-
Journal of Bacteriology Feb 1969The replication of the Salmonella typhimurium chromosome was studied. As with E. coli 15T(-), replication was sequential. After amino acid starvation, replication...
The replication of the Salmonella typhimurium chromosome was studied. As with E. coli 15T(-), replication was sequential. After amino acid starvation, replication proceeded from a unique and heritable region of the chromosome. 5-Bromouracil, when substituted for thymine, did not disturb the sequence of replication nor did it initiate extra replication cycles. By labeling the origin and the terminus of the chromosome with (3)H- and (14)C-thymine, respectively, it was possible to determine that the rate of chain elongation decreases as the growth rate decreases. No gap in the replication cycle could be observed.
Topics: Amino Acids; Autoradiography; Carbon Isotopes; Centrifugation, Density Gradient; Chromosomes, Bacterial; Conjugation, Genetic; DNA Replication; DNA, Bacterial; Escherichia coli; Nitrogen; Radioisotopes; Salmonella typhimurium; Thymine; Tritium; Uracil
PubMed: 4886297
DOI: 10.1128/jb.97.2.848-860.1969 -
The Journal of Biological Chemistry Jun 1994The synthesis of thymidylate synthase (TS) from 5-fluorouracil (FUra)- and 5-bromouracil (BrUra)-substituted mRNAs was examined to investigate the effect of...
The synthesis of thymidylate synthase (TS) from 5-fluorouracil (FUra)- and 5-bromouracil (BrUra)-substituted mRNAs was examined to investigate the effect of incorporation of uracil (Ura) analogs on translation. Human TS cDNA was transcribed in the presence of Ura-, FUra-, or BrUTP to obtain 100% substituted mRNA. The mRNAs were translated in a rabbit reticulocyte lysate system. The TS protein that was formed from each of the templates reacted identically with TS antibody in Western blots. Time courses of TS formation revealed a characteristic peak which occurred at 45 min for the Ura- and FUra-RNAs and at 2 h for the BrUra-RNA. Substitution of Ura with FUra did not alter the rate of translation, while substitution of BrU for Ura decreased the rate of translation. Substitution of Ura with FUra or BrUra enhanced the stability of the mRNAs in the rabbit reticulocyte lysate by 3- and 10-fold, respectively. Incorporation of BrUra influenced the binding and catalysis on the ribosome, resulting in a 3.5-fold greater rate of activation (Kact) and 6-fold lower Vmax than the equivalent values for the Ura- and FUra-substituted mRNAs. Nondenaturing gel electrophoresis revealed that different conformations exist among the mRNAs. These data show that translation can be influenced by the incorporation of fraudulent bases into mRNA and those bases that stabilize RNA secondary structure will have the greatest inhibitory effect on translation.
Topics: Base Sequence; Bromouracil; Cell-Free System; Fluorouracil; Humans; Kinetics; Molecular Sequence Data; Protein Biosynthesis; RNA, Messenger; Reticulocytes; Structure-Activity Relationship; Thymidylate Synthase; Transcription, Genetic
PubMed: 8206932
DOI: No ID Found -
Heliyon Mar 2023A rapid, specific and accurate high-performance liquid chromatography with tunable ultraviolet detection method was developed to simultaneously determine azathioprine...
A rapid, specific and accurate high-performance liquid chromatography with tunable ultraviolet detection method was developed to simultaneously determine azathioprine metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methyl mercaptopurine riboside (6-MMPr) in human red blood cells. Erythrocyte lysate sample was precipitated by perchloric acid under the protection of dithiothreitol, with 6-TGN and 6-MMPr being acid hydrolyzed to produce 6-thioguanine (6-TG) and 6-methymercaptopurine (6-MMP). A Waters Cortecs C column (2.1 × 150 mm, 2.7 μm) was used for chromatographic separation with a water (containing 0.01 mol/L ammonium acetate and 0.2% acetic acid)/methanol linear gradient at a flow rate of 0.45 mL/min in a 5.5 min. UV detection wavelengths were 340 nm for 6-TG, 303 nm for 6-MMP and the IS (5-bromouracil). The calibration curves fitted a least squares model (weighed 1/ ) from 0.15 to 15 μmol/L for 6-TG ( = 0.9999) and from 1 to 100 μmol/L for 6-MMP ( = 0.9998). This method was validated according to the FDA bioanalytical method validation guidance and ICH M10 bioanalytical method validation and study sample analysis guidance for industry, and successfully utilized in ten IBD patients receiving azathioprine therapy.
PubMed: 36895397
DOI: 10.1016/j.heliyon.2023.e13870 -
RNA Biology Oct 2012Changing the abundance of transcripts by regulated RNA degradation is a critical step in the control of various biological pathways. Recently, genome-wide inhibitor-free... (Review)
Review
Changing the abundance of transcripts by regulated RNA degradation is a critical step in the control of various biological pathways. Recently, genome-wide inhibitor-free technologies for determining RNA stabilities in mammalian cells have been developed. In these methods, endogenous RNAs are pulse labeled by uridine analogs [e.g., 4-thiouridine (4sU), 5-etyniluridine (EU) and 5'-bromo-uridine (BrU)], followed by purification of labeled de novo RNAs. These technologies have revealed that the specific half-life of each mRNA is closely related to its physiological function. Genes with short-lived mRNAs are significantly enriched among regulatory genes, while genes with long-lived mRNAs are enriched among housekeeping genes. This review describes the recent progress of experimental procedures for measuring RNA stability.
Topics: Alpha-Amanitin; Animals; Bromouracil; Dactinomycin; Eukaryotic Cells; Genes, Essential; Genes, Regulator; Half-Life; Humans; Nucleic Acid Synthesis Inhibitors; RNA Stability; RNA, Messenger; Thiouridine; Transcription, Genetic; Uracil; Uridine
PubMed: 23034600
DOI: 10.4161/rna.22036 -
Nucleic Acids Research Sep 2018Double-stranded RNA (dsRNA) structures form triplexes and RNA-protein complexes through binding to single-stranded RNA (ssRNA) regions and proteins, respectively, for...
Double-stranded RNA (dsRNA) structures form triplexes and RNA-protein complexes through binding to single-stranded RNA (ssRNA) regions and proteins, respectively, for diverse biological functions. Hence, targeting dsRNAs through major-groove triplex formation is a promising strategy for the development of chemical probes and potential therapeutics. Short (e.g., 6-10 mer) chemically-modified Peptide Nucleic Acids (PNAs) have been developed that bind to dsRNAs sequence specifically at physiological conditions. For example, a PNA incorporating a modified base thio-pseudoisocytosine (L) has an enhanced recognition of a G-C pair in an RNA duplex through major-groove L·G-C base triple formation at physiological pH, with reduced pH dependence as observed for C+·G-C base triple formation. Currently, an unmodified T base is often incorporated into PNAs to recognize a Watson-Crick A-U pair through major-groove T·A-U base triple formation. A substitution of the 5-methyl group in T by hydrogen and halogen atoms (F, Cl, Br, and I) causes a decrease of the pKa of N3 nitrogen atom, which may result in improved hydrogen bonding in addition to enhanced base stacking interactions. Here, we synthesized a series of PNAs incorporating uracil and halouracils, followed by binding studies by non-denaturing polyacrylamide gel electrophoresis, circular dichroism, and thermal melting. Our results suggest that replacing T with uracil and halouracils may enhance the recognition of an A-U pair by PNA·RNA2 triplex formation in a sequence-dependent manner, underscoring the importance of local stacking interactions. Incorporating bromouracils and chlorouracils into a PNA results in a significantly reduced pH dependence of triplex formation even for PNAs containing C bases, likely due to an upshift of the apparent pKa of N3 atoms of C bases. Thus, halogenation and other chemical modifications may be utilized to enhance hydrogen bonding of the adjacent base triples and thus triplex formation. Furthermore, our experimental and computational modelling data suggest that PNA·RNA2 triplexes may be stabilized by incorporating a BrUL step but not an LBrU step, in dsRNA-binding PNAs.
Topics: Base Pairing; Bromouracil; Cell Line, Tumor; Computational Biology; Computer Simulation; Halogenation; Halogens; HeLa Cells; Humans; Hydrogen Bonding; Inverted Repeat Sequences; MicroRNAs; Nucleic Acid Conformation; Peptide Nucleic Acids; RNA, Double-Stranded; RNA-Binding Proteins; Uracil
PubMed: 30011039
DOI: 10.1093/nar/gky631 -
International Archives of Allergy and... 2010Although the accepted paradigm is that the proteins stored in eosinophil crystalloid granules are translated from messenger RNA transcribed in the cell nucleus, recent...
BACKGROUND
Although the accepted paradigm is that the proteins stored in eosinophil crystalloid granules are translated from messenger RNA transcribed in the cell nucleus, recent ultrastructural evidence suggests that protein synthesis may also take place within eosinophilic granules.
METHODS
We used 2 different methods to detect the presence of DNA and RNA in eosinophil secretory granules. Using bromodeoxyuridine, a thymidine analogue, and bromouridine, a uracil analogue, we labeled the DNA and RNA in eosinophils in vivo in rabbits. Immunoelectron microscopy to localize these molecules was performed on ultrathin sections of blood and bone marrow eosinophils using monoclonal anti-bromodeoxyuridine antibody with IgG as a control. The immunogold grain density was measured in each subcellular compartment within the eosinophils and analyzed using image analysis software. A combination of DNA/CD63 immunofluorescence staining and a fluorescently labeled molecular probe that stains RNA was used to examine the presence of DNA and RNA in the secretory granules of human blood eosinophils.
RESULTS
The mean density of bromodeoxyuridine-labeled DNA and bromouridine-labeled RNA immunogold grains in the secretory granules of blood and bone marrow eosinophils were significantly higher (p < 0.0005) than cytoplasmic or background staining. We also demonstrated the existence of DNA and RNA in the CD63-positive secretory granules of human peripheral blood eosinophils by means of immunofluorescent staining and a fluorescently labeled molecular probe.
CONCLUSIONS
These results provide evidence that eosinophil granules are the site of DNA and RNA synthesis and suggest the potential for a new role(s) for eosinophil-secretory granules.
Topics: Animals; Bone Marrow Cells; Bromodeoxyuridine; Bromouracil; DNA; Eosinophils; Female; Fluorescent Dyes; Humans; Immunohistochemistry; Microscopy, Immunoelectron; Organic Chemicals; RNA; Rabbits; Secretory Vesicles; Uridine
PubMed: 19940501
DOI: 10.1159/000260079 -
Nature Protocols Aug 2015Nuclear run-on (NRO) is a method that measures transcriptional activity via the quantification of biochemically labeled nascent RNA molecules derived from nuclear...
Nuclear run-on (NRO) is a method that measures transcriptional activity via the quantification of biochemically labeled nascent RNA molecules derived from nuclear isolates. Widespread use of this technique has been limited because of its technical difficulty relative to steady-state total mRNA analyses. Here we describe a detailed protocol for the quantification of transcriptional activity in human cell cultures. Nuclei are first isolated and NRO transcription is performed in the presence of bromouridine. Labeled nascent transcripts are purified by immunoprecipitation, and transcript levels are determined by reverse-transcription quantitative PCR (RT-qPCR). Data are then analyzed using standard techniques described elsewhere. This method is rapid (the protocol can be completed in 2 d) and cost-effective, exhibits negligible detection of background noise from unlabeled transcripts, requires no radioactive materials and can be performed from as few as 500,000 nuclei. It also takes advantage of the high sensitivity, specificity and dynamic range of RT-qPCR.
Topics: Bromouracil; Cell Line; High-Throughput Nucleotide Sequencing; Humans; Immunoprecipitation; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Uridine
PubMed: 26182239
DOI: 10.1038/nprot.2015.076 -
The Journal of Physical Chemistry. B Oct 2013As a result of their inherent planarity, DNA base radicals generated by one-electron oxidation/reduction or bond cleavage form π- or σ-radicals. While most DNA base...
As a result of their inherent planarity, DNA base radicals generated by one-electron oxidation/reduction or bond cleavage form π- or σ-radicals. While most DNA base systems form π-radicals, there are a number of nucleobase analogues such as one-electron-oxidized 6-azauraci1, 6-azacytosine, and 2-thiothymine or one-electron reduced 5-bromouracil that form more reactive σ-radicals. Elucidating the availability of these states within DNA, base radical electronic structure is important to the understanding of the reactivity of DNA base radicals in different environments. In this work, we address this question by the calculation of the relative energies of π- and σ-radical states in DNA/RNA bases and their analogues. We used density functional theory B3LYP/6-31++G** method to optimize the geometries of π- and σ-radicals in Cs symmetry (i.e., planar) in the gas phase and in solution using the polarized continuum model (PCM). The calculations predict that σ- and π-radical states in one-electron-oxidized bases of thymine, T(N3-H)(•), and uracil, U(N3-H)(•), are very close in energy; i.e., the π-radical is only ca. 4 kcal/mol more stable than the σ-radical. For the one-electron-oxidized radicals of cytosine, C(•+), C(N4-H)(•), adenine, A(•+), A(N6-H)(•), and guanine, G(•+), G(N2-H)(•), G(N1-H)(•), the π-radicals are ca. 16-41 kcal/mol more stable than their corresponding σ-radicals. Inclusion of solvent (PCM) is found to stabilize the π- over σ-radical of each of the systems. U(N3-H)(•) with three discrete water molecules in the gas phase is found to form a three-electron σ bond between the N3 atom of uracil and the O atom of a water molecule, but on inclusion of full solvation and discrete hydration, the π-radical remains most stable.
Topics: Adenine; Cytosine; DNA; Electrons; Gases; Guanine; Models, Molecular; Oxidation-Reduction; RNA; Solutions; Solvents; Thymine; Uracil; Water
PubMed: 24000793
DOI: 10.1021/jp407897n -
Genome Research Oct 2020Gene expression is determined by a balance between RNA synthesis and RNA degradation. To elucidate the underlying regulatory mechanisms and principles of this,...
Gene expression is determined by a balance between RNA synthesis and RNA degradation. To elucidate the underlying regulatory mechanisms and principles of this, simultaneous measurements of RNA synthesis and degradation are required. Here, we report the development of "Dyrec-seq," which uses 4-thiouridine and 5-bromouridine to simultaneously quantify RNA synthesis and degradation rates. Dyrec-seq enabled the quantification of RNA synthesis and degradation rates of 4702 genes in HeLa cells. Functional enrichment analysis showed that the RNA synthesis and degradation rates of genes are actually determined by the genes' biological functions. A comparison of theoretical and experimental analyses revealed that the amount of RNA is determined by the ratio of RNA synthesis to degradation rates, whereas the rapidity of responses to external stimuli is determined only by the degradation rate. This study emphasizes that not only RNA synthesis but also RNA degradation is important in shaping gene expression patterns.
Topics: Bromouracil; HeLa Cells; Humans; RNA; Sequence Analysis, RNA; Thiouridine; Uridine
PubMed: 32843354
DOI: 10.1101/gr.264408.120 -
Histology and Histopathology Jan 2005Actinomycin D (AMD) inhibits DNA-dependent RNA polymerases and its selectivity depends on the concentration used; at very high concentrations it may also induce...
Actinomycin D (AMD) inhibits DNA-dependent RNA polymerases and its selectivity depends on the concentration used; at very high concentrations it may also induce apoptosis. This study investigates the effects of different concentrations (0.01 to 1 microg/ml) of AMD on RNA transcription and maturation and on the organization of nuclear ribonucleoproteins (RNPs), and their relationship with apoptosis induction. Human HeLa cells were used as a model system. At the lowest concentration used, AMD induced the segregation of the nucleolar components and impaired r-RNA synthesis, as revealed by the decreased immunopositivity for bromo-uridine incorporation and for DNA/RNA hybrid molecules. The synthesis of pre-mRNAs, on the contrary, was active, while the immunolabeling of snRNP proteins and of the SC-35 splicing factor strongly decreased on perichromatin fibrils (where they are involved in co-transcriptional splicing). This suggests that the post-transcriptional maturation of extranucleolar RNAs was also affected. Moreover, still in the absence of typical late morphological or biochemical signs of apoptosis (i.e. chromatin condensation), these cells displayed the early apoptotic features, i.e. the externalization of phosphatidylserine residues on the plasma membrane and propidium iodide exclusion in vivo. At the highest concentrations of AMD used, apoptosis massively occurred, with the typical morphological events (progressive chromatin condensation, clustering of snRNPs and SC-35 splicing factor, cell blebbing). However, transcription of hnRNAs was maintained in the residual areas of diffuse chromatin up to advanced apoptotic stages. The inhibition of rRNA synthesis and the defective pre-mRNA maturation seem to be part of the apoptotic process induced by AMD.
Topics: Antibiotics, Antineoplastic; Apoptosis; Bromouracil; Cell Nucleolus; Dactinomycin; HeLa Cells; Humans; Immunohistochemistry; Microscopy, Electron; Transcription, Genetic; Uridine
PubMed: 15578429
DOI: 10.14670/HH-20.107