Showing papers on "Metaphase published in 1993"
TL;DR: The results indicate that taxol shares a common antiproliferative mechanism with vinblastine, and at its lowest effective concentrations, taxol appears to block mitosis by kinetically stabilizing spindle microtubules and not by changing the mass of polymerizedmicrotubules.
Abstract: Taxol inhibited HeLa cell proliferation by inducing a sustained mitotic block at the metaphase/anaphase boundary. Half-maximal inhibition of cell proliferation occurred at 8 nM taxol, and mitosis was half-maximally blocked at 8 nM taxol. Inhibition of mitosis was associated with formation of an incomplete metaphase plate of chromosomes and an altered arrangement of spindle microtubules that strongly resembled the abnormal organization that occurs with low concentrations of vinblastine and other antimitotic compounds. No increase in microtubule polymer mass occurred below 10 nM taxol. The mass of microtubules increased half-maximally at 80 nM taxol and attained maximal levels (5 times normal) at 330 nM taxol. At submicromolar concentrations, taxol suppressed growing and shortening at the ends of microtubules reassembled in vitro from bovine brain tubulin in a manner that resembled suppression by vinblastine. Taxol was concentrated in HeLa cells several hundredfold to levels that were similar to those which suppressed dynamic instability in vitro. The results indicate that taxol shares a common antiproliferative mechanism with vinblastine. At its lowest effective concentrations, taxol appears to block mitosis by kinetically stabilizing spindle microtubules and not by changing the mass of polymerized microtubules.
1,066 citations
TL;DR: In this paper, immunolabeled human and mouse metaphase chromosomes with antibodies specific for the acetylated isoforms of histone H4 were labeled in regions corresponding to conventional R bands (regions enriched in coding DNA), except for a single chromosome in female cells.
711 citations
TL;DR: The behaviour of esp1 mutants shows in addition that kinase destruction can occur in the absence of anaphase completion, and the execution ofAnaphase and the destruction of CDC28 kinase activity appear to take place independently of one another.
Abstract: It is widely assumed that degradation of mitotic cyclins causes a decrease in mitotic cdc2/CDC28 kinase activity and thereby triggers the metaphase to anaphase transition. Two observations made on the budding yeast Saccharomyces cerevisiae are inconsistent with this scenario: (i) anaphase occurs in the presence of high levels of kinase in cdc15 mutants and (ii) overproduction of a B-type mitotic cyclin causes arrest not in metaphase as previously reported but in telophase. Kinase destruction is therefore implicated in the exit from mitosis rather than the entry into anaphase. The behaviour of esp1 mutants shows in addition that kinase destruction can occur in the absence of anaphase completion. The execution of anaphase and the destruction of CDC28 kinase activity therefore appear to take place independently of one another.
483 citations
TL;DR: The potential uses of FISH continue to increase with each new technical innovation, and one of the biggest impacts has been in the field of detection and diagnosis of human malignancies.
470 citations
TL;DR: It is shown that when a constitutively active mutant of calmodulin-dependent protein kinase II (CaM KII) is added to a CSF extract, cyclin degradation and Cdc2 kinase inactivation occur even in the absence of Ca2+, and the extract loses its ability to cause metaphase arrest when transferred into embryos.
Abstract: In vertebrates, unfertilized eggs are arrested at second meiotic metaphase by a cytostatic factor (CSF), an essential component of which is the product of the c-mos proto-oncogene. CSF prevents ubiquitin-dependent degradation of mitotic cyclins and thus inactivation or the M phase-promoting factor (MPF). Fertilization or parthenogenetic activation triggers a transient increase in the cytoplasmic free Ca2+ (reviewed in refs 5 and 6), inactivates both CSF and MPF, and releases eggs from meiotic metaphase arrest. A calmodulin-dependent process is required for cyclin degradation to occur in cell-free extracts prepared from metaphase II-arrested eggs (CSF extracts) when the free Ca2+ concentration is transiently raised in the physiological micromolar range. Here we show that when a constitutively active mutant of calmodulin-dependent protein kinase II (CaM KII) is added to a CSF extract, cyclin degradation and Cdc2 kinase inactivation occur even in the absence of Ca2+, and the extract loses its ability to cause metaphase arrest when transferred into embryos. Furthermore, specific inhibitors of CaM KII prevent cyclin degradation after calcium addition. Finally, the direct microinjection of constitutively active CaM KII into unfertilized eggs inactivates Cdc2 kinase and CSF, even in the absence of a Ca2+ transient. The target for Ca(2+)-calmodulin is thus CaM KII.
448 citations
TL;DR: It is shown here that CIM5 is the functional yeast homologue of the human MSS1 protein3 and that homologues of CIM3 and Cim5 are present in a highly purified preparation of the Drosophila 26S protease4.
Abstract: We isolated two mutants from the yeast Saccharomyces cerevisiae, cim3-1 and cim5-1, that arrest cell division in G2/metaphase at 37 degrees C. CIM3 (identical to SUG1; ref. 1) and CIM5 are similar to each other and are members of a family of putative ATPases that have been proposed to be 26S protease subunits. We show here that CIM5 is the functional yeast homologue of the human MSS1 protein and that homologues of CIM3 and CIM5 are present in a highly purified preparation of the Drosophila 26S protease. The short-lived ubiquitin-proline-beta-galactosidase fusion protein is stabilized in cim mutants, but Leu-beta-galactosidase is not. The CLB2 and CLB3 cyclins also accumulate in the cim mutants. Thus the 26S protease is required in vivo for the degradation of ubiquitinated substrates and for anaphase chromosome separation.
398 citations
TL;DR: It is concluded that mouse oocytes contain 42- and 44-kDa species of MAP kinase and that, after maturing oocytes enter metaphase,MAP kinase activity is stimulated by means of a process requiring protein synthesis and phosphorylation.
314 citations
TL;DR: The structural and functional homology of the KLP61F, bimC, cut7, and Eg5 kinesin-like proteins demonstrates the existence of a conserved family of kinesIn-like molecules important for spindle pole separation and mitotic spindle dynamics.
Abstract: We report here that disruption of a recently discovered kinesin-like protein in Drosophila melanogaster, KLP61F, results in a mitotic mutation lethal to the organism. We show that in the absence of KLP61F function, spindle poles fail to separate, resulting in the formation of monopolar mitotic spindles. The resulting phenotype of metaphase arrest with polyploid cells is reminiscent of that seen in the fungal bimC and cut7 mutations, where it has also been shown that spindle pole bodies are not segregated. KLP61F is specifically expressed in proliferating tissues during embryonic and larval development, consistent with a primary role in cell division. The structural and functional homology of the KLP61F, bimC, cut7, and Eg5 kinesin-like proteins demonstrates the existence of a conserved family of kinesin-like molecules important for spindle pole separation and mitotic spindle dynamics.
301 citations
TL;DR: It is reported that in mouse oocytes a CSF‐like activity is involved in the M II arrest and that the high activity of MPF is maintained through a continuous equilibrium between cyclin B synthesis and degradation, which is also dependent upon the three‐dimensional organization of the spindle.
Abstract: In unfertilized eggs from vertebrates, the cell cycle is arrested in metaphase of the second meiotic division (metaphase II) until fertilization or activation. Maintenance of the long-term meiotic metaphase arrest requires mechanisms preventing the destruction of the maturation promoting factor (MPF) and the migration of the chromosomes. In frog oocytes, arrest in metaphase II (M II) is achieved by cytostatic factor (CSF) that stabilizes MPF, a heterodimer formed of cdc2 kinase and cyclin. At the metaphase/anaphase transition, a rapid proteolysis of cyclin is associated with MPF inactivation. In Drosophila, oocytes are arrested in metaphase I (M I); however, only mechanical forces generated by the chiasmata seem to prevent chromosome separation. Thus, entirely different mechanisms may be involved in the meiotic arrests in various species. We report here that in mouse oocytes a CSF-like activity is involved in the M II arrest (as observed in hybrids composed of fragments of metaphase II-arrested oocytes and activated mitotic mouse oocytes) and that the high activity of MPF is maintained through a continuous equilibrium between cyclin B synthesis and degradation. In addition, the presence of an intact metaphase spindle is required for cyclin B degradation. Finally, MPF activity is preferentially associated with the spindle after bisection of the oocyte. Taken together, these observations suggest that the mechanism maintaining the metaphase arrest in mouse oocytes involves an equilibrium between cyclin synthesis and degradation, probably controlled by CSF, and which is also dependent upon the three-dimensional organization of the spindle.
286 citations
TL;DR: Results indicate that active MAP kinase is a component of CSF in Xenopus and suggest that the CSF activity of c-Mos(xe) is mediated byMAP kinase.
Abstract: The natural arrest of vertebrate unfertilized eggs in second meiotic metaphase results from the activity of cytostatic factor (CSF). The product of the c-mos(xe) proto-oncogene is thought to be a component of CSF and can induce metaphase arrest when injected into blastomeres of two-cell embryos. The c-Mos(xe) protein can directly activate the mitogen-activated protein kinase kinase (MAP kinase kinase) in vitro, leading to activation of MAP kinase. MAP kinase and c-Mos(xe) are active in unfertilized eggs and are rapidly inactivated after fertilization. Microinjection of thiophosphorylated MAP kinase into one blastomere of a two-cell embryo induced metaphase arrest similar to that induced by c-Mos(xe). However, only arrest with c-Mos(xe) was associated with activation of endogenous MAP kinase. These results indicate that active MAP kinase is a component of CSF in Xenopus and suggest that the CSF activity of c-Mos(xe) is mediated by MAP kinase.
271 citations
TL;DR: A role for serine and threonine phosphoprotein phosphatase type 1 in the late mitotic dephosphorylation of RB is suggested, which is consistent with the results obtained in vivo.
Abstract: The retinoblastoma gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. Pulse-chase experiments revealed that the change in RB gel electrophoretic migration which occurs near mitosis is due to enzymatic dephosphorylation (J. W. Ludlow, J. Shon, J. M. Pipas, D. M. Livingston, and J. A. DeCaprio, Cell 60:387-396, 1990). To determine the precise timing of RB dephosphorylation and whether a specific phosphatase is active in this process, we have utilized a nocodazole block and release protocol which allows a large population of cells to progress synchronously through mitosis. In such experiments, RB dephosphorylation began during anaphase and continued until complete dephosphorylation was apparent in the ensuing G1 period. In addition, late mitotic cell extracts were capable of dephosphorylating RB in vitro. This RB-specific mitotic phosphatase activity was more active in anaphase extracts than in pro- or metaphase extracts, which is consistent with the results obtained in vivo. Okadaic acid and protein phosphatase inhibitors 1 and 2 inhibited this specific RB phosphatase activity. These results suggest a role for serine and threonine phosphoprotein phosphatase type 1 in the late mitotic dephosphorylation of RB.
TL;DR: The distribution of this gamma-tubulin-related polypeptide during the complex changes in microtubule arrays that occur throughout the plant cell cycle may be significant in light of differences in the behavior and organization of plant microtubules.
Abstract: An antibody specific for a conserved gamma-tubulin peptide identifies a plant polypeptide of 58 kDa. gamma-Tubulin antibody affinity purified from this polypeptide recognizes the centrosome in mammalian cells. Using immunofluorescence microscopy, we determined the distribution of this gamma-tubulin-related polypeptide during the complex changes in microtubule arrays that occur throughout the plant cell cycle. We report a punctate association of gamma-tubulin-related polypeptide with the cortical microtubule array and the preprophase band. As cells enter prophase, gamma-tubulin-related polypeptide accumulates around the nucleus and forms a polar cap from which early spindle microtubules radiate. During metaphase and anaphase, gamma-tubulin-related polypeptide preferentially associates with kinetochore fibers and eventually accumulates at the poles. In telophase, localization occurs over the phragmoplast. gamma-Tubulin-related polypeptide appears to be excluded from the plus ends of microtubules at the metaphase plate and cell plate. Its distribution during the cell cycle may be significant in light of differences in the behavior and organization of plant microtubules. The identification of gamma-tubulin-related polypeptide could help characterize microtubule organizing centers in these organisms.
TL;DR: The staining pattern and the phenotype of ndc10-1 are consistent with Cbf2p/Ndc10p being a kinetochore protein, and provide in vivo evidence for its role in the attachment of chromosomes to the spindle.
Abstract: A mutant, ndc10-1, was isolated by anti-tubulin staining of temperature-sensitive mutant banks of budding yeast. ndc10-1 has a defect chromosome segregation since chromosomes remains at one pole of the anaphase spindle. This produces one polyploid cell and one aploid cell, each containing a spindle pole body (SPD. NDC10 was cloned and sequenced and is identical to CBF2 (Jiang, W., J. Lechnermn and J. Carbon. 1993. J. Cell Biol. 121:513) which is the 110-kD component of a centromere DNA binding complex (Lechner, J., and J. Carbon. 1991. Cell. 61:717-725). NDC10 is an essential gene. Antibodies to Ndc10p labeled the SPB region in nearly all the cells examined including nonmitotic cells. In some cells with short spindles which may be in metaphase, staining was also observed along the spindle. The staining pattern and the phenotype of ndc10-1 are consistent with Cbf2p/Ndc10p being a kinetochore protein, and provide in vivo evidence for its role in the attachment of chromosomes to the spindle.
Journal Article•
TL;DR: Data suggest that chromosomal breakpoints in 3q27 cluster in the proximity of a transcribed gene which represents a candidate protooncogene (bcl-6) involved in B-cell NHL pathogenesis.
Abstract: Chromosomal translocations involving band 3q27 and various chromosomal sites, including the sites of the immunoglobulin (Ig) loci (14q32, 2p12, 22q11), represent recurrent aberrations in non-Hodgkin's lymphoma (NHL). In order to identify the putative protooncogene involved in these translocations, we have cloned the breakpoints from two B-cell NHL cases carrying t(3;14)(q27;q32) translocations by screening genomic DNA libraries constructed from NHL biopsy samples with immunoglobulin probes. Several recombinant phages have been obtained from each case and shown to contain sequences from both 14q32 and 3q27 by fluorescence in situ hybridization mapping on metaphase chromosomes. In both cases, the translocation breakpoints were found within the switch region of the Ig heavy-chain locus on 14q32 and within the same 3-kilobase region on 3q27. When used in Southern blot hybridization, a probe from the 3q27 region detected rearrangements in an additional five NHL cases carrying 3q27 translocations with 14q32 or other genomic sites. The same probe detected a predominant 2.4-kilobase mRNA species in several lymphoid cell lines analyzed by Northern blot hybridization. These data suggest that chromosomal breakpoints in 3q27 cluster in the proximity of a transcribed gene which represents a candidate protooncogene (bcl-6) involved in B-cell NHL pathogenesis.
TL;DR: This phosphoepitope may be involved in regulating chromosome movement to the metaphase plate during prometaphase and may be part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved.
Abstract: A phosphorylated epitope is differentially expressed at the kinetochores of chromosomes in mitotic cells and may be involved in regulating chromosome movement and cell cycle progression. During prophase and early prometaphase, the phosphoepitope is expressed equally among all the kinetochores. In mid-prometaphase, some chromosomes show strong labeling on both kinetochores; others exhibit weak or no labeling; while in other chromosomes, one kinetochore is intensely labeled while its sister kinetochore is unlabeled. Chromosomes moving toward the metaphase plate express the phosphoepitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This is the first demonstration of a biochemical difference between the two kinetochores of a single chromosome. During metaphase and anaphase, the kinetochores are unlabeled. At metaphase, a single misaligned chromosome can inhibit further progression into anaphase. Misaligned chromosomes express the phosphoepitope strongly on both kinetochores, even when all the other chromosomes of a cell are assembled at the metaphase plate and lack expression. This phosphoepitope may be involved in regulating chromosome movement to the metaphase plate during prometaphase and may be part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved.
TL;DR: Immune depletion experiments confirm that CENP-F exhibits the distribution pattern in cells that was seen with the native autoimmune serum, and closely parallels that of another high molecular weight kinetochore associated protein, C ENP-E.
Abstract: We have identified a novel .ca 400 kDa cell-cycle dependent kinetochore associated protein in human cells, designated CENP-F, using human autoimmune serum. Immunofluorescence staining using the native serum, affinity purified antibodies, or antibodies raised against a cloned portion of CENP-F first reveals CENP-F homogeneously distributed throughout the nucleus of HeLa cells in the G2 stage of the cell cycle. Progression into prophase is accompanied by the localization of CENP-F to all the kinetochore regions of the karyotype. Kinetochore association is maintained throughout metaphase, but at the onset of anaphase CENP-F is no longer detected in association with the kinetochore but is found at the spindle mid-zone. By telophase, it is concentrated into a narrow band on either side of the midbody. Studies of the interaction of CENP-F with the kinetochore indicate that this protein associates with the kinetochore independent of tubulin and dissociation is dependent on events connected with the onset of anaphase. Nuclease digestion studies and immunoelectron-microscopy indicate that CENP-F is localized to the kinetochore plates and specifically to the outer surface of the outer kinetochore plate. The distribution of CENP-F closely parallels that of another high molecular weight kinetochore associated protein, CENP-E. Comparative studies indicate that there are antibodies in the CENP-F reactive autoimmune serum that recognize determinants present in the central helical rod domain of CENP-E. Immune depletion experiments confirm that CENP-F exhibits the distribution pattern in cells that was seen with the native autoimmune serum.
TL;DR: The expression of p80-coilin in quiescent cells induced to proliferate and the cyclic formation and breakdown of CBs might be consistent with the notion that CBs may be specialized centers related to the maturation of mRNA, but this evidence is indirect and needs further definitive study.
Abstract: Coiled bodies (CBs) are small, round structures found in the nucleoplasm of most eukaryotic cells. Human autoantibodies to a 80-kDa protein, p80-coilin, are immunohistologic markers for CBs. A polyclonal rabbit antiserum (R288) raised against recombinant p80-coilin was shown to have similar immunochemical properties as human autoantibodies and was used to analyze the expression of p80-coilin-associated CBs in cell cultures synchronized by double thymidine block, nocodazole arrest, serum starvation, or hormonal deprivation. By employing thymidine block and nocodazole arrest of HeLa cells, CBs were observed in immunofluorescent studies to be largest in size in the S and G2 phases of the cell cycle. These large CBs might have coalesced into one or two such structures per cell from smaller and more numerous CBs of three to eight per cell during the mid G1 phase of the cell cycle. No CB-like structures were observed in mitosis and early G1. However, immunoblotting analyses showed that the total amount of p80-coilin remained approximately the same throughout the cell cycle. When HeLa cells were separated into soluble and particulate fractions, p80-coilin was detected predominantly in the soluble fraction in mitosis and early G1, whereas it was present predominantly in the particulate fraction in late G1, S, and G2 when structurally distinct CBs were observed. In the analysis of CBs in two experimental models of cell proliferation (reversal of 3T3 serum starvation and FRTL-5 thyrotropin deprivation), proliferating cells contained larger, brighter, and more numerous CBs as well as a > 2-fold increase in the total amount of p80-coilin compared to that in quiescent cells. The expression of p80-coilin in quiescent cells induced to proliferate and the cyclic formation and breakdown of CBs might be consistent with the notion that CBs may be specialized centers related to the maturation of mRNA, but this evidence is indirect and needs further definitive study.
TL;DR: The contribution of histone H1 to mitotic chromosome condensation was examined with the use of a cell-free extract from Xenopus eggs, which transforms condensed sperm nuclei into metaphase chromosomes, and when H1 was removed from the extract, the resultant metaphase chromosome were indistinguishable from those formed in complete extract.
Abstract: The contribution of histone H1 to mitotic chromosome condensation was examined with the use of a cell-free extract from Xenopus eggs, which transforms condensed sperm nuclei into metaphase chromosomes. When H1 was removed from the extract, the resultant metaphase chromosomes were indistinguishable from those formed in complete extract. Nucleosomal spacing was the same for both. Thus, H1 is not required for the structural reorganization that leads to condensed metaphase chromosomes in this egg extract.
TL;DR: The staining pattern and the phenotype of ndc10-1 are consistent with Cbf2p/Ndc10p being a kinetochore protein, and provide in vivo evidence for its role in the attachment of chromosomes to the spindle.
TL;DR: Roberts syndrome has been interpreted as a human mitotic mutation syndrome which leads to secondary developmental defects and the RS Rating for quantitating severity is introduced.
Abstract: Roberts syndrome (RS) is a rare genetic disorder characterized by pre- and postnatal growth retardation, limb defects, and craniofacial anomalies. Affected persons have varying degrees of malformations involving symmetric reduction in the number of digits, and length or presence of bones in the arms and legs. Craniofacial malformations involve hypertelorism, hypoplastic nasal alae, and a high incidence of cleft lip and palate. Familial and sporadic cases have been reported consistent with an autosomal recessive mode of inheritance. Mitotic cells from many individuals with RS display a characteristic cytogenetic phenomenon consisting of repulsion of heterochromatic regions near centromeres, particularly of chromosomes 1, 9, 16, and splaying of the short arms of the acrocentrics and of the distal Yq. Mitosis in RS cells is abnormal in metaphase duration and anaphase progression. Specifically, anaphase figures show a higher degree of chromosomes that are outlying, lagging, or prematurely advancing toward the poles compared to normal controls. RS cells have abnormal nuclear morphology and also show a higher frequency of micronucleation than normal cells, presumably as a result of the abnormal mitotic events during anaphase. Therefore, RS has been interpreted as a human mitotic mutation syndrome which leads to secondary developmental defects. This report reviews 100 cases of RS, summarizes the phenotypic, genetic, cytogenetic, and cell biology findings in Roberts syndrome, and introduces the RS Rating for quantitating severity.
TL;DR: Observations suggest that the microtubules constituting the midbody may be nucleated on special microtubule organizing centres, active during late telophase only, and assembled on each side of the dividing plane between the daughter cells.
Abstract: Animal cells undergoing cytokinesis form an inter-cellular bridge containing two bundles of microtubules interdigitated at their plus ends, which constitute the midbody. Polyclonal antibodies raised against three specific amino acid sequences of gamma-tubulin (EEFATEGGDRKDV, NIIQGEADPTDVHKSL and EYHAATRPDYISWGTQEQ) specifically stained the centrosome in interphase, the spindle poles in all stages of mitosis, and the extremities of the midbody in mammalian cells (Potorous, human, Chinese hamster, mouse). This staining was prevented by the corresponding peptides, by Xenopus gamma-tubulin, but was not modified by purified alpha beta-tubulin heterodimer. An identical staining was obtained with affinity-purified antibodies against the carboxyl-terminal amino acid sequence of human gamma-tubulin. No gamma-tubulin could be detected in the interzone during anaphase and early telophase. Material containing gamma-tubulin first appeared in the two daughter cells on each side of the division plane in late telophase, and accumulated transiently at the minus ends of the two microtubule bundles constituting the midbody for one hour after metaphase. Micro-injection of gamma-tubulin antibodies into anaphase cells prevented the subsequent formation of the microtubule bundles between the two daughter cells. In contrast with previous views, these observations suggest that the microtubules constituting the midbody may be nucleated on special microtubule organizing centres, active during late telophase only, and assembled on each side of the dividing plane between the daughter cells.
TL;DR: Observations suggest that the 6-DMAP-sensitive kinase is involved in the control of post-fertilization events such as the formation of the interphase network of microtubules, the remodelling of sperm chromatin and pronucleus formation.
Abstract: Mouse oocyte activation is followed by a peculiar period during which the interphase network of microtubules does not form and the chromosomes remain condensed despite the inactivation of MPF. To evaluate the role of protein phosphorylation during this period, we studied the effects of the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) on fertilization and/or parthenogenetic activation of metaphase II-arrested mouse oocytes. 6-DMAP by itself does not induce the inactivation of histone H1 kinase in metaphase II-arrested oocytes, and does not influence the dynamics of histone H1 kinase inactivation during oocyte activation. However, 6-DMAP inhibits protein phosphorylation after oocyte activation. In addition, the phosphorylated form of some proteins disappear earlier in oocytes activated in the presence of 6-DMAP than in the activated control oocytes. This is correlated with the acceleration of some post-fertilization morphological events, such as sperm chromatin decondensation and its transient recondensation, formation of the interphase network of microtubules and pronuclear formation. In addition, numerous abnormalities could be observed: (1) the spindle rotation and polar body extrusion are inhibited; (2) the exchange of protamines into histones seems to be impaired, as judged by the morphology of DNA fibrils by electron microscopy; (3) the formation of a new nuclear envelope around the sperm chromatin proceeds prematurely, while recondensation is not yet completed. These observations suggest that the 6-DMAP-sensitive kinase(s) is (are) involved in the control of post-fertilization events such as the formation of the interphase network of microtubules, the remodelling of sperm chromatin and pronucleus formation.
TL;DR: Both i(12p)‐negative and ‐positive groups are characterized by an increased copy number of 12p, which is consistent with a lack of significant clinical or biological difference between them.
Abstract: The i(12p) chromosome has been shown to characterize more than 80% of male germ cell tumors (GCTs) and is an important diagnostic marker Although recent cytogenetic analyses of GCTs have defined nonrandom chromosome abnormalities in these tumors, no attempt has so far been made to compare i(12p)-positive and -negative tumors in terms of their cytogenetic, histologic, and clinical features During a 5-year period, we have ascertained 202 GCTs, of which 117 had clonally abnormal karyotypes Among the latter, 91 had one or more copies of i(12p), whereas 26 lacked an i(12p) We report here the karyotypic analysis of these 26 i(12p)-negative GCTs In this group, nonrandom sites of chromosomal rearrangements included 12p13 (9/26) and 1p11-q11 (5/26) Comparison of the cytogenetic features of i(12p)-negative tumors with i(12p)-positive tumors revealed the only significant difference to be rearrangements affecting 12p 13 in the former (35%) as compared to their absence in the latter (3%) Hybridization of metaphase preparations of 9 i(12p)-negative tumors with a chromosome 12 painting probe and with a microdissected 12p painting probe revealed extra copies of chromosome 12 segments incorporated into marker chromosomes whose composition could not otherwise be resolved by banding analysis; all were shown to be derived from 12p These data demonstrate that both i(12p)-negative and -positive groups are characterized by an increased copy number of 12p, which is consistent with a lack of significant clinical or biological difference between them An increased 12p copy number thus is a specific aberration of significance to the development of germ cell tumors © 1993 Wiley-Liss, Inc
TL;DR: Results indicate that inhibition of the polymerization dynamics of mitotic spindle microtubules and perhaps of centriole micro Tubules, rather than microtubule depolymerization, is responsible for the mitotic inhibition by vinblastine.
Abstract: Previous work from this laboratory has indicated that very low concentrations of vinblastine block HeLa cells at mitosis in the presence of a full complement of microtubules and without major disruption of spindle organization. In the present study we analyzed the structural organization of mitotic spindle microtubules, chromosomes and centrosomes by electron microscopy after incubating HeLa cells for one cell cycle with 2 nM vinblastine. We found that mitotic block of HeLa cells by vinblastine was associated with alterations of the fine structure of the spindle that were subtle but profound in their apparent consequences. The cell cycle was blocked in a stage that resembled prometaphase or metaphase; chromosomes had not undergone anaphase segregation. Neither the structure of the microtubules nor the structure of the kinetochores was detectably altered by the drug. However, the number of microtubules attached to kinetochores was decreased significantly. In addition, the centrosomes were altered; the normal close association of mother and daughter centriole was lost, numerous membranous vesicles were found in the centrosomal region, and many centrioles exhibited abnormal ultrastructure and had microtubules coursing through their interiors. These findings are consistent with our previous results and indicate that inhibition of the polymerization dynamics of mitotic spindle microtubules and perhaps of centriole microtubules, rather than microtubule depolymerization, is responsible for the mitotic inhibition by vinblastine.
TL;DR: The data show that RB-1 deletions can be diagnosed accurately by in situ hybridization on the one-cell level and the frequency detected in this study is significantly higher than previously assumed in B-CLL, and seems to be in the same range as in retinoblastoma.
TL;DR: Results indicate that optical traps are effective in holding laser-dissected chromosome fragments throughout mitosis and should be useful for studies on chromosome movement and cell genetics.
TL;DR: Molecular details of their structure and organization can now be described in conjunction with the microscopic examinations, to allow an evaluation of the various models that have been developed to explain the genesis of double‐minutes.
Abstract: Double-minute chromosomes play a critical role in tumor cell genetics where they are frequently associated with the overexpression of oncogene products. They have been observed for many years in light microscopic examinations of metaphase chromosomes from tumor cells, but their origin remains unknown and is the subject of considerable speculation. However, molecular details of their structure and organization can now be described in conjunction with the microscopic examinations, to allow an evaluation of the various models that have been developed to explain the genesis of double-minutes. The evidence now favors simple models that invoke chromosome breakage and circularization of very large acentric chromosome fragments, permitting unequal segregation of the genes on the fragment during cell division. If there is selection for overexpression of one of the genes on the fragment, daughter cells with more fragments will grow faster than daughter cells with fewer fragments, and over time the population of cells will come to contain many double-minutes per cell.
TL;DR: The cytogeneticist can combine the power of complete karyotype studies and the resolution of molecular techniques for patients suspected of having a Ph chromosome when this method is used in conjunction with conventional chromosome analysis.
TL;DR: Since proteasome accumulation appears to coincide with disappearance of cyclins A and B1 from the spindle apparatus, it is suggested that proteasomes may play a role in termination of mitosis by degrading the cyclins, which act as regulatory elements.
Abstract: We describe the isolation and characterization of proteasomes from recently established immortalized ovarian granulosa cell lines and their intracellular distribution during mitosis and during cAMP-induced differentiation, as revealed by immunofluorescence microscopy. In interphase, proteasomes were localized in small clusters throughout the cytoplasm and the nuclear matrix. In prophase, a substantial increase in proteasomal staining was observed in the perichromosomal area. A dramatic increase occurred in metaphase and in early anaphase; the chromosomes remained unstained. In late anaphase, intensive staining remained associated mainly with the spindle fibers. In telophase and early interphase of the daughter cells, intensive staining of proteasomes persisted in the nuclei. In contrast, in cells stimulated to differentiate by forskolin, which substantially elevates intracellular cAMP in these cell lines, only a weak staining of proteasomes was revealed in both the nucleus and the cytoplasm. Double staining of nondividing cells with antibodies to proteasomes and to tubulin did not show colocalization of proteasomes and microtubules. In contrast, dividing cells show a preferential concentration of proteasomes around spindle microtubules during metaphase and anaphase. The observed spatial and temporal distribution pattern of proteasomes during mitosis is highly reminiscent of the behavior of cyclins [Pines, J. & Hunter, T. (1991) J. Cell Biol. 115, 1-17]. Since proteasome accumulation appears to coincide with disappearance of cyclins A and B1 from the spindle apparatus, it is suggested that proteasomes may play a role in termination of mitosis by degrading the cyclins, which act as regulatory elements.
TL;DR: The results strongly support the contention that forces generated independently by each aster also contribute substantially to centrosome separation during anaphase, but that the process is modified by interactions between opposing astral MTs in the interzone.
Abstract: A popular hypothesis for centrosome separation during spindle formation and anaphase is that pushing forces are generated between interacting microtubules (MTs) of opposite polarity, derived from opposing centrosomes. However, this mechanism is not consistent with the observation that centrosomes in vertebrate cells continue to separate during prometaphase when their MT arrays no longer overlap (i.e., during anaphase-like prometaphase). To evaluate whether centrosome separation during prophase/prometaphase, anaphase-like prometaphase and anaphase is mediated by a common mechanism we compared their behavior in vivo at a high spatial and temporal resolution. We found that the two centrosomes possess a considerable degree of independence throughout all stages of separation, i.e., the direction and migration rate of one centrosome does not impart a predictable behavior to the other, and both exhibit frequent and rapid (4-6 microns/min) displacements toward random points within the cell including the other centrosome. The kinetic behavior of individual centrosomes as they separate to form the spindle is the same whether or not their MT arrays overlap. The characteristics examined include, e.g., total displacement per minute, the vectorial rate of motion toward and away from the other centrosome, the frequency of toward and away motion as well as motion not contributing to separation, and the rate contributed by each centrosome to the separation process. By contrast, when compared with prometaphase, anaphase centrosomes separated at significantly faster rates even though the average vectorial rate of motion away from the other centrosome was the same as in prophase/prometaphase. The difference in separation rates arises because anaphase centrosomes spend less time moving toward one another than in prophase/prometaphase, and at a significantly slower rate. From our data we conclude that the force for centrosome separation during vertebrate spindle formation is not produced by MT-MT interactions between opposing asters, i.e., that the mechanism is intrinsic to each aster. Our results also strongly support the contention that forces generated independently by each aster also contribute substantially to centrosome separation during anaphase, but that the process is modified by interactions between opposing astral MTs in the interzone.