Single Chromatid Research Articles

Aurora B and Aurora C pools at two chromosomal regions collaboratively maintain chromosome alignment and prevent aneuploidy at the second meiotic division in mammalian oocytes.

Correct chromosome segregation is essential to preserve genetic integrity. The two protein kinases, Aurora B and its meiotic homolog Aurora C, regulate attachments between chromosomal kinetochores and microtubules, thereby contributing to the accuracy of the chromosome segregation process. Here we performed a detailed examination of the localization and activity of Aurora B/C kinases, their partner Incenp and the kinetochore target Hec1, during the second meiotic division in mouse oocytes. We found that a majority of Aurora B and C changed their localization from the outer kinetochore region of chromosomes at prometaphase II to an inner central region localized between sister centromeres at metaphase II. Depletion of the Aurora B/C pool at the inner central region using the haspin kinase inhibitor 5-iodotubercidin resulted in chromosome misalignments at the metaphase II stage. To further understand the role of the Aurora B/C pool at the central region, we examined the behaviour of single chromatids, that lack a central Aurora B/C pool but retain Aurora B/C at the outer kinetochores. We found that kinetochore-microtubule attachments at single chromatids were corrected at both prometaphase II and metaphase II stages, but that single chromatids compared to paired chromatids were more prone to misalignments following treatment of oocytes with the Aurora B/C inhibitory drugs AZD1152 and GSK1070916. We conclude that the Aurora B/C pool at the inner central region stabilizes chromosome alignment during metaphase II arrest, while Aurora B/C localized at the kinetochore assist in re-establishing chromosome positioning at the metaphase plate if alignment is lost. Collaboratively these two pools prevent missegregation and aneuploidy at the second meiotic division in mammalian oocytes.

Micronucleus is not a potent inducer of the cGAS/STING pathway.

Micronuclei (MN) have been associated with the innate immune response. The abrupt rupture of MN membranes results in the accumulation of cGAS, potentially activating STING and downstream interferon-responsive genes. However, direct evidence connecting MN and cGAS activation has been lacking. We have developed the FuVis2 reporter system, which enables the visualization of the cell nucleus carrying a single sister chromatid fusion and, consequently, MN. Using this FuVis2 reporter equipped with cGAS and STING reporters, we rigorously assessed the potency of cGAS activation by MN in individual living cells. Our findings reveal that cGAS localization to membrane-ruptured MN during interphase is infrequent, with cGAS primarily capturing MN during mitosis and remaining bound to cytosolic chromatin. We found that cGAS accumulation during mitosis neither activates STING in the subsequent interphase nor triggers the interferon response. Gamma-ray irradiation activates STING independently of MN formation and cGAS localization to MN. These results suggest that cGAS accumulation in cytosolic MN is not a robust indicator of its activation and that MN are not the primary trigger of the cGAS/STING pathway.

Age-dependent loss of cohesion protection in human oocytes

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a "cohesin bridge" between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.

Preprint Highlight: Age-dependent loss of cohesion protection in human oocytes

Age-dependent loss of cohesion is a determinant of aneuploidy in human oocytes. Mechanisms for how this loss is triggered with age are not known. Using oocytes from human participants of various ages, the authors correlate loss of Sgo2, a protector of cohesins, with age-related meiosis II cohesion defects including increased inter-kinetochore distances and single chromatids. High-resolution microscopy reveals age-dependent loss of Sgo2 specifically from pericentromeric bridges, but not from centromeric regions. This study highlights the role of pericentromeric cohesins in upholding sister-chromatid cohesion, even when centromeric cohesins are protected. This work is relevant to researchers interested in chromosome biology, meiosis, aneuploidy mechanisms, and fertility. This preprint has been assigned the following badges: New Hypothesis. Read the preprint on bioRxiv ( Mihalas et al., 2023 ): https://doi.org/10.1101/2023.01.13.523952 .

Visualization of the three-dimensional structure of the human centromere in mitotic chromosomes by superresolution microscopy.

The human centromere comprises large arrays of repetitive α-satellite DNA at the primary constriction of mitotic chromosomes. In addition, centromeres are epigenetically specified by the centromere-specific histone H3 variant CENP-A that supports kinetochore assembly to enable chromosome segregation. Because CENP-A is bound to only a fraction of the α-satellite elements within the megabase-sized centromere DNA, correlating the three-dimensional (3D) organization of α-satellite DNA and CENP-A remains elusive. To visualize centromere organization within a single chromatid, we used a combination of the centromere chromosome orientation fluorescence in situ hybridization (Cen-CO-FISH) technique together with structured illumination microscopy. Cen-CO-FISH allows the differential labeling of the sister chromatids without the denaturation step used in conventional FISH that may affect DNA structure. Our data indicate that α-satellite DNA is arranged in a ring-like organization within prometaphase chromosomes, in the presence or absence of spindle's microtubules. Using expansion microscopy, we found that CENP-A organization within mitotic chromosomes follows a rounded pattern similar to that of α-satellite DNA, often visible as a ring thicker at the outer surface oriented toward the kinetochore-microtubule interface. Collectively, our data provide a 3D reconstruction of α-satellite DNA along with CENP-A clusters that outlines the overall architecture of the mitotic centromere.

Cytotoxic Evaluation of <i>Eurycoma longifolia</i> Jack Root Extract on Chromosome Aberrations in Human Lymphocytes <i>In vitro</i>

This study aimed to investigate chromosomal aberrations of Eurycoma longifolia Jack (EL) root extract in human lymphocytes in vitro. Human whole blood was cultured in medium solution that treated with distilled water, 20% DMSO, extract of EL roots at the concentration of 2.5, 5, 10, 20, 40, 80 µg/mL (extracted with distilled water and ethanol), and nontreated (blank: only culture medium and whole blood). All experiments were cultured for 72 hours in the 37°C incubator. The effects of EL roots extract on cytotoxicity were compared with the control groups including the blank, distilled water, and 20% DMSO. This study found that EL root extract significantly decreased metaphase cell number and increased chromosome aberrations dose dependent manner (p<0.01). The 7 types of chromosome aberration that were observed consisted of dicentric chromosome, single chromatid breaks, isochromatid break, isochromatid gap, single chromatid gap, fragmentation, and deletion. The dicentric chromosome was the most common chromosomal aberrations type that was treated with EL root extract both distilled water and ethanol. Moreover, the ethanolic extract of EL root was more effective to stimulate chromosome aberrations compared to the water extract of EL root (the deletion and fragmentation were not found in the water extract of EL root). This study demonstrated that the phytochemicals of EL root extract had cytotoxicity effect (decreased metaphase cells and increase cells death) and genotoxic effect (increased chromosomal aberrations. The use of EL root crude extract with distilled water is therefore safer for cells. However, when EL is used at high levels, it may lead to the inhibition of cell division process and cause side effects (toxicity). EL extracts consist of various phytochemicals with different properties and dosages, thus more studies should be conducted on the effect of those substances on cytotoxicity, especially their effects on genotoxicity humans.

Chromosomal aberration and genetic differentiation of Oreochromis niloticus affected by heavy metals from an iron ore mine area

ABSTRACT The concentrations of As, Cd, Cr, Pb and Fe in water, sediment and Oreochromis niloticus tissues and the chromosomal aberrations, genetic differentiation and genomic template stability (GTS) of O. niloticus in 2 reservoirs near iron ore mine and one reference reservoir were investigated. Genetic differentiation and %GTS were evaluated by inter simple sequence repeat analysis. The As, Cd and Cr concentrations in fish from 2 mine reservoirs exceeded the standards. Seven types of chromosomal aberrations were found in fish from the iron ore mine: single chromatid gap, iso chromatid gap, centromere gap, dicentric chromosome, fragmentation, deletion, and ring chromosome. The average percentages of aberrant chromosomes in the cells of O. niloticus from mine reservoirs 1, 2 and the reference reservoir were 23.6, 16.8 and 2.2, respectively. Genetic differentiation and subsequent dendrogram analysis separated O. niloticus into 3 clusters, corresponding to the 2 mine reservoirs and the reference reservoir. The %GTS ranges of O. niloticus were 68.75–83.75, 71.88–85.19 and 83.58–97.01, respectively. Metals accumulations in O. niloticus could induce the occurrence of chromosomal aberrations and genetic differentiation.

Haploidy in somatic cells is induced by mature oocytes in mice

Haploidy is naturally observed in gametes; however, attempts of experimentally inducing haploidy in somatic cells have not been successful. Here, we demonstrate that the replacement of meiotic spindles in mature metaphases II (MII) arrested oocytes with nuclei of somatic cells in the G0/G1 stage of cell cycle results in the formation of de novo spindles consisting of somatic homologous chromosomes comprising of single chromatids. Fertilization of such oocytes with sperm triggers the extrusion of one set of homologous chromosomes into the pseudo-polar body (PPB), resulting in a zygote with haploid somatic and sperm pronuclei (PN). Upon culture, 18% of somatic-sperm zygotes reach the blastocyst stage, and 16% of them possess heterozygous diploid genomes consisting of somatic haploid and sperm homologs across all chromosomes. We also generate embryonic stem cells and live offspring from somatic-sperm embryos. Our finding may offer an alternative strategy for generating oocytes carrying somatic genomes.

CYTOGENETIC STUDY OF MANIFESTATIONS OF THE UNIVERSAL PHENOMENON OF THE BYSTANDER RESPONSE.

to establish the level of chromosomal instability in peripheral blood lymphocytes (PBL) from healthyindividuals and cells from patients with B-cell chronic lymphocytic leukemia (CLL) under co-cultivation conditions and to determine the possibility of inducing manifestations of the universal phenomenon of bystander response in them. Cytogenetic analysis of uniformly stained chromosomes from human PBL, which differed by cytogenetic markers of sex; the presence of oncological transformation and in vitro irradiation of 137Cs in a dose of 0.50 Gy under the conditions of their joint cultivation was performed. The frequency of chromosome aberrations in PBL from healthy individuals when co-cultured with the blood from CLL patients was 3.35 per 100 cells, exceeded the control (1.48 per 100 cells, р < 0.01), did not significantly differ from the rate in non-irradiated blood T-lymphocytes from patients with B-cell CLL (3.18 per 100 cells, р > 0.05) and was lower than when co-cultivated with irradiated blood from CLL patients (5.00 per 100 cells, р < 0.01). In irradiated in vitro blood lymphocytes from CLL patients, the mean group level of chromosome aberrations under separate cultivation was 12.36 per 100 cells and exceeded the indicator during their co-cultivation with lymphocytes from healthy individuals (8.35 per 100 cells, р < 0.05). A tumor-induced bystander effect (TIBE) develops in PBL from healthy individuals when co-cultured with the blood from CLL patients and in T lymphocytes of B-cell CLL patients, the cytogenetic manifestation of which is an increase in the frequency of single chromatid fragments. The interaction of irradiated blood cells from CLL patients with lymphocytes from healthy individuals causes an increase in chromosomal instability in the latter due to the development of a radiation-induced bystander effect (RIBE) and leads to a decrease in the level of chromosomal instability in irradiated lymphocytes from CLL patients (rescue effect). An increase in the level of chromatid-type aberrations in T-lymphocytes of CLL patients during in vitro irradiation is a consequence of the development of RIBE against the background of TIBE.

Chromosome instability induced by a single defined sister chromatid fusion.

Chromosome fusion is a frequent intermediate in oncogenic chromosome rearrangements and has been proposed to cause multiple tumor-driving abnormalities. In conventional experimental systems, however, these abnormalities were often induced by randomly induced chromosome fusions involving multiple different chromosomes. It was therefore not well understood whether a single defined type of chromosome fusion, which is reminiscent of a sporadic fusion in tumor cells, has the potential to cause chromosome instabilities. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. The fused chromosome subsequently developed extra-acentric chromosomes, including chromosome scattering, indicative of chromothripsis. Live-cell imaging and statistical modeling indicated that sister chromatid fusion generated micronuclei (MN) in the first few cell cycles and that cells with MN tend to display cell cycle abnormalities. The powerful FuVis system thus demonstrates that even a single sporadic sister chromatid fusion can induce chromosome instability and destabilize the cell cycle through MN formation.

Effect of heavy metals from an electronic waste open dumping area on the cytotoxicity of climbing perch (Anabas testudineus)

ABSTRACT Abnormal chromosomes in climbing perch (Anabas testudineus) from a reservoir near an electronic waste (e-waste) open dumping area compared with reference area were investigated. The Cd, Pb, Cr, Cu, Mn, Ni and Zn concentrations in water and sediment did not exceed the standard limit of Thailand. The Cd, Pb and Cr concentrations in A. testudineus from the e-waste area exceeded the food standard. The heavy metal concentrations in the A. testudineus samples from the e-waste and the reference areas were significantly different (p < 0.05), except for Mn. There were five types of abnormal chromosomes: centromere gap, deletion, fragmentation, single chromatid gap and single chromatid decomposition. The percentages of abnormal chromosomes in the A. testudineus cells from the e-waste and the reference areas were significantly different (p < 0.05). The results indicated that Cd, Pb and Cr concentrations from the e-waste area could induce the occurrence of abnormal chromosomes in A. testudineus.

Meiotic sex chromosome cohesion and autosomal synapsis are supported byEsco2

In mitotic cells, establishment of sister chromatid cohesion requires acetylation of the cohesin subunit SMC3 (acSMC3) by ESCO1 and/or ESCO2. Meiotic cohesin plays additional but poorly understood roles in the formation of chromosome axial elements (AEs) and synaptonemal complexes. Here, we show that levels of ESCO2, acSMC3, and the pro-cohesion factor sororin increase on meiotic chromosomes as homologs synapse. These proteins are less abundant on the largely unsynapsed sex chromosomes, whose sister chromatid cohesion appears weaker throughout the meiotic prophase. Using three distinct conditional Esco2 knockout mouse strains, we demonstrate that ESCO2 is essential for male gametogenesis. Partial depletion of ESCO2 in prophase I spermatocytes delays chromosome synapsis and further weakens cohesion along sex chromosomes, which show extensive separation of AEs into single chromatids. Unsynapsed regions of autosomes are associated with the sex chromatin and also display split AEs. This study provides the first evidence for a specific role of ESCO2 in mammalian meiosis, identifies a particular ESCO2 dependence of sex chromosome cohesion and suggests support of autosomal synapsis by acSMC3-stabilized cohesion.

Analysis of chromosome abnormalities in the Asian swamp eel (Monopterus albus) affected by arsenic contamination near a gold mine area

ABSTRACT The paper reports a study of chromosome abnormalities (CA/s) in the Monopterus albus near a gold mine area compared to a non-affected area. The arsenic (As) concentrations were determined in the water, sediment and M. albus muscles. The average concentrations of As in the water and sediment samples near the gold mine were higher than allowable by Thailand’s water quality standards. The average concentrations of As in the M. albus samples were lower than the standards. The diploid chromosome number of M. albus in both areas was 2n = 24. The percentage of CA of M. albus in the gold mine area was higher than in the non-affected area. Seven types of CA were single chromatid gap (SCG), fragmentation (F), sister chromatid gap, single chromatid breaks, deletion, sister chromatid fragmentation and centric fusion. In the affected area, the most common CAs in the samples were SCG and F. The specific CA in M. albus samples was SCG on chromosome pair 1. It can be concluded that As contamination caused structural CA, but did not affect the diploid chromosome number in the Asian swamp eel from the gold mine area. The accumulation of As in M. albus has potential effects on human health in consumers of M. albus from this contaminated area.

Detection of Complex Structural Variations of X-Linked Deafness-2 (DFNX2) by Single-Molecule Sequencing

Purpose: To investigate genetic causes of X-linked deafness-2 (DFNX2) and analyse the efficiencies and advantages/disadvantages of different sequencing methods. Methods: From 2016 to 2018, 12 unrelated patients with characteristic high-resolution computed tomographic images showing incomplete partition type III (IP-III) were enrolled in this study. Targeted next-generation sequencing (NGS) and Nanopore long-read sequencing were used to analyse the genetic etiology. Results: Six variants of POU3F4 were identified in this cohort by NGS. The six variants were all located upstream of the POU-specific domain and were considered pathogenic due to truncated protein. Among six patients with negative NGS results, three patients were volunteered to choose further Nanopore sequencing and were all found to have structural variations (SVs). We detected an 870 kb deletion in J0012, located at ChrX:81,140,313-82,011,100, approximately 750 kb upstream of the POU3F4 gene. Non-homologous end joining may be a deletion mechanism. An 8 Mb inversion was found in J0011 at ChrX:82,146,071-90,188,492. The sequences of the breakpoint analysis suggested non-allelic homologous recombination occurred within a single chromatid. A 6 kb deletion at ChrX:81,096,652-81,102,705 was found in J0007, which is a region with a high density of retrotransposons. Conclusions: The most common mutations in patients with IP-III were point mutations and small INDELs or exons that can be detected by Sanger sequencing and NGS. Single-molecule long-fragment sequencing technology provides a new technique to detect the pathogenic SVs in patients with negative NGS results. Thus, the DNA diagnostic strategy for IP-III and its efficiency have been greatly improved. At the same time, the sequence characteristics around the breakpoint and the possible mechanism of SV formation should be explored further. Funding Statement: This work was supported by National Key Research and Development Program of China (2016YFC1000700, 2016YFC1000704 and 2017YFC1001800), Shanghai Key Laboratory of Translational Medicine on Ear and Nose diseases (14DZ2260300). Declaration of Interests: The authors have declared that no competing interests exist. Ethics Approval Statement: This investigation was performed with the approval of the ethics committees of the Chinese PLA General Hospital. All of the subjects or their guardians signed the informed consent forms prior to blood sampling.

Three GLI2 mutations combined potentially underlie non-syndromic cleft lip with or without cleft palate in a Chinese pedigree.

BackgroundNonsyndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial birth defect. Its etiology is complex and it has a lifelong influence on affected individuals. Despite many studies, the pathogenic gene alleles are not completely clear. Here, we recruited a Chinese NSCL/P family and explored the candidate causative variants in this pedigree.MethodsWe performed whole‐exome sequencing on two patients and two unaffected subjects of this family. Variants were screened based on bioinformatics analysis to identify the potential etiological alleles. Species conservation analysis, mutation function prediction, and homology protein modeling were also performed to preliminarily evaluate the influence of the mutations.ResultsWe identified three rare mutations that are located on a single chromatid (c.2684C > T_p.Ala895Val, c.4350G > T_p.Gln1450His, and c.4622C > A_p.Ser1541Tyr) in GLI2 as candidate causative variants. All of these three mutations were predicted to be deleterious, and they affect amino acids that are conserved in many species. The mutation c.2684C > T was predicted to affect the structure of the GLI2 protein.ConclusionOur results further demonstrate that GLI2 variants play a role in the pathogenesis of NSCL/P, and the three rare missense mutations combined are probably the potential disease‐causing variants in this family.

36. TWO HEALTHY BABIES BORN AFTER IMPLANTATION OF MULTIPLE CHROMOSOMALLY-RESCUED OOCYTES

Introduction Meiotic rescue is a well-known phenomenon but little is known about live births after oocyte rescue, because of the shortage of data on PB biopsy. PGT-A has been performed for many years to increase the chance of pregnancy, especially in older women. Embryo aneuploidy is a major cause of pregnancy failure and is largely of maternal meiotic origin (>95%), with the risk increasing exponentially from approximately 35 years of age. Molecular analysis of polar bodies provides indirect assessment of an oocyte's chromosomal status by determining whether the chromosomes correctly segregated during meiosis I and II. In about one third of cases, missegregation of one or two chromosomes in meiosis I can be rescued in meiosis II, leading to a euploid oocyte. We present two cases of healthy babies born after single and double chromosome-rescued oocytes Material and methods A 42- (case 1) and a 44-year old woman (case 2) underwent ART treatment with PGT-A PB, after histories of 3 and 2 prior ART failures respectively. Both couples underwent ICSI with first and second PB biopsy and comprehensive chromosome analysis by Array-CGH. The mechanisms leading to missegregation in PB1 and PB2 (nondisjunction or premature separation of sister chromatids (PSSC)) were analysed using chromosome-specific STRs. Results and conclusions Case 1: a single euploid fertilized oocyte was transferred, after double rescue of chromosomes 3 and 18 (PB1 -3, -18; PB2: +3, +18) at day 2, resulting in a singleton pregnancy. Case 2: a single euploid fertilized oocyte was transferred after rescue of chromosome 16 (PB1: -16; PB2:+16) at day 3. In case 1, the aneuploidy of chromosomes 3 and 18 in the first PB was caused by PSSC followed by the extrusion of 2 homologous chromosomes in the second PB. In case 2, chromosome 16 underwent PSSC and extrusion of a single chromatid in PB1, followed by nondisjunction in meiosis II and extrusion of two sister chromatids in PB2. In both cases, the rescued oocyte was the only euploid oocyte out of 12 retrieved and they were competent for generating a pregnancy. The two women gave birth at term to healthy babies (a girl of 3280 g and a boy of 3500 g). To our knowledge, this is the first report of healthy births after a multiple chromosomally-rescued oocyte. PGT-A of PBs is a valid method with the limitation of detecting only maternal aneuploidies and so is appropriate for screening of older women. Analysis of the 2 polar bodies by aCGH is valuable not only for aneuploidy screening in ART for older women, but also to understand the mechanisms leading to missegregation and rescue in oocytes.

HOW AND WHEN DO OOCYTE CHROMOSOMES FALL APART DURING FEMALE AGEING?

Mammalian female meiosis commences in utero and is marked by reciprocal exchange of DNA between parental homologues to form bivalent chromosomes. Thereafter, oocytes remain arrested in prophase of meiosis I and are recruited for growth and ovulation throughout reproductive life. Oocytes ovulated later in life are derived from a depleted stock and are characterised by an extended period of arrest in prophase of meiosis I. Extending this beyond ∼35 years in humans is associated with an increased incidence of meiotic segregation errors giving rise to aneuploid oocytes, most of which are not compatible with life. Notable exceptions are trisomies of chromosome 21 and the sex chromosomes. Bivalent chromosomes are stabilised by cohesin complexes containing the meiosis-specific alpha-kleisin subunit, Rec8. Cleavage of Rec8 by separase results in resolution of bivalents to their four constituent chromatids. This occurs in two steps: Rec8 is first cleaved on arms during anaphase I, giving rise to dyads chromosomes, and then on centromeres during anaphase II, giving rise to single chromatids. In female mammals, anaphase I occurs shortly before ovulation while/whereas anaphase II occurs after the fertilising sperm enters the egg. Studies on human oocytes indicate a strong correlation between female ageing and premature resolution of centromeric cohesin. Consistent with this, we and others have previously reported that female ageing in mice is characterised by depletion of Rec8 from oocyte chromosomes. This is accompanied by reduced recruitment of SgoL2, which protects centromeric Rec8 from cleavage by separase until anaphase II. Together, these events are sufficient to explain the prevalence of premature loss of centromeric cohesin in oocytes from older females. We are therefore interested in understanding the timing and mechanisms of cohesin depletion during female ageing. Our recent work indicates that cohesin is gradually depleted from oocyte chromosomes during the prolonged arrest at prophase of meiosis I, before oocytes are recruited for growth. Interestingly, the age-related decline in chromosome-associated Rec8 exceeds that of Smc3, which is a subunit of mitotic as well as meiotic cohesin complexes. This suggests that cohesin complexes containing alpha-kleisin subunits other than Rec8 are present on oocyte chromosomes and might be less vulnerable to depletion during female ageing. In addition, we have tested the idea that leaky inhibition of separase during the prolonged arrest at prophase of meiosis I contributes to gradual loss of Rec8. In support of this possibility, we can detect separase in oocytes before they are recruited for growth. We find, however, that deletion of separase specifically in oocytes does not prevent the decline in Rec8 levels observed during female ageing. Together, these findings indicate that cohesin loss occurs gradually during the prolonged prophase arrest and that this occurs by a separase-independent mechanism.

Полногеномное ассоциативное исследование связи полиморфных локусов с повышенной частотой хромосомных аберраций у лиц, подвергавшихся длительному радиационному воздействию

Purpose: To conduct genome wide association study of the association of 750,000 SNPs and an increased frequency of different types of chromosomal aberrations, induced by chronic irradiation in the dose range of 100–300 mSv.&#x0D; Material and methods: The study was conducted among Siberian Group of Chemical Enterprises healthy employees (n = 37) exposed to professional external γ-radiation in a dose range of 100–300 mSv. The de novo induced CNVs were previously detected in these persons. Mean dose – 188.8 ± 8.3 mSv, median – 185 mSv, interquartile range – 147.8–218.7 mSv, min – 103.4 mSv, max – 295.8 mSv. Genotyping of DNA samples from 37 employees was carried out by microarray CytoScan™ HD Array (Affymetrix, USA), containing 750,000 SNP-markers of 36,000 genes. The standard cytogenetic analysis was performed in the entire examined group.&#x0D; Results: We analyzed the association of these SNPs with the frequencies of aberrant cells and following chromosomal aberrations: single chromatid fragments, chromatid exchanges, paired fragments, dicentrics, rings, and translocations. We have found that 8 SNPs (rs10779468, rs158735, rs158710, rs158712, rs11131536, rs528170, rs9533572, rs10512439) are associated with the frequency of aberrant cells.&#x0D; Conclusion: We have discovered polymorphic variants that are associated with an increased frequency of aberrant cells in workers of Siberian Group of Chemical Enterprises exposed to irradiation at a dose of 100–300 mSv. This polymorphic variants can be considered as potential markers of individual radiosensitivity. To confirm identified associations, further validation studies on an extended sample of people exposed to radiation are needed.

Effects of electronic waste on cytogenetic and physiological changes in snakehead fish (Channa striata).

The objectives of this study were to investigate cadmium (Cd), chromium (Cr), and lead (Pb) concentrations in water and sediment as well as the muscle, gill, and liver of snakehead fish (Channa striata) and to reveal chromosomal aberrations, changes in serum biochemical parameters, and histopathological alterations of fish from a reservoir near an electronic waste dumping area. Cd, Cr, and Pb concentrations were determined using inductively coupled plasma optical emission spectrometry. Chromosomal aberrations were studied in kidney cells using a conventional technique. The biochemical parameters were measured using an automated analyzer, and histopathological photographs were obtained using a transmission electron microscope. The results showed that heavy-metal concentrations in water and sediment did not exceed the standards, whereas Cd and Pb concentrations in the gill and liver exceeded the standards. The accumulation pattern of heavy metals in organ tissues was exhibited according to the following order: gill > liver > muscle. Five types of chromosomal aberrations were a centromere gap, single chromatid break, deletion, single chromatid gap, and fragmentation. The average percentages of chromosomal aberrations in polluted and reference C. striata were 4.60% and 1.00%, respectively. The statistical analyses of chromosomal aberration and biochemical parameters indicated that total protein, aspartate aminotransferase, and alanine aminotransferase significantly differed between the polluted and reference C. striata (p < 0.05). The liver histopathological alterations revealed atypical cellular structures, such as vacuolar appearance, nucleus degeneration, rough endoplasmic reticulum disintegration, abnormal cytoplasmic mitochondria, and deposition of heavy metals. Heavy-metal contaminations from electronic waste dumping areas affect fish in terms of chromosomal aberration, serum biochemistry, and histopathology.

Absence of the Spindle Assembly Checkpoint Restores Mitotic Fidelity upon Loss of Sister Chromatid Cohesion

SummaryThe fidelity of mitosis depends on cohesive forces that keep sister chromatids together. This is mediated by cohesin that embraces sister chromatid fibers from the time of their replication until the subsequent mitosis [1, 2, 3]. Cleavage of cohesin marks anaphase onset, where single chromatids are dragged to the poles by the mitotic spindle [4, 5, 6]. Cohesin cleavage should only occur when all chromosomes are properly bio-oriented to ensure equal genome distribution and prevent random chromosome segregation. Unscheduled loss of sister chromatid cohesion is prevented by a safeguard mechanism known as the spindle assembly checkpoint (SAC) [7, 8]. To identify specific conditions capable of restoring defects associated with cohesion loss, we screened for genes whose depletion modulates Drosophila wing development when sister chromatid cohesion is impaired. Cohesion deficiency was induced by knockdown of the acetyltransferase separation anxiety (San)/Naa50, a cohesin complex stabilizer [9, 10, 11, 12]. Several genes whose function impacts wing development upon cohesion loss were identified. Surprisingly, knockdown of key SAC proteins, Mad2 and Mps1, suppressed developmental defects associated with San depletion. SAC impairment upon cohesin removal, triggered by San depletion or artificial removal of the cohesin complex, prevented extensive genome shuffling, reduced segregation defects, and restored cell survival. This counterintuitive phenotypic suppression was caused by an intrinsic bias for efficient chromosome biorientation at mitotic entry, coupled with slow engagement of error-correction reactions. Thus, in contrast to SAC’s role as a safeguard mechanism for mitotic fidelity, removal of this checkpoint alleviates mitotic errors when sister chromatid cohesion is compromised.