Mutagenesis, Vol. 17, No. 5, 399-403,
September 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
SCE formation after exposure of CHO cells prelabelled with BrdU or biotin-dUTP to various DNA-damaging agents
Institute of Genetics, Department of Molecular Genetics, 1113 Sofia, Bulgaria, 1 Institute of Nuclear Chemistry and Technology, 03-195 Warszawa, Poland, 2 Institute of Biology, Swietokrzyska Academy, 25-406 Kielce, Poland, 3 Indian Institute of Chemical Biology, 700 032 Calcutta, India and 4 Institute of Genetics, University of Essen, D-45117 Essen, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
Formation of sister chromatid exchanges (SCE) is a mechanism of repair or bypass of DNA damage during S phase. Although SCE have been studied for a long time, the types of DNA lesions involved and the role of 5-bromodeoxyuridine (BrdU) in SCE formation are a matter of debate. We have developed a novel method of differential labelling of sister chromatids with biotin-16–2'-deoxyuridine-5'-triphosphate (biotin-dUTP) and could show that a substantial proportion of radiation-induced SCE arise via damage to BrdU-moieties. The present investigations were performed to examine the role of BrdU in the formation of SCE by the endonucleases AluI and DNase I, as well as the alkylating agent mitomycin C (MMC). CHO cells unifilarily prelabelled with biotin-dUTP or BrdU were treated and the frequencies of SCE analysed in the first post-treatment mitoses. AluI induced similar frequencies of SCE in cells prelabelled with BrdU or biotin-dUTP. DNase I induced significantly more SCE in cells prelabelled with BrdU than with biotin-dUTP. MMC induced slightly more SCE in cells labelled with biotin-dUTP than BrdU, but the difference was not significant. The possible mechanisms responsible for the enhanced SCE frequency following DNase I treatment of cells prelabelled with BrdU are discussed.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Despite the fact that the phenomenon of sister chromatid exchanges (SCE) has been known for a long time (for a review see Latt, 1981
), the mechanisms of their formation are not well understood. Recent investigations have shown that RAD51-mediated homologous recombination mechanisms are involved in SCE formation (Sonoda et al., 1999). However, it appears that SCE can also be formed via alternative, RAD51-independent pathways (Lambert and Lopez, 2001). The role of topoisomerase II in SCE formation has been discussed, but remains obscure (Dominguez et al., 2001).
Uncertainties also prevail regarding the nature of lesions leading to SCE. It is known that not all types of DNA damage give rise to SCE. S phase-dependent agents such as mitomycin C (MMC) and UV light are among the most effective inducers of SCE (Latt, 1981). It is generally assumed that SCE arise during the S phase of the cell cycle, when damaged DNA is replicated (Painter, 1980). The nature of SCE following treatment of cells in G1 with agents which induce DNA double-strand breaks (dsb), like ionizing radiation, has been a matter of debate (Littlefield et al., 1979). Ionizing radiation is a poor inducer of SCE and is only effective when applied to cells in G1 with chromosomes unifilarily substituted with 5-bromodeoxyurdine (BrdU) (Littlefield et al., 1979; Bruckmann et al., 1999b). Therefore, it was proposed that SCE induced by ionizing radiation in G1 result from chromosomal aberrations, mainly inversions, and are ‘false’ SCE (Wolff et al., 1974; Mühlmann-Diaz and Bedford, 1995). In a previous study we analysed the frequencies of SCE associated with inversions in the short arm of human chromosome 3 and could show that most radiation-induced SCE do not result from inversions (Wojcik et al., 1999). We have developed a technique to visualize SCE by labelling cells with biotin-16–2'-deoxyuridine-5'-triphosphate (biotin-dUTP) (Bruckmann et al., 1999a), enabling the analysis of the role of BrdU in the formation of SCE. With this technique we have shown that X-rays induce ‘true’ SCE via radiation-induced damage to BrdU (Bruckmann et al., 1999b).
The present investigations were performed in order to study the role of BrdU in the induction of SCE by DNase I, AluI and MMC. Both DNase I and AluI have been shown to be effective inducers of SCE in CHO cells unifilarily labelled with BrdU and treated in G1 (Obe et al., 1994). The restriction enzyme AluI induces predominantly dsb (Roberts and Halford, 1993). DNase I induces single-strand breaks (ssb), which, when induced in close proximity, lead to dsb (Lutter, 1997).
Like ionizing radiation, AluI and DNase I induce chromosomal aberrations in an S phase-independent manner. Exposure in G1 phase leads to chromosome-type aberrations, such as polycentric chromosomes, and exposure in S phase leads mainly to chromatid-type aberrations, such as chromatid interchanges (Obe and Winkel, 1985; Folle et al., 1991; Obe et al., 1992, 1993, 1995). MMC is a DNA crosslinking agent (Iyer and Szybalski, 1963; Ishii, 1981; Borowy-Borowski et al., 1990) and exposure of cells in G1 phase leads to chromatid-type aberrations in the ensuing metaphase.
Here we describe results which show that following treatment with AluI, similar frequencies of SCE were observed in cells prelabelled with BrdU and biotin-dUTP. DNase I induced significantly more SCE in cells prelabelled with BrdU than with biotin-dUTP. MMC induced slightly more SCE in cells labelled with biotin-dUTP, but the difference was not significant.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Cell cultures and labelling with biotin-dUTP and BrdU
CHO-9 cells (obtained from A.T. Natarajan, Leiden, The Netherlands) were grown in Petri dishes (Greiner) and McCoy’s 5A medium (Gibco) supplemented with 10% fetal calf serum (Gibco) in the presence of 100 U/ml penicillin and 100 µg/ml dihydrostreptomycin sulphate (Gibco) at 37°C and 5% CO2.
For labelling with biotin-dUTP (Boehringer) and BrdU cells from stock cultures were subcultured for 34 h in 150 mm Petri dishes. Incorporation of biotin-dUTP was performed by electroporation principally, as described previously (Bruckmann et al., 1999a). In short, cells were trypsinized, washed once with sucrose buffer (272 mM sucrose, 7 mM KH2PO4, pH 7.4, 1 mM MgCl2) and suspended in 800 µl of sucrose buffer with 20 µM biotin-dUTP and 20 µM dTTP. Cells were electroporated with a Gene Pulser (BioRad, Germany) operating at 400 V and 25 µF. In order to enhance labelling with biotin-dUTP in G1 experiments with DNase I and AluI (Figure 1A) cells were electroporated twice, with an interval of 15 min, during which the cells remained in the electroporation buffer. After electroporation cells were kept in the cuvettes for 15 min, washed with medium and subcultured in 60 mm dishes for 14 h. All steps were performed at room temperature.
|
For labelling with BrdU (Sigma) and subsequent detection with monoclonal antibodies, cells were trypsinized, washed once with prewarmed medium and grown for 14 h in 60 mm dishes in the presence of 0.5 µM BrdU.
Treatment with AluI, DNase I and MMC
A diagrammatic scheme of the experiments is shown in Figure 1
. Treatments with AluI and DNase I were performed as described previously (Obe et al., 1994). Cells substituted with biotin-dUTP or BrdU were collected and electroporated with a Gene Pulser in 800 µl of sucrose buffer containing 40 U AluI (Gibco BRL; 10 U/µl specific activity) or 5000 U DNase I (Boehringer; 2000 U/mg), kept for 15 min at room temperature, washed with prewarmed medium (37°C) and recovered in 60 mm Petri dishes, in medium containing neither BrdU nor biotin-dUTP, for 24 h before harvest. Cells were treated either during G1 (24 h before harvest, Figure 1A) or S phase (12 h before harvest, Figure 1B). The latter treatment was performed only with cells prelabelled with BrdU.
For treatment with MMC (Sigma), medium containing biotin-dUTP or BrdU was replaced with fresh, unsubstituted medium and cells were incubated for 1 h with MMC at final concentrations of 0.1, 0.5 and 1.0 µM. Thereafter, cell monolayers were washed with PBS and recovered in fresh medium for 24 h.
In all experiments colcemid (0.08 µg/ml; Ciba Geigy) was added for the last 2 h of recovery. Cells were harvested according to a standard protocol.
The duration of labelling with biotin-dUTP and BrdU, as well as treatment times with the DNA-damaging agents, with respect to cell harvest were chosen on the basis of earlier observations to yield a maximum of cells in their second division. Furthermore, earlier experiments with X-rays revealed a predominance of chromosome-type aberrations in cells which were irradiated after a 14 h period of labeling with BrdU and which were harvested 20 h after irradiation (Bruckmann, 2000). Given this, it can be assumed that in experiments performed according to Figure 1A and C the analysed cells were treated in G1 phase of the cell cycle. Analogously, in experiments performed according to Figure 1B, cells were treated in the S phase of the cell cycle.
Detection of biotin-dUTP and BrdU
Metaphases labelled with biotin-dUTP or BrdU were detected as previously described (Bruckmann et al., 1999a,b). Biotin-dUTP was detected with TRITC-labelled avidin and biotin-labelled anti-avidin (Vector Laboratories Inc.). BrdU was detected with mouse anti-BrdU antibody and FITC-labelled goat anti-mouse antibody (CLB, The Netherlands). Cells were counterstained with DAPI (Serva) and analysed using a fluorescence microscope (Olympus) equipped with a CCD camera and filters for FITC, TRITC and DAPI. Computer images were obtained with the help of ISIS software (MetaSystems, Germany), enabling visualization of images in both single and overlaid colours.
In biotin-dUTP-labelled metaphases chromosomes X, Z10, Z12 and Z13 were excluded from analysis due to a generally incomplete labelling pattern (Bruckmann et al., 1999a,b). As these chromosomes comprise ~12% of the total chromosome length in CHO cells, SCE frequencies observed in biotin-labelled cells were divided by 0.88 in order to make the values comparable with those obtained with BrdU, in which all chromosomes were differentially labelled (Bruckmann et al., 1999a,b).
In the S phase experiments with DNase I and AluI (Figure 1B) cells in which the X chromosome was not always fully labelled with BrdU were also scored. This could possibly be a source of uncertainty of the estimated SCE frequencies, however, it is unlikely that it influenced the results significantly.
Scoring of SCE and statistical analyses
The analysis of SCE and aberrations was performed on-screen. In cells treated with DNase I and AluI heavily damaged cells (HDC) were encountered, with chromosomal aberrations and SCE frequencies >50. These cells were not considered in the SCE analysis, but their frequency was recorded. The frequencies of SCE were compared using the two-tailed t-test. The frequencies of cells with and without chromosomal damage were compared with Fisher’s exact test using a 2x2 contingency table.
The experiments with DNase I and AluI were performed twice (see Table I for the numbers of cells analysed). Three independent experiments were performed with MMC, with 30 cells scored per experiment.
|
Because we intended to compare the SCE-inducing activity of endonucleases in cells substituted with biotin-dUTP or BrdU, no concurrent controls were performed in the experiments with DNase I and AluI (Figure 1A and B
). However, we have shown previously with a similar methodology (with the exception that biotin-dUTP was electroporated once instead of twice as done here) that the baseline frequency of SCE is similar in cells electroporated with biotin-dUTP or exposed to BrdU (Bruckmann et al., 1999a,b). Hence, control values from one of these studies (Bruckmann et al., 1999b) are included in Figure 2.
|
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
In experiment A (Figure 1
) mainly chromosome type aberrations and in experiment B (Figure 1) chromatid type aberrations were found, indicating that cells were exposed to the endonucleases in G1 or S-phase of the cell cycle. In experiment C (Figure 1) mainly chromatid-type aberrations are seen, showing that MMC induces chromosomal aberrations dependent on S phase. The frequencies of cells with aberrations but not the single types of aberrations are given in Table I.
The frequencies of SCE in cells treated with DNase I and AluI are presented in Figure 2. Following treatment with DNase I a significantly higher frequency of SCE was observed in cells prelabelled with BrdU as compared with biotin-dUTP (Figure 2, upper); no such difference was seen with AluI (Figure 2, lower). A possible explanation of these results could be that following treatment with DNase I, the aberration frequency was higher in cells prelabelled with BrdU than with biotin-dUTP, resulting in more ‘false’ SCE. In order to test this, a comparative analysis was performed of the frequencies of cells with and without chromosomal aberrations (no attempt was made to enumerate the frequencies of aberrations because such an analysis would have been imprecise due to the fact that the cells were examined under a fluorescence microscope). The results are presented in Table I. Somewhat more damaged cells were indeed found following prelabelling with BrdU than with biotin-dUTP, however, this was true for both DNase I and AluI. Furthermore, the differences are not statistically significant. Thus, the higher SCE frequency observed in BrdU-labelled cells following DNase I treatment does not appear to result from ‘false’ SCE. On the other hand, significantly more SCE were found in cells with chromosomal aberrations than in those without, especially in experiments in which the cells were treated in the G1 phase of the cell cycle (Figure 2). This may be a reflection of the fact that the uptake of enzymes during electroporation is heterogeneous (Johannes and Obe, 1991). Hence, cells which took up the enzymes showed both enhanced frequencies of aberrations and SCE. No such profound differences were observed in cells treated in S phase (Figure 2A and B), although the per cent values of cells with chromosomal aberrations was only faintly lower than in the case of cells treated in G1 (Table I). These results indicate that both DNase I and AluI are effective inducers of SCE only when cells are treated in G1 and are in accordance with previously published data (Natarajan et al., 1985; Obe et al., 1994).
In order to examine whether the higher mean frequency of SCE in cells prelabelled with BrdU and treated with DNase I was representative of the whole cell population or was merely due to a few cells with high SCE numbers, the distributions of SCE were plotted. As can be seen in Figure 3A, the distributions of SCE in cells prelabelled with BrdU and biotin-dUTP differ, indicating that the higher mean frequency of SCE in cells prelabelled with BrdU is in fact representative for the whole cell population. It should be noted that the frequency of HDC (>50 SCE) is also higher in cells prelabelled with BrdU (Figure 3A). Obviously, incorporation of BrdU into DNA makes cells more susceptible to damage by DNase I leading to SCE, which is not the case for AluI (Figure 3B). No HDC were found in cells treated in S phase, supporting the concept that this cell cycle phase is relatively resistant to SCE induction by these endonucleases.
|
It was interesting to analyse the effect of BrdU and biotin-dUTP on SCE formation by MMC, an alkylating agent. The results are presented in Figure 4
. Following all treatments, slightly higher SCE frequencies were scored in cells prelabelled with biotin-dUTP, indicating that MMC was more effective in cells prelabelled with biotin-dUTP. However, a mechanistic interpretation of such an effect would be difficult. Furthermore, the difference was not significant at any MMC concentration. Also, the control SCE frequency in cells prelabelled with biotin-dUTP was somewhat higher than in cells prelabelled with BrdU (Figure 4). Therefore, we do not interpret the results as indicative of an enhancing effect of biotin-dUTP on the DNA-damaging efficiency of MMC.
|
Taken together, our results show that of the three tested compounds, only DNase I induced significantly more SCE in cells prelabelled with BrdU as compared with biotin-dUTP. Two possible mechanisms could probably account for this observation. The first one involves possible differences in steric hindrance that the biotin residue of dUTP may pose to DNase I, AluI and MMC. In contrast to AluI, which binds to the major groove of DNA, DNase I acts by interacting with the minor groove (Suck and Oefner, 1986
). It is known that biotinylated uridine is removed from DNA by the cells (Huijzer and Smerdon, 1992) and it can be hypothesized that the removal process is more effective in the major groove than in the minor groove of DNA. If that was so, DNase I would be hindered in interacting with DNA and AluI would not. Also, the hindrance could be less pertinent in the major than in the minor groove. Both phenomena should not apply to the alkylating agent MMC.
The alternative explanation relies on the fact that BrdU disintegrates spontaneously, leading to formation of highly reactive uracilyl radicals and ssb in the DNA (Morris, 1991). That this radical formation leads to SCE was shown in experiments in which cellular DNA was substituted to various degrees with BrdU: a positive correlation was observed between the extent of substitution and the frequency of spontaneous SCE (Natarajan and Mullenders, 1987). We have shown that radiation damage to BrdU is responsible for the formation of ‘true’ SCE in CHO cells (Bruckmann et al., 1999b). DNase I induces ssb which, when close together, lead to the formation of dsb (Lutter, 1997). It can be assumed that if a ssb induced by BrdU lies in close proximity to a ssb induced by DNase I, a dsb would be formed. In this sense, BrdU and DNase I would act synergistically. This assumes that dsb may be involved in SCE formation by S phase-independent agents. The fact that restriction enzymes, neocarcinostatin and bleomycin effectively induce SCE appears to support this (Natarajan et al., 1985; Obe et al., 1994; Schunck and Obe, 1995), however, the mechanism by which this occurs is not known. No synergistic effect of BrdU would be expected with the dsb-inducing agent AluI and the crosslinking agent MMC.
An interesting question in this context is why the type of DNA precursor used did not have any impact on the frequencies of cells with chromosomal aberrations following treatment with DNase I and AluI (Table I). At present no definite explanation can be given for that. As already mentioned, due to the fact that the analyses were performed under a fluorescence microscope no attempt was made to estimate the frequencies of aberrations. Given the strong overdispersion of aberrations following treatment with endonucleases, resulting probably from an inhomogeneous uptake of the enzymes (Johannes et al., 1989), the number of damaged cells may rather reflect the effectiveness of enzyme uptake than its DNA-damaging efficacy. It would be interesting to examine, with the help of chromosome painting, whether prelabelling of cells with biotin-dUTP or BrdU has a similar impact on the frequencies of aberrations as on SCE.
|
Acknowledgments |
|---|
We would like to thank Dr Wolfgang Goedecke for helpful discussions. Part of the data was used for the PhD thesis of E.B. This work was partly supported by a short-term EMBO fellowship to L.S. and a CSIR-DAAD fellowship to A.K.G.
|
Notes |
|---|
5 To whom correspondence should be addressed. Tel: 49 201 183 3388; Fax: 49 201 183; Email: guenter.obe{at}uni-essen.de
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
-
Borowy-Borowski,H., Lipman,R. and Tomasz,M. (1990) Recognition between mitomycin C and specific DNA sequences for cross-link formation. Biochemistry, 29, 2999–3004.[Medline]
Bruckmann,E. (2000) Mechanismen der Entstehung strahleninduzierter Schwesterchromatidenaustausche. PhD thesis, University of Essen, Germany.
Bruckmann,E., Wojcik,A. and Obe,G. (1999a) Sister chromatid differentiation with biotin-dUTP. Chromosome Res., 7, 185–189.[Web of Science][Medline]
Bruckmann,E., Wojcik,A. and Obe,G. (1999b) X-irradiation of G1 CHO cells induces SCE which are both true and false in BrdU-substituted cells but only false in biotin-dUTP-substituted cells. Chromosome Res., 7, 277–288.[Web of Science][Medline]
Dominguez,I., Pastor,N., Mateos,S. and Cortes,F. (2001) Testing the SCE mechanism with non-poisoning topoisomerase II inhibitors. Mutat. Res., 497, 71–79.[Web of Science][Medline]
Folle,G.A., Johannes,C. and Obe,G. (1991) Induction of chromosomal aberrations by DNase I. Int. J. Radiat. Biol., 59, 1371–1378.[Web of Science][Medline]
Huijzer,J.C. and Smerdon,M.J. (1992) Characterization of biotinylated repair regions in reversibly permeabilized human fibroblasts. Biochemistry, 31, 5077–5084.[Medline]
Ishii,Y. (1981) Nature of the mitomycin-C induced lesions causing sister-chromatid exchange. Mutat. Res., 91, 51–55.[Web of Science][Medline]
Iyer,V.N. and Szybalski,W. (1963) A molecular mechanism of mitomycin action: linking of complementary DNA strands. Proc. Natl Acad. Sci. USA, 50, 355–362.
Johannes,C. and Obe,G. (1991) Induction of chromosomal aberrations with the restriction endonuclease AluI in Chinese hamster ovary cells: comparison of different treatment methods. Int. J. Radiat. Biol., 59, 1379–1393.[Web of Science][Medline]
Johannes,C., Tuschy,S., Lamprecht,I. and Obe,G. (1989) The intercellular distributions of chromosomal aberrations induced by the restriction endonucleases Alu I and Bam HI in Chinese hamster ovary cells are overdispersed. Biol. Zentralbl., 108, 445–451.
Lambert,S. and Lopez,B.S. (2001) Role of RAD51 in sister-chromatid exchanges in mammalian cells. Ocogene, 20, 6627.
Latt,S. (1981) Sister chromatid exchange formation. Annu. Rev. Genet., 15, 11–55.[Web of Science][Medline]
Littlefield,L.G., Colyer,S.P., Joiner,E.J. and DuFrain,R.J. (1979) Sister chromatid exchanges in human lymphocytes exposed to ionizing radiation during G0. Radiat. Res., 78, 514–521.[Web of Science][Medline]
Lutter,L.C. (1997) Deoxyribonuclease I produces staggered cuts in the DNA of chromatin. J. Mol. Biol., 117, 53–69.
Morris,S.M. (1991) The genetic toxicology of 5-bromodeoxyuridine in mammalian cells. Mutat. Res., 258, 161–188.[Web of Science][Medline]
Mühlmann-Diaz,M.C. and Bedford,J.S. (1995) Comparison of gamma-induced chromosome ring and inversion frequencies. Radiat. Res., 143, 175–180.[Web of Science][Medline]
Natarajan,A.T. and Mullenders,L.H.F. (1987) Sister chromatid exchanges. In Obe,G. and Basler,A. (eds), Cytogenetics. Springer-Verlag, Berlin, Germany, pp. 339–344.
Natarajan,A.T., Mullenders,L.H.F., Meijers,M. and Mukherjee,M. (1985) Induction of sister-chromatid exchanges by restriction endonucleases. Mutat. Res., 144, 33–39.[Web of Science][Medline]
Obe,G. and Winkel,E.-U. (1985) The chromosome breaking activity of the restriction endonuclease Alu I in CHO cells is independent of the S-phase of the cell cycle. Mutat. Res., l52, 25–29.
Obe,G., Johannes,C. and Schulte-Frohlinde,D. (1992) DNA-double strand breaks induced by sparsely ionizing radiation and endonucleases as critical lesions for cell death, chromosomal aberrations, mutations and oncogenic transformation. Mutagenesis, 7, 3–12.
Obe,G., Johannes,C., Werthmann,I. and Folle,G.A. (1993) Chromatid-type aberrations induced by AluI in Chinese hamster ovary cells. Mutat. Res., 299, 305–311.[Web of Science][Medline]
Obe,G., Schunck,C. and Johannes,C. (1994) Induction of sister-chromatid exchanges by AluI, DNase I, benzon nuclease and bleomycin in Chinese hamster ovary (CHO) cells. Mutat. Res., 307, 315–321.[Web of Science][Medline]
Obe,G., Eke,P. and Johannes,C. (1995) Exposure of CHO cells to AluI: comparison of chromosomal aberrations and cell survival. Mutat. Res., 326, 171–174.[Web of Science][Medline]
Painter,R.B. (1980) A replication model for sister-chromatid exchange. Mutat. Res., 70, 337–341.[Web of Science][Medline]
Roberts,R.J. and Halford,S.H. (1993) Type II restriction endonucleases. In Linn,S.M., Lloyd,R.S. and Roberts,R.J. (eds), Nucleases. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 35–88.
Schunck,C. and Obe,G. (1995) Neocarzinostatin induces chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary cells. Mutagenesis, 10, 37–42.
Sonoda,E., Sasaki,M.S., Morrison,C., Yamaguchi-IwaI,Y., Takat,M. and Takeda,S. (1999) Sister chromatid exchanges are mediated by homologous recombination in vertebrate cells. Mol. Cell. Biol., 19, 5166–5169.
Suck,D. and Oefner,C. (1986) Structure of DNase I at 2.0 Å resolution suggets a mechanism for binding to and cutting DNA. Nature, 321, 620–625.[Medline]
Wojcik,A., Opalka,B. and Obe,G. (1999) Analysis of inversions and sister chromatid exchanges in chromosome 3 of human lymphocytes exposed to X-rays. Mutagenesis, 14, 633–637.
Wolff,S., Bodycote,J. and Painter,R.B. (1974) Sister chromatid exchanges induced in Chinese hamster cells by UV irradiation of different stages of the cell cycle: the necessity for cells to pass through S. Mutat. Res., 25, 73–81.[Web of Science][Medline]
Received on March 25, 2002; accepted on May 16, 2002.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||