Termini of human chromosomes display elevated rates of mitotic recombination

The strand-specific in situ hybridization technique of CO-FISH was used to probe telomeres of human mitotic cells in order to determine the spontaneous frequency of crossover. This approach allowed the detection of recombinational crossovers occurring anywhere along the length of individual chromosomes, including reciprocal events taking place between sister chromatids. Although the process of sister chromatid exchange (SCE) is the most prominent type of recombination in somatic mammalian cells, our results show that SCEs accounted for less than a third of the recombinational events revealed by CO-FISH. It is concluded that chromosomal regions near the termini of chromosome arms undergo extraordinarily high rates of spontaneous recombination, producing terminal crossovers whose small size precludes detection by standard cytogenetic methods. That similar results were observed for transformed epithelial cells, as well as primary fibroblasts, suggests that the phenomenon is a common characteristic of human cells. These findings are noteworthy because, although telomeric and subtelomeric DNA is known to be preferentially involved in certain types of recombination, the tips of somatic mammalian chromosomes have not previously been identified as preferred sites for crossover. Implications of these results are discussed in terms of limitations imposed on CO-FISH for its proposed use in directional hybridization mapping.     

Sequential multiple probe fluorescence in-situ hybridization analysis of human oocytes and polar bodies by combining centromeric labelling and whole chromosome painting

The incidence of chromosomal aneuploidy was analysed in 104 unfertilized human oocytes and 56 first polar bodies using a double-label fluorescence in-situ hybridization (FISH) procedure. Combinations of centromeric (or locus-specific) DNA probes and whole chromosome painting probes for chromosomes 9, 13, 16, 18, 21 and X were applied on oocyte preparations, in a sequential FISH protocol. This combined approach allowed a precise in-situ identification of both chromosomes and free chromatids, and consequently a reliable analysis of chromosomal segregation errors. Of the 104 analysed oocytes, 84 (80.7%) displayed a normal chromosome constitution. Three cases of chromosome non-disjunction (2.8%) were found, whereas seven oocytes (6.7%) presented extra single chromatids. In addition, 12 oocytes (11.5%) showed balanced pre-division of one pair of sister chromatids. Although this phenomenon was not classified as aneuploidy, it could lead to aneuploidy at anaphase II. Abnormalities were observed in all the targetted chromosomes. The present data confirm that both whole chromosome non-disjunction and premature chromatid separation constitute the two major mechanisms of aneuploidy in human female meiosis.     

Sister acts: coordinating DNA replication and cohesion establishment

The ring-shaped cohesin complex links sister chromatids and plays crucial roles in homologous recombination and mitotic chromosome segregation. In cycling cells, cohesin's ability to generate cohesive linkages is restricted to S phase and depends on loading and establishment factors that are intimately connected to DNA replication. Here we review how cohesin is regulated by the replication machinery, as well as recent evidence that cohesin itself influences how chromosomes are replicated.     

CO-FISH reveals inversions associated with isochromosome formation

Despite the likely prevalence and documented biological impactof inverted DNA sequences in humans and other species, our abilityto detect them on a routine basis is limited. The techniqueof chromosome orientation fluorescence in situ hybridization(CO-FISH) was used to detect obligate chromosome inversionsassociated with isochromo-some formation in two human cell lines.Simultaneous hybridization of a strand-specific telomeric probeallowed us to deduce the absolute orientation of repetitiveDNA sequences associated with the inverted region. These resultsshow that, in principle, CO-FISH could be used to detect virtuallyany type of inversion, including those likely to escape detectionby other methods. Prospective applications of the techniqueare discussed in relation to its principal limitation, the presentavailability of suitable single-stranded DNA probes. ³To whom correspondence should be addressed     

Nonrandom sister chromatid segregation of sex chromosomes in Drosophila male germline stem cells

Sister chromatids are the product of DNA replication, which is assumed to be a very precise process. Therefore, sister chromatids should be exact copies of each other. However, reports have indicated that sister chromatids are segregated nonrandomly during cell division, suggesting that sister chromatids are not the same, although their DNA sequences are the same. Researchers have speculated that stem cells may retain template strands to avoid replication-induced mutations. An alternative proposal is that cells may segregate distinct epigenetic information carried on sister chromatids. Recently, we found that Drosophila male germline stem cells segregate sister chromatids of X and Y chromosomes with a strong bias. We discuss this finding in relation to existing models for nonrandom sister chromatid segregation.     

Fluorescent in situ hybridization in plant polytene chromosomes

Polytene chromosomes are found in specialized tissues, with high metabolic activity, of a few angiosperm genera. They differ from Diptera polytenics in several aspects, mainly because their chromatids on each chromosome are not tightly paired, nor are they so highly endoreplicated as those of Diptera. In situ hybridization with isotopic and non-isotopic probes has been successfully used in plant polytene chromosomes, mainly in Phaseolus coccineus and Vigna unguiculata, where they have been best investigated. The results reported for mitotic and polytene chromosomes of these species, and a few others, are compared aiming to ascertain the efficiency and limitations of FISH in plant polytenics. In general, polytene chromosomes either from embryo suspensor cells of P. coccineus or from anther tapetal cells of V. unguiculata proved to be quite a suitable system for localizing DNA sequences by FISH. The partially unsynapsed chromatids, typically found in plant polytenics, seem to be the most important hindrance for a precise chromosome mapping. On the other hand, the interphase polytene nucleus is a valuable system for localizing FISH signals since they conserve a spatial organization similar to that of mitotic interphase and produce much amplified signals.     

Cohesion by topology: sister chromatids interlocked by DNA

Sister chromatid cohesion is coupled with chromosome replication and influences chromosome segregation and intra-S repair. Specialized proteins, the cohesins, together with other pathways contribute to tether sister chromatids. In this issue of Genes & Development, Wang and colleagues (pp. 2426-2433 demonstrate that TopoIV, a type II DNA topoisomerase, modulates cohesion in Escherichia coli, by removing interlocked DNA junctions between sister chromatids. They propose that DNA precatenanes, arising during replication fork progression, hold sister chromatids together.     

Correlated Positioning of Homologous Chromosomes in Daughter Fibroblast Cells

A new method of analyzing chromosome segregation in pairs of daughter human fibroblasts revealed that the positioning of chromosomes in daughter nuclei was closely correlated with their relative positions during the G₁ interphase. Two topographic values, namely distance and the angular separation between a pair of homologous chromosomes, were determined using fluorescence in-situ hybridization with four different centromeric DNA probes. These topographical values exhibited a broad distribution as a population, but, to our surprise, both were strongly correlated within each pair of cells derived from the same mother cell (daughters). This correlation was not affected by cell-to-cell distances between daughter cells. We demonstrate in this report that the positioning of chromosomes at G₁ interphase is chiefly determined by their configuration at mitosis, consistent with the nuclear architecture model in which chromosomes are immobile at a global scale in the G₁ interphase nuclei.     

Chromosome methylation patterns during mammalian preimplantation development

DNA methylation patterns were evaluated during preimplantation mouse development by analyzing the binding of monoclonal antibody to 5-methylcytosine (5-MeC) on metaphase chromosomes. Specific chromosome patterns were observed in each cell stage. A banding pattern predominated in chromosomes at the one-cell stage. Banding was replaced at the two-cell stage by an asymmetrical labeling of the sister chromatids. Then, the proportion of asymmetrical chromosomes decreased by one-half at each cell division until the blastocyst stage, and chromosomes became progressively symmetrical and weakly labeled. Our results indicate that chromosome demethylation is associated with each DNA replication and suggest that a passive mechanism predominates during early development.     

From equator to pole: splitting chromosomes in mitosis and meiosis

During eukaryotic cell division, chromosomes must be precisely partitioned to daughter cells. This relies on a mechanism to move chromosomes in defined directions within the parental cell. While sister chromatids are segregated from one another in mitosis and meiosis II, specific adaptations enable the segregation of homologous chromosomes during meiosis I to reduce ploidy for gamete production. Many of the factors that drive these directed chromosome movements are known, and their molecular mechanism has started to be uncovered. Here we review the mechanisms of eukaryotic chromosome segregation, with a particular emphasis on the modifications that ensure the segregation of homologous chromosomes during meiosis I.     

Whole chromosome loss is promoted by telomere dysfunction in primary cells

Errors in chromosome segregation during mitosis result in aneuploidy, which in humans may play a role in the onset of neoplasia by changing gene dosage. Nearly all solid tumors exhibit genomic instability at the chromosomal level, showing both structural and numerical chromosome abnormalities. Chromosomal instability occurs early in the development of cancer and may represent an important step in the initiation and/or progression of the disease. Telomere integrity appears to be a critical element in the genesis of structural chromosome imbalances, but it is still not clear whether it can also generate numerical chromosome aberrations. We investigated the possible relationship between telomere shortening and aneuploidy formation in human mammary epithelial cells using the cytokinesis‐block micronucleus assay combined with fluorescent DNA probes. In this cell system, uncapped chromosomes fuse with each other resulting in dicentric chromosomes, which are known to be a source of new structural chromosome rearrangements. Here, we show that in primary epithelial cells, the chromosomes with short telomeres are more frequently involved in missegregation events than chromosomes of normal telomere length. Whole chromosome aneuploidy occurs through both nondisjunction and anaphase lagging of dicentric chromatids, which suggests that pulling anaphase bridges toward opposite poles can generate the necessary force for detaching a chromosome from the microtubules of one or both spindle poles. Therefore, telomere‐driven instability can promote not only the appearance of chromosomal rearrangements but also the appearance of numerical chromosome aberrations that could favor cell immortalization and the acquisition of a tumor phenotype. © 2010 Wiley‐Liss, Inc.     

Analysis of the DNA content distribution of micronuclei using flow sorting and fluorescent in situ hybridization with a centromeric DNA probe

The DNA content distributions of micronuclei induced in mouse3T3 cells by ionizing radiation and chemicals was measured byflow cytometry. For a quantitative understanding of these distributions,micronuclei with increasing DNA contents were sorted and analysedfor the presence of centromeric signals using fluorescent insitu hybridization (FISH) with a mouse centromeric gamma satelliteprobe. Radiation-induced micronuclei were found to be producedmainly by chromosome fragments, whereas micronuclei inducedby the tear gas chlorobenzylidene malonitrile (CS) were foundto be produced mainly by whole chromatids. In contrast, micronucleiinduced by vinblastine (VBL) were, according to the shape oftheir DNA content distributions, produced mainly by whole chromosomesand by combinations of two or more whole chromosomes. With increasingDNA content, micronuclei induced by ionizing radiation alsocontained one or more whole chromosomes, whereas micronucleiinduced by CS or VBL were found to contain several whole chromatidsor chromosomes respectively. Computerized random breakage ofchromosomes and random combination of chromosome fragments,whole chromatids and whole chromosomes were used according tothe FISH results to simulate the measured DNA content distributionsof micronuclei. A good agreement was obtained between measuredand simulated distributions of micronuclei as well as betweenresults of the measured frequency of micronuclei showing centromericsignals as a function of their DNA content and those predictedby the simulations. These results demonstrate the usefulnessof flow cytometry and sorting combined with the FISH techniqueand computer simulations for producing a more detailed analysisof mechanisms of micronucleus induction. ⁵To whom correspondence should be addressed     

PRINS tandem labeling of satellite DNA in the study of chromosome damage.

Tandem labeling of satellite DNA was proposed a few years ago (1) for evaluating preferential chromosome breaks in the pericentromeric regions of mammalian chromosomes, and (2) for distinguishing chromosome breaks from chromosome segregation errors in interphase cells. In the presence of primers and modified nucleotides, primed in situ labeling (PRINS) tags repetitive DNA sequences, and serves as a useful alternative to fluorescence in situ hybridization (FISH). We developed a two-color method for PRINS tandem labeling of centromeric and pericentromeric sequences. The method, which appears to be more sensitive than FISH, was used to assay micronuclei in mouse splenocytes and early spermatids, and it provided insight into mechanisms of induction of chromosome damage in these cells. We compared the sensitivity of this method and of a different two-color approach, based on simultaneous labeling of centromeric and telomeric sequences.     

Application of spectral karyotyping to the analysis of the human chromosome complement of interspecies somatic cell hybrids

Mouse-human somatic cell hybrids have been extensively used in the molecular genetic dissection of human disease-related chromosome rearrangements because of their ability to selectively and randomly eliminate human chromosomes. This technology allows the isolation of structural chromosome abnormalities, which then allows determination of the precise molecular address of chromosome breakpoints associated with deletions and translocations, down to the nucleotides involved. The main confounding problem with the analysis of somatic cell hybrids is determining the exact chromosome complement unequivocally and quickly. Spectral karyotyping can identify each of the individual human chromosomes in a normal metaphase spread, as well as structural chromosome rearrangements-although, because of potential cross-hybridization between the human probe and mouse DNA sequences during the hybridization reaction, it has not been determined whether the same analysis will selectively identify human chromosomes on a mouse background. We show (to our knowledge, for the first time) that, under modified conditions of chromosomal in situ suppression hybridization, the standard spectral karyotyping probe does not cross-react with mouse chromosomes and can be used to identify subtle structurally rearranged chromosomes in hybrid cells. This analysis allows for the rapid and unequivocal identification of the human chromosome complement in these hybrids, as well as structural chromosome rearrangements that occur between mouse and human chromosomes that might otherwise confound the analysis.     

多色荧光原位杂交检测人卵细胞染色体非整倍体方法的建立

目的 建立多色荧光原位杂交技术检测人卵细胞染色体非整倍体的方法。方法 取试管婴儿助孕技术后未能受精成功的卵细胞，于取卵后13d固定，采用多色荧光原位杂交方法检测卵细胞13，16，18。21和22号染色体的情况。结果 正常未受精卵细胞中期染色体显示一个成对的杂交信号，每条染色单体显示一个单个信号；分裂相中多出或缺少一个成对杂交信号表明多余或缺少一条染色体；分裂相中多出或缺少一个单个信号表明多余或缺少一条染色单体；两个单个信号分离表明两条姐妹染色单体分离。结论 采用多色荧光原位杂交方法可以有效检测人卵细胞染色体非整倍体异常。     

First-generation physical map of the Culicoides variipennis (Diptera: Ceratopogonidae) genome

Recombinant cosmids labeled with biotin-11-dUTP or digoxigenin by nick translation were used as in situ hybridization probes to metaphase chromosomes of Culicoides variipennis (Coquillett). Paired fluorescent signals were detected on each arm of sister chromatids and were ordered along the 3 chromosomes. Thirty-three unique probes were mapped to the 3 chromosomes of C. variipennis (2n = 6): 7 to chromosome 1, 20 to chromosome 2, and 6 to chromosome 3. This work represents the first stage in generating a physical map of the genome of C. variipennis.     

用荧光原位杂交技术研究人类未受精卵细胞的染色体非整倍体

目的　探讨人类未受精卵细胞非整倍体的产生机制。方法　取未受精卵细胞固定后行多色荧光原位杂交 ,分析卵细胞中 13、16、18、2 1和 2 2号染色体的核型情况。结果　 4 7%的卵细胞核型正常 ,5 3%的卵细胞为异常核型 ,其中 18%为同源染色体不分离 ,12 %为姐妹染色单体非平衡性过早分离 ,36 %为姐妹染色单体平衡性过早分离 ;在体外培养 >2 4小时的卵细胞中 ,姐妹染色单体平衡性过早分离的发生率明显高于体外培养≤ 2 4小时的卵细胞 ( P<0 .0 1)。结论　同源染色体不分离和姐妹染色单体平衡性及非平衡性过早分离这三种机制均参与了卵细胞非整倍体的产生。姐妹染色单体平衡性过早分离与体外培养时间具有相关性