Showing papers on "Metaphase published in 1992"
TL;DR: It is proposed that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti- poleward forces, and that the nod protein provides the anti-poleward force.
Abstract: Mature Drosophila oocytes are arrested in metaphase of the first meiotic division. We have examined microtubule and chromatin reorganization as the meiosis I spindle assembles on maturation using indirect immunofluorescence and laser scanning confocal microscopy. The results suggest that chromatin captures or nucleates microtubules, and that these subsequently form a highly tapered spindle in which the majority of microtubules do not terminate at the poles. Nonexchange homologs separate from each other and move toward opposite poles during spindle assembly. By the time of metaphase arrest, these chromosomes are positioned on opposite half spindles, between the metaphase plate and the spindle poles, with the large nonexchange X chromosomes always closer to the metaphase plate than the smaller nonexchange fourth chromosomes. Nonexchange homologs are therefore oriented on the spindle in the absence of a direct physical linkage, and the spindle position of these chromosomes appears to be determined by size. Loss-of-function mutations at the nod locus, which encodes a kinesin-like protein, cause meiotic loss and nondisjunction of nonexchange chromosomes, but have little or no effect on exchange chromosome segregation. In oocytes lacking functional nod protein, most of the nonexchange chromosomes are ejected from the main chromosomal mass shortly after the nuclear envelope breaks down and microtubules interact with the chromatin. In addition, the nonexchange chromosomes that are associated with spindles in nod/nod oocytes show excessive poleward migration. Based on these observations, and the structural similarity of the nod protein and kinesin, we propose that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti-poleward forces, and that the nod protein provides the anti-poleward force.
382 citations
TL;DR: The in situ hybridization results obtained show that the H3 isochore family is localized in two coincident sets of bands of human metaphase chromosomes: telomeric bands and chromomycin A3-positive 4',6-diamidino-2-phenylindole-negative bands, the first step toward a complete compositional map of the human karyotype.
Abstract: Chromosome in situ suppression hybridization has been carried out on human metaphase chromosomes to localize the G+C-richest human DNA fraction (which only represents 3.5% of the genome), as isolated by preparative equilibrium centrifugation in Cs2SO4/3,6-bis(acetatomercurimethyl)-1,4-dioxane density gradient. This fraction essentially corresponds to isochore family H3. The rationale for carrying out this experiment is that this isochore family has, by far, the highest gene concentration, the highest concentration in CpG islands, the highest transcriptional and recombinational activity, and a distinct chromatin structure. The in situ hybridization results obtained show that the H3 isochore family is localized in two coincident sets of bands of human metaphase chromosomes: telomeric bands and chromomycin A3-positive 4',6-diamidino-2-phenylindole-negative bands. This result is the first step toward a complete compositional map of the human karyotype. Because the G+C gradient across isochore families is paralleled by a gene concentration gradient, such a map has structural, functional, and evolutionary relevance.
329 citations
TL;DR: It is concluded that Kinetochore microtubules change length predominantly through polymerization and depolymerization at the kinetochores during both metaphase and anaphase, so that flux contributes to polewards chromosome movements throughout mitosis.
Abstract: Microtubules in the mitotic spindles of newt lung cells were marked using local photoactivation of fluorescence. The movement of marked segments on kinetochore fibers was tracked by digital fluorescence microscopy in metaphase and anaphase and compared to the rate of chromosome movement. In metaphase, kinetochore oscillations toward and away from the poles were coupled to kinetochore fiber shortening and growth. Marked zones on the kinetochore microtubules, meanwhile, moved slowly polewards at a rate of approximately 0.5 micron/min, which identifies a slow polewards movement, or "flux," of kinetochore microtubules accompanied by depolymerization at the pole, as previously found in PtK2 cells (Mitchison, 1989b). Marks were never seen moving away from the pole, indicating that growth of the kinetochore microtubules occurs only at their kinetochore ends. In anaphase, marked zones on kinetochore microtubules also moved polewards, though at a rate slower than overall kinetochore-to-pole movement. Early in anaphase-A, microtubule depolymerization at kinetochores accounted on average for 75% of the rate of chromosome-to-pole movement, and depolymerization at the pole accounted for 25%. When chromosome-to-pole movement slowed in late anaphase, the contribution of depolymerization at the kinetochores lessened, and flux became the dominant component in some cells. Over the whole course of anaphase-A, depolymerization at kinetochores accounted on average for 63% of kinetochore fiber shortening, and flux for 37%. In some anaphase cells up to 45% of shortening resulted from the action of flux. We conclude that kinetochore microtubules change length predominantly through polymerization and depolymerization at the kinetochores during both metaphase and anaphase as the kinetochores move away from and towards the poles. Depolymerization, though not polymerization, also occurs at the pole during metaphase and anaphase, so that flux contributes to polewards chromosome movements throughout mitosis. Poleward force production for chromosome movements is thus likely to be generated by at least two distinct molecular mechanisms.
268 citations
TL;DR: The genomic in-situ hybridization method is fast, sensitive, accurate and informative and likely to be of great value for both cytogenetic analysis and in plant breeding programmes.
Abstract: Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Love, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.
261 citations
TL;DR: The complex patterns of expression of individual cyclin D genes and their evolutionary conservation across species suggest that each family member may play a distinct role in cell cycle progression.
255 citations
TL;DR: The folding of chromatin in interphase cell nuclei was studied by fluorescent in situ sequences chromatin according to a random walk model and it was shown that the remaining gap in the published maps of this region is negligible in size.
Abstract: The folding of chromatin in interphase cell nuclei was studied by fluorescent in situ sequences chromatin according to a random walk model. This model provides the basis for calculating the spacing of sequences along the linear DNA molecule from interphase distance measurements. An interphase mapping strategy based on this model was tested with 13 probes from a 4-megabase pair (Mbp) region of chromosome 4 containing the Huntington disease locus. The results confirmed the locations of the probes and showed that the remaining gap in the published maps of this region is negligible in size. Interphase distance measurements should facilitate construction of chromosome maps with an average marker density of one per 100 kbp, approximately ten times greater than that achieved by hybridization to metaphase chromosome. achieved by hybridization to metaphase chromosomes.
254 citations
TL;DR: DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphases.
Abstract: We have produced metaphase spindles and induced them to enter anaphase in vitro. Sperm nuclei were added to frog egg extracts, allowed to replicate their DNA, and driven into metaphase by the addition of cytoplasm containing active maturation promoting factor (MPF) and cytostatic factor (CSF), an activity that stabilizes MPF. Addition of calcium induces the inactivation of MPF, sister chromatid separation and anaphase chromosome movement. DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphase. Topoisomerase II activity towards exogenous substrates does not increase at the metaphase to anaphase transition, showing that chromosome separation at anaphase is not triggered by a bulk activation of topoisomerase II.
235 citations
TL;DR: The cyclins are synthesized continuously and are rapidly destroyed shortly before the metaphase-anaphase transition of the mitotic cell cycles, with cyclin A being degraded in advance of cyclin B.
Abstract: Fertilization of clam oocytes initiates a series of cell divisions, of which the first three--meiosis I, meiosis II, and the first mitotic division--are highly synchronous. After fertilization, protein synthesis is required for the successful completion of every division except meiosis I. When protein synthesis is inhibited, entry into meiosis I and the maintenance of M phase for the normal duration of meiosis occur normally, but the chromosomes fail to interact correctly with the spindle in meiosis II metaphase. By contrast, inhibition of protein synthesis immediately after completion of meiosis or mitosis stops cells entering the next mitosis. We describe the behavior of cyclins A and B in relation to these "points of no return." The cyclins are synthesized continuously and are rapidly destroyed shortly before the metaphase-anaphase transition of the mitotic cell cycles, with cyclin A being degraded in advance of cyclin B. Cyclin destruction normally occurs during a 5-min window in mitosis, but in the monopolar mitosis that occurs after parthenogenetic activation of clam oocytes, or when colchicine is added to fertilized eggs about to enter first mitosis, the destruction of cyclin B is strongly delayed, whereas proteolysis of cyclin A is maintained in an activated state for the duration of metaphase arrest. Under either of these abnormal conditions, inhibition of protein synthesis causes a premature return to interphase that correlates with the time when cyclin B disappears.
227 citations
TL;DR: Inhibition of myosin-II by cyclin-p34cdc2 during prophase and metaphase could delay cytokinesis until chromosome segregation is initiated and thus determine the timing of cytokineses relative to earlier events in mitosis.
Abstract: To understand how cytokinesis is regulated during mitosis, we tested cyclin-p34cdc2 for myosin-II kinase activity, and investigated the mitotic-specific phosphorylation of myosin-II in lysates of Xenopus eggs. Purified cyclin-p34cdc2 phosphorylated the regulatory light chain of cytoplasmic and smooth muscle myosin-II in vitro on serine-1 or serine-2 and threonine-9, sites known to inhibit the actin-activated myosin ATPase activity of smooth muscle and nonmuscle myosin (Nishikawa, M., J. R. Sellers, R. S. Adelstein, and H. Hidaka. 1984. J. Biol. Chem. 259:8808-8814; Bengur, A. R., A. E. Robinson, E. Appella, and J. R. Sellers. 1987. J. Biol. Chem. 262:7613-7617; Ikebe, M., and S. Reardon. 1990. Biochemistry. 29:2713-2720). Serine-1 or -2 of the regulatory light chain of Xenopus cytoplasmic myosin-II was also phosphorylated in Xenopus egg lysates stabilized in metaphase, but not in interphase. Inhibition of myosin-II by cyclin-p34cdc2 during prophase and metaphase could delay cytokinesis until chromosome segregation is initiated and thus determine the timing of cytokinesis relative to earlier events in mitosis.
198 citations
TL;DR: In this article, a reverse chromosome painting method is proposed to detect chromosome duplications in short-term blood culture preparations or cell lines by using bivariate flow karyotype analysis.
Abstract: We describe a method, termed reverse chromosome painting, which allows the rapid analysis of the content and breakpoints of aberrant chromosomes. The method involves the sorting of small numbers of the aberrant chromosome from short term blood culture preparations or cell lines by using bivariate flow karyotype analysis. The sorted chromosomes are amplified and biotin labelled enzymatically using a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), the product annealed to metaphase spreads from normal subjects, and hybridisation detected using fluorescence in situ hybridisation (FISH). We show the usefulness of this method for routine clinical cytogenetics by the analysis of cases involving an insertion, a deletion, a translocation, and two cases of a chromosome with additional material of unknown origin. The method has particular application for the rapid resolution of the origin of de novo unbalanced chromosome duplications.
198 citations
TL;DR: Results reveal a specific cell cycle-dependent redistribution of PP-1 and its involvement in reversing p34cdc2-induced effects after mid-mitosis in mammalian cells.
Abstract: We have examined the role of protein phosphatase type 1 (PP-1) in mammalian cell mitosis. Immunofluorescence using anti-PP-1 antibodies revealed that PP-1, which is mainly localized in the cytoplasm of G1 and S phase cells, accumulates in the nucleus during G2 phase and intensely colocalizes with individual chromosomes at mitosis. This increase in nuclear PP-1 in G2/M cells was confirmed by immunoblotting on subcellular fractions. Microinjection of neutralizing anti-PP-1 antibodies before division blocked cells at metaphase, whereas injection of PP-1 in one pole of an anaphase B cell accelerated cytokinesis and the reflattening of the injected cell. These results reveal a specific cell cycle-dependent redistribution of PP-1 and its involvement in reversing p34cdc2-induced effects after mid-mitosis in mammalian cells.
TL;DR: The frequency of aneuploidy in newly aspirated, uninseminated metaphase II-stage human oocytes which exhibited seven distinct forms of cytoplasmic dysmorphism is determined in the living state by DNA fluorescence followed by fixation and air-drying for karyotyping.
Abstract: The frequency of aneuploidy in 583 newly aspirated, uninseminated metaphase II-stage human oocytes which exhibited seven distinct forms of cytoplasmic dysmorphism [Van Blerkom (1990) J. Electron Microsc. Tech., 16,324] after ovarian stimulation and ovulation induction was determined in the living state by DNA fluorescence followed by fixation and air-drying for karyotyping. The findings demonstrate that as many as half of the oocytes with dysmorphic phenotypes which arise early in meiotic maturation are aneuploid, with hypohaplidy predominant. In contrast, cytoplasmic defects which occur at or after metaphase I are associated with a relatively low frequency of aneuploidy (less than 15%), which is comparable to that previously reported for human oocytes with a normal cytoplasmic appearance [Van Blerkom and Henry (1988) Hum. Reprod., 3, 777]. The aetiologies of aneuploidy in dysmorphic oocytes, as well as the clinical implications for oocyte selection in laboratory-assisted conception are discussed.
ENEA1
TL;DR: A new method is described for the isolation of large quantities of Vicia faba metaphase chromosomes and the quality and the quantity of isolated chromosomes, examined microscopically and by flow cytometry, depended on the extent of the fixation.
Abstract: A new method is described for the isolation of large quantities of Vicia faba metaphase chromo- somes. Roots were treated with 2.5 mM hydroxyurea for 18 h to accumulate meristem tip cells at the G1/S interface. After release from the block, the cells re-en- tered the cell cycle with a high degree of synchrony. A treatment with 2.5 p~M amiprophos-methyl (APM) was used to accumulate mitotic cells in metaphase. The highest metaphase index (53.9%) was achieved when, 6 h after the release from the hydroxyurea block, the roots were exposed to APM for 4 h. The chromosomes were released from formaldehyde-fixed root tips by chopping with a scalpel in LB01 lysis buffer. Both the quality and the quantity of isolated chromosomes, exam- ined microscopically and by flow cytometry, depended on the extent of the fixation. The best results were achieved after fixation with 6% formaldehyde for 30min. Under these conditions, 1.106 chromosomes were routinely obtained from 30 root tips. The chromo- somes were morphologically intact and suitable both for high-resolution chromosome studies and for flow-cyto- metric analysis and sorting. After the addition of hexy- lene glycol, the chromosome suspensions could be stored at 4 ~ C for six months without any signs of deterioration.
TL;DR: Mutations in the gene l(1)zw10 disrupt the accuracy of chromosome segregation in a variety of cell types during the course of Drosophila development, and Cytological analysis of mutant larval brain neuroblasts shows very high levels of aneuploid cells.
Abstract: Mutations in the gene l(1)zw10 disrupt the accuracy of chromosome segregation in a variety of cell types during the course of Drosophila development. Cytological analysis of mutant larval brain neuroblasts shows very high levels of aneuploid cells. Many anaphase figures are aberrant, the most frequent abnormality being the presence of lagging chromosomes that remain in the vicinity of the metaphase plate when the other chromosomes have migrated toward the spindle poles. Finally, the centromeric connection between sister chromatids in mutant neuroblasts treated with colchicine often appears to be broken, in contrast with similarly treated control neuroblasts. The 85-kD protein encoded by the l(1)zw10 locus displays a dynamic pattern of localization in the course of the embryonic cell cycle. It is excluded from the nuclei during interphase, but migrates into the nuclear zone during prometaphase. At metaphase, the zw10 antigen is found in a novel filamentous structure that may be specifically associated with kinetochore microtubules. Upon anaphase onset, there is an extremely rapid redistribution of the zw10 protein to a location at or near the kinetochores of the separating chromosomes.
TL;DR: The results indicate that morphology of prematurely condensed G1 and early S chromatin has a minor influence on chromosome constitution of manipulated embryos, however, that of late S Chromatin affects chromosome constitution in embryos and may account for reduced development of nuclear transplant embryos when late S phase donor nuclei are used.
Abstract: We investigated the influence of the cell cycle stage of the nuclear donor on prematurely condensed chromatin (PCC) and spindle morphology and on chromosome constitution in rabbit nuclear transplant embryos. The configuration of PCC following nuclear transplantation with Gl, early S, and late 5 phase donor nuclei (Gl, early S, and late S transplants, respectively) was characterized in whole mounts and chromosome spreads. In addition, the influence of the donor cell cycle stage on chromosome constitution in cleavage stage-manipulated embryos was determined. Within 2 h after fusion of the donor blastomere, the recipient oocyte cytoplasm was able to induce formation de novo of a metaphase plate associated with a spindle in G 1, early S, and late 5 transplants. Metaphase chromosomes and spindle were intact in most cases of PCC in Gi transplants. However, these structures displayed minor abnormalities in early S transplants and gross abnormalities in late S transplants, such as incomplete or absent spindle formation and incomplete chromatin condensation. Normal chromosomes were present in GI and early S transplants, whereas chromosome abnormalities were detected in late S transplants. The results indicate that morphology of prematurely condensed Gi and early S chromatin has a minor influence on chromosome constitution of manipulated embryos. That of late S chromatin, however, affects chromosome constitution in embryos and may account for reduced development of nuclear transplant embryos when late S phase donor nuclei are used.
TL;DR: The higher resolution afforded by “stretching” the centromeric heterochromatin of chromosomes 1, 9 and 16 confirmed the low level of H4 acetylation in these domains, and considers the implications of these observations in relation to chromatin conformation and activity.
Abstract: Unfixed metaphase chromosome preparations from human lymphocyte cultures were immunofluorescently labelled using antibodies to defined histone epitopes. Both mouse monoclonal antibody HBC-7, raised against the N-terminal region of H2B, and rabbit serum R5/12, which recognizes H4 acetylated at Lys-12, gave non-uniform labelling patterns, whereas control antibodies against total histone fractions H4 and H1 produced homogeneous fluorescence. HBC-7 bound approximately uniformly to the bulk of the chromosomes, but the major heterochromatic domains of chromosomes 1, 9, 15, 16 and the Y showed significantly brighter fluorescence. Serum R5/12 indicated an overall reduction in acetylation of H4 in metaphase chromosomes compared with interphase nuclei, although some specific chromosomal locations had considerably elevated acetylation levels. Acetylation levels in the major heterochromatic domains appeared extremely low. To investigate further the differences noted in heterochromatin labelling, metaphases from cultures grown in the presence of various agents known to induce undercondensation of the major heterochromatic domains were similarly immunolabelled. Decondensed heterochromatin no longer exhibited higher than normal immunofluorescence levels with HBC-7. The higher resolution afforded by "stretching" the centromeric heterochromatin of chromosomes 1, 9 and 16 confirmed the low level of H4 acetylation in these domains. We consider the implications of these observations in relation to chromatin conformation and activity.
TL;DR: The results show that interphase and metaphase FISH analyses are useful methods to detect trisomy 8 cells in BM specimens, especially for specimens with normal or uncertain conventional cytogenetic results.
TL;DR: A multicolor fluorescence in situ hybridization approach based on combinatorial probe labeling and digital imaging microscopy is demonstrated, enabling one to evaluate aberrations of multiple chromosomes in a single hybridization experiment using metaphase chromosomes and interphase nuclei from a variety of cell types, including lymphocytes and amniocytes.
Abstract: The most frequent aneuploidies in newborns involve the autosomes 13, 18 and 21 as well as both sex chromosomes. Fluorescence in situ hybridization readily allows the detection of numerical chromosomal aberrations throughout all stages of the cell cycle. Using a multicolor fluorescence in situ hybridization approach based on combinatorial probe labeling and digital imaging microscopy we demonstrate the simultaneous visualization of probe sets specific for chromosomes 13, 18, 21, X and Y. This approach enables one to evaluate aberrations of multiple chromosomes in a single hybridization experiment using metaphase chromosomes and interphase nuclei from a variety of cell types, including lymphocytes and amniocytes.
TL;DR: It is concluded that the internal free calcium level determines the transition to interphase, and the capacity of the mouse oocyte to pass into metaphase III is lost with increasing time post-ovulation.
Abstract: Nuclear maturation of the mouse oocyte becomes arrested in metaphase of the second meiotic division (MII). Fertilization or parthenogenetic activation induces meiotic completion, chromosomal decondensation and formation of a pronucleus. This completion of meiosis is probably triggered by a transient increase in cytosolic calcium ions. When activated just after ovulation by a low concentration of the calcium ionophore A23187, the majority of the mouse oocytes go through a metaphase to anaphase transition and extrude their second polar body but they do not proceed into interphase; instead their chromatids remain condensed and a microtubular metaphase spindle reforms (metaphase III). However, a high percentage of these oocytes will undergo a true parthenogenetic activation assessed by the formation of a pronucleus, when exposed to a higher concentration of the calcium ionophore. The capacity of the mouse oocyte to pass into metaphase III is lost with increasing time post-ovulation. Direct measurement of intracellular calcium with Fura-2 reveals higher levels of cytosolic calcium in aged oocytes and/or using higher concentrations of calcium ionophore for activation. It is concluded that the internal free calcium level determines the transition to interphase.
TL;DR: It is observed that a similar sequence of events takes place during these two transitions: after anaphase, a polar body is extruded, the microtubules of the midbody disappear rapidly and a new metaphase spindle forms.
Abstract: When metaphase II-arrested mouse oocytes (M II) are activated very soon after ovulation, they respond abortively by second polar body extrusion followed by another metaphase arrest (metaphase III, M III; Kubiak, 1989). The M II/M III transition resembles the natural transition between the first and second meiotic metaphases (M I/M II). We observed that a similar sequence of events takes place during these two transitions: after anaphase, a polar body is extruded, the microtubules of the midbody disappear rapidly and a new metaphase spindle forms. The MPM-2 monoclonal antibody (which reacts with phosphorylated proteins associated with the centrosome during M-phase) stains discrete foci of peri-centriolar material only in metaphase arrested oocytes; during both transitional periods, a diffuse staining is observed, suggesting that these centrosomal proteins are dephosphorylated, as in a normal interphase. However, the chromosomes always remain condensed and an interphase network of microtubules is never observed during the transitional periods. Incorporation of 32P into proteins increases specifically during the transitional periods. Pulse-chase experiments, after labeling of the oocytes in M phase with 32P, showed that a 62 kDa phosphoprotein band disappears at the time of polar body extrusion. Histone H1 kinase activity (which reflects the activity of the maturation promoting factor) drops during both transitional periods to the level characteristic of interphase and then increases when the new spindle forms. Both the M I/M II and M II/M III transitions require protein synthesis as demonstrated by the effect of puromycin. These results suggest that the two M-phase/M-phase transitions are probably driven by the same molecular mechanism.
TL;DR: Triple fluorescence in situ hybridization with a plasmid DNA library from sorted human chromosomes 8 in combination with bacteriophage clones flanking the break point in 8q24 of the Burkitt lymphoma cell line Jl was used for the specific delineation of this breakpoint in individual tumor cells.
Abstract: Triple fluorescence in situ hybridization with a plasmid DNA library from sorted human chromosomes 8 in combination with bacteriophage clones flanking the breakpoint in 8q24 of the Burkitt lymphoma cell line Jl was used for the specific delineation of this breakpoint in individual tumor cells. With this approach, tumor-specific breakpoints in translocation chromosomes can be detected at all stages of the cell cycle with high specificity.
TL;DR: The okadaic acid-induced mitotic blockage was probably due to the inhibition of a type 2A protein phosphatase that is involved in the transition from metaphase to anaphase.
Abstract: The effects of the protein phosphatase inhibitor okadaic acid were examined using the pig kidney cell line LLC-PK. At relatively low concentrations of the inhibitor (8–40 nM), cells became blocked in a metaphase-like mitotic state beginning 6–8 h after initial treatment. Spindle microtubules were present throughout the period of the mitotic block, but were not stabilized since they remained sensitive to nocodazole depolymerization. With increasing length of the mitotic block chromosome alignment at the metaphase plate was disrupted and multipolar spindles developed. Cells continued to accumulate in mitosis for at least 24 h, indicating that at these low concentrations okadaic acid was not cytotoxic, but rather acted as a cytostatic agent. Upon release of the okadaic acid block, mitotic LLC-PK cells recovered and completed anaphase. After extended periods of treatment some cells were able to escape the okadaic acid-induced mitotic block. These cells were multinucleate and had undergone cytokinesis in the absence of chromosome segregation. At higher concentrations of okadaic acid (0.5-1.0 microM), mitosis was blocked within 30–60 min of treatment. However, within 90–120 min treated cells rounded up and detached from the monolayer, regardless of whether they were in interphase or mitosis. Cytoplasmic microtubules were depolymerized in the detached cells, and these cells could not recover from the cytotoxic effects of such high concentrations of okadaic acid. Thus, differential effects of the phosphatase inhibitor could be demonstrated, depending upon the concentration of okadaic acid applied to the cultures. The okadaic acid-induced mitotic blockage was probably due to the inhibition of a type 2A protein phosphatase that is involved in the transition from metaphase to anaphase.
TL;DR: Two subfamilies located exclusively on the human acrocentric chromosomes are described and characterize and a number of monomers from these two subfam families are compared to each other and to other beta satellite monomers to assess both inter- and intrasubfamily sequence relationships for these monomers.
Journal Article•
TL;DR: In addition to the analysis of metaphase spreads, the particular usefulness of these YAC clones in combination with whole chromosome painting to analyze specific chromosome aberrations directly in the interphase nucleus is demonstrated.
Abstract: A human yeast artificial chromosome (YAC) library was screened by polymerase chain reaction with oligonucleotide primers defined for DNA sequences of the BCR gene and the protooncogenes c- raf-1 , c- fms , and c- erB-2 . Alu-PCR-generated human DNA sequences were obtained from the respective YAC clones and used for fluorescence in situ hybridization experiments under suppression conditions. After chromosomal in situ suppression hybridization to GTG-banded human prometaphase chromosomes, seven of nine initially isolated YAC clones yielded strong signals exclusively in the chromosome bands containing the respective genes. Two clones yielded additional signals on other chromosomes and were excluded from further tests. The band-specific YACs were successfully applied to visualize specific structural chromosome aberrations in peripheral blood cells from patients with myelodysplasia exhibiting del(5)(q13q34), chronic myeloid leukemia and acute lymphocytic leukemia with t(9;22)(q34;q11), acute promyelocytic leukemia (M3) with t(15;17)(q22;q21), and in a cell line established from a proband with the constitutional translocation t(3;8)(p14.2;q24). In addition to the analysis of metaphase spreads, we demonstrate the particular usefulness of these YAC clones in combination with whole chromosome painting to analyze specific chromosome aberrations directly in the interphase nucleus.
TL;DR: The regulation of the oocytes' cell cycle machinery by hormone‐mediated somatic cell signals, involving both positive and negative stimuli, ensures that meiotic cell cycle progression is synchronized with the earliest pivotal events of mammalian reproduction.
Abstract: Mammalian oocytes exhibit a series of cell cycle transitions that coordinate the penultimate events of meiosis with the onset of embryogenesis at fertilization. The execution of these cell cycle transitions, at G2/M of meiosis-I and metaphase/anaphase of meiosis I and II, involve both biosynthetic and post-translational modifications that directly modulate centrosome and microtubule behavior. Specifically, somatic cells alter the signal transduction pathways in the oocyte and influence the expression of maturation promoting factor (MPF) and cytostatic factor (CSF) activity through a microtubule-dependent mechanism. The regulation of the oocytes' cell cycle machinery by hormone-mediated somatic cell signals, involving both positive and negative stimuli, ensures that meiotic cell cycle progression is synchronized with the earliest pivotal events of mammalian reproduction.
TL;DR: In this article, the subcellular distribution of cyclin A and B1 during the somatic cell cycle was studied using immunofluorescence staining to investigate the substrate specificity of mitotic cyclin and cyclin B1.
TL;DR: Results provide direct evidence that a drop in activity of MPF in murine oocytes occurs concomitantly with the exit from metaphase I; MPF activity remains low until the cell re‐enters metaphase.
Abstract: Cell fusions have been used to determine the biological activity of the MPF complex in murine oocytes during their progression through anaphase and telophase to metaphase II. Oocytes (1) at metaphase I, (2) during the anaphase-telophase transition, or (3) at metaphase II were fused to germinal vesicle-staged (immature) oocytes. The hybrids were cultured for 1 h in the presence of db cAMP before fixation and nuclear evaluation. Metaphase I oocytes invariably induced germinal vesicle breakdown (GVBD) in the immature partner. By contrast, anaphase/telophase oocytes never induced GVBD in immature oocytes. The capacity to induce GVBD reappears after the formation of the second metaphase plate. In a second study, histone H1 kinase activity was measured during mouse oocyte maturation in single oocytes. H1 kinase activity was low in GV oocytes, increased sharply at MI, declined during anaphase and telophase and increased again at MII. After egg activation, H1 kinase activity was reduced to basal levels. These results provide direct evidence that a drop in activity of MPF in murine oocytes occurs concomitantly with the exit from metaphase I; MPF activity remains low until the cell re-enters metaphase.
TL;DR: It is shown that cdc25 protein is constitutively expressed throughout the cell cycle of nontransformed mammalian fibroblasts and does not undergo major changes in protein level, and separation in two distinct cellular compartments of the cDC25 phosphatase and its substrate p34cdc2-cyclin B may be of importance in the regulation of thecdc2 kinase activity.
Abstract: A family of proteins homologous to the cdc25 gene product of the fission yeast bear specific protein tyrosine phosphatase activity involved in the activation of the p34cdc2-cyclin B kinase. Using affinity-purified antibodies raised against a synthetic peptide corresponding to the catalytic site of the cdc25 phosphatase, we show that cdc25 protein is constitutively expressed throughout the cell cycle of nontransformed mammalian fibroblasts and does not undergo major changes in protein level. By indirect immunofluorescence, cdc25 protein is found essentially localized in the nucleus throughout interphase and during early prophase. Just before the complete nuclear envelope breakdown at the prophase-prometaphase boundary, cdc25 proteins are redistributed throughout the cytoplasm. During metaphase and anaphase, cdc25 staining remains distributed throughout the cell and excludes the condensed chromosomes. The nuclear locale reappears during telophase. In light of the recent data describing the cytoplasmic localization of cyclin B protein (Pines, J., and T. Hunter. 1991. J. Cell Biol. 115:1-17), the data presented here suggest that separation in two distinct cellular compartments of the cdc25 phosphatase and its substrate p34cdc2-cyclin B may be of importance in the regulation of the cdc2 kinase activity.
TL;DR: The results indicate that the sponge product is specifically required for the formation of actin caps and metaphase furrows, and that these structures are dispensible during the early syncytial blastoderm cell cycles.
Abstract: In the syncytial blastoderm stage of Drosophila embryogenesis, dome-shaped actin "caps" are observed above the interphase nuclei. During mitosis, this actin rearranges to participate in the formation of pseudocleavage furrows, transient membranous invaginations between dividing nuclei. Embryos laid by homozygous sponge mothers lack these characteristic actin structures, but retain other actin associated structures and processes. Our results indicate that the sponge product is specifically required for the formation of actin caps and metaphase furrows. The specificity of the sponge phenotype permits dissection of both the process of actin cap formation and the functions of actin caps and metaphase furrows. Our data demonstrate that the distribution of actin binding protein 13D2 is unaffected in sponge embryos and suggest that 13D2 is upstream of actin in cortical cap assembly. Although actin caps and metaphase furrows have been implicated in maintaining the fidelity of nuclear division and the positions of nuclei within the cortex, our observations indicate that these structures are dispensible during the early syncytial blastoderm cell cycles. A later requirement for actin metaphase furrows in preventing the nucleation of mitotic spindles between inappropriate centrosomes is observed. Furthermore, the formation of actin caps and metaphase furrows is not a prerequisite for the formation of the hexagonal array of actin instrumental in the conversion of the syncytial embryo into a cellular blastoderm.
TL;DR: The results strongly suggest that monoclonal antibody 6C6 identifies one of conserved elements of the pericentriolar material, which appeared very early during the evolution of eukaryotes.
Abstract: We have used monoclonal antibodies raised against isolated native calf thymus centrosomes to probe the structure and composition of the pericentriolar material. To distinguish prospective antibodies as specific to conserved elements of this material, we screened clones by their identification of microtubule organizing centers (MTOCs) in different animal and plant cells. Among the clonal antibodies that reacted with MTOCs in both plant and mammalian cells, we describe one (mAb 6C6) that was found to immunostain centrosomes in a variety of bovine and human cells. In cycling cells this signal persisted through the entire cell cycle. Microscopy showed that the mAb 6C6 antigen was a component of the pericentriolar material and this was confirmed by biochemical analysis of centrosomes. Using immunoblot analysis of protein fractions derived from purified components of centrosomes, we have characterized the mAb 6C6 antigen as a 180 kDa polypeptide. We conclude that we have identified a protein component permanently associated with the pericentriolar material. Surprisingly, monoclonal antibody 6C6 also stained other mitotic organelles in mammalian cells, in a cell-cycle-dependent manner. During prometaphase and metaphase the antibody stained both centrosomes and kinetochores. At the onset of anaphase the kinetochore-specific staining dissociated from chromosomes and was subsequently redistributed onto a newly characterized organelle, the telophase disc while the centrosomal stain remained intact. It is not known if the 180 kDa centrosomal protein itself redistributes during mitosis, or if the pattern observed represents other antigens with shared epitopes. The pericentriolar material is thought to be composed of conserved elements, which appeared very early during the evolution of eukaryotes. Our results strongly suggest that mAb 6C6 identifies one of these elements.