Metaphase

About: Metaphase is a research topic. Over the lifetime, 6925 publications have been published within this topic receiving 291590 citations. The topic is also known as: GO:0007091 & mitotic metaphase/anaphase transition.

Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes

Abstract: Fluorescence of the dye 33258 Hoechst bound to chromosomes is partially quenched by the introduction of BrdU into chromosomal DNA. This finding has allowed microfluorometric detection of DNA synthesis in human metaphase chromosomes. Incorporation, shortly before cell harvest, of a pulse of thymidine into chromosomes otherwise substituted with BrdU results in sharply defined foci of bright 33258 Hoechst fluorescence, corresponding to regions of late DNA replication. The latereplicating X chromosome can thereby be clearly identified, and the time course of appearance of its fluorescent bands can be compared with that of its earlier replicating homologue. Growth of cells in medium containing BrdU for two generations allows fluorometric documentation of the semiconservative distribution of newly replicated DNA between sister chromatids, and regions of sister chromated exchange are demarcated. This approach should constitute, in many instances, a convenient, high-resolution fluorometric alternative to autoradiography.

Mitotic Block Induced in HeLa Cells by Low Concentrations of Paclitaxel (Taxol) Results in Abnormal Mitotic Exit and Apoptotic Cell Death

Abstract: Paclitaxel at low concentrations (10 nm for 20 h) induces ∼90% mitotic block at the metaphase/anaphase transition in HeLa cells, apparently by suppressing dynamics of spindle microtubules (M. A. Jordan et al., Proc. Natl. Acad. Sci. USA, 90: 9552–9556, 1993). It is not known, however, whether inhibition of mitosis by such low paclitaxel concentrations results in cell death. In the present work, we found that after removal of pacli-taxel (10 nm-1 µm), blocked cells did not resume proliferation. Instead, cells exited mitosis abnormally within 24 h. They did not progress through anaphase or cytokinesis but entered an interphase-like state (chromatin decondensed, and an interphase-like microtubule array and nuclear membranes reformed). Many cells (≥55%) contained multiple nuclei. Additional DNA synthesis and polyploidy did not occur. DNA degradation into nucleosome-sized fragments characteristic of apoptosis began during drug incubation and increased after drug removal. Cells died within 48–72 h. Incubation with paclitaxel (10 nm for 20 h) resulted in high intracellular drug accumulation (8.3 µm) and little efflux after paclitaxel removal; intracellular retention of paclitaxel may contribute to its efficacy. The results support the hypothesis that the most potent chemotherapeutic mechanism of paclitaxel is kinetic stabilization of spindle microtubule dynamics.

Partial inhibition of Cdk1 in G2 phase overrides the SAC and decouples mitotic events

Abstract: Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin-dependent kinase 1 (Cdk1). In this study we utilized low doses of the Cdk1-specific inhibitor, RO3306 from early G2 phase onwards. Addition of low doses of RO3306 in G2 phase induced minor chromosome congression and segregation defects. In contrast, mild doses of RO3306 during G2 phase resulted in cells entering an aberrant mitosis, with cells fragmenting centrosomes and failing to fully disassemble the nuclear envelope. Cells often underwent cytokinesis and metaphase simultaneously, despite the presence of an active spindle assembly checkpoint, which prevented degradation of cyclin B1 and securin, resulting in the random partitioning of whole chromosomes. This highly aberrant mitosis produced a significant increase in the proportion of viable polyploid cells present up to 3 days post-treatment. Furthermore, cells treated with medium doses of RO3306 were only able to reach the threshold of Cdk1 substrate phosphorylation required to initiate nuclear envelope breakdown, but failed to reach the levels of phosphorylation required to correctly complete pro-metaphase. Treatment with low doses of Okadaic acid, which primarily inhibits PP2A, rescued the mitotic defects and increased the number of cells that completed a normal mitosis. This supports the current model that PP2A is the primary phosphatase that counterbalances the activity of Cdk1 during mitosis. Taken together these results show that continuous and subtle disruption of Cdk1 activity from G2 phase onwards has deleterious consequences on mitotic progression by disrupting the balance between Cdk1 and PP2A.

Organization of the Mitotic Chromosome

Abstract: Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two distinct three-dimensional folding states of the human genome. We show that the highly compartmentalized and cell type–specific organization described previously for nonsynchronous cells is restricted to interphase. In metaphase, we identified a homogenous folding state that is locus-independent, common to all chromosomes, and consistent among cell types, suggesting a general principle of metaphase chromosome organization. Using polymer simulations, we found that metaphase Hi-C data are inconsistent with classic hierarchical models and are instead best described by a linearly organized longitudinally compressed array of consecutive chromatin loops.

Localization of single copy DNA sequences of G-banded human chromosomes by in situ hybridization.

Abstract: Recombinant lambda bacteriophage clone H3 containing a human DNA segment of 14.9 kb present in one or two copies per haploid genome was isolated. In situ hybridization in human metaphase chromosomes of the 3H-labeled cloned DNA resulted in highly significant labeling (53% of cells) of band p36 of chromosome 1, such that 22% of all chromosomal grains were located on this region. Hybridization was dependent upon the presence of dextran sulfate in the hybridization mixture and was not affected by repetitive DNA competitor. These results demonstrate localization of a single copy sequence on human metaphase chromosomes.