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Nucleic Acids Research Nov 1986An immunological method was developed that isolates DNA fragments containing bromouracil in repair patches from unrepaired DNA using a monoclonal antibody that...
An immunological method was developed that isolates DNA fragments containing bromouracil in repair patches from unrepaired DNA using a monoclonal antibody that recognizes bromouracil. Cultured monkey cells were exposed to either UV light or the activated carcinogen aflatoxin B1 and excision repair of damage in DNA fragments containing the integrated and transcribed E. coli gpt gene was compared to that in the genome overall. A more rapid repair, of both UV and AFB1 damage was observed in the DNA fragments containing the E. coli gpt genes. The more efficient repair of UV damage was not due to a difference in the initial level of pyrimidine dimers as determined with a specific UV endonuclease. Consistent with previous observations using different methodology, repair of UV damage in the alpha sequences was found to occur at the same rate as that in the genome overall, while repair of AFB1 damage was deficient in alpha DNA. The preferential repair of damage in the gpt gene may be related to the functional state of the sequence and/or to alterations produced in the chromatin conformation by the integration of plasmid sequences carrying the gene.
Topics: Animals; Antibodies; Antigen-Antibody Complex; Base Sequence; Chlorocebus aethiops; DNA; DNA Damage; DNA Repair; Kidney; Plasmids; Ultraviolet Rays
PubMed: 3786142
DOI: 10.1093/nar/14.22.8979 -
Mutation Research Dec 1975DNA was isolated from Drosophila melanogaster larvae fed radioactive thymidine (TdR), bromodeoxyuridine (BUdR), or one of these nucleosides together with 5-fluorouracil...
DNA was isolated from Drosophila melanogaster larvae fed radioactive thymidine (TdR), bromodeoxyuridine (BUdR), or one of these nucleosides together with 5-fluorouracil (FU) under identical conditions. The four DNA samples designated T-DNA, B-DNA, T(F)-DNA, and B(F)-DNA respectively were hydrolyzed with formic acid-diphenylamine, and the pyrimidine oligonucleotides of various chain lengths (isotichs) were ractionated by DEAE-celluclose chromatography. The distributions of the labeled thymine and bromouracil (BU) residues among the pyrimidine isostichs were determined, and compared for the four DNAs. These comparisons indicated the following: (1) Similarity of T-DNA and T(F)-DNA with respect to labeled thymine distribution among the pyrimidine isostichs (using the mononucleotide to pentanucleotide tracts for statistical comparisons) showed that TdR entering DNA synthesis via the salvage pathway has the same distribution whether the synthetic pathway is operating normally or is inhibited by FU treatment. (2) Quantitative comparison of the distribution of BU among the isostichs of B-DNA and B(F)-DNA with the distribution of labeled thymine in T-DNA showed significant differences, indicating that the organism does not accept BUdR indiscriminately at all thymine sites for DNA synthesis. (3) Comparison of the distribution of BU in B-DNA and B(F)-DNA showed that isostichs 1 and 3 differ significantly. The elution profiles of the isostichs also differed qualitatively between these DNAs. This analysis confirms differential incorporation of BUdR into Drosophila DNA in the presence of a thymidylate inhibitor. The observations on BUdR incorporation in Drosophila DNA have been discussed with relation to the high frequency of somatic cell mutations induced in imaginal disc cells by treatment with the analog in the presence of inhibitors of thymidylate synthetase.
Topics: Animals; Bromouracil; DNA; Drosophila melanogaster; Larva; Mutation; Pyrimidine Nucleotides; Thymine Nucleotides
PubMed: 811984
DOI: No ID Found -
Journal of Bacteriology Aug 1966Gimlin, Dixie M. (Oklahoma State University, Stillwater), Sue D. Hardman, Betty N. Kelley, Grace C. Butler, and Franklin R. Leach. Effect of bromouracil-containing...
Gimlin, Dixie M. (Oklahoma State University, Stillwater), Sue D. Hardman, Betty N. Kelley, Grace C. Butler, and Franklin R. Leach. Effect of bromouracil-containing deoxyribonucleic acid on Bacillus subtilis. J. Bacteriol. 92:366-374. 1966.-Replacement of one-half of the thymine with bromouracil in Bacillus subtilis transforming deoxyribonucleic acid (DNA) resulted in a slight decrease in transforming activity, but, when used at high concentrations, this DNA preparation inhibited cell growth. Acid-hydrolyzed DNA, or addition of equivalent concentrations of the free base bromouracil in a transforming mixture, was without effect on cell growth. Treatment of the DNA preparation with deoxyribonuclease completely destroyed transforming activity and killing effect, whereas treatments with ribonuclease and trypsin were without effect on either transformation or killing activity. Growth of competent B. subtilis cells in test tubes was inhibited by high concentrations of both normal and bromouracil-containing DNA, with the bromouracil-containing DNA being significantly more inhibitory. This type of inhibition was also reflected in the time of division of the cells. The inhibitory effect was not due to viscosity, or to mutagenicity. The time course of killing paralleled transformation, and competency was required. These results can be interpreted as being due to uptake of homologous but imperfect DNA (containing bromouracil instead of thymine) by means of the systems involved in transformation, followed by either integration (resulting in lethal transformation, activation of a defective, nonlytic but lethal prophage) or interference with the recombination mechanism.
PubMed: 16562122
DOI: 10.1128/jb.92.2.366-374.1966 -
The Journal of Biological Chemistry Aug 2003Hypochlorous acid (HOCl), generated from H2O2 and Cl- by myeloperoxidase in activated neutrophils, causes tissue damage during inflammation. We have developed a simple,...
Hypochlorous acid (HOCl), generated from H2O2 and Cl- by myeloperoxidase in activated neutrophils, causes tissue damage during inflammation. We have developed a simple, sensitive (approximately 0.2 fmol on column) and specific GC-MS assay for the detection of 5-chlorouracil (5-ClUra), a signature product of HOCl-mediated damage to nucleobases. In this assay, 5-ClUra is released from isolated DNA by a digestion with nuclease P1, alkaline phosphatase, and thymidine phosphorylase (TP), or from chlorinated nucleosides in biological fluids by TP. The freed 5-ClUra is derivatized with 3, 5-bis-(trifluoromethyl)-benzyl bromide, which is detected by negative chemical ionization mass spectrometry. The assay can be used to simultaneously detect other halogenated uracils including bromouracil. Using this assay, we showed that 5-ClUra is generated by the reaction of low micromolar HOCl with (deoxy)cytidine, (deoxy)uridine, and DNA. In cell cultures, an increase of 5-ClUra was detected in DNA when cells were treated with sublethal doses of HOCl and allowed to proliferate. The elevation of 5-ClUra was markedly accentuated when physiologically relevant concentrations of (deoxy)uridine, (deoxy) cytidine, uracil, or cytosine were present in the medium during HOCl treatment. In the carrageenan-induced inflammation model in rats, chlorinated nucleosides was significantly increased, compared with controls, in the exudate fluid isolated from the inflammation site. Our study provides the direct evidence that chlorinated nucleosides are found in the inflammation site and can be incorporated in DNA during cell/tissue proliferation. These findings may be relevant to the carcinogenesis associated with chronic inflammation.
Topics: Alkaline Phosphatase; Animals; Biomarkers; Carrageenan; Cell Line; Chlorine; DNA; DNA Damage; Dose-Response Relationship, Drug; Electrons; Gas Chromatography-Mass Spectrometry; Hydrogen Peroxide; Hydrolysis; Hypochlorous Acid; Inflammation; Mice; Models, Chemical; Neutrophils; Nucleosides; Oxygen; Rats; Single-Strand Specific DNA and RNA Endonucleases; Thymidine Phosphorylase; Time Factors; Uracil
PubMed: 12810714
DOI: 10.1074/jbc.M304021200 -
FEBS Letters Oct 19987-Deazaxanthine (7DX) was identified as a novel inhibitor of thymidine (dThd) phosphorylase (TPase). It inhibited the TPase reaction in a concentration-dependent manner....
7-Deazaxanthine (7DX) was identified as a novel inhibitor of thymidine (dThd) phosphorylase (TPase). It inhibited the TPase reaction in a concentration-dependent manner. At 1 mM, it almost completely prevented the TPase-catalysed hydrolysis of dThd to thymine. The 50% inhibitory concentration (IC50 of 7DX was 40 microM in the presence of 100 microM of the natural substrate dThd. 7DX is also endowed with a marked inhibitory effect on angiogenesis. It significantly prevents neovascularisation in the chicken chorioallantoic membrane during development. 7DX is the first purine derivative shown to be a potent inhibitor of purified TPase and angiogenesis.
Topics: Allantois; Animals; Antibiotics, Antineoplastic; Binding Sites; Bromouracil; Chick Embryo; Chorion; Cyclohexanes; Dose-Response Relationship, Drug; Drug Design; Enzyme Inhibitors; Escherichia coli; Neovascularization, Physiologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Thymidine; Thymidine Phosphorylase; Thymine; Xanthines
PubMed: 9821965
DOI: 10.1016/s0014-5793(98)01271-x -
RNA (New York, N.Y.) Nov 2001We have determined the X-ray structures of six MS2 RNA hairpin-coat-protein complexes having five different substitutions at the hairpin loop base -5. This is a uracil...
We have determined the X-ray structures of six MS2 RNA hairpin-coat-protein complexes having five different substitutions at the hairpin loop base -5. This is a uracil in the wild-type hairpin and contacts the coat protein both by stacking on to a tyrosine side chain and by hydrogen bonding to an asparagine side chain. The RNA consensus sequence derived from coat protein binding studies with natural sequence variants suggested that the -5 base needs to be a pyrimidine for strong binding. The five -5 substituents used in this study were 5-bromouracil, pyrimidin-2-one, 2-thiouracil, adenine, and guanine. The structure of the 5-bromouracil complex was determined to 2.2 A resolution, which is the highest to date for any MS2 RNA-protein complex. All the complexes presented here show very similar conformations, despite variation in affinity in solution. The results suggest that the stacking of the -5 base on to the tyrosine side chain is the most important driving force for complex formation. A number of hydrogen bonds that are present in the wild-type complex are not crucial for binding, as they are missing in one or more of the complexes. The results also reveal the flexibility of this RNA-protein interface, with respect to functional group variation, and may be generally applicable to other RNA-protein complexes.
Topics: Adenine; Bromouracil; Capsid; Capsid Proteins; Guanine; Levivirus; Models, Molecular; Nucleic Acid Conformation; Protein Conformation; Pyrimidines; RNA, Viral; RNA-Binding Proteins; Thiouracil
PubMed: 11720290
DOI: No ID Found -
Planta Jul 2012We characterized three phases of Hyacinthus orientalis L. embryo sac development, in which the transcriptional activity of the cells differed using immunolocalization of... (Comparative Study)
Comparative Study
We characterized three phases of Hyacinthus orientalis L. embryo sac development, in which the transcriptional activity of the cells differed using immunolocalization of incorporated 5′-bromouracil, the total RNA polymerase II pool and the hypo- (initiation) and hyperphosphorylated (elongation) forms of RNA Pol II. The first stage, which lasts from the multinuclear stage to cellularization, is a period of high transcriptional activity, probably related to the maturation of female gametophyte cells. The second stage, encompassing the period of embryo sac maturity and the progamic phase, involves the transcriptional silencing of cells that will soon undergo fusion with male gametes. During this period in the hyacinth egg cell, there are almost no newly formed transcripts, and only a small pool of RNA Pol II is present in the nucleus. The transcriptional activity of the central cell is only slightly higher than that observed in the egg cell. The post-fertilization stage is related to the transcriptional activation of the zygote and the primary endosperm cell. The rapid increase in the pool of newly formed transcripts in these cells is accompanied by an increase in the pool of RNA Pol II, and the pattern of enzyme distribution in the zygote nucleus is similar to that observed in the somatic cells of the ovule. Our data, together with the earlier results of Pięciński et al. (2008), indicate post-fertilization synthesis and the maturation of numerous mRNA transcripts, suggesting that fertilization in H. orientalis induces the activation of the zygote and endosperm genomes.
Topics: Fertilization; Gene Expression Regulation, Plant; Hyacinthus; Ovule; RNA, Messenger; Seeds; Transcription, Genetic; Transcriptional Activation
PubMed: 22293855
DOI: 10.1007/s00425-012-1599-9 -
Proceedings of the National Academy of... Apr 1980Despite recent experiments showing that BrdUrd-induced mutagenesis can be independent of the level of bromouracil (BrUra) substitution [Kaufman, E.R. & Davidson, R.L....
Despite recent experiments showing that BrdUrd-induced mutagenesis can be independent of the level of bromouracil (BrUra) substitution [Kaufman, E.R. & Davidson, R.L. (1978) Proc. Natl. Acad. Sci. USA 75, 4982-4986; Aebersold, P.M. (1976) Mutat. Res. 36, 357-362], BrUra.G base mispairs are a major determinant of mutagenesis. We propose that the experiments cited above are sensitive predominantly to G . C leads to A . T transitions driven by the immeasurably small but highly mutagenic substitution of BrUra for cytosine and not by the gross substitution of BrUra for thymine in DNA. More generally, we show how accumulated evidence suggests that both BrdUrd and 2-aminopurine have two mutagenic effects intracellularly: perturbation of normal deoxyribonucleoside triphosphate pools and analogue mispairs in DNA. We propose a molecular basis for various observations of normal exogenous deoxyribonucleosides as synergists and counteragents to base analogue mutagenesis. A model is proposed to explain the antipolarity of BrdUrd and 2-aminopurine mutagenesis--i.e., why mutants at hot spots for induction by one base analogue are usually hot spots for reversion by the other. It is concluded that the configuration of the neighboring nucleotides surrounding the base analogue mispair, and not the base analogue's preference for inducing A . T leads to G . C or G . C leads to A . T errors, is responsible for the antipolarity of BrdUrd and 2-aminopurine mutagenesis.
Topics: 2-Aminopurine; Adenine; Adenosine Deaminase Inhibitors; Base Sequence; Bromodeoxyuridine; DNA; DNA Replication; Deoxyribonucleotides; Hydrogen Bonding; Mutagens; Structure-Activity Relationship
PubMed: 6929522
DOI: 10.1073/pnas.77.4.1801 -
Transcription of T7 DNA containing modified nucleotides by bacteriophage T7 specific RNA polymerase.The Journal of Biological Chemistry Jul 1978The interaction of bacteriophage T7 specific RNA polymerase with its cognate promoter sites has been probed by selectively replacing bases in one T7 promoter site with...
The interaction of bacteriophage T7 specific RNA polymerase with its cognate promoter sites has been probed by selectively replacing bases in one T7 promoter site with base analogs. Base analogs such as 2,6-diaminopurine or hypoxanthine, which alter residues appearing in the minor groove of the DNA helix, prevent utilization of the promoter by T7 RNA polymerase. These analogs do not affect transcription which starts outside of the modified region. In contrast, base analogs that have alterations that appear in the major groove of the DNA helix, such as uracil, 5-bromouracil, 5-methylcytosine, 5-hydroxymethylcytosine, and [5-HgSR]pyrimidines, do not prevent utilization of the promoter. The deoxyribonucleoside analog 5'-imino-5'-deoxythymidine, an alteration appearing in the deoxyribose-phosphodiester backbone of the DNA helix, does not prevent promoter recognition. Haemophilus aegyptius restriction endonuclease III, which cleaves DNA at the sequence 5'GGCC3', does not act at sites in which the guanine residues in one of the two DNA strands have been substituted with hypoxanthine. This implicates the guanine amino group in the minor groove of the DNA helix as a possible recognition point for this restriction endonuclease.
Topics: Base Sequence; Coliphages; DNA Repair; DNA Restriction Enzymes; DNA, Viral; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerases; Escherichia coli; Nucleic Acid Hybridization; Recombination, Genetic; Transcription, Genetic
PubMed: 353045
DOI: No ID Found -
The FEBS Journal Nov 20105-Bromodeoxyuridine (BrdU) modulates the expression of particular genes associated with cellular differentiation and senescence when incorporated into DNA instead of...
5-Bromodeoxyuridine (BrdU) modulates the expression of particular genes associated with cellular differentiation and senescence when incorporated into DNA instead of thymidine (dThd). To date, a molecular mechanism for this phenomenon remains a mystery in spite of a large number of studies. Recently, we have demonstrated that BrdU disrupts nucleosome positioning on model plasmids mediated by specific AT-tracts in yeast cells. Here we constructed a cognate plasmid that can form an ordered array of nucleosomes determined by an α2 operator and contains the BAR1 gene as an expression marker gene to examine BAR1 expression in dThd-auxotrophic MATα cells under various conditions. In medium containing dThd, BAR1 expression was completely repressed, associated with the formation of the stable array of nucleosomes. Insertion of AT-tracts into a site of the promoter region slightly increased BAR1 expression and slightly destabilized nucleosome positioning dependent on their sequence specificity. In medium containing BrdU, BAR1 expression was further enhanced, associated with more marked disruption of nucleosome positioning on the promoter region. Disruption of nucleosome positioning seems to be sufficient for full expression of the marker gene if necessary transcription factors are supplied. Incorporation of 5-bromouracil into the plasmid did not weaken the binding of the α2/Mcm1 repressor complex to its legitimate binding site, as revealed by an in vivo UV photofootprinting assay. These results suggest that BrdU increases transcription of repressed genes by disruption of nucleosome positioning around their promoters.
Topics: Antimetabolites; Aspartic Acid Endopeptidases; Blotting, Northern; Bromouracil; Gene Expression Profiling; Gene Expression Regulation, Fungal; Minichromosome Maintenance 1 Protein; Nucleosomes; Promoter Regions, Genetic; RNA, Messenger; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors; Transcription, Genetic
PubMed: 21040474
DOI: 10.1111/j.1742-4658.2010.07868.x