C-Banding as a Simple Tool to Discriminate Between Micronuclei Induced by Clastogens and Aneugens

If mouse bone marrow preparations are treated with a classical C-banding procedure, it may be possible to distinguish between micronuclei with or without centromeres. This allows discrimination between micronuclei originating from chromosome breakage and those originating from chromosome loss. Thus, using C-banding, the micronucleus test can be used not only for the detection of clastogens but also aneugens. In this way, more exhaustive methods such as immunological staining using antikinetochore antibodies may not be necessary.     

应用原位杂交技术检测X射线诱发的特定染色体的微核率

为了研究X射线与X染色体的微校率之间的关系．本实验利用原位杂交技术同时检测了经X射线诱发人双核淋巴细胞的7号和X染色体的微核率。结果发现：经2．5Gy的X射线照射后．X和7号染色体的微核率男性分别为3．4％和7．1％;女性分别为6．6％和6．0％。X和7号染色体微核率的实验观察值与理论预期值之间在统计学上无显著性差异。实验结果提示：X射线并不特异性引起X染色体的微核率增高。     

A comparison of constitutive heterochromatin staining methods in two cases of familial heterochromatin deficiencies

Summary Using DAPI staining after pretreatment with distamycin A we detected a familial deficiency of chromosome 16 heterochromatin. A distinct positively staining band, however, was seen after C-banding. Thus, by using these different heterochromatin staining methods, heterogeneity of the constitutive heterochromatin in the centromeric region of human chromosome 16 was indicated. The same C-banding procedure was also applied to a previously described familial deficiency of chromosome 9 heterochromatin evidenced using distamycin A/DAPI staining and G 11 staining (Buys et al., 1979). In this case a C-band appeared to be virtually absent on the relevant chromosome. These staining methods may be valuable tools in the study of chromosome polymorphisms.     

A case of elevated spontaneous micronucleus frequency derived from chromosome 2

This work tested the hypothesis that the content of spontaneous micronuclei in lymphocytes in an apparently healthy normal human subject, who exhibited an unusually high micronucleus frequency, was non-random. Several DNA probes were used in fluorescent in-situ hybridization (FISH), beginning with a probe generated from the subject's micronuclei. Micronuclei obtained from peripheral blood lymphocytes by microdissection were subjected to random amplification of polymorphic DNA (RAPD-PCR), and a unique PCR product was then used to isolate a cosmid clone from a human genomic library. This clone hybridized to chromosome 2. Subsequently, commercial probes were included in FISH analyses of micronuclei from the subject and age- and sex-matched controls. No significant differences were found between subject and controls in the percentages of micronuclei hybridizing with a centromere probe for the X chromosome or a painting probe for chromosome 3. However, the subject had a very highly significant increase (p<0.0001) in chromosome 2 in micronuclei over a level that might be expected to be present by chance. Characterization of micronuclei may be a promising tool in studies of mechanisms of inherited or induced chromosome instability. The strength of the strategy employed in this study is that, by characterizing the chromosomes present in micronuclei, this work has advanced from an observation of chromosomal instability to a foundation for study of the mechanism underlying the observation.     

高大山羊草Giemsa C-带的研究及对“中国春”小麦-高大山羊草异附加系、异代换系和易位系的鉴定

本文研究了高大山羊草(Aegilops longissima)的C-带带型,并对“中国春”-高大山羊草双端体异附加系(21"+t"_Bl)、双端体异代换系(20"+t"+t"_Bl)、2个二体异代换系(20"+1"_Bl)和易位系(4A/4Bl)进行了鉴定。本文还对小麦的B染色体组和4A染色体的起源进行了讨论。从带型上的明显差别可以推测高大山羊草不是B染色体组的直接供体。它们可能共同起源于一个原始的染色体组。     

Y Chromosome aneuploidy,micronuclei, kinetochores and aging in men

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.     

Standard Giemsa C-banded karyotype of Russian wildrye (Psathyrostachys juncea) and its use in identification of a deletion-translocation heterozygote.

Ten accessions of Russian wildrye, Psathyrostachys juncea (Fisch.) Nevski (2n = 2x = 14; NsNs), collected from different geographical regions were analyzed using the C-banding technique. C-banding pattern polymorphisms were observed at all levels, i.e., within homologous chromosome pairs of the same plant, among different individuals within accessions, between different accessions of the same geographic area, and among accessions of different origins. The seven homologous groups varied in the level of C-banding pattern polymorphism; chromosomes A, B, E, and F were more variable than chromosomes C, D, and G. The polymorphisms did not hamper chromosome identification in Ps. juncea, because each chromosome pair of the Ns genome had a different basic C-banding pattern and karyotypic character. A standard C-banded karyotype of Ps. juncea is proposed based on the overall karyotypes and C-bands in the 10 accessions. The C-bands on the Ns-genome chromosomes were designated according to the rules of nomenclature used in wheat. A deletion-translocation heterozygote of Russian wildrye was identified based on the karyotype and C-banding patterns established. The chromosome F pair consisted of a chromosome having the distal segment in the long arm deleted and a translocated chromosome having the distal segment of long arm replaced by the distal segment of the long arm of chromosome E. The chromosome E pair had a normal chromosome E and a translocated chromosome having the short arm and the proximal segment of the long arm of chromosome E and the distal segment of the long arm of chromosome F.     

Micronuclei and chromosome aberrations in Xenopus laevis spermatocytes and spermatids exposed to adriamycin and colcemid

Cultured testes and spermatocytes from the frog Xenopus laevis have been incubated (40-42 h) with adriamycin or colcemid followed by quantitation of chromosome aberrations in secondary spermatocytes and quantitation of micronuclei in secondary spermatocytes, early round spermatids, and round spermatids with acrosomal vacuoles (AV) at 18-162 h of culture. Micronucleus frequencies were consistently higher in secondary spermatocytes relative to round spermatids after exposure to either adriamycin or colcemid due to a higher rate of micronucleus formation during meiosis I compared to meiosis II. Also, some of the micronuclei formed during meiosis I did not survive meiosis II to form micronucleated spermatids. Micronucleus formation occurred in 3-7% of secondary spermatocytes with detectable chromosome aberrations, depending upon drug treatment. Thus, the ratio of micronuclei to total chromosome aberrations in secondary spermatocytes was always higher in colcemid-treated cells compared to adriamycin-treated cells following 18- and 42-h treatment periods. Adriamycin induced significant increases in micronuclei in both secondary spermatocytes and spermatids after 162 h of culture, the time for initial pachytene stages to develop into secondary spermatocytes and spermatids. The data show that cultured testes and spermatocytes from Xenopus may be used to quantify specific meiotic chromosome aberrations induced by both clastogens and spindle poisons using either a rapid secondary spermatocyte micronucleus assay or meiotic chromosome analysis.     

Damage to lymphocytes by X-ray and bleomycin measured with the cytokinesis-block micronucleus technique.

Chromosome damage induced by X-irradiation or bleomycin was measured using the cytokinesis-block micronucleus assay in the peripheral blood lymphocytes of 6 newborn, 8 young and 10 elderly individuals. An increase in the frequency of spontaneous micronuclei with age was observed. There was no difference in the X-irradiation-induced micronucleus frequency between the 3 groups. There was a significant increase with age in the number of micronuclei induced by bleomycin. Kinetochore-labelling studies revealed that the percentage of kinetochore-positive induced micronuclei was higher for bleomycin (36.2-43.3%) than for X-irradiation (17.1-19.7%). The age-related increase in frequency of spontaneous or bleomycin-induced micronuclei was due to increases in both kinetochore-positive and kinetochore-negative micronuclei. The frequency of kinetochore-positive or -negative micronuclei induced by X-irradiation was not different between the 3 age groups. These results suggest that bleomycin is more potent in inducing whole-chromosome loss than X-rays, and that lymphocytes from aged individuals are more sensitive to bleomycin in terms of both chromosome breakage and whole chromosome loss.     

Use of C-banding for identifying radiation induced micronuclei

Differentiation of micronuclei (MN) caused by ionizing radiation from those caused by chemicals is a crucial step for managing treatment of individuals exposed to radiation. MN in binucleated lymphocytes in peripheral blood are widely used as biomarkers for estimating dose of radiation, but they are not specific for ionizing radiation. MN induced by ionizing radiation originate predominantly as a result of chromosome breaks (clastogenic action), whereas MN caused by chemical agents are derived from the loss of entire chromosomes (aneugenic action). C-banding highlights centromeres, which might make it possible to distinguish radiation induced MN, i.e., as a byproduct of acentric fragments, from those caused by the loss of entire chromosomes. To test the use of C-banding for identifying radiation induced MN, a blood sample from a healthy donor was irradiated with 3 Gy of Co-60 gamma rays and cultured. Cells were harvested and dropped onto slides, divided into a group stained directly with Giemsa and another processed for C banding, then stained with Giemsa. The frequency of MN in 500 binucleated cells was scored for each method. In preparations stained with Giemsa directly, the MN appeared as uniformly stained structures, whereas after C banding, some MN exhibited darker regions corresponding to centromeres that indicated that they were not derived from acentric fragments. The C-banding technique enables differentiation of MN from acentric chromosomal material. This distinction is useful for improving the specificity of the MN assay as a biomarker for ionizing radiation.     

Assessment of the genotoxicity of atenolol in human peripheral blood lymphocytes: Correlation between chromosomal fragility and content of micronuclei

The antihypertensive drug atenolol was found to induce chromosome loss, detected as micronuclei in the peripheral lymphocytes of treated patients. The fundamental question which chromosomes the micronuclei were derived from remains to be answered. Analysis of structural chromosomal aberrations (CAs) and expression of fragile sites (FS) were pursued in this study. They revealed a significantly higher incidence of chromosomal aberrations (chromatid and chromosome breaks) in patients compared with controls, where 10 FS emerged as specific. Also, the band 17q12–21, where known fragile sites have not been reported, was only expressed in atenolol-treated patients. Fluorescence in situ hybridization using chromosome-specific probes revealed the preferential involvement of chromosomes 7, 11, 17 and X in the micronuclei (MN) of patients. The results also suggest a correlation between chromosomal fragility and content of MN, and support the findings for a linkage between hypertension and a locus on chromosome 17.     

Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA

We have examined the relationship between checkpoint adaptation (mitosis with damaged DNA) and micronuclei. Micronuclei in cancer cells are linked to genomic change, and may induce chromothripsis (chromosome shattering). We measured the cytotoxicity of the cancer drug cisplatin in M059K (glioma fibroblasts, IC50 15 μM). Nearly 100% of M059K cells were positive for histone γH2AX staining after 48 h treatment with a cytotoxic concentration of cisplatin. The proportion of micronucleated cells, as confirmed by microscopy using DAPI and lamin A/C staining, increased from 24% to 48%, and the total micronuclei in surviving cells accumulated over time. Promoting entry into mitosis with a checkpoint inhibitor increased the number of micronuclei in cells whereas blocking checkpoint adaptation with a Cdk inhibitor reduced the number of micronuclei. Interestingly, some micronuclei underwent asynchronous DNA replication, relative to the main nuclei, as measured by deoxy-bromo-uracil (BrdU) staining. These micronuclei stained positive for histone γH2AX, which was linked to DNA replication, suggesting that micronuclei arise from checkpoint adaptation and that micronuclei may continue to damage DNA. By contrast the normal cell line WI-38 did not undergo checkpoint adaptation when treated with cisplatin and did not show changes in micronuclei number. These data reveal that the production of micronuclei by checkpoint adaptation is part of a process that contributes to genomic change.     

Survival of aneuploid, micronucleated and/or polyploid cells: crosstalk between ploidy control and apoptosis

Microtubule inhibitors are known to block the cell cycle at M-phase, by damaging the mitotic spindle. However, under certain circumstances, cells can escape these effects and become aneuploid, polyploid and/or micronucleated. It is well known that aneuploidy can have adverse effects on human health such as pregnancy wastage, birth defects and the development of human tumours. The present paper aims at reviewing the data our laboratory has accumulated during the last years about the relation between aneuploidy/polyploidy/presence of micronuclei and the induction of apoptosis in human cells after in vitro exposure to the microtubule inhibitor nocodazole. Exposure to high doses of nocodazole results in polyploidy due to mitotic slippage in the absence of a functional spindle. Depending on their p53-status polyploid cells may eventually arrest, die or continue cycling. In these experimental conditions, our data showed that polyploidy does not constitute a strong apoptotic signal. In case of exposure to low concentrations of nocodazole, microtubule depolymerization is disturbed resulting in a spindle with damaged microtubules. This can give rise to chromosome loss and non-disjunction. Our data showed that in particular micronucleated cells, originating from chromosome loss can be eliminated by apoptosis. In addition, nocodazole-induced apoptosis involves the apical caspase-8 and -9 and the effector caspase-3. We show evidence that caspase-3, in addition to its function in apoptosis, plays a role in the formation of micronuclei.     

STERILITY BARRIERS OF SOME ARTIFICIAL F1 ORCHID HYBRIDS: MALE STERILITY. I. MICROSPOROGENESIS AND POLLEN GERMINATION

Microsporogenesis, pollen germination and fertility of males gametes were studied in 24 artificial intergeneric and interspecific F₁ hybrids of orchids. Although parental species had the same chromosome number (2n = 40), microsporogenesis of the hybrids was irregular due to the lack of homology of the chromosomes of the parental species. This led to formation of tetrads of microspores without micronuclei, tetrads with 1–8 micronuclei, triads, dyads with and without micronuclei, and monads. Chromosomes numbers found in haploid microsporocytes ranged from 7 to 40; in micronuclei the chromosome number varied between 1 and 5. In terms of pollen germination, three situations were observed: 1) hybrids whose pollen grains did not germinate in the stigma; 2) hybrids in which the pollen tubes grew down in the style, but did not penetrate into the ovary; 3) hybrids in which the pollen tubes grew down normally through the ovary, reaching the ovules. When the pollen tubes did not penetrate the ovary no fruit was formed. Therefore germination tests carried out in vitro may not indicate pollen fertility, because pollen tube growth in the style of the flower may be insufficient to induce fruit formation or to accomplish fertilization.     

Dislocation of chromatin elements in prophase induced by diethylstilbestrol: a novel mechanism by which micronuclei can arise

The in vitro micronucleus test with Syrian hamster embryo (SHE) cells assays the induction of micronuclei by chemical agents. Both chromosome fragments and lagging chromosomes can give rise to micronuclei. Nevertheless, only limited information is available on the ultrastructure of micronuclei and the mechanisms of their formation. Diethylstilbestrol (DES), a non-mutagenic carcinogen, as well as its analogue 3.3'-DES induce micronuclei in SHE cells. A comparison of the dose response of DES-induced micronucleus formation with the previously published ones for aneuploidy and transformation shows that all 3 run in parallel. Thus, a functional relationship between these endpoints, in the SHE system, may be implied. The present study is designed to address the formation of micronuclei using supravital UV microscopy, to test for the presence of defined chromosome domains within micronuclei using immunocytochemistry, and to define aspects of their ultrastructure by electron microscopy. Supravital UV microscopy showed that 3.3'-DES induces displacement of chromosomes/chromatids during prophase/anaphase and formation of micronuclei during cytokinesis. Immunocytochemistry revealed that micronuclei contain, at high frequencies, CREST antibody-reactive kinetochores, indicating the presence of whole chromosomes or centric fragments in these structures. Moreover, transmission electron microscopy showed that micronuclei exhibit ultrastructural details typical of interphase nuclei. Specifically, micronuclei exhibited morphological evidence of a nuclear lamina and segregation of karyoplasm into euchromatic and heterochromatic regions. All micronuclei examined were enclosed by a nuclear envelope of normal morphology and showed nuclear pore complexes. Together the findings provide evidence that DES interferes with the mitotic apparatus as early as prophase, resulting in the formation of micronuclei and, as a consequence, in the loss of chromatids or chromosomes.     

Banding Human Chromosomes Using a Combined C-Banding-Fluorochrome Staining Technique

Slides pretreated for C-banding and stained with DAPI or CMA3 show different banding patterns in human metaphase chromosomes compared to those obtained with either standard Giemsa C-banding or fluorochrome staining alone. Human chromosomes show C-plus DA-DAPI banding after C-banding plus DAPI and enhanced R-banding after C-banding plus Chromomycin A3 staining. If C-banding preferentially removes certain classes of DNA and proteins from different chromosome domains, C-banding pre-treatment may cause a differential DNA extraction from G- and R-bands in human chromosomes, resulting in a preferential extraction of DNA included in G-bands. This hypothesis is partially supported by the selective cleavage and removal of DNA from R-bands of restriction endonuclease HaeIII with C-banding combined with DAPI or Chromomycin A3 staining. Structural factors relating to regional differences in DNA and/or proteins could also explain these results.     

Chromosome polymorphism involving heterochromatic blocks in Chinese hamster chromosome 9

A chromosome polymorphism was detected between two early passage euploid Chinese hamster cell strains when a fluorescence shift of the small metacentric No. 9 chromosome was resolved by flow cytometry. The characteristics of the polymorphism were studied using cultures established from ear clippings taken from 16 additional hamsters from our breeding colony. Additional variants of chromosome 9 were detected using flow cytometry, and a subset of these variants were analyzed by G- and C-banding. An increase of fluorescence recorded by flow cytometry correlated with an increase of centromeric heterochromatin. Autosomal normalization of the flow karyotype from 18 different animals indicated three distinct peak positions for chromosome 9. The results indicate that a discrete block of constitutive heterochromatin may be present in one or two extra copies within the small inbred colony of hamsters studied. To determine the inheritance patterns, hamsters with known polymorphic No. 9 chromosomes were bred. The flow karyotypes derived from the offspring of these matings provide strong evidence that chromosomal polymorphisms are inherited in Mendelian fashion.     

Kinetochore identification in micronuclei in mouse bone-marrow erythrocytes: an assay for the detection of aneuploidy-inducing agents

An in vivo micronucleus assay using mouse bone marrow for identifying the ability of chemicals to induce aneuploidy and/or chromosome breaks is described. Micronucleus formation in bone-marrow erythrocytes of mice is commonly used as an index for evaluating the clastogenicity of environmental agents. However, micronuclei may also originate from intact lagging chromosomes resulting from the effect of aneuploidy-inducing agents. We have used immunofluorescent staining using anti-kinetochore antibodies to classify micronuclei for the presence or absence of kinetochores. Micronuclei positive for kinetochores are assumed to contain intact chromosomes and result from induced aneuploidy; while those negative for kinetochores contain acentric chromosomal fragments and originate from clastogenic events. The assay was evaluated using X-irradiation (a known clastogen) and vincristine sulfate (an aneuploidy-inducing agent). A dose-related response for the induction of micronuclei was observed for both agents. Micronuclei induced by X-irradiation were negative for kinetochores while the majority of the micronuclei resulting from vincristine treatment contained kinetochores. Thus, the micronucleus assay in combination with immunofluorescent staining for kinetochores may provide a useful method to simultaneously assess the ability of chemicals to induce aneuploidy and/or chromosome breaks.     

Development of Triticum aestivum-Leymus racemosus translocation lines using gametocidal chromosomes

Maan[1] and Endo[2] et al. first reported that some chromosomes from Ae. longgissima, Ae. sharonensis and Ae. triuncialis showed preferential transmission when introduced into wheat background. The mechanism for this phenomenon rests with the fact that contrary to the normal fertility of gametes with these chromosomes, chromosome structural aberrations occur seriously in the gametes without these chromosomes, causing less compatibility in selective fertilization and resulting in semi-sterilit…     

Activation status of the X chromosome in human micronucleated lymphocytes

The frequency of X chromosome aneuploidy in human female peripheral blood lymphocytes has been reported by several investigators to be significantly higher than expected based upon chance alone. Studies in our laboratory showed that 72% of the micronuclei in the peripheral blood of human females contained the X chromosome. Such a high frequency of X chromosome loss suggests that some unique mechanism may be responsible for this phenomenon. The present study was carried out to test the hypothesis that the lost or micronucleated chromsome is the inactive and not the active X. Blood samples were obtained from two unrelated females, 36 and 33 years of age, each with a different X; 9 reciprocal translocation. In each, the normal X chromosome is inactive and the translocated X is active. Isolated lymphocytes were cultured according to standard techniques and blocked with cytochalasin B. Using a modified micronucleus assay, we scored 10,000 binucleated cells from the 36 year old, while 9,500 binucleated cells were scored from the 33 year old. The slides were first labeled and the kinetochore status of each micronucleus was determined. This was followed by simultaneous hybridization with a 2.0 kilobase centromeric X chromosome-specific probe and a chromosome 9 specific whole chromosome painting probe. All micronucleated cells were relocated and scored for their probe status. A total of 217 micronuclei were scored from the two subjects, of which 96 (44.2%) contained the X chromosome. Of these 96 micronuclei, 80 (83.3%) contained the inactive X, based on the absence of chromosome 9 material in the micronucleus. These results support our hypothesis that the inactive X chromosome is preferentially included in the micronuclei, and suggest that the X chromosome hypoploidy observed at metaphase in aging women is a related phenomenon. Received: 5 May 1995 / Revised: 15 July 1995