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PubMed2000

Slk19p is necessary to prevent separation of sister chromatids in meiosis I.

BACKGROUND: A fundamental difference between meiotic and mitotic chromosome segregation is that in meiosis I, sister chromatids remain joined, moving as a unit to one pole of the spindle rather than separating as they do in mitosis. It has long been known that the sustained linkage of sister chromatids through meiotic anaphase I is accomplished by association of the chromatids at the centromere region. The localization of the cohesin Rec8p to the centromeres is essential for maintenance of sister chromatid cohesion through meiosis I, but the molecular basis for the regulation of Rec8p and sister kinetochores in meiosis remains a mystery. RESULTS: We show that the SLK19 gene product from Saccharomyces cerevisiae is essential for proper chromosome segregation during meiosis I. When slk19 mutants were induced to sporulate they completed events characteristic of meiotic prophase I, but at the first meiotic division they segregated their sister chromatids to opposite poles at high frequencies. The vast majority of these cells did not perform a second meiotic division and proceeded to form dyads (asci containing two spores). Slk19p was found to localize to centromere regions of chromosomes during meiotic prophase where it remained until anaphase I. In the absence of Slk19p, Rec8p was not maintained at the centromere region through anaphase I as it is in wild-type cells. Finally, we demonstrate that Slk19p appears to function downstream of the meiosis-specific protein Spo13p in control of sister chromatid behavior during meiosis I. CONCLUSIONS: Our results suggest that Slk19p is essential at the centromere of meiotic chromosomes to prevent the premature separation of sister chromatids at meiosis I.

Centromere↗

PubMed2003

Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase.

During anaphase identical sister chromatids separate and move towards opposite poles of the mitotic spindle. In the spindle, kinetochore microtubules have their plus ends embedded in the kinetochore and their minus ends at the spindle pole. Two models have been proposed to account for the movement of chromatids during anaphase. In the 'Pac-Man' model, kinetochores induce the depolymerization of kinetochore microtubules at their plus ends, which allows chromatids to move towards the pole by 'chewing up' microtubule tracks. In the 'poleward flux' model, kinetochores anchor kinetochore microtubules and chromatids are pulled towards the poles through the depolymerization of kinetochore microtubules at the minus ends. Here, we show that two functionally distinct microtubule-destabilizing KinI kinesin enzymes (so named because they possess a kinesin-like ATPase domain positioned internally within the polypeptide) are responsible for normal chromatid-to-pole motion in Drosophila. One of them, KLP59C, is required to depolymerize kinetochore microtubules at their kinetochore-associated plus ends, thereby contributing to chromatid motility through a Pac-Man-based mechanism. The other, KLP10A, is required to depolymerize microtubules at their pole-associated minus ends, thereby moving chromatids by means of poleward flux.

Anaphase↗

PubMed2006

Pds1/Esp1-dependent and -independent sister chromatid separation in mutants defective for protein phosphatase 2A.

Spindle disruption or DNA damage prevents sister chromatid separation through the activation of checkpoint pathways that inhibit anaphase entry by stabilizing the anaphase inhibitor Pds1. Mutation of CDC55, which encodes a B regulatory subunit of protein phosphatase 2A (PP2A), results in precocious sister chromatid separation when spindle is disrupted. Here we report that decreased Pds1 levels in Deltacdc55 mutants contribute to sister chromatid separation in the presence of nocodazole, a microtubule-depolymerizing drug. However, in the presence of DNA damage, Deltacdc55 mutant cells separate sister chromatids without noticeable decrease of Pds1 or cohesin Mcd1/Scc1 levels. Further analysis demonstrates that Deltacdc55 mutants lose cohesion along the entire chromosomes when the spindle is disrupted. In contrast, separation of sister chromatids is limited to the centromeric regions in Deltacdc55 cells after DNA damage. Moreover, mutation of TPD3, which encodes the A regulatory subunit of PP2A, also results in sister chromatid separation in DNA- or spindle-damage-arrested cells. These data suggest that PP2A regulates sister chromatid cohesion in Pds1-dependent and -independent manners.

Anaphase↗

PubMed1974

Sister chromatid exchanges, indices of human chromosome damage and repair: detection by fluorescence and induction by mitomycin C.

Sister chromatid exchanges in chromosomes from human lymphocytes grown two replication cycles in medium containing 5-bromodeoxyuridine can be detected by fluorescence microscopy after staining with the bisbenzimidazole dye 33258 Hoechst. These exchanges are much more frequent than chromosome or chromatid breaks and appear to be partly but not entirely due to 5-bromodeoxyuridine incorporation. Sister chromatid exchanges are extremely sensitive indicators of chromosome damage produced by DNA cross-linking agents such as mitomycin C. Significant increases in the sister chromatid exchange frequency occur with 3 ng/ml of mitomycin C; higher concentrations of mitomycin C induce further sister chromatid exchanges. Comparatively few gross chromosomal aberrations are seen in cells exhibiting as many as one hundred or more sister chromatid exchanges. Most of the damage caused by mitomycin C to chromosomal DNA is apparently repaired without detectable changes in chromosome morphology. Analysis of sister chromatid exchanges may permit more sensitive detection of damage to DNA caused by other agents than has previously been possible by classical cytological techniques.

Benzimidazoles↗

PubMed1999

Chromatid break rejoining and exchange aberration formation following gamma-ray exposure: analysis in G2 human fibroblasts by chemically induced premature chromosome condensation.

PURPOSE: To analyse the kinetics of chromatid break induction, rejoining, and misrejoining after y-irradiation in G2 phase human cells using premature chromosome condensation induced by calyculin A. MATERIALS AND METHODS: Human fibroblast AG1522 cells were irradiated with gamma-rays and chromosomes were then prematurely condensed by calyculin A. The number of chromatid breaks and chromatid exchanges in G2 chromosomes were scored, and fitted curves were calculated. RESULTS: Calyculin A induced premature chromosome condensation in cells immediately after irradiation. Kinetics of rejoining of chromatid breaks demonstrated two exponential components with rapid and slow time constants. Within 5 min after irradiation, the number of chromatid breaks fell rapidly to about one-half, then gradually decreased. Chromatid exchanges were formed very quickly, reaching a plateau within 20 min from exposure. CONCLUSIONS: Chemically induced premature chromosome condensation technique allows a simple, rapid and precise analysis of chromatid breakage and rejoining. The rapid kinetic component was particularly well characterized.

Cells, Cultured↗

PubMed1995

A postprophase topoisomerase II-dependent chromatid core separation step in the formation of metaphase chromosomes.

Metaphase chromatids are believed to consist of loops of chromatin anchored to a central scaffold, of which a major component is the decatenatory enzyme DNA topoisomerase II. Silver impregnation selectively stains an axial element of metaphase and anaphase chromatids; but we find that in earlier stages of mitosis, silver staining reveals an initially single, folded midline structure, which separates at prometaphase to form two chromatid axes. Inhibition of topoisomerase II prevents this separation, and also prevents the contraction of chromatids that occurs when metaphase is arrested. Immunolocalization of topoisomerase II alpha reveals chromatid cores analogous to those seen with silver staining. We conclude that the chromatid cores in early mitosis form a single structure, constrained by DNA catenations, which must separate before metaphase chromatids can be resolved.

Animals↗

PubMed1999

Sister chromatid-based DNA repair is mediated by RAD54, not by DMC1 or TID1.

In the mitotic cell cycle of the yeast Saccharomyces cerevisiae, the sister chromatid is preferred over the homologous chromosome (non-sister chromatid) as a substrate for DNA double-strand break repair. However, no genes have yet been shown to be preferentially involved in sister chromatid-mediated repair. We developed a novel method to identify genes that are required for repair by the sister chromatid, using a haploid strain that can embark on meiosis. We show that the recombinational repair gene RAD54 is required primarily for sister chromatid-based repair, whereas TID1, a yeast RAD54 homologue, and the meiotic gene DMC1, are dispensable for this type of repair. Our observations suggest that the sister chromatid repair pathway, which involves RAD54, and the homologous chromosome repair pathway, which involves DMC1, can substitute for one another under some circumstances. Deletion of RAD54 in S.cerevisiae results in a phenotype similar to that found in mammalian cells, namely impaired DNA repair and reduced recombination during mitotic growth, with no apparent effect on meiosis. The principal role of RAD54 in sister chromatid-based repair may also be shared by mammalian and yeast cells.

Cell Cycle Proteins↗

PubMed1992

Sister-chromatid misbehavior in Drosophila ord mutants.

In Drosophila males and females mutant for the ord gene, sister chromatids prematurely disjoin in meiosis. We have isolated five new alleles of ord and analyzed them both as homozygotes and in trans to deficiencies for the locus, and we show that ord function is necessary early in meiosis of both sexes. Strong ord alleles result in chromosome nondisjunction in meiosis I that appears to be the consequence of precocious separation of the sister chromatids followed by their random segregation. Cytological analysis in males confirmed that precocious disjunction of the sister chromatids occurs in prometaphase I. This is in contrast to Drosophila mei-S332 mutants, in which precocious sister-chromatid separation also occurs, but not until late in anaphase I. All three of the new female fertile ord alleles reduce recombination, suggesting they affect homolog association as well as sister-chromatid cohesion. In addition to the effect of ord mutations on meiosis, we find that in ord2 mutants chromosome segregation is aberrant in the mitotic divisions that produce the spermatocytes. The strongest ord alleles, ord2 and ord5, appear to cause defects in germline divisions in the female. These alleles are female sterile and produce egg chambers with altered nurse cell number, size, and nuclear morphology. In contrast to the effects of ord mutations on germline mitosis, all of the alleles are fully viable even when in trans to a deficiency, and thus exhibit no essential role in somatic mitosis. The ord gene product may prevent premature sister-chromatid separation by promoting cohesion of the sister chromatids in a structural or regulatory manner.

Alleles↗

PubMed1977

Automatic measurement of sister chromatid exchange frequency.

An automatic system for detecting and counting sister chromatid exchanges in human chromosomes has been developed. Metaphase chromosomes from lymphocytes which had incorporated 5-bromodeoxyuridine for two replication cycles were treated with the dye 33258 Hoechst and photodegraded so that the sister chromatids exhibited differential Giemsa staining. A computer-controlled television-microscope system was used to acquire digitized metaphase spread images by direct scanning of microscope slides. Individual objects in the images were identified by a thresholding procedure. The probability that each object was a single, separate chromosome was estimated from size and shape measurements. An analysis of the spatial relationships of the dark-chromatid regions of each object yielded a set of possible exchange locations and estimated probabilities that such locations corresponded to sister chromatid exchanges. A normalized estimate of the sister chromatid exchange frequency was obtained by summing the joint probabilities that a location contained an exchange within a single, separate chromosome over the set of chromosomes from one or more cells and dividing by the expected value of the total chromosome area analyzed. Comparison with manual scoring of exchanges showed satisfactory agreement up to levels of approximately 30 sister chromatid exchanges/cell, or slightly more than twice control levels. The processing time for this automated sister chromatid exchange detection system was comparable to that of manual scoring.

Autoanalysis↗

PubMed1986

Light and scanning electron microscopic observations on the two contrasting types of sister chromatid differential staining after ultraviolet light irradiation.

Chinese hamster cells were grown with 50 microM 5-bromodeoxyuridine (BrdU) during the penultimate S phase to obtain chromosomes with the TB-TT chromatid constitution. Chromosome preparations made by the airdrying method were used to study the sister chromatid differential staining (SCD) resulting from ultraviolet (UV) irradiation followed by Giemsa staining by light and scanning electron microscopy (SEM). When chromosomes irradiated with UV light (253.7 nm, 5.2 J/m2/s) for more than 5 h were stained with 1% to 4% Giemsa in phosphate buffered saline (PBS) or in distilled water, the resulting SCD invariably belonged to the B-light type in which the TB-chromatid stained lightly. SEM observations of these chromosomes suggested that the B-light SCD was due to the selective photolysis of the TB-chromatid. On the other hand, when chromosomes were irradiated for only 10 min, and stained with 1% Giemsa in PBS, they showed a B-dark type SCD in which the TB-chromatid stained darkly. However, when chromosomes irradiated for 10 min were stained with 4% Giemsa in PBS or 1% Giemsa in distilled water, the resulting SCD again belonged to the B-light type. These findings indicate that when the irradiation dose is small, the resultant SCD is not a simple reflection of selective photolysis in the TB-chromatids and the type of SCD depends not only on the concentration of Giemsa but also on the salinity of the staining solution.

Animals↗

PubMed2001

Colchicine promotes a change in chromosome structure without loss of sister chromatid cohesion in prometaphase I-arrested bivalents.

In somatic cells colchicine promotes the arrest of cell division at prometaphase, and chromosomes show a sequential loss of sister chromatid arm and centromere cohesion. In this study we used colchicine to analyse possible changes in chromosome structure and sister chromatid cohesion in prometaphase I-arrested bivalents of the katydid Pycnogaster cucullata. After silver staining we observed that in colchicine-arrested prometaphase I bivalents, and in contrast to what was found in control bivalents, sister kinetochores appeared individualised and sister chromatid axes were completely separated all along their length. However, this change in chromosome structure occurred without loss of sister chromatid arm cohesion. We also employed the MPM-2 monoclonal antibody against mitotic phosphoproteins on control and colchicine-treated spermatocytes. In control metaphase I bivalents this antibody labelled the tightly associated sister kinetochores and the interchromatid domain. By contrast, in colchicine-treated prometaphase I bivalents individualised sister kinetochores appeared labelled, but the interchromatid domain did not show labelling. These results support the notion that MPM-2 phosphoproteins, probably DNA topoisomerase IIalpha, located in the interchromatid domain act as "chromosomal staples" associating sister chromatid axes in metaphase I bivalents. The disappearance of these chromosomal staples would induce a change in chromosome structure, as reflected by the separation of sister kinetochores and sister axes, but without a concomitant loss of sister chromatid cohesion.

Animals↗

PubMed1994

Sister chromatid differentiation after in situ detection of ultraviolet-induced DNA breaks under electron microscopy.

Chinese hamster DON cells with 5-bromodeoxyuridine (BrdU)-substituted chromosomes were ultraviolet (UV)-exposed and processed for in situ detection of induced DNA breaks under electron microscopy. For this purpose, UV-induced breaks were amplified by an exonuclease III digestion to obtain single stranded DNA motifs which could hybridize with oligonucleotides of random sequences. These reannealed motifs could be used as primers which were extended by the Klenow polymerase, incorporating biotinylated-dUTP that was detected by a gold-tagged streptavidin. After processing, the chromatid whose DNA was BrdU-substituted in one strand showed a higher electron density than the chromatid substituted in both strands. In contrast, the unifilarly substituted chromatid showed about twice the labelling of DNA breaks as the bifilarly substituted one. This result could be the consequence of a greater loss of chromatin tracts in the bifilarly substituted chromatid, as implied by an X-ray microanalysis which showed that the amount of phosphorous lost by the bifilarly substituted chromatid was higher than that of the unifilarly substituted chromatid.

Animals↗

PubMed1991

Random segregation of chromatids at mitosis in Saccharomyces cerevisiae.

Previous experiments suggest that mitotic chromosome segregation in some fungi is a nonrandom process in which chromatids of the same replicative age are destined for cosegregation. We have investigated the pattern of chromatid segregation in Saccharomyces cerevisiae by labeling the DNA of a strain auxotrophic for thymidine with 5-bromodeoxyuridine. The fate of DNA strands was followed qualitatively by immunofluorescence microscopy and quantitatively by microphotometry using an anti-5-bromodeoxyuridine monoclonal antibody. Chromatids of the same replicative age were distributed randomly to daughter cells at mitosis. Quantitative measurements showed that the amount of fluorescence in the daughter nuclei derived from parents with hemilabeled chromosomes diminished in intensity by one half. The concentration of 5-bromodeoxyuridine used in the experiments had little effect on the frequency of either homologous or sister chromatid exchanges. We infer that the 5-bromodeoxyuridine was distributed randomly due to mitotic segregation of chromatids and not via sister chromatid exchanges.

Antibodies, Monoclonal↗

PubMed1992

Effects of adenine arabinoside and coformycin on the kinetics of G2 chromatid aberrations in X-irradiated human lymphocytes.

The kinetics of chromatid aberrations have been studied in human lymphocytes exposed to X-rays in the G2 phase of the cell cycle and incubated with or without the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara A), known to inhibit the repair of DNA double-strand breaks. In the absence of ara A an exponential decrease in frequencies of chromatid breaks occurred which we interpret as repair. Few breaks were observed if samples were harvested immediately following irradiation. The frequency of chromatid breaks at 1 h after X-irradiation (442 per 100 cells/Gy) was similar to that previously observed in Chinese hamster ovary (CHO) K1 cells. However, the exponential decrease of chromatid breaks between X-irradiation and sampling occurred with a t1/2 of 0.87 h, a faster rate than we have previously observed in CHO K1 cells and was not inhibited by 200 microM ara A alone, in contrast with our previous findings in a human fibroblast line. However, in the presence of the ADA inhibitor coformycin, inhibition of break repair was already observed at an ara A concentration of 100 microM indicating that the apparent unresponsiveness to ara A of lymphocyte chromatid break rejoining results from the deamination of this nucleoside analogue. This deamination effect was confirmed by measurements of DNA synthesis which showed stable inhibition of synthesis by ara A only when coformycin was present. Frequencies of chromatid exchanges in irradiated cells remained constant except at the sampling time directly after irradiation, consistent with the view that chromosomal radiosensitivity remained constant throughout the G2 phase, except for the period immediately prior to mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatids↗

PubMed1996

Holding chromatids together to ensure they go their separate ways.

Association between sister chromatids is essential for their attachment and segregation to opposite poles of the spindle in mitosis and meiosis II. Sister-chromatid cohesion is also likely to be involved in linking homologous chromosomes together in meiosis I. Cytological observations provide evidence that attachment between sister chromatids is different in meiosis and mitosis and suggest that cohesion between the chromatid arms may differ mechanistically from that at the centromere. The physical nature of cohesion is addressed, and proteins that are candidates for holding sister chromatids together are discussed. Dissolution of sister-chromatid cohesion must be regulated precisely, and potential mechanisms to release cohesion are presented.

Cell Cycle Proteins↗

PubMed1979

The relationship between sister chromatid exchange, chromosome aberration and gene mutation induction by several reactive polycyclic hydrocarbon metabolites in cultured mammalian cells.

The ability of three ultimate matabolites of benzo(a)-pyrene and of 7-bromomethylbenz(a)anthracene to induce 8-azaguanine mutants, sister-chromatid exchanges and chromosome aberrations has been investigated. 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was shown to be an extremely efficient inducer of both mutants and sister-chromatid exchanges at 100% survival, whereas its geometrical isomer, 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydro-benzo(a)pyrene and benzo(a)pyrene-4,5-oxide were comparatively weak. The potency of this compound, as both a mutagen and a sister-chromatid exchange inducer, gives further evidence that it may be the important carcinogenic metabolite of benzo(a)pyrene. 7-Bromomethylbenz(a)anthracene was a moderate mutagen and inducer of sister chromatid exchanges. Comparisons of the relative potencies of these four chemicals as inducers of both mutations and sister chromatid exchanges have indicated that these two phenomena are not directly related. The induction of sister chromatid exchanges also appears to be independent of the induction of chromosome aberrations.

Animals↗

PubMed1995

Genetic instability assessed by sister chromatid exchange analysis in the Dunning R-3327 rat prostatic adenocarcinoma model and its relationship to metastatic potential.

The original Dunning R-3327 tumor, described in 1961, has given rise to distinct sublines of different metastatic potentials. The different phenotypes cannot be explained by differences in chromosomal number, DNA content, or nuclear pleomorphism. Sister chromatid exchange is an interchange between two strands of DNA indicative of DNA damage. The frequency of sister chromatid exchanges is a well-accepted measure of genetic instability. To determine whether an assay of genetic instability could distinguish sublines capable of generating cells of the metastatic phenotype, cells from three sublines of low (< 10%) metastatic potential and three sublines of high (> 90%) metastatic potential were cultured in 10 microM 5-bromodeoxyuridine to label DNA. Chromosome preparations were made and sister chromatids were differentiated with Hoechst 33258 dye and Giemsa stain. Sixty metaphase spreads from each subline were scored for SCE and chromosome number. The low metastatic sublines G, AT-1, and AT-2 had 0.32 +/- standard deviation 0.10, 0.38 +/- 0.12, and 0.14 +/- 0.05 sister chromatid exchanges per chromosome, respectively. The high metastatic sublines AT-3, MAT-Lu, and MAT-LyLu had 0.55 +/- 0.17, 0.32 +/- 0.1, and 0.33 +/- 0.2 sister chromatid exchanges per chromosome, respectively. Subline differences in metastatic potentials cannot be explained by incidences of sister chromatid exchanges.

Adenocarcinoma↗

PubMed1980

Benzo[a]pyrene 4,5-oxide. Discrepancy between induction of sister chromatid exchange and binding to DNA in cultured human fibroblasts.

Benzo[a]pyrene 4,5-oxide was covalently bound to DNA of cultured human fibroblasts and caused sister chromatid exchange. The monooxygenase inhibitor alpha-napthoflavone suppressed this induction of sister chromatic exchange, but did not affect binding to DNA. Control experiments with 4-nitroquinoline 1-oxide showed that alpha-naphthoflavone does not inhibit sister chromatid exchange in general. A more likely explanation for the discrepancy between induction of sister chromatid exchange and binding to DNA is that benzo[a]pyrene 4,5-oxide itself can bind to DNA, but this binding does not lead to a significant increase in sister chromatid exchange. However benzo[a]pyrene 4,5-oxide can be oxidized by monooxygenase to yet unknown products which are potent inducers of sister chromatid exchange. An important conclusion from this is that a biological effect such as the induction of sister chromatid exchange may correlate with the exact nature of DNA binding rather than with total binding, to the point where just measuring total binding may be completely misleading if intended to detect the causes of the biological effect.

Alkylating Agents↗