Constitutive mosaic aneuploidy is a unique genetic feature widespread in the Leishmania genus

Using fluorescence in situ hybridization, we determined the ploidy of four species of Leishmania: Leishmania infantum, Leishmania donovani, Leishmania tropica and Leishmania amazonensis. We found that each cell in a strain possesses a combination of mono-, di- and trisomies for all chromosomes; ploidy patterns were different among all strains/species. These results extend those we previously described in Leishmania major, demonstrating that mosaic aneuploidy is a genetic feature widespread to the Leishmania genus. In addition to the genetic consequences induced by this mosaicism, the apparent absence of alternation between haploid/diploid stages questions the modality of genetic exchange in Leishmania sp.     

Global chromosome positions are transmitted through mitosis in mammalian cells

We investigated positioning of chromosomes during the cell cycle in live mammalian cells with a combined experimental and computational approach. By non-invasive labeling of chromosome subsets and tracking by 4D imaging, we could show that no global rearrangements occurred in interphase. Using the same assay, we also observed a striking order of chromosomes throughout mitosis. By contrast, our computer simulation based on stochastic movements of individual chromosomes predicted randomization of chromosome order in mitosis. In vivo, a quantitative assay for single chromosome positioning during mitosis revealed strong similarities between daughter and mother cells. These results demonstrate that global chromosome positions are heritable through the cell cycle in mammalian cells. Based on tracking of labeled chromosomes and centromeres during chromosome segregation and experimental perturbations of chromosomal order, we propose that chromosome specific timing of sister chromatid separation transmits chromosomal positions from one cell generation to the next.     

A telomere-mediated chromosome fragmentation approach to assess mitotic stability and ploidy alterations of Leishmania chromosomes

We have used a telomere-associated chromosome fragmentation strategy to induce internal chromosome-specific breakage of Leishmania chromosomes. The integration of telomeric repeats from the kinetoplastid Trypanosoma brucei into defined positions of the Leishmania genome by homologous recombination can induce chromosome breakage accompanied by the deletion of the chromosomal part that is distal to the site of the break. The cloned telomeric DNA at the end of the truncated chromosomes is functional and it can seed the formation of new telomeric repeats. We found that genome ploidy is often altered upon telomere-mediated chromosome fragmentation events resulting in large chromosomal deletions. In most cases diploidy is either preserved, or partial trisomic cells are observed, but interestingly we report here the generation of partial haploid mutants in this diploid organism. Partial haploid Leishmania mutants should facilitate studies on the function of chromosome-assigned genes. We also present several lines of evidence for the presence of sequences involved in chromosome mitotic stability and segregation during cell cycle in this parasitic protozoan. Telomere-directed chromosome fragmentation studies in Leishmania may constitute a useful tool to assay for centromere function.     

Lack of cell cycle checkpoints in human cleavage stage embryos revealed by a clonal pattern of chromosomal mosaicism analysed by sequential multicolour FISH

Multicolour fluorescence in situ hybridisation (FISH) analysis of interphase nuclei in cleavage stage human embryos has highlighted a high incidence of postzygotic chromosomal mosaicism, including both aneuploid and ploidy mosaicism. Indeed, some embryos appear to have a chaotic chromosomal complement in a majority of nuclei, suggesting that cell cycle checkpoints may not operate in early cleavage. Most of these studies, however, have only analysed a limited number of chromosomes (3-5), making it difficult to distinguish FISH artefacts from true aneuploidy. We now report analysis of 11 chromosomes in five sequential hybridisations with standard combinations of two or three probes and minimal loss of hybridisation efficiency. Analysis of a series of arrested human embryos revealed a generally consistent pattern of hybridisation on which was superimposed frequent deletion of one or both chromosomes of a specific pair in two or more nuclei indicating a clonal origin and continued cleavage following chromosome loss. With a binucleate cell in a predominantly triploid XXX embryo, the two nuclei remained attached during preparation and the chaotic diploid/triphoid status of every chromosome analysed was the same for each nucleus. Furthermore, in each hybridisation the signals were distributed as a mirror-image about the plane of attachment, indicating premature decondensation during anaphase consistent with a lack of checkpoint control.     

Micromechanics of human mitotic chromosomes

Eukaryote cells dramatically reorganize their long chromosomal DNAs to facilitate their physical segregation during mitosis. The internal organization of folded mitotic chromosomes remains a basic mystery of cell biology; its understanding would likely shed light on how chromosomes are separated from one another as well as into chromosome structure between cell divisions. We report biophysical experiments on single mitotic chromosomes from human cells, where we combine micromanipulation, nano-Newton-scale force measurement and biochemical treatments to study chromosome connectivity and topology. Results are in accord with previous experiments on amphibian chromosomes and support the 'chromatin network' model of mitotic chromosome structure. Prospects for studies of chromosome-organizing proteins using siRNA expression knockdowns, as well as for differential studies of chromosomes with and without mutations associated with genetic diseases, are also discussed.     

Functional analysis of bacterial artificial chromosomes in mammalian cells: mouse Cdc6 is associated with the mitotic spindle apparatus

Bacterial artificial chromosomes (BACs) provide a well-characterized resource for studying the functional organization of genes and other large chromosomal domains. To facilitate functional studies in cell cultures, we have developed a simple approach for generating stable cell lines with variable copy numbers of any BAC. Here we describe hamster cell lines with BAC transgenes that express mouse Cdc6 at levels that correlate with BAC copy number; show that mouse Cdc6 is regulated normally during the cell cycle, binds chromatin, and is degraded during apoptosis; and report a novel fraction of Cdc6 that associates with the spindle apparatus during mitosis. With RNA interference to assess genetic complementation by BAC alleles, this system will facilitate functional studies on large chromosomal domains at variable copy number in mammalian cell models.     

Coordination of Chromosome Alignment and Mitotic Progression Chromosome-Based Ran Signal

Mitotic spindle assembly and chromosome segregation are controlled by the cell cycle machinery and by the guanosine triphosphatase Ran (RanGTPase). We developed a spatial model that allows us to simulate RanGTP production with different degrees of chromosome alignment in mitosis. Aided by this model, we defined three factors that modulate mitotic RanGTP gradients and mitotic progression in somatic cells. First, the concentration of RanGTPtransport-receptor (represented by RanGTP-importin β) and its spatial distribution are very sensitive to the level of RanBP1. Reduction of RanBP1 leads to an elevated RanGTP-transport receptor concentration throughout the cell, which disrupts spindle assembly and weakens spindle checkpoint control. Second, the completion of chromosome alignment at the metaphase plategenerates highest local RanGTP concentrations on chromosomes that could lead to spindle checkpoint silencing and metaphase-anaphase transition. Finally, chromosomal RanGTP production could be dampened by a reduction of RCC1 phosphorylation in mitosis. Our spatialsimulation of RanGTP production using individual chromosomes should provide means to further understand how the Ran system and the cell cycle machinery coordinately regulate mitosis.     

Tissue-specific placental mosaicism for autosomal trisomies in spontaneous human abortuses: mechanisms of formation and phenotypic effects]

The frequencies of autosomal trisomies in extraembryonic human tissues were estimated in the cases of different abnormalities of prenatal development, from the confined placental mosaicism (CPM) with either relatively normal embryogenesis or restricted intrauterine growth to spontaneous abortion. A tissue-specific compartmentalization was found to be characteristic of cell lines with trisomies for individual autosomes. Analysis of various phenotypical effects of chromosomal aberrations associated with mosaicism is necessarily required to understand the mechanisms and factors responsible for tissue chromosomal mosaicism. Based on analysis of the cell karyotype during prenatal diagnosing of chromosome aberrations in tissues of both extraembryonic and embryonic origin, in 1996, Wolstenholme proposed a model of CPM for individual chromosomes. According to the model, the distribution of cell lines with autosomal trisomies between extraembryonic tissues depends on the ratio between meiotic and mitotic mutations early in embryonic development. However, the model cannot be used to study tissue chromosomal mosaicism in spontaneous abortions, because little information is available on cell karyotype in embryonic tissues themselves after intrauterine fetal death. In this work, a model of tissue-specific chromosomal mosaicism was suggested based on the data on cell karyotype determined in extraembryonic tissues alone, which can be helpful in evaluating the contribution of tissue chromosomal differences into the etiology of early intrauterine death. Along with the experimental evidence, comparative analysis of the two models indicated that the meiotic chromosome nondisjunction plays the major role in trisomy formation and the resultant spontaneous arrest of embryonic development. Other factors responsible for tissue-specific distribution of chromosomal aberrations are also discussed. These are differences in cell proliferative activity, as well as changes in compartmentalization and migration of cells with abnormal karyotypes.     

Tissue-Specific Placental Mosaicism for Autosomal Trisomies in Human Spontaneous Abortuses: Mechanisms of Formation and Phenotypical Effects

The frequencies of autosomal trisomies in extraembryonic human tissues were estimated in the cases of different abnormalities of prenatal development, from the confined placental mosaicism (CPM) with either relatively normal embryogenesis or restricted intrauterine growth to spontaneous abortion. A tissue-specific compartmentalization was found to be characteristic of cell lines with trisomies for individual autosomes. Analysis of various phenotypical effects of chromosomal aberrations associated with mosaicism is necessarily required to understand the mechanisms and factors responsible for tissue chromosomal mosaicism. Based on analysis of the cell karyotype during prenatal diagnosing of chromosome aberrations in tissues of both extraembryonic and embryonic origin, in 1996, Wolstenholme proposed a model of CPM for individual chromosomes. According to the model, the distribution of cell lines with autosomal trisomies between extraembryonic tissues depends on the ratio between meiotic and mitotic mutations early in embryonic development. However, the model cannot be used to study tissue chromosomal mosaicism in spontaneous abortions, because little information is available on cell karyotype in embryonic tissues themselves after intrauterine fetal death. In this work, a model of tissue-specific chromosomal mosaicism was suggested based on the data on cell karyotype determined in extraembryonic tissues alone, which can be helpful in evaluating the contribution of tissue chromosomal differences into the etiology of early intrauterine death. Along with the experimental evidence, comparative analysis of the two models indicated that the meiotic chromosome nondisjunction plays the major role in trisomy formation and the resultant spontaneous arrest of embryonic development. Other factors responsible for tissue-specific distribution of chromosomal aberrations are also discussed. These are differences in cell proliferative activity, as well as changes in compartmentalization and migration of cells with abnormal karyotypes.     

Chromosomal mosaicism in spontaneous abortions: Analysis of 650 cases

It is known that up to 50% spontaneous abortions (SA) in the first trimester of pregnancy are associated with chromosomal abnormalities. We studied mosaic forms of chromosomal abnormalities in 650 SA specimens using interphase MFISH and DNA probes for chromosomes 1, 9, 13/21, 14/22, 15, 16, 18, X, and Y. Numerical chromosomal abnormalities were discovered in 58.2% (378 cases). They contained combined chromosomal abnormalities (aneuploidy of several chromosomes or aneuploidy in combination with polyploidy in the same specimen) in 7.7% (29 cases) or 4.5% of the entire SA sample; autosomal trisomy, in 45% (18.2% in chromosome 16, 8.9% in chromosomes 14/22, 7.9% in chromosomes 13/21, 3.1% in chromosome 18, and 1.4% in chromosome 9). Chromosome X aneuploidy was found in 27% cases, among which 9.6% represented chromosome X monosomy. Polyploidy was observed in 22.9% cases. In 5.1% cases, we observed mosaic form of autosomal monosomy. Among the SA cases with chromosomal abnormalities mosaicism was observed in 50.3% (∼ 25% of the entire SA sample). The results of the present study indicate that significant amount of chromosomal abnormalities in SA cells are associated with disturbances in mitotic chromosome separation, which represents the most common cause of intrauterine fetal death. It was also shown that original collection of DNA probes and the technique of interphase MFISH could be useful for detection of chromosomal mosaicism in prenatal cell specimens.     

Leishmania: sex, lies and karyotype

The exploration of the genome of the tryponosomotid protozoan Leishmania has been difficult until recently owing to a number of obstacles, not least our ignorance of the ploidy and of the number of chromosomes (as in many other protozoa, the latter do not condense during mitosis), the uncertainty of the species concept in these allegedly asexual protozoa and the absence of classical genetic studies. Here, Patrick Bastien, Christine Bloineou and Michel Pages discuss the advances in this field brought about by the advent of molecular biology and its techniques, with on emphasis on ploidy and genetic exchange. In particular, they discuss the data from pulsed field gel electrophoresis (PFGE). When coupled with DNA restriction analysis, PFGE constitutes a powerful tool for the direct examination o f chromosomes of protozoa.     

Role of the Number of Microtubules in Chromosome Segregation during Cell Division

Faithful segregation of genetic material during cell division requires alignment of chromosomes between two spindle poles and attachment of their kinetochores to each of the poles. Failure of these complex dynamical processes leads to chromosomal instability (CIN), a characteristic feature of several diseases including cancer. While a multitude of biological factors regulating chromosome congression and bi-orientation have been identified, it is still unclear how they are integrated so that coherent chromosome motion emerges from a large collection of random and deterministic processes. Here we address this issue by a three dimensional computational model of motor-driven chromosome congression and bi-orientation during mitosis. Our model reveals that successful cell division requires control of the total number of microtubules: if this number is too small bi-orientation fails, while if it is too large not all the chromosomes are able to congress. The optimal number of microtubules predicted by our model compares well with early observations in mammalian cell spindles. Our results shed new light on the origin of several pathological conditions related to chromosomal instability.     

Low level chromosome instability in embryonic cells of primary aneuploid mice

Karyotypic analyses of Down syndrome patients have identified a low level of chromosome mosaicism, suggesting that the primary aneuploid status of the cells promotes further chromosomal segregation errors. Sycp3-null female mice produce aneuploid oocytes, which after fusion with normal haploid sperm, result in offspring with systemic whole chromosome, aneuploid embryo cells. Using the Sycp3-null female as a model, we observe an increase in the number of embryonic cells at E7.0 that exhibit abnormal chromosomal bridges at the anaphas estage of mitosis. This result suggests that global changes in gene expression patterns resulting from primary aneuploidy can affect mitotic chromosome segregation, resulting in a low level of chromosomal instability. The increased level of chromosomal instability could in the absence of mitotic checkpoints, lead to chromosomal mosaicism within the adult organism, as seen in Down syndrome patients.     

Differential role of CENP-A in the segregation of holocentric C. elegans chromosomes during meiosis and mitosis

Two distinct chromosome architectures are prevalent among eukaryotes: monocentric, in which localized centromeres restrict kinetochore assembly to a single chromosomal site, and holocentric, in which diffuse kinetochores form along the entire chromosome length. During mitosis, both chromosome types use specialized chromatin, containing the histone H3 variant CENP-A, to direct kinetochore assembly. For the segregation of recombined homologous chromosomes during meiosis, monocentricity is thought to be crucial for limiting spindle-based forces to one side of a crossover and to prevent recombined chromatids from being simultaneously pulled towards both spindle poles. The mechanisms that allow holocentric chromosomes to avert this fate remain uncharacterized. Here, we show that markedly different mechanisms segregate holocentric chromosomes during meiosis and mitosis in the nematode Caenorhabditis elegans. Immediately prior to oocyte meiotic segregation, outer-kinetochore proteins were recruited to cup-like structures on the chromosome surface via a mechanism that is independent of CENP-A. In striking contrast to mitosis, both oocyte meiotic divisions proceeded normally following depletion of either CENP-A or the closely associated centromeric protein CENP-C. These findings highlight a pronounced difference between the segregation of holocentric chromosomes during meiosis and mitosis and demonstrate the potential to uncouple assembly of outer-kinetochore proteins from CENP-A chromatin.     

Cytological and functional aspects of telomere maintenance

The fact that eukaryotic chromosomes are linear poses a special problem for their maintenance: the natural ends of chromosomes must be distinguished from ends generated by chromosomal breakage and somehow, the chromosome ends must also be fully replicated to maintain their integrity. Telomeres, the complex structures at the ends of chromosomes are thought to be instrumental for both of these functions. However, recent insights in telomere biology suggest that these terminal structures do much more than just fulfill these two basic functions. Cytological data demonstrate that telomeres may play leading roles in chromatin organization and nuclear architecture during mitosis and meiosis. Moreover, non-functional telomeres may lead to genetic instability, a common prelude to cancer. Here, we review the basic functions of telomeres during chromosome replication and discuss the cytological aspects of telomere function during mitosis and meiosis.     

Aneuploidy in the human cleavage stage embryo

The cleavage stage embryo (days 1-3) stands out due to the high level of chromosomal anomalies, especially mosaicism that arises prior to global embryonic genome activation. Molecular cytogenetic studies show that an average of 60% of in vitro derived embryos have at least one aneuploid cell by the time they are 3 days old. However, comprehensive studies of the chromosome content of individual cells have revealed that 25% of these embryos have no aneuploid cells, a fact that sits well with the knowledge that at most 1 in 5 have the capacity to implant. The evidence is that extensive mosaicism, affecting several chromosomes, interferes with development to a greater extent than does uniform aneuploidy. Follow-up studies on embryos after pre-implantation genetic aneuploidy screening indicate that the frequency of meiotic errors varies according to the referral reason, with the highest frequency being recorded for the recurrent miscarriage category and the lowest in the repeated implantation failure group where younger women have a good response to ovarian stimulation. The exceptionally high incidence of pre- and post-zygotic chromosomal anomalies seen in early human embryos is thus the product of several mechanisms. Firstly, the error-prone cell cycle during the embryonic cleavage stage and secondly, parental susceptibility to meiotic and mitotic chromosomal instability together with their general genetic background.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts

During cell division, condensation and resolution of chromosome arms and the assembly of a functional kinetochore at the centromere of each sister chromatid are essential steps for accurate segregation of the genome by the mitotic spindle, yet the contribution of individual chromatin proteins to these processes is poorly understood. We have investigated the role of embryonic linker histone H1 during mitosis in Xenopus laevis egg extracts. Immunodepletion of histone H1 caused the assembly of aberrant elongated chromosomes that extended off the metaphase plate and outside the perimeter of the spindle. Although functional kinetochores assembled, aligned, and exhibited poleward movement, long and tangled chromosome arms could not be segregated in anaphase. Histone H1 depletion did not significantly affect the recruitment of known structural or functional chromosomal components such as condensins or chromokinesins, suggesting that the loss of H1 affects chromosome architecture directly. Thus, our results indicate that linker histone H1 plays an important role in the structure and function of vertebrate chromosomes in mitosis.     

Ploidy mosaicism in well-developed nuclear transplants produced by transfer of adult somatic cell nuclei to nonenucleated eggs of medaka (Oryzias latipes)

Chromosomal abnormalities such as ploidy mosaicism have constituted a major obstacle to the successful nuclear transfer of adult somatic cell nuclei in lower vertebrates to date. Euploid mosaicism has been reported previously in well-developed amphibian transplants. Here, we investigated ploidy mosaicisms in well-developed transplants of adult somatic cell nuclei in medaka fish (Oryzias latipes). Donor nuclei from primary cultured cells from the adult caudal fin of a transgenic strain carrying the green fluorescent protein gene (GFP) were transferred to recipient nonenucleated eggs of a wild-type strain to produce 662 transplants. While some of the transplants developed beyond the body formation stage and several hatched, all exhibited varying degrees of abnormal morphology, limited growth and subsequent death. Twenty-one transplants, 19 embryos and two larvae, were selected for chromosomal analysis; all were well-developed 6-day-old or later embryonic stages exhibiting slight morphological abnormalities and the same pattern of GFP expression as that of the donor strain. In addition, all exhibited various levels of euploid mosaicism with haploid-diploid, haploid-triploid or haploid-diploid-triploid chromosome sets. No visible chromosomal abnormalities were observed. Thus, euploid mosaicism similar to that observed in amphibians was confirmed in well-developed nuclear transplants of fish.