Nondisjunction Of Sister Chromatids Research Articles

PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis.

PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural abnormalities, and hypersensitivity to an inhibitor of Topoisomerase II (Topo II), ICRF-193. ICRF-193-treated PICH−/− cells undergo sister chromatid non-disjunction in anaphase, and frequently abort cytokinesis. PICH co-localizes with Topo IIα on UFBs and at the ribosomal DNA locus, and the timely resolution of both structures depends on the ATPase activity of PICH. Purified PICH protein strongly stimulates the catalytic activity of Topo II in vitro. Consistent with this, a human PICH−/− cell line exhibits chromosome instability and chromosome condensation and decatenation defects similar to those of ICRF-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis.

90 DESTABILIZATION OF COHESIN REC8 CAUSES ANEUPLOIDY AFTER THE SECOND MEIOSIS IN MURINE POST-OVULATORY AGED OOCYTES

We have previously reported that the early embryos derived from post-ovulatory aged oocytes frequently exhibit aneuploidy, resulting from abnormalities in the cleavage apparatus of MII oocytes. Other studies have also described a potential mechanism that results in aneuploidy, which is attributed to the failure of a cell cycle checkpoint. The spindle assembly checkpoint (SAC), which acts during metaphase, is a monitoring system that equally distributes sister chromatids by correctly attaching spindle fibres to the appropriate centromere. Cohesin, a functional protein complex of the SAC, includes the REC8 subunit, and acts as an adhesion factor for sister chromatids. Segregation of sister chromatids occurs following the degradation of the cohesin complex by the separase enzyme. The segregation process can be mediated by meiosis-specific REC8, which contains a recognition site for separase. In this study, we examined the expression of meiosis-specific REC8 protein in murine post-ovulatory aged oocytes, and verified the association with aneuploidy induced during second meiosis. Superovulated oocytes from the ICR mouse strain were aged by culture for 3 to 24 h in vitro. To eliminate the male genome factor, chromosomal analysis was performed using oocytes activated by SrCl2, without fertilization. The expression level of REC8 in oocytes, before and after activation, was analysed by Western blot, using a rabbit anti-REC8 antibody (primary) and horseradish peroxidase-conjugated anti-rabbit antibody (secondary). In the 6- and 12-h aged groups, 23.8% and 40.3% of oocytes, respectively, exhibited aneuploidy after the second meiosis. The rate of aneuploidy in the 12-h aged group was significantly higher than that in the fresh oocyte group (10.3%; P < 0.05). It could be speculated from our previous data that this fact contributed to the occurrence of aneuploidy in early embryos derived from the aged oocytes. In 3-, 6-, 12-, and 24-h aged groups, the results of semiquantitative analysis of REC8 levels in MII oocytes (non-activated) were 1.32 ± 0.38, 1.30 ± 0.58, 1.15 ± 0.21, and 0.98 ± 0.14, respectively. REC8 levels in the 24-h aged group were significantly lower than in the fresh group (1.86 ± 0.56, P < 0.05). The expression levels of REC8 in activated oocytes at 3 and 6 h were 0.53 ± 0.01 and 0.55 ± 0.04, respectively. REC8 levels in the 12-h (1.00 ± 0.03) and 24-h (0.95 ± 0.04) groups were significantly higher than in the fresh group (0.49 ± 0.09; P < 0.05). The significant reduction of REC8 levels at anaphase, after oocyte activation, was not observed in oocytes aged 12 h or more. In MII oocytes, REC8 levels tend to decrease gradually with post-ovulatory age. Destabilisation of the cohesin REC8 subunit may contribute to the nondisjunction of sister chromatids during second meiosis in post-ovulatory aged oocytes, ultimately resulting in aneuploidy.

The Cdk/Cdc14 Module Controls Activation of the Yen1 Holliday Junction Resolvase to Promote Genome Stability

Faithful genome transmission during cell division requires precise, coordinated action of DNA metabolic enzymes, including proteins responsible for DNA damage detection and repair. Dynamic phosphorylation plays an important role in controlling repair enzymes during the DNA damage response (DDR). Cdc14 phosphatases oppose cyclin-dependent kinase (Cdk) phosphorylation and have been implicated in the DDR in several model systems. Here, we have refined the substrate specificity of budding yeast Cdc14 and, using this insight, identified the Holliday junction resolvase Yen1 as aDNA repair target of Cdc14. Cdc14 activation atanaphase triggers nuclear accumulation and enzymatic activation of Yen1, likely to resolve persistent recombinational repair intermediates. Consistent with this, expression of a phosphomimetic Yen1 mutant increased sister chromatid nondisjunction. In contrast, lack of Cdk phosphorylation resulted in constitutive activity and elevated crossover-associated repair. The precise timing of Yen1 activation, governed by core cell-cycle regulators, helps coordinate DNA repair with chromosome segregation and safeguards against genome destabilization.

Structure-specific endonucleases: guardians of fragile site stability

Fragile sites are conserved loci predisposed to form breaks in metaphase chromosomes. The inherent instability of these loci is associated with chromosomal rearrangements in cancers and is a feature of cells from patients with chromosomal instability syndromes. One class of fragile sites, the common fragile sites (CFSs), have previously been shown to recruit several DNA repair proteins after the completion of bulk DNA synthesis in the cell, probably indicative of their inability to complete timely DNA replication. CFS loci are also prone to trigger mitotic non-disjunction of sister chromatids, leading to the formation of ultra-fine anaphase bridges (UFBs) and micronuclei. We discuss recent developments in the CFS field; in particular, the role of DNA structure-specific endonucleases in promoting cleavage at CFSs.

The Cohesion Protein SOLO Associates with SMC1 and Is Required for Synapsis, Recombination, Homolog Bias and Cohesion and Pairing of Centromeres in Drosophila Meiosis

Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome cores.

In vitro induction of a trisomic of Agave tequilana Weber var. Azul (Agavaceae) by para-fluorophenylalanine treatment

The effect of para-fluorophenylalanine (PFP) on the production of trisomic plants of Agave tequilana Weber var. Azul produced through somatic embryogenesis was investigated. Normal diploid plants with 2n = 2x = 60 were obtained in the control treatment and with 4 mg L−1 PFP exposure, while use of 8 and 12 mg L−1 PFP led to production of trisomics with 2n = 2x = 61. Normal diploid plants showed a bimodal karyotype with five pairs of large chromosomes and 25 pairs of small chromosomes. Trisomic plants also had a bimodal karyotype with a group of three chromosomes in position five of the chromosome set. More than 13 homologous chromosome pairs exhibited structural changes. Differences in chromosome arm ratio (long arm/short arm) were also found in eight chromosome pairs; all these aberrations in the chromosome complement of trisomic plants were probably caused by inversions, deletions, and/or duplications produced by high concentrations of PFP. The gross chromosome structural changes and the presence of a single extra chromosome could have been induced by the effect of PFP on the mitotic spindle by inducing nondisjunction of sister chromatids, resulting in hyperploids (2n + x) and hypoploids (2n − x). Flow cytometric analysis of nuclear DNA content was performed using nuclei isolated from young leaves of normal and trisomic plants. The 2C DNA content of 8.635 pg (1Cx = 4,223 Mbp of trisomic plants was different (p < 0.001) than that of normal plants (2C DNA = 8.389 pg (1Cx = 4,102 Mbp). The difference in genome size was correlated with the large structural changes in the trisomic plant genomes.

Mechanisms of Whole Chromosome Gains in Tumors – Many Answers to a Simple Question

Whole chromosome gain is the most common type of gross genomic abnormality observed in human tumors. It is particularly frequent in lympho-haematopoietic and embryonic neoplasms, where trisomies and tetrasomies are typically present together with few or no other cytogenetic imbalances, resulting in hyperdiploid chromosome numbers. Despite the high prevalence of whole chromosome gains in neoplastic cells, their mechanism of origin remains disputed. Here, 4 potential models for the generation of whole chromosome gains are reviewed: (1) loss of chromosomes from the tetraploid level, (2) sequential sister chromatid non-disjunction, (3) multipolar mitosis coupled to sister chromatid non-disjunction, and (4) multipolar mitosis coupled to incomplete cytokinesis. Each of these mechanisms may in theory result in the generation of hyperdiploid neoplastic clones, but none of them were single-handedly able to reproduce the scenario of chromosome copy number alterations in tumors when cell populations resulting from these models were simulated in silico and compared to published cytogenetic data. To develop models for the generation of whole chromosome gains further, it is critical to improve our knowledge of the principles of clonal selection in tumors and of the baseline rate of chromosome segregation errors in human cells. To illustrate this, a model combining multipolar mitosis coupled to incomplete cytokinesis with a low rate of baseline sister chromatid non-disjunction was shown readily to reproduce copy number distributions in hyperdiploid karyotypes from human tumors.

Distinct Mitotic Segregation Errors Mediate Chromosomal Instability in Aggressive Urothelial Cancers

Chromosomal instability (CIN) is believed to have an important role in the pathogenesis of urothelial cancer (UC). The aim of this study was to evaluate whether disturbances of mitotic segregation contribute to CIN in UC, if these processes have any effect on the course of disease, and how deregulation of these mechanisms affects tumor cell growth. We developed molecular cytogenetic methods to classify mitotic segregation abnormalities in a panel of UC cell lines. Mitotic instabilities were then scored in biopsies from 52 UC patients and compared with the outcome of tumor disease. Finally, UC cells were exposed in vitro to a telomerase inhibitor to assess how this affects mitotic stability and cell proliferation. Three distinct chromosome segregation abnormalities were identified: (a) telomere dysfunction, which triggers structural rearrangements and loss of chromosomes through anaphase bridging; (b) sister chromatid nondisjunction, which generates discrete chromosomal copy number variations; and (c) supernumerary centrosomes, which cause dramatic shifts in chromosome copy number through multipolar cell division. Chromosome segregation errors were already present in preinvasive tumors and a high rate mitotic instability was an independent predictor of poor survival. However, induction of even higher levels of the same segregation abnormalities in UC cells by telomerase inhibition in vitro led to reduced tumor cell proliferation and clonogenic survival. Several distinct chromosome segregation errors contribute to CIN in UC, and the rate of such mitotic errors has a significant effect on the clinical course. Efficient tumor cell proliferation may depend on the tight endogenous control of these processes.

The mal2p protein is an essential component of the fission yeast centromere.

Precise segregation of chromosomes requires the activity of a specialized chromatin region, the centromere, that assembles the kinetochore complex to mediate the association with spindle microtubules. We show here that Mal2p, previously identified as a protein required for genome stability, is an essential component of the fission yeast centromere. Loss of functional Mal2p leads to extreme missegregation of chromosomes due to nondisjunction of sister chromatids and results in inviable cells. Mal2p associates specifically with the central region of the complex fission yeast centromere, where it is required for the specialized chromatin architecture as well as for transcriptional silencing of this region. Genetic evidence indicates that mal2(+) interacts with mis12(+), encoding another component of the inner centromere core complex. In addition, Mal2p is required for correct metaphase spindle length. Our data imply that the Mal2p protein is required to build up a functional fission yeast centromere.

Centromere mapping functions for aneuploid meiotic products: Analysis of rec8, rec10 and rec11 mutants of the fission yeast Schizosaccharomyces pombe.

Recent evidence suggests that the position of reciprocal recombination events (crossovers) is important for the segregation of homologous chromosomes during meiosis I and sister chromatids during meiosis II. We developed genetic mapping functions that permit the simultaneous analysis of centromere-proximal crossover recombination and the type of segregation error leading to aneuploidy. The mapping functions were tested in a study of the rec8, rec10, and rec11 mutants of fission yeast. In each mutant we monitored each of the three chromosome pairs. Between 38 and 100% of the chromosome segregation errors in the rec8 mutants were due to meiosis I nondisjunction of homologous chromosomes. The remaining segregation errors were likely the result of precocious separation of sister chromatids, a previously described defect in the rec8 mutants. Between 47 and 100% of segregation errors in the rec10 and rec11 mutants were due to nondisjunction of sister chromatids during meiosis II. In addition, centromere-proximal recombination was reduced as much as 14-fold or more on chromosomes that had experienced nondisjunction. These results demonstrate the utility of the new mapping functions and support models in which sister chromatid cohesion and crossover position are important determinants for proper chromosome segregation in each meiotic division.

The CHL 1 (CTF 1) gene product of Saccharomyces cerevisiae is important for chromosome transmission and normal cell cycle progression in G2/M.

We have analyzed the CTF1 gene, identified in a screen for mutants with decreased chromosome transmission fidelity and shown to correspond to the previously identified chl1 mutation. Chl1 null mutants exhibited a 200-fold increase in the rate of chromosome III missegregation per cell division, and near wild-type rates of marker homozygosis on this chromosome by mitotic recombination. Analysis of the segregation of a marker chromosome indicated that sister chromatid loss (1:0 segregation) and sister chromatid non-disjunction (2:0 segregation) contributed equally to chromosome missegregation. A genomic clone of CHL1 was isolated and used to map its physical position on chromosome XVI. Nucleotide sequence analysis of CHL1 revealed a 2.6 kb open reading frame with a 99 kd predicted protein sequence that contained two PEST sequences and was 23% identical to the coding region of a nucleotide excision repair gene, RAD3. Domains of homology between these two predicted protein sequences included a helix-turn-helix motif and an ATP binding site containing a helicase consensus. Mutants lacking the CHL1 gene product are viable and display two striking, and perhaps interrelated, phenotypes: extreme chromosome instability and a delay in cell cycle progression in G2/M. This delay is independent of the cell cycle checkpoint that requires the function of the RAD9 gene.

Mitotic non-disjunction of sister chromatids and anomalous mitosis induced by low temperatures in HeLa cells

An anomalous mitosis was observed in HeLa cells within 24 hr after a shift in growth temperature from 37 °C to 29 °C. The anomalous mitosis was characterized by ( a) the presence of a mitotic spindle, ( b) the formation of a “metaphase plate” which passed through a number of diffusions and reformations prior to the onset of “anaphase”, ( c) the failure of the separation of sister chromatids in the kinetochore region, ( d) the asynchronous movement and random distribution of (undivided) chromosomes towards the poles. The induction of the anomalous mitosis appeared to be dependent upon the time spent by the interphase cell at 29 °C rather than on the specific phase of the cell cycle during which the temperature shift occurred.

THE EFFECT OF ULTRAVIOLET LIGHT ON RECOMBINATION IN YEAST.

tion, diploid segregants which are homozygous for one or more of these genes. PONTECORVO, ROPER, HEMMONS, MACDONALD and BUFTON (1953) and PONTECORVO and =FER (1958) have shown that such segregations of homozygous diploids in Aspergillus niduluns are the results of either of two mechanisms: mitotic crossing over, as first discovered in Drosophilu melunoguster by STERN (1936) , in which crossing over occurs at the four-strand stage as in meiosis but the centromeres divide as in mitosis; or nondisjunction of sister chromatids at mitosis without crossing over. In untreated A. niduluns the frequency of mitotic crossing over is less than meiotic crossing over by a factor of about lo4 (PRITCHARD 1960). The frequency of mitotic nondisjunction is even rarer than the frequency of mitotic crossing over (but the relative frequencies of these two mechanisms depends upon the selective method used [PONTECORVO et al. 19531). Various treatments given to induce somatic segregation have increased the frequency of segregation. FRATELLO, MORPURGO and SERMONTI (1960) found that in Aspergillus nidulans increasing doses of ultraviolet light (UV) and nitrogen mustard increased the frequency of mitotic nondisjunction but had no effect on mitotic crossing over. However, HOLLIDAY (1961) working with Ustilugo maydis obtained a 90-fold increase over the spontaneous rate of mitotic crossing over with UV treatment. A further important difference in mitotic crossing-over in different organisms has been found when intragenic recombination is analysed. PRITCHARD ( 1960), working with the ad-20 gene in A. niduluns, found the reciprocal products, the 4- allele and the double mutant, in the same diploid nucleus in the proportion expected from the mechanism of reciprocal mitotic crossing over. ROMAN and JACOB (1958), on the other hand, working with a diploid heteroallelic for is-1 in Saccharomyces, did not find the reciprocal double mutant and concluded that the recombination was not the result of a reciprocal crossover (see also KAKAR 1963). Spontaneous mitotic crossing over in Aspergillus nidulans and spontaneous and induced mitotic crossing over in Ustilugo maydis are so rare that in general only segregants for homozygosis in one arm of one chromosome are found in any one nucleus. In Saccharomyces cerevisiue, on the other hand, a high frequency of simultaneous homozygosis of unlinked genes on different chromosomes after UV treatment was described by WILKIE and HAWTHORNE (1961 ) . These results

XXY Mice

Welshons &amp; Russell (1959) have presented data to show that the XO chromosomal constitution in the mouse is female. This conclusion was based on results of genetical tests with sex-linked markers and on chromosome counts. All XO females were matroclinous, that is, they had inherited their X-chromosome from their mother. Females of this type will arise when non-disjunction occurs in the meiotic divisions of the father and results in spermatozoa without a sex-chromosome. Alternatively, the paternal sex-chromosome may be lost from the fertilized ovum if non-disjunction of sister-chromatids occurs during the first cleavage division. This latter explanation has been urged by Ohno, Kaplan &amp; Kinosita (1959), who found no evidence for non-disjunction of the X-and Y-chromosome in an extensive cytolosical examination of the mouse testis.