cohesiveness required for maintenance of chiasmata and proper chromosomal disjunction. Cor1 protein accumulates in juxtaposition to pairs of sister centromeres during metaphase I. Presumably, independent movement of sister centromeres at anaphase I is restricted by Cor1 anchorage. That reductional separation of sister centromeres is mediated by Cor1, is supported by the dissociation of Cor1 from separating sister centromeres at anaphase II and by its absence from mitotic anaphases.     

Electron microscopy of differentially BrdU-substituted sister chromatids treated with sodium phosphate

Electron microscopy of unstained BrdU-substituted chromosomes treated with 1.0 M NaH₂PO₄ at high pH and high temperature has demonstrated that there is a structural basis for the light microscopic observation of differentially Giemsa-stained unifilarly and bifilarly BrdU-substituted chromatids and the appearance of chromosome dots. At progressively higher treatment temperatures, sequential structural changes occurred in the chromosomes. After treatment with NaH₂PO₄ at 70–80° C, unifilarly BrdU-substituted chromatids were much more electron opaque than bifilarly substituted chromatids, and the overall data suggest that this difference in electron opacity is a result of the preferential extraction of chromosomal DNA from the bifilarly BrdU-substituted chromatids. NaH₂PO₄ treatment of the BrdU-substituted chromosomes at 80–90° ° C resulted in the formation of highly electron opaque spots (dots) on one or both chromatids. Dots first appeared on the electron lucent bifilarly BrdU-substituted chromatid, indicating that the chromatin with the greatest substitution of BrdU in its DNA is most susceptible to dot formation. At a slightly higher temperature, dots also appeared on the unifilarly BrdU-substituted chromatid concomitant with a disappearance of the electron opacity characterizing this chromatid at the lower treatment temperature. The dots may be formed by an extreme reorganization of residual chromatin or by some kind of interaction or reaction between the chromatin and the salts in the incubation medium. G-band regions may serve as focal points for dot formation.     

Studies on the mitotic chromosome scaffold of Allium sativum

An argentophilic structure is present in the metaphase chromosomes of garlic(Allium sativum),Cytochemical studies indicate that the main component of the structure is non-histone proteins(NHPs).The results of light and electron microscopic observations reveal that the chromosme NHP scaffold is a network which is composed of fibres and granules and distributed throughout the chromosomes.In the NHP network,there are many condensed regions that are connected by redlatively looser regions.The distribution of the condensed regions varies in individual chromosomes.In some of the chromosomes the condensed regions are lognitudinally situsted in the central part of a chromatid while in others these regions appear as coillike transverse bands.At early metaphase.scaffolds of the sister chromatids of a chromosome are linked to each other in the centromeric region,meanwhile,they are connected by scafold materials along the whole length of the chromosome.At late metaphase,however,the connective scaffold materials between the two sister chromatids disappear gradually and the chromatids begin to separate from one another at their ends.but the chromatids are linked together in the centromeric region until anaphase.This connection seems to be related to the special structure of the NHP scaffold formed in the centromeric region.The morphological features and dynamic changes of the chromosome scaffold are discussed.     

Centromeric organization in the chloromonadophycean alga Vacuolaria virescens

Observations with light and electron microscopes indicate the presence of a localized kinetochore in the phytoflagellate Vacuolaria virescens Cienk. (class Chloromonadophyceae). Thus, metaphase chromosomes have a clearly defined constriction which stains less densely than the remainder of the chromosome; at late metaphase the chromatids separate at definite points; acentric chromosome fragments may be formed. Ultrastructurally the kinetochore is recognizeable as a specialized area on the poleward surface of the chromosome into spindle microtubules are inserted.     

Identification of the Meiotic Division of Malarial Parasites

Zygotes of Plasmodium berghei were cultured 15–25 h in vitro to yield mature infective ookinetes. Samples taken in the first 5 h of culture were examined by electron microscopy. Meiotic figures were detected in the nuclei of the zygotes. Threadlike leptotene chromatids (chromosomes) condensed from attachment plaques on the nuclear envelope; chromatid pairing followed (zygotene), with synaptonemal complexes subsequently appearing (pachytene). These complexes persisted into metaphase but dissociated when the chromatids rapidly decondensed during anaphase. At telophase of the first meiotic division the kinetochores were retracted toward two small spindle complexes, which were found at widely separated poles in the nuclear envelope. The observations are consistent with a haploid genome of 8–10 chromosomes.     

THE STRUCTURE OF THE CENTROMERIC REGION OF CHO CHROMOSOMES

CHO chromosomes, prepared for fluorescence microscopy, or for scanning electron microscopy, sometimes show a splitting of the centromere proper into two sister centromeres, with a space between them, while the sister chromatids are joined in the most proximal regions of the chromosome arms. It is suggested that this might represent the final stage of chromatid splitting before the anaphase separation of chromatids.     

A new chromosome model

We present a new model of the three-dimensional structure of chromosomes. With DNA and protein staining it could be shown by high-resolution scanning electron microscopy that metaphase chromosomes are mainly composed of DNA packed in "chromomeres" (coiled solenoides) and a dynamic matrix formed of parallel protein fibers. In the centromeric region, the chromomeres are less densely packed, giving insight into the matrix fibers. We postulate that chromosome condensation is achieved by the binding of solenoids to matrix fibers which have contact sites to one another and move antiparallel to each other. As condensation progresses, loops of solenoids accumulate to form additional chromomeres, causing chromosomes to become successively shorter and thicker as more chromomeres are formed. For sterical reasons, a tension vertical to the axial direction forces the chromatids apart. The model can simply explain the enormous variety of chromosome morphology in plant and animal systems by varying only a few cytological parameters. Primary and secondary constrictions and deletions are defined as regions devoid of chromomeres. Even in the highly condensed metaphase, all genes would be easily accessible.     

Differential immunolocalization of a putative Rec8p in meiotic autosomes and sex chromosomes of triatomine bugs

Hemipteran chromosomes are holocentric and show regular, special behavior at meiosis. While the autosomes pair at pachytene, have synaptonemal complexes (SCs) and recombination nodules (RNs) and segregate at anaphase I, the sex chromosomes do not form an SC or RNs, divide equationally at anaphase I, and their chromatids segregate at anaphase II. Here we show that this behavior is shared by the X and Y chromosomes of Triatoma infestans and the X(1)X(2)Y chromosomes of Triatoma pallidipennis. As Rec8p is a widely occurring component of meiotic cohesin, involved in meiotic homolog segregation, we used an antibody against Rec8p of Caenorhabditis elegans for immunolocalization in these triatomines. We show that while Rec8p is colocalized with SCs in the autosomes, no Rec8p can be found by immunolabeling in the sex chromosomes at any stage of meiosis. Furthermore, Rec8p labeling is lost from autosomal bivalents prior to metaphase I. In both triatomine species the sex chromosomes conjoin with each other during prophase I, and lack any SC, but they form "fuzzy cores", which are observed with silver staining and with light and electron microscopy during pachytene. Thin, serial sectioning and electron microscopy of spermatocytes at metaphases I and II reveals differential behavior of the sex chromosomes. At metaphase I the sex chromosomes form separate entities, each surrounded by a membranous sheath. On the other hand, at metaphase II the sex chromatids are closely tied and surrounded by a shared membranous sheath. The peculiar features of meiosis in these hemipterans suggest that they depart from the standard meiotic mechanisms proposed for other organisms.     

Continuous reorganization leads to extensive polymorphism in a monkey centromeric satellite

Previously we reported the existence of a highly polymorphic satellite, deca-satellite, in the African green monkey genome; deca-satellite probe anneals to complex sets of repeated restriction endonuclease fragments that differ from individual to individual in the monkey population. Here we present experiments aimed at clarifying the structure and organization of deca-satellite sequences and investigating the mechanisms that generate the polymorphisms. Deca-satellite represents less than 1% of the monkey genome but the percentage varies from one monkey to another. The core sequence 5'-C-C-G-G within the ten base-pair deca-satellite repeat unit is well conserved and the central 5'-C-G is sometimes but not always methylated. Restriction endonuclease analysis with BamHI and EcoRI defines separate satellite domains that have evolved in an independent manner. In situ hybridization shows deca-satellite to be located at the centromeric regions of some but not all monkey chromosomes. This location is independently confirmed by a high frequency, in monkey libraries, of segments containing junctions between deca-satellite and alpha-satellite, the main monkey centromeric satellite. The total number of metaphase chromosomes that show centromeric grains after in situ hybridization with a deca-satellite probe varies from one monkey to another. Moreover, in situ hybridization to endoreduplicated diplochromosomes showed that deca-satellite is occasionally distributed asymmetrically on one or the other of the two pairs of sister chromatids in one diplochromosome. This indicates that major reorganization of the satellite can occur frequently in somatic cells. We discuss several possible mechanisms by which deca-satellite sequences could be either amplified or deleted during a single replicative cycle. Also, on the basis of the marked fluidity of deca-satellite abundance and organization and other well-known attributes of centromeric satellites, we suggest that the existence and maintenance of centromeric satellite rests on the role of the tandem repeats themselves and not on any particular nucleotide sequence, repeat length or organization.     

Mitotic chiasmata in human diplochromosomes

Summary Three placental tissue cultures of spontaneous human abortions showed an unusually high frequency of metaphases with diplochromosomes. In 62 such cells, nine configurations were interpreted as mitotic chiasmata between the two sister chromosomes of a diplochromosome. One U-type exchange between two sister chromosomes was also found. This differs significantly from the 1:1 ratio of adjacent and alternate exchanges in translocations, thus supporting the idea that mitotic chiasmata are in principle different from chromatid translocations. The hypothesis is put forward that the frequency of homologous exchanges is determined by the intimacy of pairing which ranges from meiotic pairing through sister chromatid association, through sister chromosome association in diplochromosomes to accidental pairing of homologous regions in diploid cells.     

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.     

Observations on dicentrics in living cells

Summary In previously irradiated endosperm cells of Haemanthus katherinae studied in vitro by means of micro-cinematography, two-kinetochore chromatids and dicentric chromosomes have been observed. Breaking of such dicentric chromatids and chromosomes has been analysed. Behaviour of some of the dicentric chromosomes during anaphase deserves special attention: interlocking dicentrics cut one through another and rejoin in a few minutes. In this way from a metaphase interlocking dicentric, two sister anaphase dicentrics are formed. Interlocked dicentrics can also uncoil and not break at all. In this case no activity was observed in one kinetochore of one dicentric in later stages of anaphase (two kinetochores were active in one dicentric and only one in its sister). Analysis of chromosome movements in two-kinetochore chromatids and dicentrics is also presented.