Induction of sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA under conditions of nucleotide-pool imbalance

The induction of sister-chromatid exchanges (SCEs) by the replication of 5-bromouracil(BrUra)-containing DNA under conditions of nucleotide-pool imbalance was investigated. A modification of a protocol developed for the induction of mutations under these conditions (E.R. Kaufman, Mol. Cell. Biol., 4, 2449-2454, 1984) was used. To induce SCEs, Chinese hamster ovary cells were grown under non-mutagenic conditions which allowed the uniform incorporation of BrUra into their DNA at specific levels of substitution for thymine residues (25, 50 and 75% BrUra substitution). After 4 and 5 days of growth, the cells, which had incorporated BrUra into their DNA, were washed free of 5-bromodeoxyuridine (BrdUrd) and provided with fresh culture medium supplemented with various concentrations of thymidine (10 microM to 3 mM) and no BrdUrd. The cells were allowed to replicate their BrUra-containing DNA under these conditions, in the absence of BrdUrd, for two rounds of DNA synthesis to achieve sister-chromatid differentiation, and second-division metaphases were scored for SCEs. The results of these studies indicated that the SCEs observed were proportional to the level of BrUra substituted for thymine in the cellular DNA, were induced by increasing concentrations of thymidine in the culture medium during replication of the BrUra-containing DNA, correlated well with the induction of mutations to thioguanine resistance and to ouabain resistance, correlated with increases in the intracellular levels of dTTP and dGTP generated by the high concentrations of thymidine. These findings provide direct evidence for the induction of SCEs by the replication of BrUra-containing DNA and for the importance of the pools of nucleoside triphosphate precursors for DNA replication in these processes. When the effects of 3-aminobenzamide, a potent inhibitor of poly(ADP-?ibose) synthesis, were tested, it was found that 3-aminobenzamide significantly increased SCEs, but it had no effect on mutations induced.     

Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells.

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.     

The role of deoxyribonucleotide metabolism in 5-bromo-2′-deoxyuridine mutagenesis in mammalian cells

The effects of deoxyribonucleoside triphosphate (dNTP) pool imbalance on the induction of mutations and siste-chromatid exchanges (SCEs) by 5-bromo-2′-deoxyuridine (BrdUrd) in mammalian cells is reviewed. The INC BrdUrd mutagenesis protocol involves the incorporation of BrdUrd into DNA under conditions of specific dNTP pool imbalance, while the REP BrdUrd mutagenesis protocol involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of specific (but different) dNTP pool imbalance. Biochemical and genetic analyses of both the INC and REP mutagenesis protocols provided evidence that (1) INC mutagenesis resulted from errors of incorporation due to the mispairing of BrdUTP with a guanine residue in replicating DNA leading to GC to AT transitions and (2) REP mutagenesis resulted from errors of replication due to the mispairing of dGTP with a BrURA residue in replicating DNA leading to AT to GC transitions. Further analyses involving different cell lines has led to an hypothesis describing the role of mismatch repair in the induction of mutations and SCEs.     

DNA synthesis in cytokinin-autotrophic tobacco cells

DNA isolated from various Nicotiana tabacum cell types, differing in their degree of hormone autotrophy and incubated in the presence of bromodeoxyuridine (BrdUrd), was analyzed by isopycnic CsCl gradient centrifugation. All cell types incorporate BrdUrd into DNA in such a way that hybrid DNA is formed with 60–80% of thymine (Thy) residues replaced by bromouracil (BrUra) in the newly synthesized strand. This DNA is not replicated further under ordinary culture conditions. Whereas in normal hormone-dependent cells this state is final and cells necrotize, in tumor (cytokinin-auxin autotrophic) and cytokinin-autotrophic cells a mechanism is induced leading to the reduction of BrUra content in DNA. As a result a decrease in the buoyant density (in CsCl) of BrUra DNA can be observed. In the case of cytokinin-autotrophic cells supplemented with kinetin, the buoyant density of the whole DNA decreases gradually to the value of that of unsubstituted DNA, but specific radioactivities of different DNA fractions reflect the retention of the pyrimidine ring of BrUra in DNA. This is interpreted as debromination of DNA in situ. The process can be inhibited by fluorodeoxyuridine (FdUrd) and deoxycytidine (dCyd). Moreover, FdUrd (but not dCyd) allows replication of hybrid DNA in tumor cells in such a way that HH DNA with all Thy residues replaced by BrUra is formed. For cytokinin-autotrophic cells FdUrd and kinetin are required. In hormone-dependent cells replication of hybrid DNA cannot be induced under any conditions. Most of these conclusions complement our previous findings that BrdUrd tolerance in hormone-autotrophic tobacco cells in hormone controlled. It is postulated that a modulation of thymidylate synthetase specificity is one factor affecting the level of BrUra substitution in DNA. The possibility of cytokinins being involved in the control of DNA synthesis is discussed.Abbreviations BrdUrd 5-bromo-2-deoxyuridine - BrUra 5-bromouracil - dCyd 2-deoxycytidine - FdUrd 5-fluoro-2-deoxyuridine - dThd thymidine - Thy thymine - EDTA Na₂-ethylenedia-minotetraacetate - IAA idole-3-acetic acid (auxin) - SDS Na-dodecylsulphate - LL, HL, HH DNA light-light (unsubstituted), heavy-light (unifilarly BrUra substituted), heavy-heavy (bifilarly BrUra substituted) DNAs, respectively     

Analysis of mutagenesis and sister-chromatid exchanges induced by 5-bromo-2′-deoxyuridine in somatic hybrids derived from Syrian hamster melanoma cells and Chinese hamster ovary cells

Somatic cell hybrids were derived from the fusion of Chinese hamster ovary (CHO) cells and Syrian hamster melanoma cells (2E). These two cell lines had previously been shown to differ in their response to the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2′-deoxyuridine (BrdUrd) (Kaufman, 1987). The parental cells and a number of representative, independent hybrid clones were tested for their response to both the INC and REP mutagenesis protocols. INC mutagenesis involves the incorporation of BrdUrd into DNA under conditions of deoxyribonucleoside triphosphate (dNTP) pool imbalance, while REP mutagenesis involves the replication of 5-bromouracil-substituted DNA in the presence of dNTP pool imbalance. When tested for the toxic effects of high concentrations of BrdUrd and for the induction of mutations by the INC protocol, the hybrid clones all expressed the 2E phenotype, i.e., sensitivity to relatively low concentrations of BrdUrd and thymidine for the induction of mutations, dNTP pool perturbation, and the toxic effects of BrdUrd. When the hybrid clones were tested for the induction of mutations and SCEs by the REP protocol, it was found that they expressed the 2E phenotype for the induction of mutations and the CHO phenotype for the induction of SCEs. Thus, various aspects of the 2E phenotype, such as high mutation frequencies associated with large dNTP pool perturbations, appeared to be dominantly expressed in the cell hybrids, while the lack of induction of SCEs by these mutagenic conditions in 2E cells was found to be a recessive characteristic.     

Analysis of mutagenesis and sister-chromatid exchanges induced by 5-bromo-2'-deoxyuridine in somatic hybrids derived from Syrian hamster melanoma cells and Chinese hamster ovary cells

Somatic cell hybrids were derived from the fusion of Chinese hamster ovary (CHO) cells and Syrian hamster melanoma cells (2E). These two cell lines had previously been shown to differ in their response to the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2'-deoxyuridine (BrdUrd) (Kaufman, 1987). The parental cells and a number of representative, independent hybrid clones were tested for their response to both the INC and REP mutagenesis protocols. INC mutagenesis involves the incorporation of BrdUrd into DNA under conditions of deoxyribonucleoside triphosphate (dNTP) pool imbalance, while REP mutagenesis involves the replication of 5-bromouracil-substituted DNA in the presence of dNTP pool imbalance. When tested for the toxic effects of high concentrations of BrdUrd and for the induction of mutations by the INC protocol, the hybrid clones all expressed the 2E phenotype, i.e., sensitivity to relatively low concentrations of BrdUrd and thymidine for the induction of mutations, dNTP pool perturbation, and the toxic effects of BrdUrd. When the hybrid clones were tested for the induction of mutations and SCEs by the REP protocol, it was found that they expressed the 2E phenotype for the induction of mutations and the CHO phenotype for the induction of SCEs. Thus, various aspects of the 2E phenotype, such as high mutation frequencies associated with large dNTP pool perturbations, appeared to be dominantly expressed in the cell hybrids, while the lack of induction of SCEs by these mutagenic conditions in 2E cells was found to be a recessive characteristic.     

Altered CTP synthetase activity confers resistance to 5-bromodeoxyuridine toxicity and mutagenesis

A V79 Chinese hamster fibroblast cell line selected for resistance to the toxic effects of 5-fluorouracil (Kaufman, 1984b) was found to be cross-resistant to the toxic effects of the thymidine analog 5-bromodeoxyuridine (BrdUrd). When tested for sensitivity to BrdUrd mutagenesis, the fluorouracil-resistant cells were found to be resistant to mutagenesis induced by high concentrations of BrdUrd in the medium (INC mutagenesis) but not to mutagenesis induced by the replication of DNA containing 5-bromouracil (REP mutagenesis). Analyses of deoxyribonucleoside triphosphate pools indicated that high endogenous dCTP levels in the mutant prevented the high BrdUTP/dCTP ratio associated with INC mutagenesis. However, the mutant phenotype had no effect on the nucleotide pool imbalance associated with REP mutagenesis. This mutant provides further genetic evidence for the existence of two independent mechanisms for BrdUrd mutagenesis in mammalian cells.     

Uncoupling of the induction of mutations and sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA

The REP mutagenesis protocol, which involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of deoxyribonucleoside triphosphate (dNTP) pool imbalance, has been shown to induce both mutations and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. However, when a Syrian hamster melanoma-derived cell line, called 2E, which was selected for its ability to replace all of the thymine residues in DNA with BrUra, was subjected to the REP mutagenesis protocol, the correlation between the induction of mutations and SCEs was no longer observed. The 2E cells were found to be much more sensitive to the induction of mutations by REP mutagenesis than were the CHO cells. This increased sensitivity to REP mutagenesis was found to correlate with increased perturbations of the dNTP pools that have been shown to be involved in the mutagenic mechanism of this protocol. In contrast, when the induction of SCEs by the REP protocol was measured, it was found that although a baseline level of SCEs was detected in 2E cells, no significant induction of SCEs due to dNTP pool perturbation was observed. It was shown that high levels of SCEs were readily induced in 2E cells by other agents, e.g. mitomycin C. A model, which discusses the fate of mismatched bases thought to be generated by the REP mutagenesis protocol as the determining factor for the induction of mutations of SCEs, is proposed to explain the uncoupling of mutagenesis and SCE induction in 2E cells.     

On the mechanism of 5-bromodeoxyuridine induction of prolactin synthesis in rat pituitary tumor cells

GH12C1, a clonal strain of rat pituitary tumor cells in culture (GH cells), does not produce detectable amounts of prolactin. 5-Bromodeoxyuridine (BrdUrd), the thymidine analogue, at sublethal concentrations (3-5 microgram/ml) induces prolactin synthesis in these cells. BrdUrd also induces prolactin synthesis in F1BGH12C1 cells, a BrdUrd resistant (BrdUrdr) substrain isolated from GH12C1 cells. The F1BGH12C1 strain is not drug dependent, but its resistance to BrdUrd is a stable phenotype. The significant features of the induction of prolactin synthesis in the BrdUrdr strain are the increased net synthesis of prolactin and the shortening of the lag period of prolactin induction. As BrdUrd concentration in the growth medium is increased, the rise in prolactin synthesis parallels the increased incorporation of BrdUrd into DNA. Prolactin synthesis is first detected when BrdUrd replaces 20-25% of the thymidine in DNA. BrdUrd can replace up to 75-80% of the thymidine within 2 d of treatment. Partial starvation of these cells under specified growth conditions does not affect the general growth pattern of the cells, general protein synthesis, and thymidine uptake, but does affect DNA synthesis. When cells are cultured under conditions in which DNA synthesis is preferentially inhibited, BrdUrd does not induce prolactin synthesis, suggestive of a DNA-mediated mechanism of action for the drug.     

Analysis of bromodeoxyuridine incorporation into DNA: comparison of gas chromatographic/mass spectrometric, CsCl gradient sedimentation, and specific radioactivity methods

A sensitive new method for the quantitation of 5-bromodeoxyuridine (BrdUrd) incorporated into DNA by GC/MS analysis of enzymatically released Thy and bromouracil (BrUra) is presented. The hydrolysis procedure was characterized and found to give uniform results when sample size was 1-10 micrograms DNA and incubation time for DNA digestion was between 40 min and 16 h. Samples of DNA containing 3H-labeled BrdUrd were analyzed in parallel by the GC/MS technique and by specific radioactivity and buoyant density measurements, in order to compare the three methods. The GC/MS procedure gave values for percentage replacement of Thy by BrUra which were higher than those obtained by specific activity and lower than those obtained by buoyant density. This GC/MS method can detect 1% replacement in a 1-microgram DNA sample, equivalent to approximately 10(5) cells or 0.1 mg tissue, and will permit sensitive and quantitative analysis of the presence of this chemotherapeutic agent/radiosensitizer in cellular DNA from biopsy samples of normal or tumor tissue.     

Uracil-DNA glycosylase causes 5-bromodeoxyuridine photosensitization in Escherichia coli K-12.

An Escherichia coli uracil-DNA glycosylase-defective mutant (ung-1 thyA) was more resistant than its wild-type counterpart (ung+ thyA) to the killing effect of UV light when cultured in medium containing 5-bromouracil or 5-bromo-2'-deoxyuridine (BrdUrd). The phenotype of resistance to BrdUrd photosensitization and the uracil-DNA glycosylase deficiency appeared to be 100% cotransduced by P1 phage. During growth with BrdUrd, both strains exhibited similar growth rates and 5-bromouracil incorporation into DNA. The resistant phenotype of the ung-1 mutant was observed primarily during the stationary phase. In cells carrying 5-bromouracil-substituted DNA, mutations causing resistance to rifampin and valine were induced by UV irradiation at a higher frequency in the wild type than in the ung-1 mutant. This Ung-dependent UV mutagenesis required UmuC function. These results suggest that the action of the uracil-DNA glycosylase on UV-irradiated 5-bromouracil-substituted DNA produces lethal and mutagenic lesions. The BrdUrd photosensitization-resistant phenotype allowed us to develop a new, efficient method for enriching and screening ung mutants.     

5-Azacytidine inhibits the induction of transient TK-deficient cells by 5-bromodeoxyuridine. A novel hypothesis for the facilitation of hypermethylation by 5-bromodeoxyuridine.

This paper examines the mechanism by which 5-bromodeoxyuridine (BrdUrd) induces a high frequency of transient trifluorothymidine (F3TdR)-resistant variants in the TK6 human lymphoblast cell line (a TK +/- heterozygote). This phenomenon has previously been termed 'pseudomutation' (Liber et al., 1985). We now report that 5-azacytidine (5-AzaC), an inhibitor of DNA methylation, reverses BrdUrd-induced pseudomutation in a dose-dependent manner. The inhibition by 5-AzaC is highly specific and does not appear to involve nucleotide pool perturbations. 5-AzaC inhibits the pseudomutagenic effect (transient trifluorothymidine resistance in a thymidine kinase heterozygote), but not the stable mutagenic effect (stable 6-thioguanine resistance or trifluorothymidine resistance in a hypoxanthine-guanine phosphoribosyltransferase-proficient cell) induced by BrdUrd. 5-AzaC did not affect the induction nor expression of mutation induced by several other chemical mutagens at either the tk or hgprt loci. Inhibition of pseudomutation by 5-AzaC did not appear to be caused by a number of potential confounding factors. Although significant changes in the levels of DNA methylation were detected by HPLC analysis in BrdUrd-treated cells, the dose response for inhibition of pseudomutation by 5-AzaC was correlated with a significant decrease in 5-methylcytidine levels. These results and additional data in the literature have led us to postulate a novel mechanism in which the substitution of BrdUrd in a TpG dinucleotide(s) may serve as a substrate for non-heritable methylation and hence transiently inactivate tk gene expression.     

Bromodeoxyuridine mutagenesis in mammalian cells is related to deoxyribonucleotide pool imbalance.

The relationship between bromodeoxyuridine (BrdUrd) mutagenesis in mammalian cells and the effects of BrdUrd on deoxyribonucleoside triphosphate pools was analyzed. It was found that the exposure of Syrian hamster melanoma cells to mutagenic concentrations of BrdUrd resulted in the formation of a large bromodeoxyuridine triphosphate (BrdUTP) pool, which remained at a high level for several days. In contrast, the size of the deoxycytidine triphosphate (dCTP) pool dropped rapidly after the addition of BrdUrd, reached a minimum at about 6 h, and then expanded gradually to nearly its original level over the next 3 days. The addition of lower concentrations of BrdUrd, which had less of a mutagenic effect, resulted in the formation of a smaller BrdUTP pool and a slightly smaller drop in the dCTP pool. When a high concentration of deoxycytidine was added at the same time as a normally mutagenic concentration of BrdUrd, the drop in the dCTP pool was prevented, as was BrdUrd mutagenesis. In all of these experiments, mutagenesis was related to the ratio of BrdUTP to dCTP in the cells. In addition, it was shown that mutagenesis occurred primarily during the first 24 h of BrdUrd exposure, when the BrdUTP/dCTP ratio was at its highest level. It appears that there is a critical ratio of BrdUTP to dCTP that must be attained for high levels of mutagenesis to occur and that the extent of mutagenesis is related to the ratio of the BrdUrd and dCTP pools.     

Mutational and pseudomutational effects of 5-bromodeoxyuridine in human lymphoblasts

We have studied the effects of 5-bromodeoxyuridine (BrdUrd) at two genetic loci in diploid human lymphoblast cells. In thymidine kinase heterozygotes (tk +/-), a 2-h dose of BrdUrd induced a transient, non-heritable resistance to the thymidine analogue, trifluorothymidine (F3TdR). We have called this phenomenon pseudomutation and have shown that affected cells acquire the ability to survive in the presence of F3TdR and then, after degradation of F3TdR in the medium, return to an apparently normal wild-type state. Our data suggest that BrdUrd incorporation into DNA as a thymidine analogue is responsible for the effect, which we interpret as a temporary loss of thymidine kinase activity. This effect is not seen in tk +/+ homozygotes. In contrast, at the hypoxanthine-guanine phosphoribosyl transferase locus in tk +/- heterozygotes, BrdUrd did not induce a permanent, heritable resistance to 6-thioguanine (gene locus mutation). We detected such mutations only in the tk +/+ homozygote and only at external BrdUrd concentrations considerably higher than those which saturate the uptake of BrdUrd into DNA as a thymidine analogue. We postulate that the reduced TK enzyme levels (30%) in the heterozygote prevent the build-up of a sufficiently high intracellular BrdUrd triphosphate pool to promote the misincorporations as deoxycytidine triphosphate which may be responsible for gene locus mutation.     

The mechanism of mutation induction by a hydrogen bond ambivalent, bicyclic N4-oxy-2'-deoxycytidine in Escherichia coli.

The triphosphate of the nucleoside deoxyribosyl dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is known to be incorporated into DNA efficiently by Taq polymerase and is a useful tool for polymerase-mediated in vitro mutagenesis. It is shown here that dP is a potent mutagen in Escherichia coli and Salmonella typhimurium . In E.coli , this deoxycytidine analog induces both GC-->AT and AT-->GC transitions. No induced transversions are observed. It is highly mutagenic in wild-type E.coli, but this is much reduced in a strain lacking thymidine kinase. Mutagenesis induced by dP is efficiently inhibited by the addition of thymidine. Partially purified thymidine kinase from E.coli catalyzes phosphorylation of dP to its 5'-monophosphate. When E.coli was grown in the presence of dP, the nucleoside analog was incorporated into its DNA. The content of dP in DNA was dependent on the concentration of dP added to the medium. The incorporation characteristics of the 5'-triphosphate of dP (dPTP) were also studied using E.coli DNA polymerase I large fragment. The results confirm that this triphosphate can be incorporated opposite A and G in the template with similar efficiencies. This indicates that dP is metabolized as a thymidine analog and that the resulting triphosphate induces a high rate of mutagenesis through replicational errors.     

Ribonucleotide reductase: A determinant of 5-bromodeoxyuridine mutagenesis in phage T4

Summary Mutagenesis by 5-bromodeoxyuridine (BrdUrd) can result from base-pairing errors either during replication of a BrdUrd-containing template or at the nucleotide incorporation step. Replication errors give rise predominantly to AT-to-GC transitions, while incorporation errors, in which 5-bromo-dUTP competes with dCTP at a template guanine site, should give rise to GC-to-AT transitions. The latter pathway should be sensitive to deoxyribonucleoside triphosphate (dNTP) pool fluctuations. Since dNTP pools are regulated through allosteric control of ribonucleotide reductase, the control of this enzyme should be a determinant of BrdUrd mutagenesis — if mutagenesis results largely from incorporation errors. Since T4 phage-encoded ribonucleotide reductase is insensitive to feedback inhibition, we established conditions under which phage DNA replication is dependent upon ribonucleotide reductase of the host, Escherichia coli. We examined BrdUrd mutagenesis of rII mutants known to revert to wild type either by AT-to-GC or GC-to-AT transition pathways. While both reversion pathways were stimulated under all conditions analyzed, the AT-to-GC pathway was stimulated more when the E. coli reductase was functioning, while the GC-to-AT pathway was more specifically enhanced when the T4 reductase was active. These results confirm that ribonucleotide reductase is a determinant of BrdUrd mutagenesis, but our observations, plus experiments showing that BrdUrd has relatively small effects upon dNTP pool sizes, indicate that the relationship between deoxyribonucleotide metabolism and BrdUrd mutagenesis is more complex than anticipated.     

In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe

Summary An in vivo 5-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%–20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.     

Analysis of bromodeoxyuridine-induced single and twin sister chromatid exchanges in tetraploid Chinese hamster ovary cells

Culture of cells in high exogenous levels (>10^–4 M) of bromodeoxyuridine (BrdUrd) or thymidine will increase the baseline sister chromatid exchange (SCE) frequency. The effect is thought to be related to the balance of the DNA precursors thymidine and deoxycytidine. Exogenous addition of deoxycytidine will reverse this effect. Single and twin SCEs were analysed in Colcemid-induced tetraploid Chinese hamster ovary cells exposed to different concentrations of BrdUrd to determine at what stage SCEs are induced by high levels of BrdUrd. In cells exposed to low concentrations of BrdUrd (10^–5 M), equal numbers of SCEs were induced in each of the two cell cycles. With increasing concentrations of BrdUrd (10^–4 to 2×10^–4 M), SCE frequency increased in both cell cycles, but far more SCEs were induced in the second cell cycle. Deoxycytidine (2×10^–4 M) reduced the frequency of SCEs primarily by reducing the frequency of SCEs induced in the second cell cycle. Treatment with 3-aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) polymerase, produced effects similar to exposure to high levels of BrdUrd including inducing SCEs in the second replication cycle. This suggests a similar mechanism of action. Deoxycytidine had no effect on 3AB-induced SCEs, however, and there was no interaction between 3AB and high exogenous levels of BrdUrd in SCE induction. Thus these two agents probably act through different mechanisms.     

Deoxycytidine reverses the suppression of pigmentation caused by 5-BrdUrd without changing the amount of 5-BrdUrd in DNA.

5-Bromodeoxyuridine (BrdUrd) suppresses pigmentation in Syrian hamster melanoma cells in vitro, and this suppression is reversed by the addition of deoxycytidine (dCyd) to the culture medium. Exogenous dCyd also decreases the incorporation of BrdUrd into nuclear DNA. Over a certain range of dCyd concentrations, however, increasing the concentration of dCyd does not lead to any significant further decrease in the level of BrdUrd incorporation. With 0.5 μM BrdUrd in the medium, increasing the dCyd concentration from 0.4 mM to 1.0 mM produces essentially no detectable change in the amount of BrdUrd incorporated. This is the range of dCyd concentrations, however, in which the addition of dCyd has the greatest effect in reversing the suppression of pigmentation caused by BrdUrd. The effects of dCyd on pigmentation are prevented by aminopterin, which blocks the conversion of dCyd to thymidine nucleotides. The results suggest that dCyd reverses the suppression of pigmentation caused by BrdUrd through a mechanism that does not involve changes in the amount of BrdUrd in nuclear DNA, and that the effect of dCyd on pigmentation depends upon the conversion of the exogenously supplied dCyd to thymidine nucleotides.     

Breakage of Parental DNA Strands in Haemophilus influenzae by 313 nm Radiation after Replication in the Presence of 5-Bromodeoxyuridine

Haemophilus influenzae was labeled with thymidine-³H (dThd), then grown in the presence of 5-bromodeoxyuridine (BrdUrd), and then irradiated with 313 nm light (a wavelength that selectively photolyzes DNA containing 5-bromouracil [BrUra]). Irradiation with 313 nm light induced breaks in the ³H-labeled strands in cells grown with BrdUrd at a much higher frequency than in ¹⁴C-labeled DNA of cells not exposed to BrdUrd. Breakage of the ³H-labeled strands was about 0.6% as efficient as that of fully BrUra-substituted DNA. During growth in the presence of BrdUrd, susceptibility to 313 nm-induced breakage of the ³H-labeled DNA strands increased, reaching a maximum in about one generation, and it decreased to zero during subsequent growth for one generation in medium containing dThd instead of BrdUrd. Heat denaturation of DNA extracted from dThd-³H-labeled cells grown in the presence of BrdUrd eliminated 313 nm-induced breakage of the ³H-labeled strands. It is concluded that breakage of the ³H-labeled DNA strands resulted from reaction with photoproducts in the base-paired, BrUra-containing strands, rather than from photolysis of BrdUrd incorporated into parental strands. It may be possible to utilize the phenomenon of interstrand breakage in physical studies of DNA replication.