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At least 19 recordsLinked to original sources

Relationships among chromatid interchanges, sister chromatid exchanges, and meiotic recombination in Drosophila melanogaster.

Repair- and recombination-defective mutations at two loci (mei-9 and mei-41) of Drosophila melanogaster have been examined for their effects on the induction of chromosome aberrations by x-rays and the formation of sister chromatid exchanges (SCEs). Irradiation of larval neuroblast cells during the S phase with x-rays showed that mutants at both of these loci are about 10 times more sensitive than wild type to the induction of chromosome aberrations. The pattern of induced aberrations was characteristic for each mutant locus: in cells bearing mei-9 mutations most breaks were chromatid deletions, whereas in the presence of mei-41 mutations similar frequencies of chromatid and isochromatid deletions were observed. Furthermore, chromatid interchanges could not be induced in cells carrying mei-9 alleles; therefore these mutations define a step necessary for chromatid rejoining. mei-41 alleles also define a function involved in the formation of chromatid interchanges; total exchanges were less frequent than expected from nonmutant controls; and the proportion of exchanges arising by symmetrical rejoining was markedly reduced. These data indicate that chromatid and isochromatid deletions have different molecular steps in their formation, and that different molecular mechanisms are also involved in the symmetrical and unsymmetrical rejoining in chromatid interchanges. Neuroblast cells of larvae bearing mei-9 and mei-41 alleles were also treated for 13 hr with 5-bromodeoxyuridine at 9 mug/ml in order to differentiate sister chromatids for the scoring of SCEs. Whereas mei-41 had a normal level of SCEs, mei-9 exhibited a frequency of SCEs that was about 70% that of the control. Because both mei-9 and mei-41 mutations result in defective meiotic recombination, these data suggest that they define steps shared by symmetrical interchange formation and meiotic recombination that do not participate in the formation of most SCEs.

Animals↗

Induction by alkylating agents of sister chromatid exchanges and chromatid breaks in Fanconi's anemia.

Sister chromatid exchanges, which may reflect chromosome repair in response to certain types of DNA damage, provide a means of investigating the increased chromosome fragility characteristic of Fanconi's anemia. By a recently developed technique using 33258 Hoechst and 5-bromodeoxyuridine, it was observed that the baseline frequency of sister chromatid exchanges in phytohemagglutinin-stimulated lymphocytes from four males with Fanconi's anemia differed little from that of normal lymphocytes. However, addition of the bifunctional alkylating agent mitomycin C (0.01 or 0.03 mug/ml) to the Fanconi's anemia cells during culture induces less than half of the increase in exchanges found in identically treated normal lymphocytes. This reduced increment in exchanges in accompanied by a partial suppression of mitosis and a marked increase in chromatid breaks and rearrangements. Many of these events occur at sites of incomplete chromatid interchange. The increase in sister chromatid exchanges induced in Fanconi's anemia lymphocytes by the monofunctional alkylating agent ethylmethane sulfonate (0.25 mg/ml) was slightly less than that in normal cells. Lymphocytes from two sets of parents of the patients with Fanconi's anemia exhibited a normal response to alkylating agents, while dermal fibroblasts from two different patients with Fanconi's anemia reacted to mitomycin C with an increase in chromatid breaks, but a nearly normal increment of sister chromatid exchanges. The results suggest that chromosomal breaks and rearrangements in Fanconi's anemia lymphocytes may result from a defect in a form of repair of DNA damage.

Alkylating Agents↗

Segregation of DNA polynucleotide strands into sister chromatids and the use of endoreduplicated cells to track sister chromatid exchanges induced by crosslinks, alkylations, or x-ray damage.

The method of Matsumoto and Ohta [Matsumoto, K. & Ohta, T. (1992) Chromosoma 102, 60-65; Matsumoto, K. & Ohta, T. (1995) Mutat. Res. 326, 93-98] to induce large numbers of endoreduplicated Chinese hamster ovary cells has now been coupled with the fluorescence-plus-Giemsa method of Perry and Wolff [Perry, P. & Wolff, S. (1974) Nature (London) 251, 156-158] to produce harlequin endoreduplicated chromosomes that after the third round of DNA replication are composed of a chromosome with a light chromatid and a dark chromatid in close apposition to its sister chromosome containing two light chromatids. Unless the pattern is disrupted by sister chromatid exchange (SCE), the dark chromatid is always in the center, so that the order of the chromatids is light-dark light-light. The advent of this method, which permits the observation of SCEs in endoreduplicated cells, makes it possible to determine with great ease in which cell cycle an SCE occurred. This now allows us to approach several vexing questions about the induction of SCEs (genetic damage and its repair) after exposure to various types of mutagenic carcinogens. The present experiments have allowed us to observe how many cell cycles various types of lesions that are induced in DNA by a crosslinking agent, an alkylating agent, or ionizing radiation, and that are responsible for the induction of SCEs, persist before being repaired and thus lose their ability to inflict genetic damage. Other experiments with various types of mutagenic carcinogens and various types of cell lines that have defects in different DNA repair processes, such as mismatch repair, excision repair, crosslink repair, and DNA-strand-break repair, can now be carried out to determine the role of these types of repair in removing specific types of lesions.

Alkylating Agents↗

Sister-chromatid cohesion mediated by the alternative RF-CCtf18/Dcc1/Ctf8, the helicase Chl1 and the polymerase-alpha-associated protein Ctf4 is essential for chromatid disjunction during meiosis II.

Cohesion between sister chromatids mediated by a multisubunit complex called cohesin is established during DNA replication and is essential for the orderly segregation of chromatids during anaphase. In budding yeast, a specialized replication factor C called RF-C(Ctf18/Dcc1/Ctf8) and the DNA-polymerase-alpha-associated protein Ctf4 are required to maintain sister-chromatid cohesion in cells arrested for long periods in mitosis. We show here that CTF8, CTF4 and a helicase encoded by CHL1 are required for efficient sister chromatid cohesion in unperturbed mitotic cells, and provide evidence that Chl1 functions during S-phase. We also show that, in contrast to mitosis, RF-C(Ctf18/Dcc1/Cft8), Ctf4 and Chl1 are essential for chromosome segregation during meiosis and for the viability of meiotic products. Our finding that cells deleted for CTF8, CTF4 or CHL1 undergo massive meiosis II non-disjunction suggests that the second meiotic division is particularly sensitive to cohesion defects. Using a functional as well as a cytological assay, we demonstrate that CTF8, CHL1 and CTF4 are essential for cohesion between sister centromeres during meiosis but dispensable for cohesin's association with centromeric DNA. Our finding that mutants in fission yeast ctf18 and dcc1 have similar defects suggests that the involvement of the alternative RF-C(Ctf18/Dcc1/Ctf8) complex in sister chromatid cohesion might be highly conserved.

Chromatids↗

Evidence that sister chromatid exchanges and chromatid breaks are two independent events.

The relative frequencies of sister chromatid exchanges (SCE) and chromatid breaks in BrdU (5-bromodeoxyuridine) - sensitive site (lq22 leads to lq23) in Chinese hamster cells after BrdU incorporation were studied. The results show that chromatid breaks do not follow the "exchange hypothesis" and provide evidence that chromatid breaks and SCEs are two independent events despite some common features.

Animals↗

Localisation of 7-12-dimethylbenz(a)anthracene induced chromatid breaks and sister chromatid exchanges in chromosomes 1 and 2 of bone marrow cells of rat in vivo.

The frequency of chromatid breaks associated with sister chromatid exchanges at the break point was determined in rat bone marrow cells treated in vivo with 7-12 DMBA, during the late S phase of the cell cycle. The chromosomal aberrations and SCEs were scored in the same cells. Under the experimental conditions employed, more than 40% of the chromatid breaks were found to be associated with an SCE, a frequency expected according to Revell's hypothesis for the formation of chromatid breaks.

9,10-Dimethyl-1,2-benzanthracene↗

Effect of novobiocin on the frequencies of chromatid-type aberrations and sister-chromatid exchanges following gamma-irradiation.

The effect of novobiocin on the frequencies of chromatid-type aberrations and SCEs was examined in Chinese hamster V79 cells which were exposed to gamma-rays and post-treated with novobiocin. While no chromatid aberrations were induced in the unirradiated cells by novobiocin, the frequency of SCEs was slightly increased by treatment with novobiocin alone. Irradiation of G2 cells produced multiple chromatid-type aberrations and post-treatment of the irradiated cells with novobiocin resulted in a significant increase of the aberrations, including chromatid gaps and breaks. In contrast, novobiocin failed to increase the frequency of SCEs induced by gamma-rays when the irradiated cells were post-treated with novobiocin.

Animals↗

Effects of repair inhibition in the G1 phase of clastogen-treated human lymphocytes on the frequencies of chromosome-type and chromatid-type aberrations and sister-chromatid exchanges.

It has been shown that certain types of DNA lesions induced by an S-dependent clastogen are converted to chromosome-type aberrations when their repair is inhibited in the G1 phase of the cell cycle. The purpose of the present study was to investigate which kinds of repair inhibitors have the ability to induce chromosome-type aberrations in cells having DNA lesions and which kinds of DNA lesions will be converted to chromosome-type aberrations when their repair is inhibited. For this purpose, human peripheral blood lymphocytes, which were treated with a clastogen in their G0 phase, were post-treated with one of several kinds of repair inhibitors in the G1 phase, and resulting frequencies of both chromosome-type and chromatid-type aberrations as well as of sister-chromatid exchanges (SCEs) were compared with those of the control cultures: chromatid-type aberrations and SCEs were adopted as cytogenetic indicators of lesions remaining in S and G2 phases. Chemicals used for the induction of DNA lesions were 4-nitroquinoline 1-oxide (4NQO), methyl methanesulfonate (MMS) and mitomycin C (MMC); inhibitors used were excess thymidine (dThd), caffeine, hydroxyurea (HU), 5-fluoro-2'-deoxyuridine (FdUrd), 1-beta-D-arabinofuranosylcytosine (ara C), 9-beta-D-arabinofuranosyladenine (ara A), 1-beta-D-arabinofuranosylthymine (ara T) and aphidicolin (APC). Induction of chromosome-type aberrations was observed in cells pretreated with 4NQO or MMS followed by ara C, ara A, ara T or APC, whereas other combinations of a clastogen and an inhibitor did not induce them. Among the inhibitors, ara C alone induced chromosome-type aberrations in cells without pretreatment. Chromatid-type aberrations were increased only in cells pretreated with MMC and their frequency was enhanced further by post-treatment with ara C. All of the clastogens used in the present experiments induced SCEs. Most inhibitors did not modify the SCE frequencies except for ara C which synergistically increased the frequency in MMC-treated cells. The present study offers further evidence that the lesions responsible for chromosome-type aberrations are those which are repaired quickly, and that they are converted to chromosome-type aberrations when repair by polymerase alpha is inhibited. The effects of ara C on MMC-induced lesions are considered residual effects of ara C treatment in the S or G2 phases rather than repair inhibition in the G1 phase.

Cell Cycle↗

Chromatid lesions and chromatid core morphology.

A silver-staining technique revealed the core morphology of metaphase chromosomes of irradiated CHO cells with chromatid lesions (breaks, gaps). These cells were photographed before and after silver staining. As a rule, the core was not continuous in chromatid gaps, suggesting that the chromatid is broken in many so-called gaps. Ten cytogeneticists who were asked to classify chromatid gaps and breaks from photographs of chromosome lesions before silver core staining agreed in only 19 of 53 cases.

Animals↗

Cytogenetic effects of etoposide (VP-16) on human lymphocytes; with special reference to the relation between sister chromatid exchange and chromatid breakage.

The effects of etoposide (VP-16) on sister chromatid exchange (SCE) and chromosome abnormalities were studied by using human peripheral lymphocytes. This drug produced a significant increase in the SCE frequency and chromosomal aberrations such as breaks, exchanges, and tetraploidy with or without endoreduplication. Analysis of the breakpoints of chromatid deletion with respect to their association with SCE in the metaphases from the second division demonstrated that VP-16-induced deletions arose with little dependence on the site of SCE. It is suggested that chromatid deletions induced by this drug derive from unrepaired chromatid breaks rather than from incomplete exchanges.

Cells, Cultured↗

Differential effects of cordycepin on the induction of sister-chromatid exchange and chromatid breaks in BALB/Mo mouse lymphocytes treated with mitomycin C.

BALB/Mo mice lymphocytes, carrying endogenous Moloney murine leukaemia virus (M-MuLV), show significantly higher in vitro baseline frequencies of sister chromatid exchange (SCE) than lymphocytes from control (M-MuLV free) BALB/c mice. In vitro treatment of lymphocytes with cordycepin (10 micrograms/ml), an antiviral substance, lowers the level of SCE in BALB/Mo cells to the same value of BALB/c cells. The drug also reduces the higher sensitivity of BALB/Mo compared to BALB/c lymphocytes to the induction of SCE by mitomycin C (MMC) administered either in vitro (3 x 10(-8), 10(-7) M) or in vivo (0.3, 3 mg/kg). BALB/Mo lymphocytes treated in vivo with a high dose of MMC (10 mg/kg) show reduced susceptibility to the induction of SCE but increased frequencies of chromatid breaks and micronuclei. In this situation, cordycepin increases the level of SCE in BALB/Mo lymphocytes to exactly the level seen in BALB/c cells, but it does not affect the frequency of chromosomal aberrations. Since cordycepin is known to inhibit poly(A) synthesis, thus blocking RNA maturation, it is suggested that M-MuLV proviral integration is not the sole factor responsible for the altered susceptibility of BALB/Mo lymphocytes to SCE induction, but that it is most likely viral gene expression that is needed for this effect to occur. On the contrary, the high susceptibility of BALB/Mo lymphocytes to the induction of chromatid aberrations by a high dose of MMC administered in vivo seems to be independent of viral maturation.

Animals↗

The relation between chemically induced sister-chromatid exchanges and chromatid breakage.

Studies of classical chromosome aberrations and sister-chromatid exchanges (SCES) suggest independent mechanisms for the two events despite some common features. Examination of chromosome breakage caused by X-rays, visible light, and viruses has shown that few chromatid breaks are accompanied by SCEs at the sites of breaks. No similar observations were available for chemically induced breaks, but it has been reported that rat chromosomes exposed to dimethylbenzanthracene (DMBA) contained a preponderance of both aberrations and SCEs in certain specific regions, implicating a common process in their formation. These conclusions were drawn from a comparison of breaks induced in vivo with SCEs induced in vitro. However, we used 7 chemical mutagens to induce both chromatid breaks and SCEs in "harlequin" chromosomes of cultured rat and Chinese hamster ovary (CHO) cells and found that 25% of the 914 breaks scored were associated with SCEs. The proportion of breaks accompanied by SCEs is related to the overall SCE frequency and falls into the range predicted on the basis that breaks and SCEs occur independently. The reported association between sites for SCEs and aberrations also reflects secondary factors, such as induction of SCEs and aberrations during DNA synthesis in late replicating regions of the chromosomes.

9,10-Dimethyl-1,2-benzanthracene↗

Increased frequency of sister-chromatid exchange and chromatid breaks in lymphocytes after treatment of human volunteers with therapeutic doses of paracetamol.

Paracetamol was given to 10 healthy human volunteers in 3 doses of 1 g each during a period of 8 h. Blood samples for lymphocyte cultures were taken before and 24 h after paracetamol administration. A small but significant increase was found in the frequency of sister-chromatid exchanges (SCE) after intake of paracetamol (0.187 +/- 0.030 per chromosome before and 0.208 +/- 0.024 per chromosome after). After exposure the mean frequency of chromatid breaks per 100 cells was significantly increased (2.16 +/- 1.33 versus 0.33 +/- 0.50 before exposure). Exposure of human lymphocytes in vitro showed that concentrations of paracetamol above 0.1 mM induced inhibition of replicative DNA synthesis. Increased SCE was found in lymphocytes exposed to 1-10 mM paracetamol for 2 h. Furthermore, 0.75-1.5 mM paracetamol exposure for 24 h increased the frequency of chromatid and chromosome breaks in the lymphocytes. The paracetamol-induced SCE and chromosome aberrations may be secondary effects of paracetamol-induced inhibition of DNA synthesis or due to covalent binding of paracetamol metabolite(s) to DNA.

Acetaminophen↗

Random single chromatid and nonrandom double chromatid type segregation of human acrocentric chromosomes in BrdU-labeled mitoses.

Chromatid segregation was analyzed using satellite association of 5-bromodeoxyuridine (BrdU) differentially stained acrocentric chromosomes of human leukocytes. Data were classified into cis and trans configurations in second and third division cycles. It was found that single chromatid types have random segregation (1:1) while nonrandom segregation was noted for double chromatid types. The nonrandom segregation hypothesis of earlier investigators needs to be reexamined.

Bromodeoxyuridine↗

Sister chromatid exchange induction and the course of DNA duplication, two mechanisms of sister chromatid exchange induction by ENU and the role of BrdU.

The aims of the present study were to establish the following: (i) the course of sister chromatid exchange (SCE) induction by ethylnitrosourea (ENU) in the first, second and third divisions as a function of the exposure time; (ii) the persistence of SCE-inducing lesions and the determination of whether or not they are always involved in SCE formation; (iii) the effect of bromodeoxyuridine (BrdU) incorporation on the induction and persistence of SCE. Three-way differential staining of sister chromatids in murine bone marrow cells in vivo was used in the present study. The results indicate the following: (i) SCE induction in each cell division depends on the course of DNA duplication, suggesting that SCE occurs at the replication fork; (ii) the cell population under study could be considered synchronous and had a cell cycle duration of nearly 9 h; (iii) in the second and third cell divisions ENU preferentially induced SCE in the cycle in which the exposure occurred; (iv) lesions induced by exposure to ENU did not cause SCE at the same site in subsequent divisions; (v) ENU was also capable of producing a long-lasting induction of SCE in BrdU-unsubstituted DNA; (vi) the sensitivity to SCE induction by the mutagen increases nearly proportionally to BrdU incorporation into DNA.

Animals↗

Characterization of mitoses with sister chromatid differentiation (SCD) and consequences for the analysis of proliferation kinetics and sister chromatid exchanges in asynchronously growing cells.

Labeling cells with bromodeoxyuridine (BrdU) permits the differentiation of mitoses of the first, second, and third generation after the addition of BrdU. The term "second mitoses" is used for those cells which have incorporated BrdU for two S-phases and which exhibit sister chromatid differentiation (SCD). However, SCD can also be obtained if the cell was in S-phase at the time of BrdU-addition and had already replicated part of its DNA. Such cells with incomplete BrdU-substitution in the first S-phase can only be differentiated from completely substituted ones by the quality of the SCD and are usually also grouped as "second mitoses" in the evaluation of experiments. Due to the heterogeneity of the evaluated "second mitoses", the determination of proliferation delay and the incidence of sister chromatid exchange-induction can depend on the time of chromosome preparation.

Animals↗

Spontaneous sister-chromatid exchange frequencies in human B and T lymphocytes at BrdU borderline concentrations for sister-chromatid differentiation.

Human peripheral blood B and T lymphocytes, highly purified by immunologic methods, were supplemented with gamma-irradiated unseparated autologous mononuclear cells to restore helper functions and stimulated with pokeweed mitogen and phytohemagglutinin, respectively. Spontaneous sister-chromatid exchange (SCE) frequencies were investigated in proliferating B and T lymphocyte cultures labeled with the cell-type-specific borderline concentrations of 5-bromodeoxyuridine (BrdU) for sister-chromatid differentiation (SCD). B lymphocytes from 6 different donors showed mean values of 3.28-3.72 SCE events/cell. In T lymphocytes, mean values of 6.30-7.28 SCEs/cell were observed. The differences between the SCE distributions of the cell populations are highly significant. The results show that the differences in the spontaneous SCE frequencies between human B and T lymphocytes were not due to a difference in the uptake of BrdU.

B-Lymphocytes↗