Showing papers on "Metaphase published in 1995"
TL;DR: The three dimensional organization of microtubules in mitotic spindles of the yeast Saccharomyces cerevisiae has been determined by computer- aided reconstruction from electron micrographs of serially cross- sectioned spindes, and interpretation of these reconstructed spindle offers some insights into the mechanisms of mitosis in this yeast.
Abstract: The three dimensional organization of microtubules in mitotic spindles of the yeast Saccharomyces cerevisiae has been determined by computer-aided reconstruction from electron micrographs of serially cross-sectioned spindles. Fifteen spindles ranging in length from 0.6-9.4 microns have been analyzed. Ordered microtubule packing is absent in spindles up to 0.8 micron, but the total number of microtubules is sufficient to allow one microtubule per kinetochore with a few additional microtubules that may form an interpolar spindle. An obvious bundle of about eight interpolar microtubules was found in spindles 1.3-1.6 microns long, and we suggest that the approximately 32 remaining microtubules act as kinetochore fibers. The relative lengths of the microtubules in these spindles suggest that they may be in an early stage of anaphase, even though these spindles are all situated in the mother cell, not in the isthmus between mother and bud. None of the reconstructed spindles exhibited the uniform populations of kinetochore microtubules characteristic of metaphase. Long spindles (2.7-9.4 microns), presumably in anaphase B, contained short remnants of a few presumed kinetochore microtubules clustered near the poles and a few long microtubules extending from each pole toward the spindle midplane, where they interdigitated with their counterparts from the other pole. Interpretation of these reconstructed spindles offers some insights into the mechanisms of mitosis in this yeast.
476 citations
TL;DR: It is concluded that CDC27 and CDC16 are evolutionarily conserved components of the centrosome and mitotic spindle that control the onset of postmetaphase events during mitosis.
362 citations
TL;DR: It is shown here that mutations in the Drosophila gene fizzy (fzy) block the mitotic degradation of these cyclins, indicating that fzy plays a crucial role in the metaphase/anaphase transition.
Abstract: While entry into mitosis is triggered by activation of cdc2 kinase, exit from mitosis requires inactivation of this kinase. Inactivation results from proteolytic degradation of the regulatory cyclin subunits during mitosis. At least three different cyclin types, cyclins A, B and B3, associate with cdc2 kinase in higher eukaryotes and are sequentially degraded in mitosis. We show here that mutations in the Drosophila gene fizzy (fzy) block the mitotic degradation of these cyclins. Moreover, expression of mutant cyclins (delta cyclins) lacking the destruction box motif required for mitotic degradation affects mitotic progression at distinct stages. Deltacyclin A results in a delay in metaphase, deltacyclin B in an early anaphase arrest and deltacyclin B3 in a late anaphase arrest, suggesting that mitotic progression beyond metaphase is ordered by the sequential degradation of these different cyclins. Coexpression of deltacyclins A, B and B3 allows a delayed separation of sister chromosomes, but interferes wit chromosome segregation to the poles. Mutations in fzy block both sister chromosome separation and segregation, indicating that fzy plays a crucial role in the metaphase/anaphase transition.
308 citations
TL;DR: The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.
Abstract: We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process The movement of the activated zone was also measured along with chromosome movement and spindle elongation To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase We determined values for the turnover of kMTs (t1/2 = 71 +/- 24 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 375 +/- 85 min at 30 degrees C) Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16% These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point
304 citations
TL;DR: Comparative genomic hybridization analysis was applied to the identification of genomic imbalances in 26 bladder cancers in order to gain information on the genetic events underlying the development and progression of this malignancy.
Abstract: Comparative genomic hybridization (CGH) makes it possible to detect losses and gains of DNA sequences along all chromosomes in a tumor specimen based on the hybridization of differentially labeled tumor and normal DNA to normal human metaphase chromosomes. In this study, CGH analysis was applied to the identification of genomic imbalances in 26 bladder cancers in order to gain information on the genetic events underlying the development and progression of this malignancy. Losses affecting 11p, 11q, 8p, 9, 17p, 3p, and 12q were all seen in more than 20% of the tumors. The minimal common region of loss in each chromosome was identified based on the analysis of overlapping deletions in different tumors. Gains of DNA sequences were most often found at chromosomal regions distinct from the locations of currently known oncogenes. The bands involved in more than 10% of the tumors were 8q21, 13q21-q34, 1q31, 3q24-q26, and 1p22. In conclusion, these CGH data highlight several previously unreported genetic alterations in bladder cancer. Further detailed studies of these regions with specific molecular genetic techniques may lead to the identification of tumor suppressor genes and oncogenes that play an important role in bladder tumorigenesis.
233 citations
TL;DR: A role for CKII in regulation and/or execution of the eukaryotic cell cycle is established and flow cytometry of pheromone-synchronized cells confirmed that CKII is required in G1, at a point which must lie at or beyond Start but prior to DNA synthesis.
221 citations
Journal Article•
TL;DR: Interphase FISH, by which chromosomal aneuploidy was detected in almost 90% of patients with MM, represents an approach for evaluating the clinical significance of specific chromosomal abnormalities in MM.
Abstract: Because metaphase cytogenetic studies in multiple myeloma (MM) are hampered by a low proliferative activity of myeloma cells in vitro, interphase cytogenetics by means of fluorescence in situ hybridization (FISH) should improve the detection of chromosomal abnormalities in MM. We therefore investigated chromosomal aneuploidy in 36 patients with MM using interphase FISH and alpha-satellite DNA probes for chromosomes 1, 3, 7, 8, 11, 12, 16, 17, 18, and X. By FISH, myeloma cells from 32 patients (88.9%) were aneuploid for at least one of the chromosomes examined. In 24 patients (66%), aberrations of > or = 3 chromosomes were observed. Aneuploidy was predominantly characterized by a gain of chromosome numbers, with involvement of chromosomes 3, 7, and 11 occurring in > 50% of patients. Loss of a centromeric signal suggesting monosomy was most frequently observed for chromosomes 17 (22.2% of patients) and X (monosomic in 42.3% of female patients, but loss of chromosome X was never observed in males, P < 0.05). Dual-color FISH studies provided evidence for marked heterogeneity of aneuploid cells in 8 patients (22.8%). Occurrence of chromosomal aneuploidy was independent of stage and pretreatment status. Gain of chromosome 3 was significantly correlated with an IgA paraprotein (P < 0.05). In 12 patients, the direct comparison of metaphase cytogenetics and FISH showed that FISH detected aneuploidy of chromosomes in 9 patients that was missed by metaphase analysis. In conclusion, interphase FISH, by which chromosomal aneuploidy was detected in almost 90% of patients with MM, represents an approach for evaluating the clinical significance of specific chromosomal abnormalities in MM.
213 citations
TL;DR: It is shown that Ca2+ transients cease during entry into interphase, at the time when pronuclei are forming, which suggests that the ability of the sperm to trigger repetitive Ca2- transients in oocytes is modulated in a cell cycle-dependent manner.
Abstract: Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca2+ transients triggered by the sperm at fertilization. These Ca2+ transients have been widely reported to last for a number of hours but when, or why, they cease is not known. Here we show that Ca2+ transients cease during entry into interphase, at the time when pronuclei are forming. In fertilized oocytes arrested at metaphase using colcemid, Ca2+ transients continued for as long as measurements were made, up to 18 hours after fertilization. Therefore sperm is able to induce Ca2+ transients during metaphase but not during interphase. In addition metaphase II oocytes, but not pronuclear stage 1-cell embryos showed highly repetitive Ca2+ oscillations in response to microinjection of inositol trisphosphate. This was explored further by treating in vitro maturing oocytes at metaphase I for 4-5 hours with cycloheximide, which induced nuclear progression to interphase (nucleus formation) and subsequent re-entry to metaphase (nuclear envelope breakdown). Fertilization of cycloheximide-treated oocytes revealed that continuous Ca2+ oscillations in response to sperm were observed after nuclear envelope breakdown but not during interphase. However interphase oocytes were able to generate Ca2+ transients in response to thimerosal. This data suggests that the ability of the sperm to trigger repetitive Ca2+ transients in oocytes is modulated in a cell cycle-dependent manner.
180 citations
TL;DR: Both ataxia telangiectasia cell lines showed more robust telomerase activity than the normal cell lines, precluding defective enzymatic capacity as the basis for the chromosome end associations.
Abstract: Cells derived from individuals with ataxia telangiectasia (AT) show enhanced spontaneous levels of chromosomal abnormalities and are sensitive to ionizing radiations and radiomimetic drugs, as evidenced by decreased survival and increased chromosome aberration frequencies at mitosis when compared with normal cell lines. The higher base line frequencies of chromosome aberrations in part involve chromosome end-to-end associations as seen at metaphase. Since telomeres of tumor cells and aging tissues are often reduced in length, chromosome end associations may be due to loss of telomeric repeats. We studied the chromosome behavior and telomeres of two ataxia telangiectasia lymphoblastoid cell lines compared to two normal control cell lines. The ataxia telangiectasia cell lines showed higher frequencies of chromosome end associations both at metaphase and in interphase, determined in prematurely condensed chromosomes of G1 and G2 cells. They also showed higher frequencies of chromosomal breaks at metaphase and fewer telomeric signals determined using fluorescent in situ hybridization with a (TTAGGG)n probe. The frequency of telomeric repeats was variable in the ataxia telangiectasia cell lines (4.3 and 8.2 kb) compared to the normal cell lines (9.6 and 12 kb) and an inverse correlation between telomere length and chromosome end associations was observed. Both ataxia telangiectasia cell lines showed more robust telomerase activity than the normal cell lines, precluding defective enzymatic capacity as the basis for the chromosome end associations. It is possible that chromatin structure in the form of telomere-nuclear matrix interactions are variant in ataxia telangiectasia cells negatively influencing telomerase function and contributing to telomere associations.
173 citations
TL;DR: Time-lapse video images of single cells support the localization shift of p93dis1 to the SPBs in metaphase and spindle microtubules in anaphase, and it is proposed that these localization shifts of p 93dis1 in mitosis facilitates sister chromatid separation by affecting SPB and anaphases spindle function.
Abstract: Fission yeast cold-sensitive (cs) disl mutants are defective in sister chromatid separation. The dis1 + gene was isolated by chromosome walking. The null mutant showed the same phenotype as that of cs mutants. The d/s/+ gene product was identified as a novel 93-kD protein, and its localization was determined by use of anti-disl antibodies and green fluorescent protein (GFP) tagged to the carboxyl end of p93 dis1. The tagged p93 dlsl in living cells localizes along cytoplasmic microtubule arrays in interphase and the elongating anaphase spindle in mitosis, but association with the short metaphase spindle microtubules is strikingly reduced. In the spindle, the tagged p93 ais~ is enriched at the spindle pole bodies (SPBs). Time-lapse video images of single cells support the localization shift of p93 dis~ to the SPBs in metaphase and spindle microtubules in anaphase. The carboxy-terminal fragment, which is essential for Disl function, accumulates around the mitotic SPB. We propose that these localization shifts of p93 dis~ in mitosis facilitates sister chromatid separation by affecting SPB and anaphase spindle function.
164 citations
TL;DR: Chromosomes have multiple roles both in controlling the cell assembly and structure of the spindle and in determining chromosomal position in many meiotic cells and in some types of mitotic cells.
Abstract: Chromosomes have multiple roles both in controlling the cell assembly and structure of the spindle and in determining chromosomal position on the spindle in many meiotic cells and in some types of mitotic cells. Moreover, functionally significant chromosome-microtubule interactions are not limited to the kinetochore but are also mediated by proteins localized along the arms of chromosomes. Finally, chromosomes also play a crucial role in control of the cell cycle.
TL;DR: This is the first demonstration of how a complete set of mouse chromosome paints can be produced, separated using flow cytometry into 18 distinguishable peaks.
Abstract: Using peripheral blood lymphocyte cultures and duallaser flow cytometry, we have routinely obtained high-resolution bivariate flow karyotypes of the dog in which 32 peaks are resolved. To allow the identification of the chromosome types in each peak, chromosomes were flow sorted, amplified and labelled by polymerase chain reaction with partially degenerate primers and hybridized onto metaphase spreads of a male dog. The chromosome paints from 22 of the 32 peaks each hybridized to single homologue pairs and eight peaks each hybridized to two pairs. Paints from the remaining two peaks hybridized to only one homologue each in the male metaphase spread, thus corresponding to the sex chromosomes X and Y. All of the 38 pairs of autosomes and the two sex chromosomes of the dog could be accounted for in these painting experiments. The positions of chromosomes 1–21 were assigned to the flow karyotype (only chromosomes 1–21 have as yet been officially designated). The high-resolution flow karyotype and the chromosome paints will facilitate further standardization of the dog karyotype. The ability to sort sufficient quantities of dog chromosomes for the production of chromosome-specific DNA libraries has the potential to accelerate the physical and genetic mapping of the dog genome.
TL;DR: The results indicate that the presence of a univalent chromosome causes severe meiotic disruption during mammalian meiosis, affecting the alignment and segregation of other chromosomes in the complement, and suggests that cell cycle control differs between male and female meiosis and that monitoring of meiotic chromosome behavior is less efficient in the female.
Abstract: To monitor the behavior of specific chromosomes at various stages of mammalian female meiosis, we have combined immunofluorescence staining and fluorescence in situ hybridization (FISH) on intact oocytes. We have utilized this technique to evaluate the behavior of the single X chromosome in oocytes from XO female mice, providing the first observations on segregation of an achiasmate chromosome during mammalian female meiosis and its effect on the meiotic process. As has been described in other species, we found that the univalent chromosome could either segregate as an intact chromosome to one pole or divide equationally at the first meiotic division. Our results also indicate that the presence of a univalent chromosome causes severe meiotic disruption during mammalian meiosis, affecting the alignment and segregation of other chromosomes in the complement. Despite these meiotic abnormalities, the vast majority of oocytes from XO females were able to resume and successfully complete the first meiotic division. This is in contrast to previous studies of male mice with sex chromosome abnormalities where the presence of a univalent acts to arrest meiosis at metaphase of the first meiotic division. This sex-specific difference in the ability of a cell with a univalent chromosome to initiate anaphase suggests that cell cycle control differs between male and female meiosis and that monitoring of meiotic chromosome behavior is less efficient in the female. The combined use of immunofluorescence staining and FISH on intact oocytes has obvious application to the study of meiotic chromosome non-disjunction in the human female. Simultaneous study of the meiotic cell cycle, protein components of the meiotic apparatus, and chromosome-specific behaviors during mammalian female meiosis provides a new approach to defining age-related changes in the meiotic process that result in increased chromosome malsegregation.
TL;DR: It is shown that during prometaphase nod protein is localized on oocyte chromosomes and is not restricted to either specific chromosomal regions or to the kinetochore, providing a molecular explanation for the polar ejection force.
TL;DR: The results of this Noah's Ark approach show that olomoucine arrests cells both at the G1S and the G2M boundaries, consistent with the hypothesis of a prevalent effect on the cdk2 and cdc2 kinases, respectively.
TL;DR: It is proposed that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetchore-microtubule attachment or is a signaling component of the checkpoint pathway regulating the metaphase to anaphase transition.
Abstract: The transition from metaphase to anaphase is regulated by a checkpoint system that prevents chromosome segregation in anaphase until all the chromosomes have aligned at the metaphase plate. We provide evidence indicating that a kinetochore phosphoepitope plays a role in this checkpoint pathway. The 3F3/2 monoclonal antibody recognizes a kinetochore phosphoepitope in mammalian cells that is expressed on chromosomes before their congression to the metaphase plate. Once chromosomes are aligned, expression is lost and cells enter anaphase shortly thereafter. When microinjected into prophase cells, the 3F3/2 antibody caused a concentration-dependent delay in the onset of anaphase. Injected antibody inhibited the normal dephosphorylation of the 3F3/2 phosphoepitope at kinetochores. Microinjection of the antibody eliminated the asymmetric expression of the phosphoepitope normally seen on sister kinetochores of chromosomes during their movement to the metaphase plate. Chromosome movement to the metaphase plate appeared unaffected in cells injected with the antibody suggesting that asymmetric expression of the phosphoepitope on sister kinetochores is not required for chromosome congression to the metaphase plate. In antibody-injected cells, the epitope remained expressed at kinetochores throughout the prolonged metaphase, but had disappeared by the onset of anaphase. When normal cells in metaphase, lacking the epitope at kinetochores, were treated with agents that perturb microtubules, the 3F3/2 phosphoepitope quickly reappeared at kinetochores. Immunoelectron microscopy revealed that the 3F3/2 epitope is concentrated in the middle electronlucent layer of the trilaminar kinetochore structure. We propose that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetochore-microtubule attachment or is a signaling component of the checkpoint pathway regulating the metaphase to anaphase transition.
TL;DR: It is found that in earlier stages of mitosis, silver staining reveals an initially single, folded midline structure, which separates at prometaphase to form two chromatid axes, constrained by DNA catenations.
Abstract: Metaphase chromatids are believed to consist of loops of chromatin anchored to a central scaffold, of which a major component is the decatenatory enzyme DNA topoisomerase II Silver impregnation selectively stains an axial element of metaphase and anaphase chromatids; but we find that in earlier stages of mitosis, silver staining reveals an initially single, folded midline structure, which separates at prometaphase to form two chromatid axes Inhibition of topoisomerase II prevents this separation, and also prevents the contraction of chromatids that occurs when metaphase is arrested Immunolocalization of topoisomerase II alpha reveals chromatid cores analogous to those seen with silver staining We conclude that the chromatid cores in early mitosis form a single structure, constrained by DNA catenations, which must separate before metaphase chromatids can be resolved
TL;DR: It is suggested that the progression of meiosis is arrested at metaphase I in both partially competent growing oocytes and fully grown LT/Sv oocytes because of continued p34cdc2 kinase activity sustained, at least in part, by restricted degradation of cyclin B.
Abstract: Fully grown mouse oocytes are normally competent to progress from prophase I to metaphase II without interruption. However, growing mouse oocytes initially become only partially competent to undergo meiotic maturation. Meiotic maturation in these oocytes does not progress beyond metaphase I. In contrast to the oocytes of most strains of mice, most oocytes of strain LT/Sv mice become arrested at metaphase I even when they are fully grown. The initiation of oocyte maturation is correlated with an increase in p34cdc2 kinase activity that continues to rise until metaphase I. The transition into anaphase I is normally correlated with a decrease in p34cdc2 kinase activity. This study demonstrated that metaphase I arrest in both partially competent growing oocytes and fully grown LT/Sv oocytes is correlated with a sustained elevation of p34cdc2 kinase activity. In fact, p34cdc2 activity continued to increase during the time when activity normally decreased. In normally maturing oocytes, some, but not all, of the cyclin B, the regulatory protein associated with p34cdc2, became degraded in oocytes that entered anaphase I. In contrast, the amount of cyclin B present in the metaphase I-arrested oocytes continued to increase at the time when it was being degraded in normal oocytes progressing to metaphase II. These results suggest that the progression of meiosis is arrested at metaphase I in both groups of oocytes because of continued p34cdc2 kinase activity sustained, at least in part, by restricted degradation of cyclin B.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The cause of micronucleus formation from a lagging Y chromosome appears probably to be either a faulty or a diminished amount of kinetochore protein.
Abstract: This investigation was conducted to determine the relationship between Y chromosome loss and increased micronucleus formation with age. We also investigated the status of kinetochore proteins in the micronuclei. Umbilical cord blood samples were obtained from 18 newborn males, and peripheral blood was obtained from 35 adult males ranging in age from 22 to 79 years. Isolated lymphocytes from all 53 donors were cultured and blocked with cytochalasin B. Two thousand binucleate cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. This assay showed 23.8% of the micronuclei to be kinetochore-positive, while 76.2% of the micronuclei were kinetochore-negative. Cells were then hybridized with a 3.56-kb biotinylated Y chromosome-specific probe. All micronucleate cells were relocated and their Y probe status was determined. A significant mcrease in Y-bearing micronuclei with age was observed. Metaphase cells from the same samples were analyzed for the presence or absence of Y chromosome. The relationship between Y chromosome-positive micronuclei and Y chromosome-negative metaphase cells was highly significant, suggesting that Y chromosome-deficient metaphase cells result from cells which had previously lost a Y chromosome due to micronucleation. The cause of micronucleus formation from a lagging Y chromosome appears probably to be either a faulty or a diminished amount of kinetochore protein.
TL;DR: This unit provides protocols for CGH, for preparation of metaphase chromosomes, tumor and normal DNAs for FISH and for the microscopy and image analysis of CGH experiments.
Abstract: Comparative Genomic Hybridization (CGH) is a powerful molecular cytogenetic technique that permits assessment of DNA copy number on a genome-wide scale. Of note, this methodology uses tumor DNA as a probe for fluorescence in situ hybridization (FISH) to normal metaphase chromosomes and does not require dividing cells from the tumor specimen. This unit provides protocols for CGH, for preparation of metaphase chromosomes, tumor and normal DNAs for FISH and for the microscopy and image analysis of CGH experiments.
TL;DR: Ca2+ spikes and signals from integrin‐activated focal adhesion complexes and Ca2+ receptors on the cell surface along with cyclic AMP begin the cycle of cyclin‐dependent protein kinases (PKs), which stimulate the coordinate expression of DNA‐replicating enzymes and stimulates cytoplasmic division.
Abstract: Ca2+ and Ca(2+)-binding proteins are involved in running the cell cycle. Ca2+ spikes and signals from integrin-activated focal adhesion complexes and Ca2+ receptors on the cell surface along with cyclic AMP begin the cycle of cyclin-dependent protein kinases (PKs). These transiently expressed PKs stimulate the coordinate expression of DNA-replicating enzymes, activate replication enzymes, inactivate replication suppressors (e.g., retinoblastoma susceptibility protein), activate the replicator complexes at the end of the G1 build-up, and when replication is complete they and a Ca2+ spike trigger mitotic prophase. Another Ca2+ surge at the end of metaphase triggers the destruction of the prophase-stimulating PKs and starts anaphase. Ca2+ finally stimulates cytoplasmic division (cytokinesis). However, Ca2+ does more than this in epithelial cells, such as those lining the colon, and skin keratinocytes. These cells also need Ca2+, integrin signals, and only a small amount (e.g., 0.05-0.1 mM) of external Ca2+ to start DNA replication. Signals from their surface Ca2+ receptors trigger a combination of differentiation and apoptosis ("diffpoptosis") when external Ca2+ concentration reaches their setpoints. The skin's steep, upwardly directed, Ca2+ gradient has a low concentration in the basal layer to allow stem and precursor keratinocytes to proliferate, and higher concentrations in the suprabasal layers to trigger the differentiation-apoptosis ("diffpoptosis") mechanism that converts granular cells into protective, hard-shelled, dead corneocytes. A similar Ca2+ gradient may exist in the colon crypt allowing the stem cell and its amplifying transit or precursor offspring to cycle in the lower parts of the crypt, while stopping proliferation and stimulating terminal differentiation in the upper crypt and flat mucosa. Raising the amount of Ca2+ in fecal water above a critical level reduces proliferation and thus colorectal carcinogenesis in normal rats and some high-risk humans. But during carcinogenesis the Ca2+ sensors malfunction or their signals become ineffective: high Ca2+ does not stop, and may even stimulate, the proliferation of initiated mutants. Therefore, Ca2+ may either not affect, or even promote, the growth of epithelial cells in carcinogen-initiated rat colon and human adenoma patients. Clearly, a much greater understanding of how Ca2+ controls the proliferation and differentiation of epithelial cells and why initiated cells lose their responsiveness to Ca2+ are needed to assess the drawbacks and advantages of using Ca2+ as a chemopreventor.
TL;DR: A sensitive and rapid means of identifying the t(2;13) in rhabdomyosarcoma specimens, and it will be a useful adjunct for the diagnosis of pediatric small round cell tumors.
Abstract: Cytogenetic studies of the pediatric solid tumor alveolar rhabdomyosarcoma have demonstrated the presence of a consistent chromosomal translocation, t(2;13)(q35;q14). We recently identified PAX3 and FKHR as the genes on chromosomes 2 and 13, respectively, that are juxtaposed by this translocation. As one means of detecting the t(2;13) translocation in clinical specimens, we have developed a fluorescence in situ hybridization (FISH) assay that may be used for both interphase and metaphase cells. Translocation of the 5' region of the FKHR gene to the derivative chromosome 2, and retention of the 3' region of FKHR on the derivative chromosome 13 [(der(13)], were demonstrated in metaphase cells from a rhabdomyosarcoma cell line with a previously identified t(2;13) translocation. A 5' PAX3 cosmid probe was shown to localize to 2q35 in normal cells, and to translocate to the der(13) in the rhabdomyosarcoma cell line. In order to detect the der(13) in interphase nuclei, we labeled the 3'FKHR and the 5'PAX3 cosmid probes with digoxigenin and biotin, respectively, and used these in a two-color FISH assay. The presence of the der(13) was visualized as juxtaposed or overlapping red and green signals in metaphase and interphase tumor cells. The PAX3-FKHR FISH assay was then applied to a series of cytogenetically characterized pediatric sarcoma cell lines. The presence of the der(13) was demonstrated by FISH in all cases containing a cytogenetically detectable t(2;13). The FISH assay was then applied to a series of 20 embryonal and alveolar rhabdomyosarcoma samples. All 10 of the alveolar rhabdomyosarcoma specimens demonstrated a der(13) with the FISH assay.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The results suggest that cyclin B1 levels in porcine oocytes differ from those in starfish, clam, and Xenopus oocytes in which considerably higher concentrations of cyclIn B1 appear to be present during the GV stage and that the nature of the association between p34cdc2 and cyclinB1 changes between the first and the second metaphase.
TL;DR: It appears that in addition to their function in motility, kinetochores act as dynamic and adaptable centres for regulating cell cycle progression through mitosis.
TL;DR: These results demonstrate that nucleolar components involved in pre-rRNA processing, including rRNA itself, probably in an incompletely processed form, are transferred from the parental to the daughter cell nucleoli by means of transient structures, such as the perichromosomal sheath and prenucleolar bodies.
Journal Article•
TL;DR: Moderate hyperthermia appears to induce a novel morphological pattern of cell injury and death in HeLa S3 cell lines that may be useful as a means of screening cell lines for nonmorphological analyses of hyperthermic injury.
Abstract: Hyperthermia is an efficient means of inducing cell death in vivo and in vitro. Among human neoplastic cells, HeLa S3 cells are susceptible to heat injury when exposed to long duration moderate hyperthermia (41.5 C), conditions that are reproducible and sustainable in the clinical setting. Hence, HeLa S3 cells are a useful substrate for evaluation of hyperthermic injury in human neoplasia. Previous studies have demonstrated a consistent response of HeLa S3 cells to moderate hyperthermia: spontaneous premature condensation of chromosomes during heat exposure in S phase followed by apparent nuclear fragmentation and, inevitably, cell death. To further characterize the morphological features of this process, HeLa S3 cells grown in suspension at 37 C were heated for 4, 8, 12, or 16 hours at 41.5 C and harvested in glutaraldehyde for electron microscopic evaluation. Compared with untreated controls, heated samples exhibited a characteristic pattern of chromosome condensation that mimicked mitotic prophase but was followed by haphazard asymmetric segregation of chromatid clusters in abnormal metaphase/anaphase and premature reformation of nuclear membrane, resulting not in nuclear fragmentation, but in multiple micronuclei. This pattern of nuclear morphology was not observed in controls. The fraction of cells with micronuclear morphology increased with time in heated samples (from 3.6% at 4 hours to 16.6% at 16 hours), consistent with previous light microscopic analyses of nuclear fragmentation. Cells with multiple micronuclei subsequently exhibited features similar to necrotic cell death. Apoptosis was never observed. Moderate hyperthermia appears to induce a novel morphological pattern of cell injury and death in HeLa S3 cell lines that may be useful as a means of screening cell lines for nonmorphological analyses of hyperthermic injury.
TL;DR: Interphase FISH provides a rapid diagnostic procedure to detect 14q32 translocations in B-cell malignancies and allowed us to unequivocally detect tumor-specific breakpoints of 14q 32 translocations.
TL;DR: The hypothesis that CENP‐E may act as a minus end‐directed microtubule motor during mitosis with vinblastine is strongly supported, strongly supporting the idea that dynein and kinesin are major culprits in mitosis.
Abstract: A minus end-directed microtubule motor activity from extracts of HeLa cells blocked at prometaphase/metaphase of mitosis with vinblastine has been partially purified and characterized. The motor activity was eliminated by immunodepletion of Centromere binding protein E (CENP-E). The CENP-E-associated motor activity, which was not detectable in interphase cells, moved microtubules at mean rates of 0.46 micron/s at 37 degrees C and 0.24 micron/s at 25 degrees C. The motor activity co-purified with CENP-E through several purification procedures. Motor activity was clearly not due to dynein or to kinesin. The microtubule gliding rates of the CENP-E-associated motor were different from those of dynein and kinesin. In addition, the pattern of nucleotide substrate utilization by the CENP-E-associated motor and the sensitivity to inhibitors were different from those of dynein and kinesin. The CENP-E-associated motor had an apparent native molecular weight of 874,000 Da and estimated dimensions of 2 nm x 80 nm. This is the first demonstration of motor activity associated with CENP-E, strongly supporting the hypothesis that CENP-E may act as a minus end-directed microtubule motor during mitosis.
TL;DR: It is shown that crossovers between homologs attached to the same centromere do not induce metaphase arrest, and exchanges induce metaphases arrest only when they physically conjoin two separate kinetochores.
Abstract: In normal Drosophila melanogaster oocytes, meiosis arrests at metaphase I and resumes after oocyte passage through the oviduct. Thus, metaphase arrest defines a control point in the meiotic cell cycle. Metaphase arrest only occurs in oocytes that have undergone at least one meiotic exchange. Here it is shown that crossovers between homologs attached to the same centromere do not induce metaphase arrest. Hence, exchanges induce metaphase arrest only when they physically conjoin two separate kinetochores. Thus, the signal that mediates metaphase arrest is not the exchange event per se but the resulting tension on homologous kinetochores.
TL;DR: Using fluorescencein situ hybridization (FISH), four different seed protein genes were physically mapped on metaphase chromosomes oficia faba L using a mixture of polymerase chain reaction-amplified DNA fragments of the coding region of up to 1 kb in size.
Abstract: Using fluorescencein situ hybridization (FISH), four different seed protein genes were physically mapped on metaphase chromosomes ofVicia faba L. dropped on slides. FISH with a 2.8 kb genomic probe of a legumin B4 gene resulted in reproducible signals on the long arm of chromosome III near the centromere. The same clone cross-hybridized at a lower frequency to the short arm of chromosome II, where the closely related legumin B3 gene family is located. The locus for legumin A-genes could be detected in the distal half of the long arm of chromosome V using a 1.7 kb cDNA clone. The locus of an unknown seed protein gene was mapped to the long arm of chromosome I using a mixture of polymerase chain reaction-amplified DNA fragments of the coding region of up to 1 kb in size.