Distribution of sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac

The frequency of sister chromatid exchanges (SCEs) was determined for the chromosomes (except Y2) of the Indian muntjac stained by the fluorescence plus Giemsa (FPG) or harlequin chromosome technique. The relative DNA content of each of the chromosomes was also measured by scanning cytophotometry. After growth in bromodeoxyuridine (BrdU) for two DNA replication cycles. SCEs were distributed according to the Poisson formula in each of the chromosomes. The frequency of SCE in each of the chromosomes was directly proportional to DNA content. A more detailed analysis of SCEs was performed for the three morphologically distinguishable regions of the X-autosome composite chromosome. The SCE frequency in the euchromatic long arm and short arm were proportional to the amount of DNA. In contrast, the constitutive heterochromatin in the neck of this chromosome contained far fewer SCEs than expected on the basis of the amount of DNA in this region. A high frequency of SCE, however, was observed at the point junctions between the euchromatin and heterochromatin.     

The effects of incorporated tritium and bromodeoxyuridine on the frequency of sister chromatid exchanges

Cells in third mitosis treated during the first cell cycle with ³H-TdR and during the next two cycles with BrdU (without ³H-TdR) show a typical pattern of chromosome differentiation which allows identification of sister chromatid exchanges occurring during the first (SCE₁, second (SCE₂) and third cycles (SCE₃). Chromosomes labeled only with ³H-TdR had the most SCEs; those labeled only with BrdU, the second highest number; and those labeled with ³H-TdR plus BrdU, the fewest. Since BrdU and ³H-TdR are well known inducers of SCEs, the relatively low frequency of exchanges produced by the combined action of these two compounds is paradoxical. — It is assumed that SCEs are generated by the abnormal recombination of double-strand DNA breaks occurring at the junctions between completely and partially duplicated replicon clusters. Thus, agents that induce absolute blocks to DNA fork displacement will favor the appearance of SCEs because double-strand breaks have more time to occur at junctions. Conversely, agents that inhibit the initiation of replication will decrease the probability of SCEs. Ionizing radiation delays the onset of cluster replication. Therefore, in ³H-TdR plus BrdU-substituted chromosomes the radiation from tritium may inhibit the appearance of BrdU-induced SCEs. Since the inhibition does not exist in chromosomes substituted only with BrdU, the frequency of SCEs in these elements is higher than in double-substituted chromosomes. During the first cell cycle the onset of cluster replication is normal. However, the incorporation of ³H-TdR in the replication fork may enhance the appearance of double-strand breaks, thus inducing a high frequency of SCEs.     

The distribution of mitomycin C-induced sister chromatid exchanges in the euchromatin and heterochromatin of the Indian Muntjac

The frequency of sister chromatid exchanges (SCEs) induced by mitomycin C (MMC) in Indian Muntjac chromosomes was determined by the fluorescence plus Giemsa (FPG) technique. Using scanning cytophotometry the relative DNA content of each chromosome was measured with and without acid or alkali pretreatments for C-banding. During acid and alkali treatments, euchromatin lost 20 to 30% of its DNA, while heterochromatin lost less than 5%; an intermediate DNA loss was observed for the short arm of the X chromosome. After growth of cells in the presence of MMC during the first cycle and in the presence of bromodeoxyuridine (BrdU) during the first and second cycles of DNA replication, SCEs in the euchromatin were proportional to DNA content. SCEs at the junctions between the neck of the X chromosome and the long and short arms occurred more frequently than expected. A threshold effect for the induction of SCEs was observed in regions resistant to DNA extraction by acid and alkali treatments (i.e., the neck and short arm of the X chromosome). At high concentrations of MMC, the frequency of SCE at each junction appears to plateau at 0.5.     

Relationship between sister-chromatid exchanges and heterochromatin or DNA replication in chromosomes of Crepis capillaris

The C-band patterns, DNA late replication patterns and distribution patterns of spontaneous and γ-ray-induced SCEs in Crepis capillaris chromosomes were studied. The fluorescence plus Giemsa (FPG) technique was used for detection of SCEs and late-replicating chromosome regions after unifilar incorporation of BrdU into DNA. An asynchronous replication of both euchromatic and heterochromatic chromosome regions was established. The frequency of SCEs is increased about 2-fold by 1.5 Gy γ-rays. The localization of the sites of SCEs was analyzed with special reference to eu- and heterochromatin and early- and late-replicating regions. The data obtained showed that SCEs were distributed nonrandomly along the chromosomes. Preferential occurrence of SCEs was observed in the following chromosome regions: at the junction between eu- and heterochromatic regions, the latter being rich in late-replicating DNA; at the junction between early- and late-replicating regions, the latter not being C-band positive. Certain heterochromatic regions were more rarely involved in SCEs than expected on the basis of their length. The lowest incidence of SCEs was found in the centromeric regions. Very similar distribution patterns of spontaneous and γ-ray-induced SCEs were observed. The possible role of the differences in the time of replication of the different chromosome regions in the formation of SCEs is discussed.     

Human-hamster hybrid cells used as models to investigate species-specific factors modulating the efficiency of repair of UV-induced DNA damage

The human-Chinese hamster hybrid cell line XR-C1#8, containing human chromosome 8, was used as a model system to investigate the relative importance of cellular enzymatic environment and chromosomal structure for modulating the efficiency of repair of UV-induced DNA damage. The hybrid cells were irradiated with UVC light and the extent of cytogenetic damage, detected as frequencies of sister chromatid exchanges (SCEs), was compared between the human and the hamster chromosomes. The combination of immunofluorescent staining for SCEs and chromosome painting with fluorescence in situ hybridization allowed the simultaneous analysis of SCEs in the human and hamster chromosomes. The aim of the present study was to determine if the differences in biological response to comparable UV treatments observed between human and hamster cells were maintained in the hybrid cells in which human and hamster chromosomes are exposed in the same cellular environment. The analysis of replication time of human chromosome 8 indicated the active status of this chromosome in XR-C1#8 hybrid cells. The frequencies of SCEs for human chromosome 8 and a hamster chromosome of comparable size were 0.35 +/- 0.52, 0.80 +/- 0.73, 1.24 +/- 2.24 and 0.36 +/- 0.12, 0.71 +/- 0.2, 0.97 +/- 0.27, respectively, after irradiation with 0, 5, and 10 J/m2. The persistence of UV-induced SCEs after three cell cycles was also analyzed, both for the human and hamster chromosomes. The observed frequencies of SCEs were 0.40 +/- 0.57, 0.62 +/- 1.05, 0.58 +/- 0.83 and 0.26 +/- 0.08, 0.67 +/- 0.18, 0.69 +/- 0.24, in human and hamster chromosomes respectively, after treatment with 0, 10, and 20 J/m2 of UVC light. No significant differences could be observed between the human and hamster chromosomes. These results suggest that the enzymatic environment of human and hamster cells has the main role, in comparison to the structural organization of human and hamster chromosomes, for determining the different level of repair of UV-induced DNA damage observed in these two species.     

Approximation of baseline and BrdU-induced SCE frequencies

In the present paper we have developed a new rationale and an experimental schedule to approximate the frequency of SCEs which occur independently of BrdU incorporation, namely, the baseline frequency of SCEs. The method used includes the analysis of SCE yields in second and third division chromosomes after BrdU-substitution for 1, 2, and/or 3 successive replication rounds in the presence of this thymidine analogue, leading to a set of ten different experimental results. As a result of formulating various mathematical equations and applying them to the data, an accurate estimation of the frequency of baseline (BrdU-independent) and BrdU-induced SCEs, can be made, thus avoiding the difficulties inherent in the current extrapolation methods. The conclusions are that 1) SCEs seem to be formed after DNA synthesis (by exchanging post-replicative DNA portions), but, obviously, very near to the replication fork and 2) that under our experimental conditions about 0.065 SCEs per picogram of DNA per cell cycle occur as a consequence of chromosome replication, this frequency being increased by BrdU-substitution. The methodology seems to be reliable enough to be used in other species and systems in order to compare baseline SCE frequencies.Abbreviations SCEs sister-chromatid exchanges - BrdU(BrdUrd) 5-bromodeoxyuridine - dTh(dThd) thymidine - ³H-dTh(³H-dThd) tritiated thymidine - FdU(FdUrd) 5-fluorodeoxyuridine - Urd uridine - FPG fluorescent plus Giemsa     

Strand breaks arising from the repair of the 5-bromodeoxyuridine-substituted template and methyl methanesulphonate-induced lesions can explain the formation of sister chromatid exchanges

5-Bromodeoxyuridine (BrdU)-induced sister chromatid exchanges (SCEs) are mainly determined during replication on a BrdU-substituted template. The BrdU, once incorporated, is rapidly excised as uracil (U), and the gap is repaired with the incorporation of BrdU from the medium, which leads to further repair. During the second S period in BrdU medium, this process continues as the strand acts as template. Experiments suggest that 3-amino-benzamide (3AB) delays the ligation of the gaps formed after U excision, resulting in enhanced SCE levels during the second cycle of BrdU incorporation. When normal templates of G1 cells are treated before BrdU introduction with methyl methanesulphonate (MMS), 3AB in the first cycle doubles the MMS-induced SCEs but has no effect on them during the second cycle. When the BrdU-substituted template is treated with MMS in G1 of the second cycle, 3AB again doubles the SCEs due to MMS and also enhances the SCEs resulting from delays in ligation of the gaps following U excision in the BrdU-substituted template. The repair processes of MMS lesions that are sensitive to 3AB and lead to SCEs take place rapidly, while the repair process of late repairing lesions that lead to SCEs appear to be insensitive to 3AB. A model for SCE induction is proposed involving a single-strand break or gap as the initial requirement for SCE initiation at the replicating fork. Subsequent events represent natural stages in the repair process of a lesion, ensuring replication without loss of genetic information. G1 cells treated with methylnitrosourea (MNU) and grown immediately in BrdU medium rapidly lose the O⁶-methylguanine from their DNA and the rate of loss is BrdU-dose dependent. The rapid excision of the U lesions can explain the effect of BrdU concentration on SCE reduction following both MNU or MMS treatment.     

SCEs are induced by replication of BrdU-substituted DNA templates, but not by incorporation of BrdU into nascent DNA

After 3 rounds of DNA replication in the presence of BrdU, third-division metaphase cells can be scored for the frequencies of SCEs that occurred during cycles 1 and 2, and also for the frequency of SCE during cycle 3. This procedure was used to resolve the issue of SCE induction by replication of BrdU-substituted DNA templates versus induction by BrdU incorporation into nascent DNA. It was observed that third-cycle SCE frequencies in CHO are dependent upon the amount of BrdU that was present during cycles 1 and 2 and are independent of the BrdU concentration during the third cycle. It is therefore BrdU serving as a template, rather than BrdU being incorporated, that initiates the SCE event. A model is proposed that produces reasonable fits to the observed data. It also predicts a true background or spontaneous SCE frequency of 3 per cell per cycle as previously reported by Heartlein et al. (Mutation Res., 107 (1983) (103-109). The predicted single twin ratio is higher than that reported by Wolff and Perry (Exp. Cell Res., 93 (1975) 23-30), and possible explanations for this discrepancy are discussed.     

Chromosomal aberrations and SCEs in Allium cepa root-tip cells treated with caffeine and pyronin Y

The effectiveness of caffeine and pyronin Y in the induction of both chromosomal aberrations and sister-chromatid exchanges (SCEs) in root meristematic cells of A. cepa was studied.The rate of SCEs proved to be increased when 5-bromo-2′-deoxyuridine- (BrdU) substituted chromosomes were allowed to replicate in thymidine (dT) for a second S period simultaneously with caffeine or pyronin Y. In contrast, only caffeine was able to induce aberrations in BrdU-substituted chromosomes, while pyronin Y seemed to be ineffective at the doses employed.     

Persistence of DNA lesions and the cytological cancellation of sister chromatid exchanges

The ability of UV light, mitomycin C and ionizing radiation to induce the formation of sister chromatid exchanges (SCEs) at the same locus in successive cell generations was investigated in human lymphocytes. Cells were exposed to the DNA damaging agents after they had completed their first round of DNA replication, and SCEs were examined at the third division in chromosomes that had been differentially stained three ways. Although some of these treatments induced long-lived lesions that increased the frequency of SCEs in successive cell generations, none of the lesions led to the formation of consecutive SCEs at the same locus in successive cell generations. This observation seriously challenges the hypothesis that SCE cancellation results as a consequence of persistence of the lesions induced by these agents.     

Yield of SCEs and translocations produced by 3 aminobenzamide in cultured Chinese hamster cells

Different concentrations of 3-aminobenzamide (3AB), a strong inhibitor of poly(ADP-ribose) polymerase (PARP), were used to study their effect on the BrdU-substituted DNA of the Chinese hamster AA8 cell line. The frequencies of sister chromatid exchanges (SCEs) and translocations were determined using the fluorescence plus Giemsa (FPG) and fluorescence in situ hybridization (FISH) techniques, respectively. The results indicate that 3AB effectively induced a dose-dependent increase in the frequency of SCEs, but this enhancement in the yield of SCEs was not paralleled by an increase in translocations. These results are discussed in terms of the as yet poorly understood molecular mechanisms of action of the enzyme PARP.     

Mechanisms for sister chromatid exchanges and their relation to the production of chromosomal aberrations

By taking advantage of the fact that fluorescent light (FL) induces strand breaks only in bromodeoxyuridine(BrdU)-substituted DNA, and that those breaks eventually lead to the formation of sister chromatid exchanges (SCEs), the response of SCEs to FL was studied carefully in Chinese hamster chromosomes in which, out of four DNA strands, BrdU-substitution had occurred either in one or three strands. The FL-induced SCE frequency did not differ greatly between these two types of chromosomes. However, when they were submitted to caffeine treatment, a drastic increase in the frequency was detected in the trifilarly-substituted chromosomes while a significant decrease occurred in the unifilarly-substituted chromosomes. Based on these results, a working hypothesis was developed that the SCE can arise by at least two different mechanisms, one operating at replicating points probably utilizing the machinery of DNA replication, and the other acting only in the post-replicational DNA portion, probably in a similar fashion as assumed in a general model of crossing over in the eukaryote. These dual mechanisms may account for the discrepancy encountered in the explanations of the induction of SCEs by various exogenous agents as well as spontaneous SCEs. The present study also showed that some, but clearly not all, of chromatid deletions are the outcome of the failure to complete SCEs arising through these mechanisms.     

The relationship between the cell time available for repair and the effectiveness of a damaging treatment in provoking the formation of sister-chromatid exchanges

The effectiveness of a given dosage of visible light in inducing increased yields of SCEs was studied in Allium cepa L. meristems. Cells were first grown for one cycle time in the presence of BrdUrd and then irradiated at different times throughout the second cell cycle. The effectiveness of this treatment in provoking the formation of SCEs increases the closer the irradiation time is to the beginning of the S phase, and then decreases rapidly as cells progress through the S period. The largest increase in SCEs is obtained when irradiation coincides with early S phase. These results strongly suggest that SCEs arise at the time of DNA replication due to the presence of unrepaired lesions. Since repair appears to be a time-dependent process, the shorter the interval between damage induction and DNA replication, the greater the number of lesions that remain unrepaired, and as a consequence, the higher the effectiveness of the damaging treatment in provoking the formation of SCEs.     

Effect of 5-bromodeoxyuridine substitution on sister chromatid exchange induction by chemicals

The fluorescence-plus-Giemsa (FPG) technique for analysis of sister chromatid exchange (SCE) is widely used as an assay for mutagenic carcinogens. There is very little information, however, on whether incorporation of the bromodeoxyuridine (BrdU) necessary for visualization of SCEs affects the sensitivity of the SCE test system to different chemical agents. We have investigated the effect of BrdU incorporation on SCE induction by labeling cells with BrdU for either the first cell cycle or the first and second cell cycles. The cells were then treated with bleomycin, which produces DNA strand breakage; proflavine, which intercalates into DNA; mitomycin C, which produces monoadducts and DNA crosslinks; or aphidicolin, which inhibits DNA polymerase . Chemicals were added before BrdU exposure or during the first, second, or both cell cycles. Only mitomycin C, which induces long-lived lesions, elevated the SCE frequency when cells were treated before BrdU labeling. When bleomycin, proflavine, or mitomycin C was present concurrently with BrdU, the frequency of SCEs was increased independently of the BrdU labeling protocol. Aphidicolin, on the other hand, induced more SCEs when present for the second cell cycle, when DNA replicates on a template DNA strand containing BrdU. We also examined the induction of SCEs in the first cell cycle (twins) and in the second cell cycle (singles) after continuous treatment of cells with BrdU and the test chemicals. Only aphidicolin increased SCE frequency in the second cell cycle. These results indicate that aphidicolin, but not bleomycin, proflavine, or mitomycin C, affects BrdU-substituted DNA and unsubstituted DNA differently. This type of interaction should be taken into consideration when the SCE test is used as an assay system.     

Relation between the SCE points and the DNA replication bands

A method for obtaining a combination of differential sister chromatid staining and DNA replication banding is described. Using this method the SCE points can be precisely localized to particular bands of individual chromosomes. It was shown, that SCEs occur not only in the regions of early DNA replication bands (=euchromatic segments=negative G-bands), but also in the regions of late DNA replication bands (=heterochromatic segments=positive G-bands). SCEs occurred about three times more frequently in the euchromatic segments than in the heterochromatic segments. Furthermore, more SCEs were observed in the early replicating X-chromosome than in the late replicating X-chromosome.     

Sister chromatid differentiation and isolabeling of chromosomes

Summary Isolabeling observed during sister chromatid differentiation (SCD) was studied from human skin fibroblasts by the fluorescence-plus-Giemsa (FPG) technique. Bromodeoxyuridine (BrdU) was fed to exponentially dividing cells for 52 h to enable completion of two consecutive cycles of DNA replication. During this period, the late-replicating regions of some chromosomes were able to go through three replication cycles. These chromosome regions had evidently incorporated BrdU bifiliarly in both chromatids and hence, on staining with FPG, appeared isostained (isolabeled). Thus, incubation of exponentially dividing cells with BrdU for a period longer than that required for two cell cycles appears to be a suitable method for revealing the late-replicating regions of the genome, such as the X chromosome in a human female, as isolated.In another experiment with Indian muntjac chromosomes, isolabeled segments were darkly stained, which suggested unifilar incorporation of BrdU. In this case, unequal crossing-over or an unequal distribution of thymine residues probably is responsible for the isolabel.     

Considerations on the mechanism of differential Giemsa staining of BrdU-substituted chromosomes

Summary The staining properties of unifilarly bromodeoxyuridine (BrdU)-substituted chromatids were compared using fluorescent-plus-Giemsa (FPG) staining methods. It was found that the staining intensity of chromatids which had incorporated BrdU in the next to last S-phase is less than that of chromatids whose BrdU-containing strand came from the last cell cycle. Thus, FPG-staining is not a function of the number of BrdU-substituted DNA strands alone. These findings lead to the conclusion that the primary point of action of PFG staining leading to sister chromatid differentiation (SCD) are chromosomal proteins which have been altered in the replication of BrdU-substituted DNA and that the demonstration of the SCD and replication patterns with the same staining procedure is based on different mechanisms.     

Distribution of sister chromatid exchanges in chromosomes of normal Chinese hamster and its cell lines exposed to BrdU and MMC

Sister chromatid exchanges (SCEs) were investigated in chromosomes from normal male Chinese hamster (CH) and its cell lines (CHW, 1102 and 1103). The fibroblasts were grown for two replication cycles in medium containing BrdU and mitomycin C (MMC) at concentrations of 0.01, 0.02 and 0.03 micrograms/ml of medium. The difference in SCEs/cell between male CH and CHW was negligible, but the difference between CHW and 1102 was about 2.6-fold. It is suggested from karyotypic differences between CHW and 1102, that the control of SCEs might be due partly or completely to chromosome 5 in Chinese hamster. The lines CHW and 1102 were less responsive than normal Chinese hamster cells when exposed to different MMC concentrations. It is suggested that the lines CHW and 1102 might be slightly resistant to MMC. The frequency of SCEs decreased with the decrease of chromosome size. SCEs are not preferentially distributed on any autosomal chromosomes. No SCEs were found in normal X-chromosomes. The majority of exchanges appear to be either interband regions or very near band-interband junctions.     

Distribution of sister chromatid exchanges on the mouse chromosomes in vivo with reference to the replication properties of the X chromosome

Frequency of sister chromatid exchanges (SCE) were recorded separately for different chromosomes from bone marrow cells of female mice of the two genetic strains (C3H/S and C57BL/6J). SCEs were evaluated following different doses of 5-bromo-2'-deoxyuridine (BrdU) as nine hourly i.p. injections. The SCE per cell increased with increasing BrdU doses which was slightly higher in C3H/S than in the C57BL/6J. SCEs per cell were variable at every treatment-strain combination, possibly reflecting the heterogeneous nature of the bone marrow cells. In general, there is a positive correlation between SCE per chromosome and the relative chromosome length. Total SCEs on one of the large chromosomes (most likely the X chromosome), however, are significantly higher than expected on the basis of relative length alone. Most of this increase is attributable to one of the homologues of this chromosome, which is not in synchrony with the rest of the chromosomes and may represent the late-replicating X. These results when viewed in the light of replication properties of the heterochromatinized X, suggest a direct involvement of DNA replication in SCE formation and may argue against the replication point as the sole site for the SCEs.     

Sister chromatid exchanges in barley

Summary A mean frequency of 20.6 sister chromatid exchanges (SCEs) per cell has been observed in a reconstructed karyotype of Hordeum vulgare by application of the FPG technique after unifilar incorporation of BrdU into chromosomes. The involvement in SCEs of the 48 segments into which the chromosome set had been subdivided was, with a single deviation, length proportional and independent of the segment's heterochromatin content. Asymmetric bands, indicative of an uneven distribution of adenine and thymidine between the DNA strands in adenine (A)-thymidine (T) rich chromosome regions, could not be detected after incubation of the cells in BrdU for one cycle of DNA replication.