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1.
Summary Cells containing X-ray induced micronuclei were treated for a few hours before fixation with inhibitors of DNA synthesis (cytosine arabinoside; azathioprine; thymidine; trenimon), of RNA synthesis (actinomycin D; ethidium bromide), and of protein synthesis (puromycin). Only the inhibitors of DNA synthesis lead to a significant suppression of the frequencies of mitoses with micronucleus derived premature chromosome condensation (PCC). We tend to interprete the result as follows: Micronuclei that are in the G1 phase of their cell cycles are accumulated at the G1/S border or in the early S phase of their cell cycles under the influence of the inhibitors of the DNA synthesis. Micronuclei blocked in this way cannot be induced to undergo PCC and seem to disappear from the cells. 相似文献
2.
Primary roots ofVicia faba were grown for 24 h in the presence of tritiated thymidine (1.85–18.5 kBq ml−1) and then irradiated with a dose of 1.5 Gy of60Co-gamma- rays. The aim of these experiments was to determine whether low-level endogenous beta-irradiation from incorporated
radioactive thymidine could influence the frequencies of sister chromatid exchanges (SCEs) and the numbers of micronuclei
induced by subsequent external irradiation with high doses of gamma-rays. The results demonstrated that the pretreatment with3H-dTh had no significant effect on the frequencies of SCEs in gamma-irradiated root tip cells ofVicia faba. In contrast to SCEs, the yields of micronuclei in the3H-dTh pretreated cells were altogether less than the yield induced by gamma-rays alone (protective effects). 相似文献
3.
4.
The Chinese hamster bone marrow was used as a test system in vivo to analyse the chromosome-danaging effect of bleomycin. Both chromosome and chromatid aberrations were found. Mitoses with aberrations (Ma) show a linear dose-effect relationship after a recovery time of 24 h, the same hold true for cells with micronuclei (Cm) and for mitoses with premature chromosome condensation (PCC). The dose-effect relationships for Ma, Cm and PCC run parallel to each other with Ma at the highest and PCC at the lowest level (Ma greater than Cm greater than PCC). The time-effect relationships for Ma, Cm and PCC show that after 12 h recovery time there are no PCCs but the highest frequencies of Ma and Cm indicating that most cells are in their first post-treatment mitoses or Gi-phases at this fixation time. In addition to the frequency determinations autoradiographic analysis were performed to clarigy the nature of the PCCs. The results are interpreted as follows: bleomycin induces chromosomal aberrations that in turn give rise to micronuclei by means of lagging chromatin, main and micronuclei eventually become asynchronous in their cell cycles and mitosing main nuclei induce PCC in the micronuclei. 相似文献
5.
Chromosomes of dividing and non-dividing aging cells were examined by fusing senescent WI38 cells with mitotic HeLa cells to induce premature chromosome condensation (PCC). Exposure of the WI38 cells to 3H-thymidine 48 h prior to fusion allowed autoradiographic identification of cells that did not synthesize DNA (non-dividing cells). Ninety-six percent of the non-dividing cells, diploid or tetraploid, induced into PCC had single chromatids and were therefore blocked in the G1 phase of the cell cycle. Anomalous centromeric pairing of chromatids was noted in the remaining 4% of the non-dividing cells. Typical G2 configurations (double chromatids) were observed only among labeled (dividing) cells. The efficiency of PCC induction was independent of culture age. In addition, the efficiency of PCC induction was independent of phase in the cell cycle, as shown by comparison of observed frequencies with expected frequencies. 相似文献
6.
Fluorescence in situ hybridization using simultaneously a combination of DNA probes for the telomeric hexamer repeat (TTAGGG) and the centromerically repeated murine gamma-satellite DNA was applied to analyze the nature of radiation-induced micronuclei in mouse NIH 3T3 fibroblasts. After subtraction of spontaneously occurring micronuclei independent from the dose and time after irradiation, approximately 22% of the radiation-induced micronuclei did not reveal any hybridization signal. Approximately 17% showed one centromeric hybridization signal and about four telomeric signals, suggesting their origin from whole chromosomes. Almost 60% of radiation-induced micronuclei had telomeric signals only, suggesting their origin from acentric fragments. A fraction of micronuclei were found to contain two or more acentric fragments. Micronuclei derived from whole chromosomes or from multiple acentric fragments might, together with DNA synthesis in micronuclei, explain the occurrence of radiation-induced micronuclei with DNA contents greater than the largest chromosome arm. 相似文献
7.
The influence of aminopterin (AP), tritiated thymidine ([3H] TdR) and "cold" thymidine (TdR) on production of chromosomal aberrations in meristematic cells of Crepis capillaris irradiated in different stages of the mitotic cycle with 300 rad of 63Co gamma-rays was studied. All the chemical treatments increased most of all the frequency of aberrations induced during two "critical periods" localized before the stage of DNA synthesis (fixation 9 h after irradiation) and before that of mitosis (4 h). Treatments with TdR and [3H]TdR increased most of all the frequency of chromatid aberrations when irradiation was performed in G1, and the frequency of gaps when irradiated in G2. Treatment with AP increased the yield of different types of aberration more uniformly. The modifying effect of the chemicals tested appeared to be independent of replicative synthesis. The "critical periods" are suggested to be the stages when regular "proof reading" and correction of spontaneous errors takes place [9,13]. In addition to this regular mechanism, radiation induces an "emergency" mechanism of repair. AP inhibits the mechanism of regular repair; in addition TdR and [3H] TdR suppress the lateral spread of primary injuries across the chromosome. 相似文献
8.
A modified mouse splenocyte culture system was standardized after testing different mitogens (i.e., phytohemagglutinin (PHA), concanavalin A (Con A)). The mitotic index was determined for comparison between different mitogens. Following selection of appropriate mitogen (PHA 16, Flow), a series of experiments were conducted to evaluate the application of a cytokinesis-block for scoring micronuclei and assays for chromosomal aberrations produced by treatment in G0 and G2 for the purposes of biological dosimetry following in vivo and/or in vitro exposure to X-rays, fission neutrons and bleomycin. In the X-irradiation studies, the frequencies of micronuclei and chromosomal aberrations (i.e., dicentrics and rings) increased in a dose-dependent manner. These data could be fitted to a linear-quadratic model. No difference was observed between irradiation in vivo and in vitro, suggesting that measurement of dicentrics and micronuclei in vitro after X-irradiation can be used as an in vivo dosimeter. Following in vivo irradiation with 1 MeV fission neutrons and in vitro culturing of mouse splenocytes, linear dose-response curves were obtained for induction of micronuclei and chromosomal aberrations. The lethal effects of neutrons were shown to be significantly greater than for a similar dose of X-rays. The relative biological effectiveness (RBE) was 6-8 in a dose range of 0.25-3 Gy for radiation-induced asymmetrical exchanges (dicentrics and rings), and about 8 for micronuclei in a dose range of 0.25-2 Gy. Furthermore, the induction of chromosomal aberrations by bleomycin was investigated in mouse G0 splenocytes (in vitro) and compared with X-ray data. Following bleomycin treatment (2 h) a similar pattern of dose-response curve was obtained as with X-rays. In this context a bleomycin rad equivalent of 20 micrograms/ml = 0.50 Gy was estimated. 相似文献
9.
The DNA replication patterns of the terminal S phase of three species of Mus were analyzed by tritiated thymidine autoradiography. The centromeric heterochromatin of M. fulvidiventris is the latest component to finish DNA synthesis. The Y chromosome finishes replication earlier than the centromeric heterochromatin. The centromeric heterochromatin of M. musculus, on the other hand, is not the latest component to finish DNA synthesis. At the very late S phase, grains are found in the euchromatic arms instead of the heterochromatic areas. The hot X and the hot Y can be identified in the majority of, but not all, cases. The heterochromatic short arms of the autosomes in M. dunni finish DNA replication earlier than many areas in the euchromatic long arms and the heterochromatin of the sex chromosomes. This indicates that in M. dunni there are at least two types of heterochromatin. The late-replicating zones in the euchromatic long arms are distinctly banded. This banded grain pattern can be seen in all Mus species observed, but in M. dunni it is most exaggerated. Late-replicating chromosome segments can be demonstrated also by 2+ cycles of BUdR incorporation and Giemsa staining. 相似文献
10.
Meta-analysis of increases in micronuclei in peripheral blood lymphocytes after angiography or excretory urography 总被引:5,自引:0,他引:5
Norman, A., Cochran, S. T. and Sayre, J. W. Meta-analysis of Increases in Micronuclei in Peripheral Blood Lymphocytes after Angiography or Excretory Urography. Radiat. Res. 155, 740-743 (2001). Meta-analysis of 10 studies confirms a significant increase in the frequency of micronuclei in peripheral blood lymphocytes after angiography or excretory urography; the weighted average increase is 4.2 (95% confidence interval 2.8-5.6) per 1000 binucleate lymphocytes, about the same increase in micronuclei as that produced in vitro by a diagnostic X-ray dose of 4 cGy. The analysis failed to reveal a significant effect of the specific contrast medium used in the X-ray examinations on the increased frequency of micronuclei. These results are consistent with the hypothesis that the effect of the contrast media is limited to the enhancement, by the photoelectric effect, of the X-ray dose absorbed by the lymphocytes irradiated while suspended in the contrast medium. Therefore, an estimate of increased cancer risk based on elevated frequencies of micronuclei or chromosome aberrations in peripheral blood lymphocytes may be greatly exaggerated whenever the radiation damage is largely confined to the cells circulating in the blood, as it is in people who have recently had X-ray examinations that use intravenous injections of contrast medium. Such examinations include angiography, excretory urography and CT scans, which are received annually by millions of people. 相似文献
11.
A new flow cytometric method is presented that quantifies the frequency of radiation-induced micronuclei in mammalian cell cultures with high precision. After preparing a suspension of main nuclei and micronuclei stained with ethidium bromide and Hoechst 33258, both types of particles are measured simultaneously in a flow cytometer using forward light scatter and three fluorescence emission intensities excited by UV, 488 nm, and by energy transfer from Hoechst 33258 to ethidium bromide. Nonspecific debris overlapping the micronucleus distribution especially in the low fluorescence intensity region was discriminated from micronuclei by calculating ratios of the different fluorescences. The frequencies of radiation-induced micronuclei measured with this new technique agreed well with results obtained by conventional microscopy. The lower limit of the DNA content of micronuclei identified by this technique was found to be about 0.5%-0.75% of the DNA content of G1-phase nuclei. Dose effect curves and the time-dependent induction of micronuclei were measured for two different mouse cell lines. 相似文献
12.
Surprising deficiency of CENP-B binding sites in African green monkey alpha-satellite DNA: implications for CENP-B function at centromeres. 总被引:8,自引:1,他引:7
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I G Goldberg H Sawhney A F Pluta P E Warburton W C Earnshaw 《Molecular and cellular biology》1996,16(9):5156-5168
Centromeres of mammalian chromosomes are rich in repetitive DNAs that are packaged into specialized nucleoprotein structures called heterochromatin. In humans, the major centromeric repetitive DNA, alpha-satellite DNA, has been extensively sequenced and shown to contain binding sites for CENP-B, an 80-kDa centromeric autoantigen. The present report reveals that African green monkey (AGM) cells, which contain extensive alpha-satellite arrays at centromeres, appear to lack the well-characterized CENP-B binding site (the CENP-B box). We show that AGM cells express a functional CENP-B homolog that binds to the CENP-B box and is recognized by several independent anti-CENP-B antibodies. However, three independent assays fail to reveal CENP-B binding sites in AGM DNA. Methods used include a gel mobility shift competition assay using purified AGM alpha-satellite, a novel kinetic electrophoretic mobility shift assay competition protocol using bulk genomic DNA, and bulk sequencing of 76 AGM alpha-satellite monomers. Immunofluorescence studies reveal the presence of significant levels of CENP-B antigen dispersed diffusely throughout the nuclei of interphase cells. These experiments reveal a paradox. CENP-B is highly conserved among mammals, yet its DNA binding site is conserved in human and mouse genomes but not in the AGM genome. One interpretation of these findings is that the role of CENP-B may be in the maintenance and/or organization of centromeric satellite DNA arrays rather than a more direct involvement in centromere structure. 相似文献
13.
Mammalian cell fusion 总被引:5,自引:1,他引:4
The behaviour of heterochromatin during premature chromosome condensation (PCC) was studied in a cell line of Microtus agrestis after fusion with mitotic HeLa cells. In the G1- and G2-PCC, the heterochromatic nature of the X-chromosomes was detectable by their intense staining. The pulverized appearance of the S-phase PCC was correlated with incorporation of 3H TdR into the DNA. Three types of S-PCC were observed. PCC with a pulverized appearance of: (a) only the autosomes (early S); (b) autosomes and X-chromosomes (mid S); and (c) only the X-chromosomes (late S). The behaviour of heterochromatin during replication, as observed by the PCC method, was no different from that of euchromatin. The data on the sequence of chromosome replication indicate that the centromeric regions of the X-chromosomes were the last segments to replicate. The completion of DNA synthesis in the X-chromosomes appears to be followed by progressive chromosome condensation during G2 even before the actual initiation of prophase. 相似文献
14.
Summary Polymorphism of constitutive heterochromatin has been studied in a series of 30 normal individuals. A high frequency of C-band variants were observed. Twenty-six of the 30 individuals studied had at least one polymorphic variant of the C band. A total of 42 variants were recorded which were predominately localized near the centromeric heterochromatin block of chromosome 9 (26.19%), chromosome 16 (19.05%), and chromosome 1 (16.66%). These results are discussed together with the findings revealed by different studies.Aided by U.G.C. grant No. 9-32/75 X (RF). 相似文献
15.
Clia Carvalho Henrique M. Pereira Joo Ferreira Cristina Pina Denise Mendona Agostinho C. Rosa Maria Carmo-Fonseca 《Molecular biology of the cell》2001,12(11):3563-3572
Gene expression can be silenced by proximity to heterochromatin blocks containing centromeric alpha-satellite DNA. This has been shown experimentally through cis-acting chromosome rearrangements resulting in linear genomic proximity, or through trans-acting changes resulting in intranuclear spatial proximity. Although it has long been been established that centromeres are nonrandomly distributed during interphase, little is known of what determines the three-dimensional organization of these silencing domains in the nucleus. Here, we propose a model that predicts the intranuclear positioning of centromeric heterochromatin for each individual chromosome. With the use of fluorescence in situ hybridization and confocal microscopy, we show that the distribution of centromeric alpha-satellite DNA in human lymphoid cells synchronized at G(0)/G(1) is unique for most individual chromosomes. Regression analysis reveals a tight correlation between nuclear distribution of centromeric alpha-satellite DNA and the presence of G-dark bands in the corresponding chromosome. Centromeres surrounded by G-dark bands are preferentially located at the nuclear periphery, whereas centromeres of chromosomes with a lower content of G-dark bands tend to be localized at the nucleolus. Consistent with the model, a t(11; 14) translocation that removes G-dark bands from chromosome 11 causes a repositioning of the centromere, which becomes less frequently localized at the nuclear periphery and more frequently associated with the nucleolus. The data suggest that "chromosomal environment" plays a key role in the intranuclear organization of centromeric heterochromatin. Our model further predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centromere localization. 相似文献
16.
Light- and electron-microscope autoradiography have been used to follow the incorporation of [3H]thymidine at different stages during the interphase of synchronously growing populations of Amoeba proteus. Two main patterns were found for tritiated thymidine incorporation, i.e. DNA synthesis. The major incorporation was in the central region of the nucleus, but a lesser degree of incorporation occurred in the nucleolar region. The bulk of this nucleolar DNA was found to be late replicating, i.e. it replicated during the G2 phase. 相似文献
17.
Madin-Darby bovine kidney (MDBK) cells were treated with the bifunctional DNA cross-linker, L-7, to examine the generation of micronuclei and other nuclear abnormalities. The preceding paper demonstrates that L-7 treatment induces the formation of triradial and quadriradial chromosomes in MDBK cells. These chromosomes are believed to result from interduplex DNA cross-links formed between G-C rich centromeric satellite DNA regions on non-sister chromatids. Treatment produces a majority of centromere-positive micronuclei. In addition, many daughter cells remain attached by chromatin bridges which are sometimes beaded with micronuclei. Up to 15% of cell nuclei become lobular and fused with numerous micronuclear-like structures attached to their membranes. These attached structures are classified as attached micronuclear-like structures (AMNLS). Fluorescence in situ hybridization (FISH) using a centromeric satellite sequence was performed on treated cells. Hybridization reveals that intercellular bridges are composed of centromeric sequences and initiate at centromeric foci in daughter cells. Furthermore, the majority of junctions between AMNLS and nuclei contain an enhancement of centromeric signal. The frequency of AMNLS appears dependent on the concentration of L-7 and the duration of treatment. Similar results were found for the generation of cross-linked chromosome products in the previous paper. We suggest that AMNLS result from the abnormal mitotic segregation of cross-linked chromosome products. 相似文献
18.
Distinct roles for Sir2 and RNAi in centromeric heterochromatin nucleation,spreading and maintenance
Alessia Buscaino Erwan Lejeune Pauline Audergon Georgina Hamilton Alison Pidoux Robin C Allshire 《The EMBO journal》2013,32(9):1250-1264
Epigenetically regulated heterochromatin domains govern essential cellular activities. A key feature of heterochromatin domains is the presence of hypoacetylated nucleosomes, which are methylated on lysine 9 of histone H3 (H3K9me). Here, we investigate the requirements for establishment, spreading and maintenance of heterochromatin using fission yeast centromeres as a paradigm. We show that establishment of heterochromatin on centromeric repeats is initiated at modular ‘nucleation sites’ by RNA interference (RNAi), ensuring the mitotic stability of centromere‐bearing minichromosomes. We demonstrate that the histone deacetylases Sir2 and Clr3 and the chromodomain protein Swi6HP1 are required for H3K9me spreading from nucleation sites, thus allowing formation of extended heterochromatin domains. We discovered that RNAi and Sir2 along with Swi6HP1 operate in two independent pathways to maintain heterochromatin. Finally, we demonstrate that tethering of Sir2 is pivotal to the maintenance of heterochromatin at an ectopic locus in the absence of RNAi. These analyses reveal that Sir2, together with RNAi, are sufficient to ensure heterochromatin integrity and provide evidence for sequential establishment, spreading and maintenance steps in the assembly of centromeric heterochromatin. 相似文献
19.
Disruption of centromere assembly during interphase inhibits kinetochore morphogenesis and function in mitosis 总被引:19,自引:0,他引:19
The relationship between the kinetochore and the centromeric heterochromatin that surrounds it is unknown. Anti-centromere autoantibodies (ACAs) that recognize antigens found in the heterochromatin beneath the kinetochore disrupt mitotic events when microinjected into human cells. We show here that ACAs interfere with two different stages of centromere assembly during interphase, resulting in abnormal kinetochore structures during mitosis. Antibody injection prior to late G2 results in the subsequent failure to assemble a trilaminar kinetochore. Such chromosomes bind microtubules but are incapable of movement. Antibody disruption of events during G2 produces unstable kinetochores that prevent the normal transition into anaphase. These experiments present a novel way to examine events in the pathway of kinetochore assembly that occur during interphase, at a time when this structure cannot be visualized directly. 相似文献
20.
M O Krause 《Journal of cellular physiology》1967,70(2):141-154
Exponentially growing L -cells were synchronized by the double thymidine-block method and exposed to high specific activities of tritiated thymidine. DNA, RNA, and protein synthetic rates were measured through one cell cycle with 1-hour pulses of the appropriate C14-labelled precursors. Equivalent doses of tritiated water were substituted for tritiated thymidine in some experiments. Total amounts of DNA and histones per nucleus were determined photometrically in Feulgen and fast-green stained cells. It was observed that incorporated tritiated thymidine has an effect distinct from that of tritiated water and that it enhances the incorporation of the precursors at specific stages of the cell cycle, to a degree roughly proportional to the dose. Photometric data indicated an increase in DNA net synthesis and a metabolic instability of histones in the H3-thymidine-treated cells, resulting in higher DNA:histone ratios. 相似文献