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Type I topoisomerase activity is required for proper chromosomal segregation in Escherichia coli.

Type I DNA topoisomerases are ubiquitous enzymes involved in many aspects of DNA metabolism. Escherichia coli possesses two type I topoisomerase activities, DNA topoisomerase I (Topo I) and III (Topo III). The gene encoding Topo III (topB) can be deleted without affecting cell viability. Cells possessing a deletion of the gene encoding Topo I (topA) are only viable in the presence of an additional compensatory mutation. In the presence of compensatory mutations, Topo I deletion strains grow normally; however, if Topo III activity is repressed in these cells, they filament extensively and possess an abnormal nucleoid structure. These defects can be suppressed by the deletion of the recA gene, suggesting that these enzymes may be involved in RecA-mediated recombination and may specifically resolve recombination intermediates before partitioning.

Anaphase↗

The influence of postovulatory ageing on the retardation of mouse oocyte maturation and chromosome segregation induced by vinblastine.

Certain compounds can induce ovulated metaphase I (MI) oocytes. To study if these MI oocytes can overcome this blockage, ICR mice were given human chorionic gonadotrophin and 0.6 mg/kg vinblastine sulfate (VBS). Their ovulated oocytes were collected at 17, 19, 21, 23 and 25 h later. The results showed that the frequencies of MI oocytes decreased, the proportions of diploid metaphase II (MII) oocytes increased and the frequencies of hyperploid MII oocytes did not significantly differ (P > 0.05) among the five harvest or postovulatory times. We also found that the proportions of MII oocytes exhibiting premature anaphase II and premature centromere separation increased with postovulatory ageing, and that these frequencies were consistently higher in controls than in the VBS groups. These findings indicate that some of the oocytes blocked in MI can overcome this inhibition and produce primarily diploid MII oocytes. The ultimate fate of ovulated MI and diploid oocytes on aneuploid production hinges upon the formation of a functioning meiotic spindle, which is affected by both dosage and the specific chemical.

Aneuploidy↗

A dependent pathway of gene functions leading to chromosome segregation in Saccharomyces cerevisiae.

Methyl-benzimidazole-2-ylcarbamate (MBC) inhibits the mitotic cell cycle of Saccharomyces cerevisiae at a stage subsequent to DNA synthesis and before the completion of nuclear division (Quinlan, R. A., C. I. Pogson, and K, Gull, 1980, J Cell Sci., 46: 341-352). The step in the cell cycle that is sensitive to MBC inhibition was ordered to reciprocal shift experiments with respect to the step catalyzed by cdc gene products. Execution of the CDC7 step is required for the initiation of DNA synthesis and for completion of the MBC-sensitive step. Results obtained with mutants (cdc2, 6, 8, 9, and 21) defective in DNA replication and with an inhibitor of DNA replication (hydroxyurea) suggest that some DNA replication required for execution of the MBC-sensitive step but that the completion of replication is not. Of particular interest were mutants (cdc5, 13, 14, 15, 16, 17, and 23) that arrest cell division after DNA replication but before nuclear division since previous experiments had not been able to resolve the pathway of events in this part of the cell cycle. Execution of the CDC17 step was found to be a prerequisite for execution of the MBC-sensitive step; the CDC13, 16 and 23 steps are executed independently of the MBC-sensitive step; execution of the MBC-sensitive step is prerequisite for execution of the MBC-sensitive step; execution of the MBC-sensitive step is prerequisite for execution of the CDC14 and 23 steps. These results considerably extend the dependent pathway of events that constitute the cell cycle of S. cerevisiae.

Benzimidazoles↗

Segregation of chromosomes in sperm of reciprocal translocation carriers: a review.

Reciprocal translocations, the most frequent structural aberration in humans, are mainly transmitted by one of the parents. In order to analyze the chromosomal content of the spermatozoa from carriers of chromosomal reorganizations, two methods have been used, karyotyping of sperm chromosomes by the human-hamster system and fluorescence in situ hybridization (FISH) in decondensed sperm nuclei. In this work, we review 92 sperm chromosome segregation studies from 85 different reciprocal translocation carriers, including a triple translocation carrier. Using the human-hamster method, a total of 5,818 spermatozoa from 44 reciprocal translocation carriers have been analyzed, 43 of them carrying a single reciprocal translocation and one was a carrier of a double reciprocal translocation. A segregation analysis in a carrier of a t(2;22;11) has been also reported. Carrying out FISH in sperm nuclei, a total of 237,042 spermatozoa from 46 reciprocal translocation carriers have been analyzed. Six of these were also analyzed by the human-hamster system. Taking into account both methods, a total of 76 different reciprocal translocations have been studied. In 74 of these 76 translocations, the reorganization occurs between autosomes, and in the other two, the Y chromosome is involved. Although along general lines, there are similarities between the results obtained by the two methods of analysis, variations are observed when the distribution of the different types of segregations that produce imbalances is compared. As a general rule reciprocal translocation carriers produce more unbalanced sperm than normal or balanced sperm. The results reported also corroborate that the proportion of unbalanced forms depends on the characteristics of the reorganization and that it varies widely. Thus the importance of performing a detailed meiotic behavior analysis for each particular translocation in order to obtain enough information to give adequate genetic counseling is stressed. Aspects as to the possible overestimation of 3:1 segregations or the presence of interchromosomal effects still need to be elucidated.

Chromosome Segregation↗

Asymmetrical segregation of chromosomes with a normal metaphase/anaphase checkpoint in polyploid megakaryocytes.

During differentiation, megakaryocytes increase ploidy through a process called endomitosis, whose mechanisms remain unknown. As it corresponds to abortive mitosis at anaphase and is associated with a multipolar spindle, investigation of chromosome segregation may help to better understand this cell-cycle abnormality. To examine this variation, a new method was developed to combine primed in situ labeling to label centromeres of one chromosome category and immunostaining of tubulin. Human megakaryocytes were obtained from normal bone marrow culture. By confocal microscopy, this study demonstrates an asymmetrical distribution of chromosomes (1 or 7) either between the spindle poles at anaphase stage of endomitosis and between the different lobes of interphase megakaryocyte nuclei. The metaphase/anaphase checkpoint appears normal on the evidence that under nocodazole treatment megakaryocytes progressively accumulate in pseudo-metaphase, without spontaneous escape from this blockage. Immunostaining of p55CDC/hCDC20 with similar kinetochore localization and dynamics as during normal mitosis confirms this result. HCdh1 was also expressed in megakaryocytes, and its main target, cyclin B1, was normally degraded at anaphase, suggesting that the hCdh1-anaphase-promoting complex checkpoint was also functional. This study found the explanation for these unexpected results of an asymmetrical segregation coupled to normal checkpoints by careful analysis of multipolar endomitotic spindles: whereas each aster is connected to more than one other aster, one chromosome may segregate symmetrically between 2 spindle poles and still show asymmetrical segregation when the entire complex spindle is considered.

Anaphase↗

Dynactin targets Pavarotti-KLP to the central spindle during anaphase and facilitates cytokinesis in Drosophila S2 cells.

The dynactin complex cooperates with the dynein complex in various systems for mitotic completion. Here we analysed the mitotic phenotype of Drosophila S2 cells following the knockdown of the dynactin subunit p150(Glued). We found that p150(Glued)-depleted cells were delayed in metaphase and that the centrosomes were poorly connected to mitotic spindle poles. In addition, anaphase occurred with asynchronous chromosome segregation. Although cyclin B was degraded in these anaphase cells, Aurora B, MEI-S322 and BubR1 were not released from the non-segregating chromosomes. We also found that the density and organisation of the central spindle were compromised, with Aurora B and polo kinases absent from the diminished number of microtubules. Pavarotti-KLP, a component of the centralspindlin complex required for the formation of stable microtubule bundles, was not immediately targeted to the plus ends of the microtubules following anaphase onset as happened in controls. Instead, it accumulated transiently at the cell cortex during early anaphase and its targeting to the central spindle was delayed. These data suggest that the dynactin complex contributes to cytokinesis by promoting stable targeting of the centralspindlin complex to microtubule plus ends at anaphase onset. The contribution of the dynein-dynactin complex to synchronous chromosome segregation and cytokinesis is discussed.

Anaphase↗

Aneuploidy in germ cells: disruption of chromosome mover components.

The task of the Workgroup on "Disruption of Chromosome Mover Components" was to establish what cellular structures are involved in chromosome segregation and how disruption of these could occur. Recent research on the mechanism of action of the cellular components that segregate chromosomes accurately during mitosis or meiosis has served to highlight the number of potential targets for disruption. The process of chromosome segregation represents an orchestrated chain of events centered on the activities of cellular motors, kinesins and dyneins. These motors are involved in arranging chromosomes at the metaphase plate, providing the spindle tension necessary for progression, and the actual segregation of the chromosomes to the poles. The Workgroup determined that there is a lack of information on the effects of chemical exposure to cell motors and other chromosome mover components, and that there is a clear need for further research. This article describes the discussions of the Workgroup and highlights areas of future research into chromosome movement, particularly in human meiotic and mitotic cells. The Workgroup emphasized that obtaining mechanistic data on the induction of aneuploidy will allow for extrapolation of the dose response curves for chemical exposures below the level of observation and for using aneuploidy data for quantitative risk assessment for adverse health effects.

Age Factors↗

Two distinct types of mitochondrial DNA segregation in mouse-rat hybrid cells. Stochastic segregation and chromosome-dependent segregation.

Two distinct patterns of mitochondrial DNA (mtDNA) segregation were found in different mouse-rat hybrid cell lines. On mouse-rat hybrid cell line, H2, retained complete sets of chromosomes and mtDNAs of both mouse and rat. Even after cultivation for about one year after cloning, the H2 cell population still retained both parental mtDNAs. However, when mtDNAs of H2 subclones were examined, it was found that some individual cells in the H2 cell population contained only mouse or only rat mtDNA, although they still retained complete sets of both kinds of parental chromosomes. This type of mtDNA segregation, named stochastic segregation, is bidirectional and may be caused by the repetition of random sharing of mouse and rat mtDNAs with daughter cells. This segregation occurred spontaneously during long-term cultivation. The second type of mtDNA segregation, named chromosome-dependent segregation, was found in the other mouse-rat hybrid cell lines that segregated either mouse or rat chromosomes. In these hybrid cells, chromosomes and mtDNA of the same species co-segregated. This second type of segregation is unidirectional. The types of mtDNA segregation appear to depend on the stability of the parental chromosomes in the hybrid cells. When both mouse and rat chromosomes retain stably, mtDNA shows stochastic segregation. On the contrary, when either species of chromosomes is segregated from the cells, mtDNA shows chromosome-dependent segregation.

Animals↗

DNA segregation: putting chromosomes in their place.

Recent studies provide evidence that bacterial chromosomes are replicated by an enzyme factory, the replisome, located at a fixed position at the center of the cell; the fixed replisome could be a major factor in determining chromosome order in the cell, and may provide the force that drives chromosome segregation.

Bacteria↗

Involvement of cell shape in the replication and segregation of chromosomes in Escherichia coli.

Chromosome replication appears to initiate in E. coli when the dnaA boxes in oriC become filled with DnaA protein, which could simultaneously mediate both the unwinding of the origin for the start of polymerization and the attachment of oriC to the cell envelope (Bramhill and Kornberg, 1988; Løbner-Olesen et al., 1989; Pierucci et al., 1989). The attachment takes place somewhere within the cell half in which the oriC resides. The boundaries of this attachment/replication zone, which cannot include the polar cap, could be demarcated by the polar and centrally located periseptal annuli (Rothfield, this Forum). Since attachment and polymerization are two aspects of the same process, the attachment probably takes place via the polymerizing strand. Once polymerization begins, the oriC with the older template strand moves away from the younger one, by mechanisms unknown, to eventually take up residence in the equivalent domain of the complementary sister cell. Thus, the template strand that stays within its domain corresponds to the strand that was attached during the previous round of replication, and the template that moves away is the one that was not attached. The driving force for this translocation is not specified by our model, but a number of plausible alternatives have been proposed by others (reviewed in Leonard and Helmstetter, 1990). Throughout the ensuing replication and cell division, the chromosomes are located (or can move freely) within the attachment/replication zone of the developing daughter cell (lateral cylinder and septum). At some time during the course of this process, but before the next initiation event, the replication origins must be released from the attachment sites so that the entire process can be repeated. Thus, the probabilistic non-random chromosome segregation is due to the asymmetry of the attachment/replication zone in the cell, whereas the partitioning system itself must possess a mechanism to discriminate between template strands of different ages. This apparent mechanistic relationship between chromosome replication, chromosome partitioning and the maintenance of cell shape may provide an interesting framework for future experiments.

Biomechanical Phenomena↗

Chromosomal malsegregation and micronucleus induction in vitro by the DNA topoisomerase II inhibitor fisetin.

The plant flavonol fisetin is a common dietary component that has a variety of established biological effects, one of which is the inhibition of the enzyme DNA topoisomerase II (topo II). Compounds that inhibit topo II can exert genotoxic effects such as DNA double strand breaks, which can lead to the induction of kinetochore- or CREST-negative micronuclei. Despite reports that fisetin is an effective topoisomerase II inhibitor, its genotoxic effects have not yet been well characterized. Genotoxicity testing of fisetin was conducted in TK6 and HL60 cell lines and the cells were analyzed for malsegregating chromosomes as well as for the induction of micronuclei. Using the cytokinesis-blocked CREST micronucleus assay to discriminate between micronuclei formed from chromosomal breakage (CREST-negative) and chromosomal loss (CREST-positive), a statistically significant increase in CREST-positive micronuclei was seen for all doses tested in both cell lines. CREST-negative micronuclei, however, were significantly increased at the higher test concentrations in the TK6 cell line. These data indicate that at low concentrations fisetin is primarily exerting its genotoxic effects through chromosomal loss and that the induction of DNA breaks is a secondary effect occurring at higher doses. To confirm these results, the ability of fisetin to inhibit human topoisomerase II-alpha was verified in an isolated enzyme system as was its ability to interfere with chromosome segregation during the anaphase and telophase periods of the cell cycle. Fisetin was confirmed to be an effective topo II inhibitor. In addition, significant increases in the number of mis-segregating chromosomes were observed in fisetin-treated cells from both cell lines. We conclude that fisetin is an aneugen at low concentrations capable of interfering with proper chromosomal segregation and that it is also an effective topo II inhibitor, which exerts clastogenic effects at higher concentrations.

Chromosomes, Human↗

Bacterial chromosome dynamics.

Bacterial chromosomes are highly compacted structures and share many properties with their eukaryote counterparts, despite not being organized into chromatin or being contained within a cell nucleus. Proteins conserved across all branches of life act in chromosome organization, and common mechanisms maintain genome integrity and ensure faithful replication. The principles that underlie chromosome segregation in bacteria and eukaryotes share similarities, although bacteria segregate DNA as it replicates and lack a eukaryote-like mitotic apparatus for segregating chromosomes. This may be because the distances that newly replicated bacterial chromosomes move apart before cell division are small as compared to those in eukaryotes. Bacteria specify positional information, which determines where cell division will occur and which places the replication machinery and chromosomal loci at defined locations that change during cell cycle progression.

Bacterial Physiological Phenomena↗

The mitotic spindle matrix: a fibro-membranous lamin connection.

The mitotic spindle apparatus has attracted the attention of cell biologists for decades. Whereas the main function of this microtubule-based system is to segregate chromosomes, spindle morphogenesis and chromosome segregation must also coordinate with the segregation of the whole cell. The finding that RanGTPase stimulates the assembly of a lamin B-containing membranous matrix in mitosis(1) may provide a connection between the segregation of mitotic chromosomes and the partitioning of membrane systems during cell division.

Chromosome Segregation↗

The spindle: a dynamic assembly of microtubules and motors.

In all eukaryotes, a microtubule-based structure known as the spindle is responsible for accurate chromosome segregation during cell division. Spindle assembly and function require localized regulation of microtubule dynamics and the activity of a variety of microtubule-based motor proteins. Recent work has begun to uncover the molecular mechanisms that underpin this process. Here we describe the structural and dynamic properties of the spindle, and introduce the current concepts regarding how a bipolar spindle is assembled and how it functions to segregate chromosomes.

Animals↗