A schedule to demonstrate radiation-induced sister chromatid exchanges in human lymphocytes

Summary The reciprocal interchange between the chromatids of a chromosome, termed sister chromatid exchange (SCE), is considered to be one of the most sensitive and accurate cytogenetic parameters and respond to toxic chemicals at very low doses. But the response of SCE to ionizing radiation is very poor. Human lymphocytes fail to give SCE response when irradiated at G₀. Probably the primary lesions induced at G₀ do not remain available long enough to find expression as SCEs. Based on this assumption a schedule was developed using caffeine to demonstrate radiation induced SCEs. Following this schedule a dose-dependent increase in the frequency of radiation induced SCEs has been observed.     

Induction of the fragile X on BrdU-substituted chromosomes with direct visualization of sister chromatid exchanges on banded chromosomes

Summary After incorporation of BrdU for one or more replication cycles, the fragile site at Xq27 [fra(X)] was induced by a late pulse with excess thymidine (dT), resulting in the simultaneous visualization of G bands and differentially stained sister chromatids. The degree of BrdU substitution (uni- vs bifilarly substituted DNA) did not affect the expression of the fra(X). Without addition of dT, expression was the same in M1, M2, and M3 cells. With the addition of dT, expression was reduced in M1 cells and increased in M2 and M3 cells. One way to explain this fact would be an increased repair of the fragile site in M1 cells by illegitimate G:BrdU pairing under dCTP-deficient conditions. A preferential depletion of M3 cells, and to a lesser extent also M2 cells, could suggest a synergistic toxic effect of BrdU substitution and dCTP depletion. With this technique, sister chromatid exchanges (SCEs) could be directly localized at band level, facilitating a more detailed study of SCEs at the Xq27 fragile site.     

小麦愈伤组织细胞的姐妹染色单体交换

用BrdU标记,改良的FPG法染色,建立了一种植物愈伤组织细胞姐妹染色单体分染的方法。并对培养基的不同附加成分对SCE的影响进行了研究。所有培养基上愈伤组织细胞的SCE率都显著高于正常根尖分生组织细胞。6-BA,AgNO_3,高浓度的2,4-D,蔗糖均可诱发SCE。     

Sister chromatid exchanges in Allium cepa

Differential staining of sister chromatids with Giemsa after BrdU incorporation into DNA was performed in Allium cepa L. chromosomes. A treatment solution containing 10^–7 M FdU, 10^–4 M BrdU and 10^–6 M Urd was found to ensure BrdU incorporation without affecting cell cycle duration. After several procedures before staining the slides with Giemsa had been tested, treatment with the fluorochrome compound 33258 Hoechst, exposure to UV light and heating at 55° C in 0.5×SSC, were found to be essential for good differentiation. The distribution of SCEs per chromosome agrees with the expected Poisson distribution. The mean value of SCEs per chromosome occurring when cells were exposed to the treatment solution for two consecutive rounds of replication (=5.5) was double the mean value observed when cells were exposed to the same treatment for only one round of replication (=2.8). SCEs were found to occur more frequently in those chromosome regions corresponding neither to C-bands nor to late replicating DNA-rich regions. Finally, the occurrence of SCEs involving less than the width of a chromatid is discussed.     

Comparisons of in vivo BrdU labeling methods and spontaneous sister chromatid exchange frequencies in regenerating murine liver and bone marrow cells

BrdU and BrdC have been employed as DNA labeling agents for differentiation of sister chromatids and for extension of sister chromatid exchange (SCE) methods to regenerating murine liver cells in vivo. Comparisons were made between bone marrow and liver cells isolated simultaneously from mice following DNA labeling with either BrdC or BrdU. Although the total mitotic yield of bone marrow cells was considerably greater than in liver, a higher percentage of second division metaphases was observed in liver cell preparations. The percentages of second division c-metaphase cells observed were 31.5% in bone marrow and 73% in liver cell preparations. Utilizing either BrdU or BrdC, no significant difference in percentage of second division metaphases was discerned. The number of spontaneous SCEs per cell was distributed according to the Poisson probability function. No significant differences in mean numbers of SCEs per cell were found in comparisons of bone marrow (1.40) and liver cells (1.65) or of cells which had incorporated BrdU or BrdC.     

Three-way differentiation of sister chromatids in endoreduplicated (M3) chromosomes of Bloom syndrome B-lymphoid cell line

Summary The three-way differentiation of sister chromatids (3-way SCD) in M₃ endoreduplicated chromosomes in a Bloom syndrome (BS) B-lymphoid cell line, suggested that in addition to exchanges between sister chromatids (intra-exchanges), non-sister chromatid exchanges (inter-exchanges) also occur, especially in BS high SCE cells. In BS diploid chromosomes such inter-exchanges probably get confused with intra-exchanges when total SCEs are accounted for. Bloom syndrome high SCE cells probably do not follow the same bromodeoxyuridine (BrdU) uptake pattern over three cell cycles as normal cells. The 3-way SCD in M₃ endoreduplicated chromosomes can be explained on the basis of Schvartzman's second model (1979) as well as Miller's model (1976), depending on the pattern of uptake of BrdU over three cell cycles. An interference in the previous events of exchanges in the following cell cycle (i.e., cancellation of SCEs) in BS chromosomes was observed in some regions, though not in high numbers.     

In vivo sister chromatid differentiation and baseline sister chromatid exchanges in a mouse ascites tumour model.

Induction of differentially stained sister chromatids at G2/M and determination of baseline sister chromatid exchanges (SCEs) in ascites form of mouse sarcoma 180 cell line have been done by in vivo incorporation of 5-bromodeoxyuridine (BrdU) for two consecutive DNA replication cycles. The baseline SCE frequency is 6.24 at log phase of tumour growth.     

Differential fluorescence of sister chromatids in chicken embryos exposed to 5-bromodeoxyuridine

Differential fluorescence of sister chromatids (SCD) and sister chromatid exchanges (SCE) were visualized in chromosomes obtained directly from growing chicken embryos. SCD was obtained by exposing 3-day embryos to BrdU (12.5-50 mug) in ovo for 26 hours and staining air dried chromosome preparations with 33258 Hoechst. Bright, stable fluorescence and continued SCD were achieved if slides were mounted in McIlvaine's pH 4.4 buffer. Embryo growth, mitotic activity and gross chromosome morphology were not adversely altered by the BrdU treatments. The SCE rate was estimated to be 0.07 SCEs per macrochromosome and 0.75 SCEs per metaphase for two cell cycles.     

The diplochromosome of endoreduplicated cells: A new approach to highlight the mechanism of sister chromatid exchange

Chinese hamster lung embryonic cells (CL1) were treated with colchicine in order to induce endoreduplication and subsequently with mitomycin-C (MMC) to induce exchanges within the diplochromosome. The use of chromosomal differential staining through incorporation of 5-bromodeoxyuridine, resulting in only one stained chromatid, has allowed the analysis of all classes of exchanges among the four chromatids of the diplochromosome. Three classes of exchanges may occur: intradiplochromatid exchanges (ICEs) between the two inner chromatids, cousin chromatid exchanges (CCEs) between one inner and one outer chromatid, and sister chromatid exchanges (SCEs) between the two sister chromatids of the diplochromosome. The results show that MMC treatment, in the last cell cycle of endoreduplication, as expected, significantly increases only the frequency of SCEs, whereas the frequency of ICEs and CCEs remains unchanged. This result supports replication models of formation of SCEs. Furthermore the fact that the number of ICEs does not increase means that the molecular mechanism of somatic crossing over is not related to that of SCE formation, or very rarely. The results also indicate a statistically significant lower induction of SCEs in endoreduplicated metaphases as compared with diploid ones both in control and MMC-treated cells. Such a result may be due to structural restrictions within the diplochromosome. Received: 29 December 1995; in revised form; 4 March 1996 / Accepted: 24 March 1996     

Sister chromatid exchanges in garlic (Allium sativum L.) callus cells

A technique is described for differential staining of sister chromatids and the study of sister chromatid exchanges (SCEs) in garlic (Allium sativum L.) callus cells. BrdU incorporation into newly synthesized DNA was ensured by culturing calli on medium containing 100 M BrdU+0.01 M FudR+1 M Urd. SCEs were visualized by FPG staining technique and their frequency was analysed. Mean frequency of SCEs in callus cells was higher than that in meristem root-tip cells. Using the same staining method, cell cycle time of callus cells was analysed. It was found that it ranges from 48 to 132 hrs. The method described represents a new approach in the study of genetic instability of plant cells cultured in vitro.Abbreviations BrdU 5-bromo-2-deoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - FPG fluorescent-plus-Giemsa - FudR 5-fluoro-2-deoxyuridine - SCE sister chromatid exchange - SSC 0.15 M NaCl + 0.015 M Na-citrate - T thymidine-containing strand of the DNA duplex - B 5-bromo-2-deoxyuridine-containing strand of the DNA duplex - Urd uridine     

Lateral asymmetry in human constitutive heterochromatin

Human lymphocytes were grown for one replication cycle in BrdU, stained with 33258 Hoechst, exposed to UV light and subsequently treated with 2 x SSC and stained with Giemsa. This technique differentially stains the constitutive heterochromatin of chromosomes 1, 9, 15, 16, and the Y. In the heterochromatin of chromosome 9 both sister chromatids stained darkly and symmetrically but in the other four chromosomes the heterochromatin showed lateral asymmetry, one chromatid being darkly stained while its sister chromatid was as pale or paler than the rest of the chromosome. The lateral asymmetry is presumed to reflect an underlying asymmetry in distribution of thymine between the two strands of the DNA duplex in the satellite DNA component of the chromosomes. In some number 1 chromosomes compound lateral asymmetry was seen; darkly staining material was present on both sister chromatids although at any given point lateral asymmetry was maintained so that if one chromatid stained darkly the corresponding point on the sister chromatid was very pale. The pattern of compound lateral asymmetry varied among the number 1 chromosomes studied but was constant for any one homologue from one individual. This technique reveals a previously unsuspected type of polymorphism within the constitutive heterochromatin of man.     

Analysis of the frequency of sister chromatid exchange in different regions of chromosomes of the kangaroo rat (Dipodomys ordii)

The frequency of sister chromatid exchanges (SCEs) has been determined for C band and non-C band regions of chromosomes of the kangaroo rat after staining with the fluorescence plus giemsa (FPG) technique. After one complete round of DNA synthesis in the presence of bromodeoxyuridine (BrdU) staining of the C band regions revealed simple or complex asymmetries between chromatids. After two complete rounds of DNA synthesis in the presence of BrdU harlequin chromosomes were observed. Analysis of the distribution of SCE in chromosomes at their 1st and 2nd mitosis showed that relatively few exchanges occur within C band regions, although the frequency of SCEs is high at the junction between C band and non-C band chromosome regions.     

Differential giemsa staining of sister chromatids after extraction with acids

Chromosomes of Chinese hamster strain cells were air-dried on slides after BrdU substitution for two or three rounds of replication. The preparations were treated with 20% PCA at 55 degrees C for 20-30 min, or 5N HCl at 55 degrees C for 15-20 min. After staining with Giemsa, unifilarly BrdU-substituted chromatids stained faintly and bifilarly substituted chromatids stained darkly. Such a pattern of sister chromatid differential staining was confirmed by the examination of metaphase cells grown with BrdU for three rounds of replication.     

Murine bone marrow culture system for cytogenetic analysis

A mouse bone marrow culture system for examining genotoxicity of agents by first exposing animals in vivo then growing cells in vitro is presented. This assay can also be used for in vitro and/or for the in vivo and in vitro comparative cytogenetic studies. The protocol involves culturing of approximately 1,000,000 nucleated cells obtained from mice tibia and femora in 5 ml of Ham's F-12 medium containing 20% fetal bovine serum, 10% whole uterus extract from pregnant mice and 1% penicillin-streptomycin. The use of flasks and mouse uterus extract for culturing are important steps for higher mitotic yield. The addition of 20 microM BrdU for 24 h helps in the differentiation of sister chromatids for sister-chromatid exchange (SCE) analysis. Cyclophosphamide, given to mice through intraperitoneal injection, induced significant dose-related SCEs in culture. Trinitrofluorenone, a direct-acting mutagen, caused dose-related SCEs in in vitro bone marrow cell culture.     

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.     

Cytotoxic effect and induction of sister chromatid exchange in exponentially growing rat 9L gliosarcoma cells after brief exposure to BrdU

The magnitude of DNA modulation in rat 9L gliosarcoma cells after a brief exposure to bromodeoxyuridine (BrdU) was studied by assaying colony-forming efficiency (CFE) and the number of sister chromatid exchanges (SCEs) per metaphase. The CFE assay showed that a 1-hr exposure to BrdU, at concentrations ranging from 10 to 1000 microM, produced a maximum cell kill of 5%. After a 2-hr exposure to 20 microM BrdU, the surviving fraction was 0.99, and even at a BrdU concentration of 1000 microM, 77% of the 9L cells survived. Compared with control cultures, the relative number of SCEs per metaphase in treated cultures was increased after a 1-hr exposure to BrdU at concentrations of 100 microM or more and after a 2-hr exposure to concentrations of 20 microM or more; no increase was observed in cells treated for 30 min with BrdU at concentrations up to 1000 microM. When the treated cells were allowed to grow in BrdU-free growth medium, the number of SCEs per metaphase returned to the control level within 24 hr, even after exposure to BrdU at concentrations as high as 1000 microM. These results demonstrate that exposure to BrdU at concentrations of up to 1000 microM for 30 min, 100 microM for 1 hr, and 20 microM for 2 hr causes little modulation of DNA.     

A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae

We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle. We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division. SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants. These data show that sister chromatids have two different states prior to nuclear division. We suggest that the sister chromatids of cir. III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B). Other interpretations are also discussed. SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation.     

A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae

We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle. We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division. SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants. These data show that sister chromatids have two different states prior to nuclear division. We suggest that the sister chromatids of cir. III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B). Other interpretations are also discussed. SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation. Received: 3 July 1996 / Accepted: 4 October 1996     

Induction of endoreduplication by hydrazine in Chinese hamster V 79 cells and reduced incidence of sister chromatid exchanges in endoreduplicated mitoses

Hydrazine in high concentrations very effectively induces endoreduplication in Chinese hamster V 79 cells. The addition of 5-bromodeoxyuridine (BrdU) for the duration of one cell cycle prior to the induction of endoreduplication produces diplochromosomes with sister chromatid differentiation (SCD) after differential chromatid staining. The fact that diplochromosomes with complete SCD are obtained shows that endoreduplication was induced in cells that were in G2-phase. The analysis of sister chromatid exchanges (SCEs) showed that hydrazine treatment rarely led to increased SCE frequencies in mitoses after endoreduplication, but that it caused a strong SCE induction in diploid second division metaphases in the same culture. Neither catalase nor cysteine had an effect on the induction of endoreduplication or the incidence of SCEs. Treatment of the cells with mitomycin C prior to addition of BrdU led to increased SCE frequencies. Compared with the normal mitoses from the same preparation, the mitoses after endoreduplication showed a significantly reduced induction of SCEs. In contrast to these findings, SCE induction was not reduced in the common tetraploid V 79 cells after colcemid-induced polyploidization.     

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.