Chromosome Segregation Pattern Research Articles

Chromosome segregation pattern in day 3 and spare embryos in carriers of chromosome translocation

Chromosome segregation pattern in day 3 and spare embryos in carriers of chromosome translocation

Meiosis I Is Established through Division-Specific Translational Control of a Cyclin

In budding yeast, key meiotic events such as DNA replication, recombination, and the meiotic divisions are controlled by Clb cyclin-dependent kinases (Clb-CDKs). Using a novel synchronization procedure, we have characterized the activity of these Clb-CDKs and observed a surprising diversity in their regulation during the meiotic divisions. Clb1-CDK activity is restricted to meiosis I, and Clb3-CDK activity to meiosis II, through 5'UTR-mediated translational control of its transcript. The analysis of cells inappropriately producing Clb3-CDKs during meiosis I furthermore defines Clb3 as an inhibitor of the meiosis I chromosome segregation program. Our results demonstrate an essential role for Clb-CDK regulation in establishing the meiotic chromosome segregation pattern.

Genetic intratumour heterogeneity in high‐grade brain tumours is associated with telomere‐dependent mitotic instability

Glioblastoma multiforme (GBM) and other high-grade brain tumours are typically characterized by complex chromosome abnormalities and extensive intratumour cytogenetic heterogeneity. The mechanisms behind this diversity have been little explored. In this study, we analysed the pattern of chromosome segregation at mitosis in 20 brain tumours. We found an abnormal segregation of chromatids at mitosis through anaphase bridging (10-25% of anaphase cells) in all 10 GBMs. Anaphase bridging was also found in two medulloblastomas (7-15%), one anaplastic astrocytoma (17%) and one oligodendroglioma (6%). These tumours showed a relatively high degree of cytogenetic complexity and heterogeneity. In contrast, cell division abnormalities were not found in low-grade brain tumours with less complex karyotypes, including two pilocytic astrocytomas and two ependymomas. Further analysis of two GBMs by fluorescence in situ hybridization with telomeric repeat probes revealed excessive shortening of TTAGGG repeats, indicating dysfunctional protection of chromosome ends. In xenografts established from these GBMs, there was a gradual reduction in cytogenetic heterogeneity through successive passages as the proportion of abnormally short telomeres was reduced and the frequency of anaphase bridges decreased from >25% to 0. However, bridging could be reintroduced in late-passage xenograft cells by pharmacological induction of telomere shortening, using a small-molecule telomerase inhibitor. Telomere-dependent abnormal segregation of chromosomes at mitosis is thus a common phenomenon in high-grade brain tumours and may be one important factor behind cytogenetic intratumour diversity in GBM.

DNA and origin region segregation are not affected by the transition from rod to sphere after inhibition of Escherichia coli MreB by A22

The bacterial actin homologue MreB forms a helix underneath the cytoplasmic membrane and was shown to be essential in the morphogenesis of the rod-shaped cells. Additionally, MreB was implicated to be involved in DNA segregation. However, in our hands the mreBCD deletion strain (PA340-678) grew without apparent DNA segregation defect, suggesting that the reported chromosome segregation inhibition could be caused by a temporarily effect of MreB inhibition or depletion. To assess the involvement of MreB in DNA segregation during the transition from rod to sphere, we compared the effect of A22 and the PBP2 inhibitor mecillinam on the percentage of cells with segregated nucleoids and the number of oriC foci in wild-type Escherichia coli cells. Cells became spherical in the same time window during both treatments and we could not detect any difference in the chromosome or oriC segregation between these two treatments. Additionally, flow cytometric analyses showed that A22 and mecillinam treatment gave essentially the same chromosome segregation pattern. We conclude that MreB is not directly involved in DNA segregation of E. coli.

An Analysis of Univalent Segregation in Meiotic Mutants ofArabidopsis thaliana: A Possible Role for Synaptonemal Complex

During first meiotic prophase, homologous chromosomes are normally kept together by both crossovers and synaptonemal complexes (SC). In most eukaryotes, the SC disassembles at diplotene, leaving chromosomes joined by chiasmata. The correct co-orientation of bivalents at metaphase I and the reductional segregation at anaphase I are facilitated by chiasmata and sister-chromatid cohesion. In the absence of meiotic reciprocal recombination, homologs are expected to segregate randomly at anaphase I. Here, we have analyzed the segregation of homologous chromosomes at anaphase I in four meiotic mutants of Arabidopsis thaliana, spo11-1-3, dsy1, mpa1, and asy1, which show a high frequency of univalents at diplotene. The segregation pattern of chromosomes 2, 4, and 5 was different in each mutant. Homologous univalents segregated randomly in spo11-1-3, whereas they did not in dsy1 and mpa1. An intermediate situation was observed in asy1. Also, we have found a parallelism between this behavior and the synaptic pattern displayed by each mutant. Thus, whereas spo11-1-3 and asy1 showed low amounts of SC stretches, dsy1 and mpa1 showed full synapsis. These findings suggest that in Arabidopsis there is a system, depending on the SC formation, that would facilitate regular disjunction of homologous univalents to opposite poles at anaphase I.

Mad2-independent Spindle Assembly Checkpoint Activation and Controlled Metaphase–Anaphase Transition inDrosophilaS2 Cells

The spindle assembly checkpoint is essential to maintain genomic stability during cell division. We analyzed the role of the putative Drosophila Mad2 homologue in the spindle assembly checkpoint and mitotic progression. Depletion of Mad2 by RNAi from S2 cells shows that it is essential to prevent mitotic exit after spindle damage, demonstrating its conserved role. Mad2-depleted cells also show accelerated transit through prometaphase and premature sister chromatid separation, fail to form metaphases, and exit mitosis soon after nuclear envelope breakdown with extensive chromatin bridges that result in severe aneuploidy. Interestingly, preventing Mad2-depleted cells from exiting mitosis by a checkpoint-independent arrest allows congression of normally condensed chromosomes. More importantly, a transient mitotic arrest is sufficient for Mad2-depleted cells to exit mitosis with normal patterns of chromosome segregation, suggesting that all the associated phenotypes result from a highly accelerated exit from mitosis. Surprisingly, if Mad2-depleted cells are blocked transiently in mitosis and then released into a media containing a microtubule poison, they arrest with high levels of kinetochore-associated BubR1, properly localized cohesin complex and fail to exit mitosis revealing normal spindle assembly checkpoint activity. This behavior is specific for Mad2 because BubR1-depleted cells fail to arrest in mitosis under these experimental conditions. Taken together our results strongly suggest that Mad2 is exclusively required to delay progression through early stages of prometaphase so that cells have time to fully engage the spindle assembly checkpoint, allowing a controlled metaphase-anaphase transition and normal patterns of chromosome segregation.

Paternal chromosome segregation during the first mitotic division determinesWolbachia-induced cytoplasmic incompatibility phenotype

The most common Wolbachia-induced phenotype in insects is cytoplasmic incompatibility (CI), which occurs when sperm from infected males fertilize eggs from uninfected females. CI produces distinct phenotypes in three closely related haplo-diploid species of the genus Nasonia: mortality in N. longicornis and N. giraulti, and conversion to male development in N. vitripennis. We demonstrate that the majority of CI-induced mortality occurs during embryogenesis and that the pattern of paternal chromosome segregation during the first mitosis is a good predictor of CI phenotype. In N. giraulti and N. longicornis, the paternal chromosomes mis-segregate, producing abnormal nuclei connected by chromatin bridges. Consequently, these embryos arrest development with very few and abnormal nuclei. In contrast, the paternal genome in N. vitripennis is either not segregated or mis-segregates to one of the two daughter nuclei. Consequently, these embryos continue development utilizing the maternally derived haploid nuclei, resulting in male offspring. The latter class is the first documented example of asymmetric mitotic segregation of abnormal chromosomes. We conclude that in haplo-diploids, CI-induced embryonic lethality occurs only when abnormal paternal genome segregation affects both products of the first mitotic division. This is the first study to associate differences in types of CI with specific cytological defects.

Rec8 phosphorylation and recombination promote the step-wise loss of cohesins in meiosis

During meiosis, cohesins--protein complexes that hold sister chromatids together--are lost from chromosomes in a step-wise manner. Loss of cohesins from chromosome arms is necessary for homologous chromosomes to segregate during meiosis I. Retention of cohesins around centromeres until meiosis II is required for the accurate segregation of sister chromatids. Here we show that phosphorylation of the cohesin subunit Rec8 contributes to step-wise cohesin removal. Our data further implicate two other key regulators of meiotic chromosome segregation, the cohesin protector Sgo1 and meiotic recombination in bringing about the step-wise loss of cohesins and thus the establishment of the meiotic chromosome segregation pattern. Understanding the interplay between these processes should provide insight into the events underlying meiotic chromosome mis-segregation, the leading cause of miscarriages and mental retardation in humans.

Meiotic segregation patterns and ICSI pregnancy outcome of a rare (13;21) Robertsonian translocation carrier: a case report

t(13;21) is an uncommon Robertsonian translocation (RT) with limited information in the literature. Hereby, we assessed the meiotic segregation and interchromosomal effect (ICE) in sperm nuclei from a t(13;21) carrier. The pregnancy outcome following ICSI was also included as reference for physicians and patients. Dual-colour fluorescent in situ hybridization (FISH) was carried out to analyse the segregation pattern of chromosomes 13 and 21, while triple-colour FISH was used to investigate the possible concurrence of ICE. With respect to chromosomal constitutions of 13 and 21, 88.39% of the spermatozoa were normal or balanced due to alternative segregations, and 11.08% showed nullisomy or disomy as a result of adjacent segregations. However, for chromosome 18 and sex chromosomes, the proportion of normal haploids was 98.79%. The rate of disomy was not significantly higher than the controls for either chromosome 18 or X/Y. The rare t(13;21) case exhibited a similar pattern of meiotic segregation as in the common RTs. ICEs were not observed in the current case.

Sex Chromosome Meiotic Drive in Hybrid Males of the Common Shrew (Sorex araneus)

Patterns of sex chromosome segregation in six homozygous males of the common shrew (Sorex araneus LINNAEUS, 1758) belonging to two chromosomal races, as well as in 16 interracial hybrids were studied. Based on their karyotypes the hybrids can be subdivided into two groups: (a) complex heterozygotes, which form meiotic quadrivalents in chain and chain + ring configurations, and (b) complex heterozygotes, which form meiotic pentavalents in chain configurations. Random (1:1) segregation of sex chromosomes was found in homozygous as well as those heterozygous males which form meiotic complexes of four chromosomes. However, in some hybrids with meiotic pentavalents we observed a strong preferential segregation in favour of X chromosomes.

A Meiosis-Specific Cyclin Regulated by Splicing Is Required for Proper Progression through Meiosis

The meiotic cell cycle is modified from the mitotic cell cycle by having a premeiotic S phase which leads to high levels of recombination, a reductional pattern of chromosome segregation at the first division, and a second division with no intervening DNA synthesis. Cyclin-dependent kinases are essential for progression through the meiotic cell cycle, as for the mitotic cycle. Here we show that a fission yeast cyclin, Rem1, is present only during meiosis. Cells lacking Rem1 have impaired meiotic recombination, and Rem1 is required for premeiotic DNA synthesis when Cig2 is not present. rem1 expression is regulated at the level of both transcription and splicing, with Mei4 as a positive and Cig2 a negative factor of rem1 splicing. This regulation ensures the timely appearance of the different cyclins during meiosis, which is required for the proper progression through the meiotic cell cycle. We propose that the meiosis-specific B-type cyclin Rem1 has a central role in bringing about progression through meiosis.

Cohesins determine the attachment manner of kinetochores to spindle microtubules at meiosis I in fission yeast.

During mitosis, sister kinetochores attach to microtubules that extend to opposite spindle poles (bipolar attachment) and pull the chromatids apart at anaphase (equational segregation). A multisubunit complex called cohesin, including Rad21/Scc1, plays a crucial role in sister chromatid cohesion and equational segregation at mitosis. Meiosis I differs from mitosis in having a reductional pattern of chromosome segregation, in which sister kinetochores are attached to the same spindle (monopolar attachment). During meiosis, Rad21/Scc1 is largely replaced by its meiotic counterpart, Rec8. If Rec8 is inactivated in fission yeast, meiosis I is shifted from reductional to equational division. However, the reason rec8Delta cells undergo equational rather than random division has not been clarified; therefore, it has been unclear whether equational segregation is due to a loss of cohesin in general or to a loss of a specific requirement for Rec8. We report here that the equational segregation at meiosis I depends on substitutive Rad21, which relocates to the centromeres if Rec8 is absent. Moreover, we demonstrate that even if sufficient amounts of Rad21 are transferred to the centromeres at meiosis I, thereby establishing cohesion at the centromeres, rec8Delta cells never recover monopolar attachment but instead secure bipolar attachment. Thus, Rec8 and Rad21 define monopolar and bipolar attachment, respectively, at meiosis I. We conclude that cohesin is a crucial determinant of the attachment manner of kinetochores to the spindle microtubules at meiosis I in fission yeast.

Occurrence of numerically unreduced (2n) gametes in Alstroemeria interspecific hybrids and their significance for sexual polyploidisation

The F1 hybrids of seven diploid Alstroemeria species (2n=2x=16) were investigated for the production of numerically unreduced (2n) gametes and their mode of origin. Based on a survey of 17 interspecific hybrid combinations,consisting of 119 genotypes, it was found that the F1 hybrids of Chilean-Brazilian species mostly produced first division restitution (FDR) 2n gametes. These F1 hybrids were self-pollinated in order to obtain F2 seeds, which was an indication that the F1 plants also produced 2neggs simultaneously. All the F2 progeny plants were typical allotetraploids, most of which formed 16 bivalents and a small proportion formed multivalents during metaphase I stages of meiosis. Through genomic in situ hybridisation (GISH) it was proved that multivalent formation in F2plants, derived from A. inodora ×A. pelegrina hybrid, was due to homoeologous recombination but not from reciprocal translocations. In order to test the segregation pattern of the recombinant chromosomes, an F3 population from one genotype, P6C49-6, was investigated. The recombinant chromosomes assorted independently from each other supporting the hypothesis that the segregation of chromosomes in ring quadrivalents did not behave like those in translocation heterozygotes. It was concluded that in allopolyploids of Alstroemeria,bilateral sexual polyploidisation could accomplish genetic recombination by both homoeologous crossing-over as well as through the assortment of chromosomes.

Cosegregation of Robertsonian metacentric chromosomes in the first meiotic division of multiple heterozygous male mice as revealed by FISH analysis of spermatocyte II metaphases

Contrasting results (random segregation or cosegregation of isomorphic chromosomes) have been reported up to now on the segregation pattern of Robertsonian metacentric chromosomes of Mus musculus domesticus in multiple heterozygotes, using different approaches (karyotypical analysis of the progeny or of second meiotic metaphases). In the present contribution data are presented based on FISH (Fluorescence In Situ Hybridisation) analysis with telomeric probes, which allowed us to distinguish metacentric chromosomes from pairs of acrocentric chromosomes with their centromeric regions close to each other. Probes were hybridized to DAPI stained metaphases of spermatocytes II of mice heterozygous for two, three or four Robertsonian metacentrics in an all-acrocentric background, the karyotype of which has been reconstructed starting from laboratory strains. Isomorphic chromosomes tend to cosegregate (metacentrics with metacentrics, acrocentrics with acrocentrics); the values found for cosegregation have a clear even if moderate effect on the reproductive isolation caused by underdominant chromosomal rearrangements.

Embryonic hybrid cells: a powerful tool for studying pluripotency and reprogramming of the differentiated cell chromosomes.

The properties of embryonic hybrid cells obtained by fusion of embryonic stem (ES) or teratocarcinoma (TC) cells with differentiated cells are reviewed. Usually, ES-somatic or TC-somatic hybrids retain pluripotent capacity at high levels quite comparable or nearly identical with those of the pluripotent partner. When cultured in vitro, ES-somatic- and TC-somatic hybrid cell clones, as a rule, lose the chromosomes derived from the somatic partner; however, in some clones the autosomes from the ES cell partner were also eliminated, i.e. the parental chromosomes segregated bilaterally in the ES-somatic cell hybrids. This opens up ways for searching correlation between the pluripotent status of the hybrid cells and chromosome segregation patterns and therefore for identifying the particular chromosomes involved in the maintenance of pluripotency. Use of selective medium allows to isolate in vitro the clones of ES-somatic hybrid cells in which "the pluripotent" chromosome can be replaced by "the somatic" counterpart carrying the selectable gene. Unlike the TC-somatic cell hybrids, the ES-somatic hybrids with a near-diploid complement of chromosomes are able to contribute to various tissues of chimeric animals after injection into the blastocoel cavity. Analysis of the chimeric animals showed that the "somatic" chromosome undergoes reprogramming during development. The prospects for the identification of the chromosomes that are involved in the maintenance of pluripotency and its cis- and trans-regulation in the hybrid cell genome are discussed.

Higher-order chromatin structure of human granulocytes.

The structural organisation of chromatin in eukaryotes plays an important role in a number of biological processes. Our results provide a comprehensive insight into the nuclear topography of human peripheral blood granulocytes, mainly neutrophils. The nuclei of granulocytes are characterised by a segmented shape consisting of two to five lobes that are in many cases connected by a thin DNA-containing filament. The segregation of chromosomes into the nuclear lobes was studied using fluorescence in situ hybridisation (FISH). We were able to distinguish different topographic types of granulocytes on the basis of the pattern of segregation. Five topographic types were detected using dual-colour FISH in two-lobed nuclei. The segregation of four sets of genetic structures could be studied with the aid of repeated FISH and a large number of topographic types were observed. In all these experiments a non-random distribution of chromosomes into nuclear lobes was found. The painting of a single type of chromosome in two-lobed nuclei showed the prevalence of symmetric topographic types (on average in 65.5% of cases) with significant variations among individual chromosomes. The results of analysis of five topographic types (defined by two chromosomes in two-lobed nuclei) showed that the symmetric topographic types for both chromosomes are significantly more frequent than predicted. Repeated hybridisation experiments confirmed that the occurrence of certain patterns of chromosome segregation is much higher than that predicted from the combination of probabilities. The frequency of symmetric topographic types for chromosome domains was systematically higher than for genes located on these chromosomes. It appears that the prevalence of symmetric segregation patterns is more probable for large objects such as chromosome domains than for genes located on chromatin loops extending outwards from the surface of the domain defined by specific chromosome paints. This means that one chromosome domain may occur in different lobes of granulocytic nuclei. This observation is supported by the fact that both genes and centromeres were observed on filaments joining different lobes. For all chromosomes, the distances between the membrane and fluorescence gravity centre of the chromosome were measured and correlated with the segregation patterns. A higher percentage of symmetric topographic types was found in those chromosomes that were located closer to the nuclear membrane. Nuclear positioning of all genetic elements in granulocytic nuclei was studied in two-dimensional projection; however, the results were verified using three-dimensional analysis.

Fission yeast Bub1 is essential in setting up the meiotic pattern of chromosome segregation.

In meiosis, sister-chromatids move to the same spindle pole during the first division (MI) and to opposite poles during the second division (MII). This requires that MI sister kinetochores are co-orientated and form an apparent single functional unit that only interacts with microtubules from one pole, and that sister-chromatids remain associated through their centromeres until anaphase II. Here we investigate the function of Bub1 and Mad2, which are components of the mitotic-spindle checkpoint, on chromosome segregation during meiosis. Both proteins are required to prevent the occurrence of non-disjunction events in MI, which is consistent with recent findings that components of the mitotic-spindle checkpoint also operate during meiosis. However, Bub1 has several functions that are not shared with Mad2. When the bub1 gene is deleted, sister chromatids often move to opposite spindle poles during MI, indicating that sister kinetochores are disunited. Furthermore, the cohesin Rec8 is never retained at centromeres at anaphase I and sister-chromatid cohesion is lost. Our results show that Bub1, besides its functions in monitoring chromosome attachment, is essential for two other significant aspects of MI - unification of sister kinetochores and retention of centromeric cohesion.

Pre-meiotic S phase is linked to reductional chromosome segregation and recombination.

Meiosis is initiated from G1 of the cell cycle and is characterized by a pre-meiotic S phase followed by two successive nuclear divisions. The first of these, meiosis I, differs from mitosis in having a reductional pattern of chromosome segregation. Here we show that meiosis can be initiated from G2 in fission yeast cells by ectopically activating the meiosis-inducing network. The subsequent meiosis I occurs without a pre-meiotic S phase and with decreased recombination, and exhibits a mitotic pattern of equational chromosome segregation. The subsequent meiosis II results in random chromosome segregation. This behaviour is similar to that observed in cells lacking the meiotic cohesin Rec8 (refs 3, 4), which becomes associated with chromosomes at G1/S phase, including the inner centromere, a region that is probably critical for sister-centromere orientation. If the expression of Rec8 is delayed to S phase/G2, then the centromeres behave equationally. We propose that the presence of Rec8 in chromatin is required at the pre-meiotic S phase to construct centromeres that behave reductionally and chromosome arms capable of a high level of recombination, and that this explains why meiosis is initiated from G1 of the cell cycle.

Sperm chromosome analysis and outcome of IVF in patients with non-mosaic Klinefelter’s syndrome

Objective: The aim of the study was to determine the potential risk for fetal chromosomal anomalies in non-mosaic Klinefelter’s syndrome patients undergoing IVF and intracytoplasmic sperm injection. Design: Individually collected spermatozoa were isolated from wet testicular tissue preparations and fixed on glass slides using micromanipulation. Their nuclei were analyzed for chromosomes X, Y, and 18 by fluorescent in situ hybridization. Setting: Assisted reproductive technology program. Patient(s): Consenting patients with non-mosaic Klinefelter’s syndrome undergoing testicular biopsy and IVF (fresh specimens) or following such treatment (cryopreserved specimens). Intervention(s): None. Main Outcome Measure(s): The rates of numerical chromosome abnormalities for chromosomes X, Y, and 18 among spare testicular sperm and the pregnancy outcome following treatment. Result(s): Testicular sperm were found in 8 of 20 patients. Four couples became pregnant following embryo replacement. Sperm chromosomes were analyzed in five patients. One hundred and five sperm of 112 analyzed (93.7%) were normal with X to Y ratio of 50:55 (NS) respectively. Among the 112 sperm tested, seven (6.3%) demonstrated chromosomal abnormalities, of which five were related to the sex chromosomes and two to chromosome 18. One set of triplets, one set of twins, and two singletons (four males and three females) with normal karyotypes were born. Conclusion(s): Most of the testicular sperm retrieved from Klinefelter’s syndrome patients demonstrates a normal pattern of sex chromosome segregation. Therefore, the risk of transmitting numerical sex chromosome abnormalities is relatively low and probably comparable with the rates found in other severe male factor infertility patient groups.

A Monte Carlo Simulation of the Escherichia coliCell Cycle

A Monte-Carlo simulation of the division cycle of an individual bacterium has been developed to test theories concerning the control of cell cycle events and for the analysis of cell cycle data. The model is based on the work of Cooper and Helmstetter and other theories concerning the control of DNA replication initiation, chromosome segregation and cell division. Variability in the molecular events that initiate a particular cell cycle event is incorporated using a Monte Carlo approach. The model results are compared with experimental data from a number of different sources and collected using a number of different analytical techniques. This model is able to accurately represent the cell size distributions of an exponentially growing population as determined by a Coulter Counter and the DNA distributions as determined by flow cytometry. The model is also able to accurately simulate the cell age distribution and chromosome replication and segregation patterns as determined by the membrane-elution technique. Results from parameter sensitivity analysis indicate that variability in the symmetry of cell division and in the cell size at initiation of chromosome replication have the most significant impact on the cell size, age and DNA distributions. Results from the membrane-elution simulation support the hypothesis that total cell protein is synthesized exponentially during the division cycle and that cell size control is a plausible explanation for control of DNA replication. The simulation results for chromosome segregation agree with experimental data and support chromosome strand segregation models. In the future, this simulation should be useful in testing complex theories about molecular control of cell cycle events against experimental data.