Bromodeoxyuridine substitution in mammalian DNA can both stimulate and inhibit restriction cleavage

Total replacement of thymidine by 5-bromodeoxyuridine in mammalian DNA causes a 5-fold stimulation in the rate of DNA cleavage by Mbo I. This is the first report of the stimulation of restriction endonuclease activity by 5-bromodeoxyuridine and is in contrast to the inhibition found with other restriction enzymes. We propose a hypothesis to rationalize these results.     

Effect of nucleotide analogs on the cleavage of DNA by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI

The cleavage of specific DNA sequences by the restriction endonucleases AluI, DdeI, HinfI, RsaI, and TaqI has been studied by monitoring the effect of various nucleotide modifications on the rate of DNA digestion. Bacteriophage fd DNA was completely substituted in one strand with a single nucleotide analog, using an in vitro primed DNA synthesis reaction on a single-stranded viral DNA template. Twelve deoxynucleotide analogs were incorporated into these DNA substrates: 2-aminopurine, 2,6-diaminopurine, deoxytubercidin, deoxyuridine, 5-bromodeoxyuridine, 5-allylamine deoxyuridine, 5-biotinyl deoxyuridine, deoxypseudouridine, deoxyinosine, 8-azadeoxyguanosine, 5-iododeoxycytidine, and 5-bromodeoxycytidine. The restriction enzymes tested varied considerably in their ability to digest hemi-substituted DNAs containing these modified nucleotides. Structural alterations in the base pairs immediately adjacent to the phosphodiester bonds cleaved by the enzyme reduced the rate of enzyme activity most dramatically, and in most cases more than a single determinant on each base pair altered activity. Interactions with nucleotides outside the recognition site seem to have little importance in the binding or catalytic activity of these enzymes.     

Different mechanisms for the induction of acetylcholinesterase in neuroblastoma cells.

Neuroblastoma cells, incliding clones selected for resistance to dibutyryl-cAMP or for their ability to survive and multiply at 40°C, were used to study differences in the induction of acetylcholinesterase activity by dibutyryl-cAMP and 5-bromodeoxyuridine. In nonselected neuroblastoma cells both of these compounds induced this enzyme activity. Actinomycin D inhibited induction by 5-bromodeoxyuridine but did not inhibit inducion by dibutyryl-cAMP. Enzyme activity in dibutyryl-cAMP-resistant cells was induced by 5-bromodeoxyuridine and not by dibutyryl-cAMP. In the temperature-resistant cells, induction by 5-bromodeoxyuridine was lower at 40 than at 37°C and induction by dibutyryl-cAMP was higher at 40 than at 37°C. This difference in induction at the two temperatures was associated with a higher inhibition of cell multiplication at 40°C by both compounds. The results indicate that 5-bromodeoxyuridine and dibutyryl-cAMP induce acetylcholinesterase activity in neuroblastoma cells by different mechanisms.     

Effect of 5-bromodeoxyuridine on the appearance of the liver isoenzyme of pyruvate kinase

Hepatocyte cultures derived from 15-day foetal rats produce the liver form of pyruvate kinase (EC 2.7.1.40) only after 3 days of culture. The appearance of the liver form of the enzyme can be blocked by the addition of 5-bromodeoxyuridine on day 2 of culture, but not by addition on day 3 of culture. The reversibility of the action of 5-bromodeoxyuridine was shown when the inhibitor was added on day 2 and removed on day 4. By day 6 of culture the liver form of pyruvate kinase was detectable. The specificity of the action of 5-bromodeoxyuridine was monitored by following changes in the closely related embryonic form of the enzyme as a control. This was unaltered by the inhibitor.     

Superoxide dismutase activity in nerve cell culture

Superoxide dismutase (EC 1.15.1.1) activity was assayed in preparations obtained from several clonal lines of nerve cell culture, by enzymatic and nonenzymatic assays. The enzyme was found in dialyzed homogenates of washed cells and in partially purified fractions. The enzyme was also found in cells which had been grown in media containing 5-bromodeoxyuridine, where cell differentiation was observed.     

Resistance to mutagenesis of cells biochemically transformed by herpesvirus DNA fragments

LM(TK-) mouse fibroblast cells that were biochemically transformed to the dThd kinase-positive phenotype by restriction nuclease fragments of herpes simplex virus or marmoset herpesvirus DNA, all of which contained the virus dThd kinase coding region, or by HeLa S3 DNA were more resistant to mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine or 5-bromodeoxyuridine than were dThd kinase-positive LM and LM(TK-) cells. Measurements of dNTP pool sizes did not reveal relative imbalances for representative cell lines under several conditions of growth.     

Role of 5-bromodeoxyuridine in the formation of sister chromatid exchanges in human lymphocytes exposed to gamma-irradiation at the presynthetic stage of the mitotic cycle

The formation of SCE was studied in human lymphocytes irradiated at the presynthetic stage of the cell cycle in the presence or absence of 5-bromodeoxyuridine. The results obtained showed that 5-bromodeoxyuridine present in the culture medium at the time of exposure or during the postirradiation period increased the yield of SCE. It is suggested that 5-bromodeoxyuridine exerts its effect through interfering with DNA repair.     

Random Segregation of Chromatids at Mitosis in Saccharomyces Cerevisiae

Previous experiments suggest that mitotic chromosome segregation in some fungi is a nonrandom process in which chromatids of the same replicative age are destined for cosegregation. We have investigated the pattern of chromatid segregation in Saccharomyces cerevisiae by labeling the DNA of a strain auxotrophic for thymidine with 5-bromodeoxyuridine. The fate of DNA strands was followed qualitatively by immunofluorescence microscopy and quantitatively by microphotometry using an anti-5-bromodeoxyuridine monoclonal antibody. Chromatids of the same replicative age were distributed randomly to daughter cells at mitosis. Quantitative measurements showed that the amount of fluorescence in the daughter nuclei derived from parents with hemilabeled chromosomes diminished in intensity by one half. The concentration of 5-bromodeoxyuridine used in the experiments had little effect on the frequency of either homologous or sister chromatid exchanges. We infer that the 5-bromodeoxyuridine was distributed randomly due to mitotic segregation of chromatids and not via sister chromatid exchanges.     

Characterization of the thymidine kinase of Physarum polycephalum

Thymidine kinase [ATP: thymidine 5'-phosphotransferase, EC 2.7.1.21] has been purified more than 3,500 fold from microplasmodia of Physarum polycephalum. Properties of the enzyme were determined on preparations purified 1,400 fold. Thymidine was transformed to dTMP while a stoichiometric quantity of ATP was transformed to ADP. 5-Iododeoxyuridine, 5-bromodeoxyuridine, and 5-fluorodeoxyuridine acted as competitive inhibitors for the thymidine substrate while 5-bromodeoxyuridine could be used as a substrate. In contrast uridine did not inhibit the enzymatic activity while deoxyuridine was a very poor competitive inhibitor in agreement with the observation that deoxyuridine could not be used as a substrate. Two apparent Michaelis constants were found for thymidine. Only the highest Michaelis constant could be decreased in the presence of increasing concentrations of ATP. Among the various nucleoside mono, di, or triphosphates studied only ATP and to a less extent dATP could be used as phosphate donors. A non competitive inhibition for thymidine was observed with dTTP. dTMP, dTDP, and dTTP acted as competitive inhibitors for ATP. None of the nucleoside mono, di, or triphosphates studied showed an activatory effect at low concentrations of ATP, even in the presence of dTTP. However, dUTP and dGDP acted as competitive inhibitors for ATP.     

Effect of 5-bromodeoxyuridine on incorporation of RNA precursors in sea urchin embryos

Exposure of early sea urchin embryos to 5-bromodeoxyuridine (at concentrations up to 100 μg per ml) severely decreases the uptake of exogenous ³H-uridine into RNA. However, the actual gross rate of DNA or RNA synthesis in these embryos appears not to be affected by the presence of 5-bromodeoxyuridine.     

Two Phases in Adventitious Root Formation in Phaseolus mungo Hypocotyl Cuttings, a Phase Sensitive to an Inhibitor of RNA or Protein Synthesis and a Phase Sensitive to an Inhibitor of DNA Synthesis

The development of adventitious roots in Phaseolus mungo cuttingswas inhibited by 2-thiouracil, cycloheximide, and 5-bromodeoxyuridine.The stage of rooting blocked by 2-thiouracil and cycloheximidewas different from that blocked by 5-bromodeoxyuridine. Somecell division in the basal rooting region occurred with 5-bromodeoxyuridine,but not with 2-thiouracil and cycloheximide. Radioactivity from labelled 2-thiouracil appeared in RNA fractionsbut the amount was reduced by simultaneously applied uracil.5-Bromodeoxyuridine inhibited incorporation of thymidine intoDNA fractions. 2-Thiouracil and 5-bromodeoxyuridine act as antimetabolitesof uracil and thymidine, respectively. Cycloheximide, an inhibitorof protein synthesis, prevented the incorporation of radioactivityfrom labelled leucine into the trichloroacetic acid-insolublefraction. RNA synthesis inhibitors (2-thiouracil and actinomycin D) andprotein synthesis inhibitors (cycloheximide and blasticidinS) increased roots effectively when dosed at the beginning ofincubation. Inhibitors of DNA synthesis (5-bromodeoxyuridineand mitomycin C) were effective when applied after several hours'pre-incubation in water. It is suggested that there are at leasttwo phases in adventitious root formation, a phase sensitiveto an inhibitor of RNA or protein synthesis and a phase sensitiveto an inhibitor of DNA synthesis.     

ELISA detection of restriction site polymorphisms in the pig ryanodine receptor locus

We compare two strategies for ELISA detection of restriction site polymorphisms (EDRSP) that are suitable for high-throughput genotyping of the pig ryanodine receptor point mutation (RYR1 ^hal ). In both procedures, target DNA is amplified by PCR with one primer that is 5′ biotinylated and a second primer that is 5′ fluoresceinylated. PCR products are captured in duplicate wells on a streptavidin-coated, 96-well plate. The duplicates may be treated in two ways. In a single restriction enzyme assay, one duplicate is exposed to a restriction enzyme that cuts one allele specifically, and the second duplicate is exposed to no restriction enzyme. In a dual restriction enzyme assay, the second replicate is exposed to a second restriction enzyme that cuts the alternate allele specifically. Thereafter, the two procedures are similar; anti-fluorescein antibodies conjugated to peroxidase are allowed to bind to the fluoresceinylated ends, the plate is washed, and a substrate is converted to a colored end product. The ratio of the absorbances in the two wells is used to classify subjects by genotype. When the dual restriction enzyme assay is run, three genotype groups are easily distinguishable. When the single restriction enzyme assay is run, heterozygotes generate values that may overlap with those of the homozygotes that are not cut by the restriction enzyme. Dual restriction enzyme assays are more accurate than single restriction enzyme assays; however, single restriction enzyme assays are sufficient for identifying pigs that carry RYR1 ^hal . Received: 30 December 1997 / Accepted: 20 April 1998     

5-Bromodeoxyuridine suppresses position effect variegation of transgenes in HeLa cells

An ectopic gene integrated in the host genome is occasionally silenced due to a position effect of its adjacent chromatin structure. We found that 5-bromodeoxyuridine clearly activated such a transgene in HeLa cells. The transgene was also activated to various degrees by inhibitors of histone deacetylase, DNA topoisomerases, or DNA methyltransferase. The peptide antibiotic distamycin A potentiated markedly the effect of 5-bromodeoxyuridine. Transient expression of an artificial AT-hook protein termed MATH20 also potentiated its effect although significantly activated the transgene alone. Since distamycin A and MATH20 are able to displace histone H1 and other DNA-binding proteins bound to specific AT-rich sequences by a dominant, mutually exclusive fashion, these results suggest that 5-bromodeoxyuridine targets such an AT-rich sequence located adjacent to the silenced transgene, resulting in chromatin accessibility.     

Variation of thymidine incorporation patterns in the alternating vegetative and sexual life cycles of Chlamydomonas reinhardtii

The cellular distribution of thymidine kinase activities in Chlamydomonas reinhardtii, as manifested by the in vivo incorporation of exogenous thymidine and 5-bromodeoxyuridine into different DNA species, appeared to be organelle specific and varied with different developmental stages in the life cycle of this organism. During vegetative growth and gametogenic differentiation, thymidine and 5-bromodeoxyuridine were shown to be selectively incorporated into chloroplast but not nuclear DNA. On the other hand, during zygotic germination in which meiosis occurs and the ensuing vegetative divisions of meiotic products, thymidine as well as 5-bromodeoxyuridine were incorporated into both nuclear and chloroplast DNA. These results suggest that, in addition to the thymidine kinase activity that is constantly present in the chloroplast, a cytoplasmic thymidine kinase is derepressed only during the sexual reproductive cycle of C. reinhardtii.     

Isolation of Mutants of Bacteriophage T4 Unable to Induce Thymidine Kinase Activity

New mutants of T4 have been isolated by using a strain of Escherichia coli lacking thymidine kinase activity. These T4 mutants, designated tk, are able to grow on this E. coli strain under light on plates containing 5-bromodeoxyuridine and were all found to be unable to induce thymidine kinase (ATP: thymidine 5'-phosphotransferase, EC 2.7.1.21). All of these tk mutants fall into one complementation group which maps just to the right of rI on the standard T4 genetic map, far from most other genes coding for enzymes involved in pyrimidine metabolism. The tk mutants grow as well as wild-type T4, indicating that thymidine kinase is a non-essential enzyme.     

Increases in the number of adventitious roots caused by 2-thiouracil and 5-bromodeoxyuridine in Phaseolus mungo cuttings

A cutting of Phaseolus mungoproduced about 4 adventitious rootsat the basal 1 mm region when the basal part of the cuttingwas dipped in water. Rootlets became visible after a 36 hr lagperiod in untreated cuttings. Treatment with 2-thiouracil or5-bromodeoxyuridine increased the number of roots formed onthe cutting and prolonged the lag period. Effects of 2-thiouraciland 5-bromodeoxyuridine were reversed by simultaneously applieduracil and thymidine. The number of roots decreased and thelength of lag period was shortened. The increases in the numberof roots by 2-thiouracil or 5-bromodeoxyuridine was reducedby gibberellic acid, which did not cause a decrease in the numberof roots to be formed on control cuttings. Roots formed at thebasal region seem to suppress further root formation at theupper part of the hypocotyl. Inhibitors used here probably workby blocking the formation of these bottommost roots. (Received April 30, 1971; )     

5-Iododeoxyuridine as well as 5-bromodeoxyuridine induce the formation of specific dicentric chromosomes in cells with micronuclei

5-iododeoxyuridine used instead of 5-bromodeoxyuridine for the induction of delayed disruption of telomeric links between chromosomes in polykaryocytes consisting of micronuclei was studied. It has been shown that treatment with 5-iododeoxyuridine (20 micrograms/ml) and colcemide (0.1 microgram/ml) for 42 hours induced the dicentric formation. Dicentrics were tested in 27% of all metaphases. This index was lower in comparison with 5-bromodeoxyuridine used for the same purpose.     

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. VI. The binding and cleavage of substrates containing nucleotide analogs.

The present study deals with the binding and cleavage by EcoRII endonuclease of concatemer DNA duplexes containing EcoRII recognition sites (formula; see text) in which dT is replaced by dU or 5-bromodeoxyuridine, or 5'-terminal dC in the dT-containing strand is methylated at position 5. The enzyme molecule is found to interact with the methyl group of the dT residue of the DNA recognition site and to be at least in proximity to the H5 atom of the 5'-terminal dC residue in dT-containing strand of this site. Modification of any of these positions exerts an equal effects on the cleavage of both DNA strands. Endonuclease EcoRII was found to bind the substrate specifically. At the same time modification of the bases in recognized sequence may result in the formation of unproductive, though stable, enzyme-substrate complexes.