The role of the repair of double-stranded DNA breaks in the radioresistance of yeast cells

The role of DNA double-strand break (DSB) repair in radioresistance of Saccharomyces cerevisiae G1 cells is discussed. The contribution of rapid and slow DNA DSB repair to radioresistance of diploid yeast has been estimated. The contribution of the DNA DSB repair involving no homologous chromosome interaction is shown to be insignificant in comparison with the recombinational repair. The rapid DNA DSB repair efficiency calculation method based on the proposed yeast radiation inactivation model is given. The calculations are in a satisfactory agreement with the experimental data. Possible mechanisms of radiation induction of lethal sectoring in yeast are discussed. This phenomenon is supposed to be due to the DNA DSB processing during vegetative division of irradiated cells. A general scheme of radiation inactivation of yeast cells is proposed.     

Generation of a novel isogenic trisomy panel in human embryonic stem cells via microcell-mediated chromosome transfer

Aneuploidy is the gain or loss of a chromosome. Down syndrome or trisomy (Ts) 21 is the most frequent live-born aneuploidy syndrome in humans and extensively studied using model mice. However, there is no available model mouse for other congenital Ts syndromes, possibly because of the lethality of Ts in vivo, resulting in the lack of studies to identify the responsible gene(s) for aneuploid syndromes. Although induced pluripotent stem cells derived from patients are useful to analyse aneuploidy syndromes, there are concerns about differences in the genetic background for comparative studies and clonal variations. Therefore, a model cell line panel with the same genetic background has been strongly desired for sophisticated comparative analyses. In this study, we established isogenic human embryonic stem (hES) cells of Ts8, Ts13, and Ts18 in addition to previously established Ts21 by transferring each single chromosome into parental hES cells via microcell-mediated chromosome transfer. Genes on each trisomic chromosome were globally overexpressed in each established cell line, and all Ts cell lines differentiated into all three embryonic germ layers. This cell line panel is expected to be a useful resource to elucidate molecular and epigenetic mechanisms of genetic imbalance and determine how aneuploidy is involved in various abnormal phenotypes including tumourigenesis and impaired neurogenesis.     

Aneuploidy detection with a short-term hexaploid wheat assay

A new type of assay for the identification of agents causing aneuploidy is described. This assay takes advantage of allohexaploid wheat in which monosomic and nullisomic cell lineages can be genetically detected. The wheat strain used (Neatby's virescens) was homozygous for a pair of recessive alleles (v1) which in homozygous condition interfere with normal pigmentation of the leaves at low temperature whereas at higher temperature nearly normal green color formation is permitted. In a single dose this allele cannot suppress the formation of green color even at low temperature, i.e., it is hemizygous ineffective. This locus is in the short arm of chromosome 3B near the centromere. As a consequence of non-disjunction of this chromosome twin sectors may be detected, in which the monosomic cell lineages appear green whereas the trisomic sectors display with color on a cream-colored background at low temperature. This genetic system can also be used for the detection of deletions or duplications involving the short arm of chromosome 3B, and to some extent the A- and the D-genome homeologues. We have determined the pattern of differentiation of the shoot apex and on that basis we can separate the independent genetic events from reappearance of the sectors of common origin in the successive leaves. Such an understanding of development of the leaf sectors permits a quantitative estimation of the genetic response of the plants to mutagenic factors. We have found that X-rays, gamma-rays, p-fluorophenylalanine, 3-aminotriazole, caffeine, vinblastin sulfate, benzo[a]pyrene and auramine significantly increased aneuploidy, and diethylstilbestrol, sulfacetamide, safrole and dichlorvos caused some increase of sectoring. Cytological data on root tips of irradiated seeds support the interpretation of the mechanism of sector formation in the leaves. The test is simple, fast, inexpensive, and it does not require elaborate facilities or highly trained technicians. The trials were well reproducible during a period of 3 years in 2 laboratories. Therefore we consider the new assay a useful complement to other tests of chemicals or physical agents that may cause non-disjunction and other chromosomal aberrations in human populations.     

Interrogating cell division errors using random and chromosome-specific missegregation approaches

Accurate segregation of the duplicated genome in mitosis is essential for maintaining genetic stability. Errors in this process can cause numerical and/or structural chromosome abnormalities – hallmark genomic features commonly associated with both tumorigenesis and developmental disorders. A cell-based approach was recently developed permitting inducible missegregation of the human Y chromosome by selectively disrupting kinetochore assembly onto the Y centromere. Although this strategy initially requires several steps of genetic manipulation, it is easy to use, highly efficient and specific for the Y without affecting the autosomes or the X, and does not require cell cycle synchronization or mitotic perturbation. Here we describe currently available tools for studying chromosome segregation errors, aneuploidy, and micronuclei, as well as discuss how the Y-specific missegregation system has been used to elucidate how chromosomal micronucleation can trigger a class of extensive rearrangements termed chromothripsis. The combinatorial use of these different tools will allow unresolved aspects of cell division defects and chromosomal instability to be experimentally explored.     

Mechanisms of aneuploidy induction in human oogenesis and early embryogenesis

The mechanisms of aneuploidy induction in human oogenesis mainly involve nondisjunction arising during the first and second meiotic divisions. Nondisjunction equally affects both whole chromosomes and chromatids, in the latter case it is facilitated by "predivision" or precocious centromere division. Karyotyping and CGH studies show an excess of hypohaploidy, which is confirmed in studies of preimplantation embryos, providing evidence in favour of anaphase lag as a mechanism. Preferential involvement of the smaller autosomes has been clearly shown but the largest chromosomes are also abnormal in many cases. Overall, the rate of chromosomal imbalance in oocytes from women aged between 30 and 35 has been estimated at 11% from recent karyotyping data but accruing CGH results suggest that the true figure should be considerably higher. Clear evidence has been obtained in favour of germinal or gonadal mosaicism as a predisposing factor. Constitutional aneuploidy in embryos is most frequent for chromosomes 22, 16, 21 and 15; least frequently involved are chromosomes 14, X and Y, and 6. However, embryos of women under 37 are far more likely to be affected by mosaic aneuploidy, which is present in over 50% of 3-day-old embryos. There are two main types, diploid/aneuploid and chaotic mosaics. Chaotic mosaics arise independently of maternal age and may be related to centrosome anomalies and hence of male origin. Aneuploid mosaics most commonly arise by chromosome loss, followed by chromosome gain and least frequently by mitotic nondisjunction. All may be related to maternal age as well as to lack of specific gene products in the embryo. Partial aneuploidy as a result of chromosome breakage affects a minimum of 10% of embryos.     

Germ-cell nondisjunction in testes biopsies of men with idiopathic infertility.

Intracytoplasmic sperm injection (ICSI) has been used in combination with testicular sperm extraction to achieve pregnancies in couples with severe male-factor infertility, yet many of the underlying genetic mechanisms remain largely unknown. To investigate nondisjunction in mitotic and meiotic germ cells, we performed three-color FISH to detect numeric chromosome aberrations in testicular tissue samples from infertile men confirmed to have impaired spermatogenesis of unknown cause. FISH was employed to determine the rate of sex-chromosome aneuploidy in germ cells. Nuclei were distinguished as haploid or diploid, respectively. The overall incidence of sex-chromosome aneuploidy in germ cells was found to be significantly higher (P<.00001) in all three abnormal histopathologic patterns (range 39.0%-43.5%) as compared with normal controls (29.1%). The relative ratio of normal to aneuploid nuclei in the diploid cells of patients with impaired spermatogenesis was approximately 1.0, a >300% decrease when compared with the 4.42 ratio detected in patients with normal spermatogenesis. These results provide direct evidence of an increased incidence of sex-chromosome aneuploidy observed in germ cells of men with severely impaired spermatogenesis who might be candidates for ICSI with sperm obtained directly from the testis. The incidence of aneuploidy was significantly greater among the diploid nuclei, which suggests that chromosome instability is a result of altered genetic control during mitotic cell division and proliferation during spermatogenesis.     

Monitoring meiosis in gametogenesis

Meiotic recombination is essential to hold homologous chromosomes together so that they can separate accurately in the formation of gametes, thus preventing fetal loss due to aneuploidy. How do germ cells know when they have finished genetic recombination and that it is time to enter the meiotic division phase, and what are the elements that signal the onset of the division phase? During spermatogenesis there is no arrest at the end of meiotic prophase (as there is in oogenesis) and signals for progress into the meiotic division phase may be closely related to events of chromosome pairing and recombination. Methods for culture of male germ cells have been used to show that spermatocytes become competent for some aspects of the division phase by the early pachytene stage, long before they would normally enter division. Evidence suggests that establishment of homologous chromosome pairing is one aspect of acquiring competence. Activation of the cell cycle regulator MPF also appears to be important, and there is a requirement for activity of topoisomerase II in order for spermatocytes to exit prophase and enter the meiotic division phase. Understanding how these molecular entities tie into monitoring the completion of recombination and meiotic progress will be instructive about important gametic safeguards preventing aberrant chromosome segregation and resultant aneuploidy.     

A monochromosomal hybrid cell assay for evaluating the genotoxicity of environmental chemicals

The development and utilization of a monochromosomal hybrid cell assay for detecting aneuploidy and chromosomal aberrations are described. The monochromosomal hybrid cell lines were produced by a two-step process involving transfer of a marker bacterial gene to a human chromosome and then by integration of that human chromosome into a mouse complement of chromosomes through microcell fusion. For chemically induced aneuploidy, the segregation of a single human chromosome among mouse chromosomes is used as a cytogenetic marker. The genetic assay for aneuploidy is based on the ability of the cells to grow in a medium that selects for the loss of the human chromosome. The assay for clastogenicity is based on survival of the cells after treatment with the chemicals in medium that selects for retention of the human chromosome but loss of its segment containing diphtheria toxin locus. The assays greatly simplify the detection of chromosomal aberrations induced by environmental factors at low-dose levels.     

Guilt by association? p53 and the development of aneuploidy in cancer

Aneuploidy is one of the most frequent genetic alterations in solid tumors. It is commonly caused by cell division errors that are induced by oncogene activation or loss of tumor suppressor functions. In addition, certain viral oncoproteins have been implicated in the induction of chromosome copy number changes. Aneuploidy and inactivation of p53 frequently coincide in human cancers but there is increasing evidence that loss of p53 by itself is not a primary cause of aneuploidy. Nonetheless, p53 inactivation synergizes with additional oncogenic events to promote aneuploidy and may facilitate chromosomal imbalances through indirect mechanisms. This review summarizes the current knowledge about the association between aneuploidy and p53, and discusses two of the most controversial mechanisms that have been implicated in genomic instability associated with loss of p53: subversion of ploidy control and aberrant centrosome duplication.     

Genetic variation in aneuploidy prevalence and tolerance across Saccharomyces cerevisiae lineages

Individuals carrying an aberrant number of chromosomes can vary widely in their expression of aneuploidy phenotypes. A major unanswered question is the degree to which an individual’s genetic makeup influences its tolerance of karyotypic imbalance. Here we investigated within-species variation in aneuploidy prevalence and tolerance, using Saccharomyces cerevisiae as a model for eukaryotic biology. We analyzed genotypic and phenotypic variation recently published for over 1,000 S. cerevisiae strains spanning dozens of genetically defined clades and ecological associations. Our results show that the prevalence of chromosome gain and loss varies by clade and can be better explained by differences in genetic background than ecology. The relationships between lineages with high aneuploidy frequencies suggest that increased aneuploidy prevalence emerged multiple times in S. cerevisiae evolution. Separate from aneuploidy prevalence, analyzing growth phenotypes revealed that some genetic backgrounds—such as the European Wine lineage—show fitness costs in aneuploids compared to euploids, whereas other clades with high aneuploidy frequencies show little evidence of major deleterious effects. Our analysis confirms that chromosome gain can produce phenotypic benefits, which could influence evolutionary trajectories. These results have important implications for understanding genetic variation in aneuploidy prevalence in health, disease, and evolution.     

Tests which distinguish induced crossing-over and aneuploidy from secondary segregation in Aspergillus treated with chloral hydrate or gamma-rays

A system of tests with the ascomycete Aspergillus nidulans was devised that can detect 3 primary effects of genotoxic agents: (1) increases in mitotic crossing-over; (2) induced aneuploidy; and (3) clastogenic effects which cause chromosomal imbalance. Conidia of a new diploid tester strain, heterozygous for 4 recessive markers which alter conidial color, are treated and plated onto nonselective media. In cases of induced crossing-over, large color segments are found in normal green colonies, frequently adjacent to reciprocal twin segments. In contrast, both malsegregation and chromosome breakage produce unbalanced types which grow poorly and segregate further. Cases with yellow segregants are replated and their secondary diploid sectors tested for markers which are located on both chromosome arms in coupling with yA. Induced aneuploidy can be distinguished from chromosome breakage by the pattern of marker segregation. Any aneuploid type will produce euploid sectors solely by segregation of whole chromosomes; trisomic colonies (yA / yA / +) will show 1:2 ratios for yellow (homozygous yA) to parental green (yA/+) sectors and have characteristic phenotypes. Other induced unbalanced types, if heterozygous for deletions or aberrations may produce yellow diploid sectors by secondary crossing-over as well as by nondisjunction and such cases show unique patterns of genetic segregation and non- predictable phenotypes. As a complementary test, haploid strains are treated and induced abnormally growing types are replated and classified by phenotype. Aneuploids are unstable and produce many normal sectors, and some of these disomic or trisomic types can be visually identified.In contrast, induced deletions are lethal, and duplications or 'morphological' mutants show much more stable abnormal phenotypes. This test system was used to characterize the primary effects of gamma-rays and chloral hydrate. Results and evidence were as follows: (1) A dose-dependent increase of color segments resulting from reciprocal crossing-over was found after treatment of dividing nuclei in germinating diploid conidia with gamma-rays, but not with chloral hydrate. (2) Highly aneuploid and polyploid types were induced in diploid and haploid germinating conidia by chloral hydrate but not to any significant extent by gamma-rays. (3) gamma-Rays caused a dose- dependent increase off abnormally growing colonies when dormant or germinating diploid conidia were treated. These colonies produced secondary euploid sectors by spontaneous nondisjunction and frequently also by crossing-over, which provided evidence for induced semidominant and recessive lethal mutations of many types.     

Aneuploidy: a matter of bad connections

Proper chromosome segregation is required to maintain the appropriate number of chromosomes from one cell generation to the next and to prevent aneuploidy, the condition in which a cell has gained or lost one or several chromosomes during cell division. Aneuploidy is a hallmark associated with birth defects and cancer, and is observed at relatively high frequencies in human somatic cells. Recent studies in mammalian tissue culture cells suggest that the persistence of kinetochore-microtubule misattachments through mitosis is a major cause of chromosome mis-segregation and aneuploidy. Furthermore, studies in mice and humans suggest that small changes in the expression, rather than complete inactivation, of genes encoding specific proteins might be associated with aneuploidy in living organisms. In this article (which is part of the Chromosome Segregation and Aneuploidy series), we survey the outcome of these studies, focusing on the importance of kinetochore misattachments in producing aneuploid cells.     

The maternal-effect gene futile cycle is essential for pronuclear congression and mitotic spindle assembly in the zebrafish zygote

Embryos have been successfully used for the general study of the cell cycle. Although there are significant differences between the early embryonic and the somatic cell cycle in vertebrates, the existence of specialised factors that play a role during the early cell cycles has remained elusive. We analysed a lethal recessive maternal-effect mutant, futile cycle (fue), isolated in a maternal-effect screen for nuclear division defects in the zebrafish (Danio rerio). The pronuclei fail to congress in zygotes derived from homozygous fue mothers. In addition, a defect in the formation of chromosomal microtubules prevents mitotic spindle assembly and thus chromosome segregation in fue zygotes. However, centrosomal functions do not appear to be affected in fue embryos, suggesting this mutant blocks a subset of microtubule functions. Cleavage occurs normally for several divisions resulting in many anucleate cells, thus showing that nuclear- and cell division can be uncoupled genetically. Therefore, we propose that in mitotic spindle assembly chromosome-dependent microtubule nucleation is essential for the coupling of nuclear and cell division.     

Mammalian X Chromosome Dosage Compensation: Perspectives From the Germ Line

Sex chromosomes are advantageous to mammals, allowing them to adopt a genetic rather than environmental sex determination system. However, sex chromosome evolution also carries a burden, because it results in an imbalance in gene dosage between females (XX) and males (XY). This imbalance is resolved by X dosage compensation, which comprises both X chromosome inactivation and X chromosome upregulation. X dosage compensation has been well characterized in the soma, but not in the germ line. Germ cells face a special challenge, because genome wide reprogramming erases epigenetic marks responsible for maintaining the X dosage compensated state. Here we explain how evolution has influenced the gene content and germ line specialization of the mammalian sex chromosomes. We discuss new research uncovering unusual X dosage compensation states in germ cells, which we postulate influence sexual dimorphisms in germ line development and cause infertility in individuals with sex chromosome aneuploidy.     

Meiosis-driven genome variation in plants

Meiosis includes two successive divisions of the nucleus with one round of DNA replication and leads to the formation of gametes with half of the chromosomes of the mother cell during sexual reproduction. It provides a cytological basis for gametogenesis and nheritance in eukaryotes. Meiotic cell division is a complex and dynamic process that involves a number of molecular and cellular events, such as DNA and chromosome replication, chromosome pairing, synapsis and recombination, chromosome segregation, and cytokinesis. Meiosis maintains genome stability and integrity over sexual life cycles. On the other hand, meiosis generates genome variations in several ways. Variant meiotic recombination resulting from specific genome structures induces deletions, duplications, and other rearrangements within the genic and non-genic genomic regions and has been considered a major driving force for gene and genome evolution in nature. Meiotic abnormalities in chromosome segregation lead to chromosomally imbalanced gametes and aneuploidy. Meiotic restitution due to failure of the first or second meiotic division gives rise to unreduced gametes, which triggers polyploidization and genome expansion. This paper reviews research regarding meiosis-driven genome variation, including deletion and duplication of genomic regions, aneuploidy, and polyploidization, and discusses the effect of related meiotic events on genome variation and evolution in plants. Knowledge of various meiosis-driven genome variations provides insight into genome evolution and genetic variability in plants and facilitates plant genome research.     

Test of a semiselective screen for induced aneuploidy in germ cells of Drosophila melanogaster females with structurally normal chromosomes

A new semiselective screen (only female progeny survive) for induced aneuploidy in germ cells of Drosophila melanogaster (referred to as 20/Q56 for the X-chromosome mutation markers in the parental females) has been validated by recovering cold, colchicine and N,N-dimethylnitrosamine (DMN) induced chromosome gain and loss events in females that contain structurally normal chromosomes. In addition, the spontaneous and induced results from the 20/Q56 assay, which identifies gain events at division I and loss events at divisions I and II of meiosis, were compared with a nonselective (all progeny survive) modified mating scheme that identifies gains and losses at both divisions of oogenesis. Females with the same genotypes are treated in the two mating schemes and are then mated with males that contain different marked Y chromosomes. The spontaneous rates of chromosome gains and losses were not significantly different in the two mating schemes (these rates ranged from 0.008 to 0.022%), supporting previous reports that spontaneous aneuploidy occurs at a higher frequency at division I of meiosis in females of D. melanogaster than at division II. Both the 20/Q56 and modified screens were able to identify significant increases in aneuploidy after adult treatments with cold shock (10 degrees C and 5 degrees C), colchicine (5 ppm and 10 ppm), and DMN (100 ppm). Brood analysis (five 2-day or five 3-day broods) showed that the largest increases in aneuploidy after cold treatment occurred in the first brood, which contains a high proportion of stage 14 oocytes, whereas colchicine induced the highest frequencies in the latter broods and DMN was effective in all but the last brood. Although the 20/Q56 mating scheme identifies gain events only in division I of meiosis whereas the modified mating scheme identifies gains in both divisions, the 20/Q56 scheme is just as effective in identifying induced aneuploidy as is the modified scheme. There were no significant differences in the frequencies of induced gains or losses in the two schemes. These results also suggest that the 3 treatments induced chromosome gain events mainly at division I of oogenesis. Taken together, the results from this study suggest that the 20/Q56 mating scheme in D. melanogaster, which is semiselective and therefore less expensive and time-consuming to perform, is an appropriate test system to screen for chemical induced aneuploidy in germ cells of a higher organism.     

Merotelic kinetochore orientation, aneuploidy, and cancer

Accurate chromosome segregation in mitosis is crucial to maintain a diploid chromosome number. A majority of cancer cells are aneuploid and chromosomally unstable, i.e. they tend to gain and lose chromosomes at each mitotic division. Chromosome mis-segregation can arise when cells progress through mitosis with mis-attached kinetochores. Merotelic kinetochore orientation, a type of mis-attachment in which a single kinetochore binds microtubules from two spindle poles rather than just one, can represent a particular threat for dividing cells, as: (i) it occurs frequently in early mitosis; (ii) it is not detected by the spindle assembly checkpoint (unlike other types of mis-attachments); (iii) it can lead to chromosome mis-segregation, and, hence, aneuploidy. A number of studies have recently started to unveil the cellular and molecular mechanisms involved in merotelic kinetochore formation and correction. Here, I review these studies and discuss the relevance of merotelic kinetochore orientation in cancer cell biology.     

Novel insights into genome plasticity in Eukaryotes: mosaic aneuploidy in Leishmania

Leishmania are unicellular eukaryotes that have many markedly original molecular features compared with other uni‐ or multicellular eukaryotes like yeasts or mammals. Genome plasticity in this parasite has been the subject of many publications, and has been associated with drug resistance or adaptability. Aneuploidy has been suspected by several authors and it is now confirmed using state‐of‐the‐art technologies such as high‐throughput DNA sequencing. The analysis of genome contents at the single cell level using fluorescence in situ hybridization (FISH) has brought a new light on the genome organization: within a cell population, every chromosome, in every cell, may be present in at least two ploidy states (being either monosomic, disomic or trisomic), and the chromosomal content varies greatly from cell to cell, thus generating a constitutive intra‐strain genomic heterogeneity, here termed ‘mosaic aneuploidy’. Mosaic aneuploidy deeply affects the genetics of these organisms, leading, for example, to an extreme degree of intra‐strain genomic diversity, as well as to a clearance of heterozygous cells in the population without however affecting genetic heterogeneity. Second, mosaic aneuploidy might be considered as a powerful strategy evolved by the parasite for adapting to modifications of environment conditions as well as for the emergence of drug resistance. On the whole, mosaic aneuploidy may be considered as a novel mechanism for generating phenotypic diversity driven by genomic plasticity.