Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 5-thio and 5-methylmercurithio derivatives of UTP, dUTP and dCTP have been synthesized and tested as substrates for nucleic acid polymerases. The 5-thio-nucleotides were polymerized inefficiently by both RNA polymerase and DNA polymerase I of Escherichia coli. The 5-methylmercurithio derivatives of dUTP and dCTP were, however, utilized by DNA polymerase I, an enzyme insensitive to mercurial compounds, although they were potent inhibitors of all other polymerases tested. While polymers containing the 5-thio substituent possess structural abnormalities, most likely interstrand disulfide bridges, polymers containing 5-methylmercurithio groups appear normal. The latter polynucleotides are readily separated from non-sulfated polymers by chromatography on mercuriagarose.
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PMID:The synthesis and enzymatic polymerization of 5-thio- and 5-methylmercurithio-pyrimidine nucleotides. 79 73

The synthesis of 2'-deoxyuridine 5'-triphosphate analogues with fluorescent residues of fluorescein and rhodamine nature at C5 of the uracil base was performed. Reverse transcriptase of avian myeloblastosis virus, DNA polymerase beta of rat liver, terminal deoxynucleotidyl transferase of calf thymus and E. coli DNA polymerase I, Klenow fragment, were shown to be capable to incorporate a nucleotide residue with fluorescent label into 3'-terminus of oligonucleotide. These fluorescent labeled oligonucleotides were used as primers for synthesis of (-)-chain of M13mp10 phage. Fluorescently labeling template-primer complexes were used for DNA sequencing.
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PMID:[Fluorescent analogs of nucleoside-5'-phosphates for the study of nucleic acids by nonradioactive methods]. 170 Dec 17

A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N3dUTP is the nitration of dUMP in 98% yield in 5 min at 25 degrees C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH2dUMP). Diazotization of 5-NH2dUMP with HNO2 followed by the addition of NaN3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N3dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N3dUTP through a chemical coupling of pyrophosphate to 5-N3dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used as the starting compound, 5-N3UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [gamma-32P]-5-N3dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing gamma-32P-labeled pyrimidine nucleotides was developed. [gamma-32P]-5-N3dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA. 354 18

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. The dUTPase gene of the retrovirus equine infectious anemia virus (EIAV) was cloned and overexpressed in Escherichia coli using the T7 RNA polymerase expression system. The recombinant vector (pET-3a/EDU), constructed by mutagenic PCR, was transformed into E. coli BL21 (DE3) pLysS cells, resulting in expression of EIAV dUTPase at about 40% of the extracted protein. This level of overproduction is very high compared to previous reports on heterologous expression of dUTPases in E. coli. A one-step purification procedure using phosphocellulose chromatography results in a homogeneous preparation of the enzyme in a yield of 45 mg liter-1 of bacterial culture. The purified EIAV dUTPase, run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows an apparent molecular mass of 15.1 kDa in accordance with the gene structure. The isoelectric point (pI) was determined to 5.6. Gel filtration under nondenaturating conditions gives a retention volume corresponding to a molecular mass of 40.6 kDa, suggesting a trimeric organization of the enzyme. The amino acid composition and amino-terminal sequence of the recombinant dUTPase are in agreement with predictions from the DNA sequence.
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PMID:dUTPase from the retrovirus equine infectious anemia virus: high-level expression in Escherichia coli and purification. 766 76

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), widespread in nature with a crucial role in the nucleotide metabolism, catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate. The enzyme from herpes simplex virus type 1 (HSV-1 dUTPase) was overproduced in Escherichia coli by using the T7 RNA polymerase expression system. The coding region of the HSV-1 dUTPase gene, UL 50, was positioned downstream of the promoter and the ribosome-binding site of the phage T7 gene 10 on the expression vector pET-3a. The resulting recombinant plasmid, pET-3a/UL50, was transformed into E. coli BL21(DE3)pLysS cells, conferring expression of HSV-1 dUTPase as 2-3% of the soluble protein inducible by isopropyl thiogalactoside. By chromatography on phosphocellulose and Mono S (Pharmacia LKB) columns a nearly homogeneous preparation of the enzyme with a high specific activity (49 mumol per minute per milligram) was obtained. The recombinant protein was compared with the native dUTPase similarly purified from HSV-1-infected Vero cells (African green monkey kidney fibroblasts). The two proteins showed the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the amino-terminal sequences were found to be identical. The molecular mass (39 kDa) and the amino acid composition of the recombinant enzyme are also in accordance with predictions from the DNA sequence. Thus, the overproducing system described here appears suitable for providing HSV-1 dUTPase for detailed studies of molecular properties.
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PMID:dUTPase from herpes simplex virus type 1; purification from infected green monkey kidney (Vero) cells and from an overproducing Escherichia coli strain. 838 36

Foot and mouth disease virus RNA was visualized in infected primary tissue culture cells by in situ PCR incorporating digoxigenin-labeled dUTP. The viral RNA polymerase gene was used as a target for amplification. Infected cells revealed cytoplasmic staining, predominantly perinuclear. The intensity of staining was in proportion to the degree of cytopathology observed and similar to the results obtained using immunoperoxidase staining. The in situ PCR technique for FMDV detection could be applied to formalin-fixed samples and be useful for the study of persistent infections.
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PMID:Localization of foot and mouth disease virus RNA in tissue culture infected cells via in situ polymerase chain reaction. 853 May 68

The deoxyuridine triphosphatase gene of vaccinia virus, encoded by the open reading frame F2L, was cloned into Escherichia coli and expressed under the control of a bacteriophage T7 promoter. After induction of T7 RNA polymerase by isopropyl beta-D-thiogalactopyranoside, a 16.5-kDa peptide accumulated to high levels. This 16.5-kDa protein was purified to homogeneity and characterized. Gel filtration of the purified protein revealed a trimeric native structure. Biochemical analysis revealed the enzyme to be a metalloenzyme; enzymatic activity is inhibited by EDTA. This inhibition was reversed by the addition of Mg2+, Mn2+, or Zn2+. While the enzyme activity was highly specific for dUTP with an apparent Km of 0.94 microM, inhibition studies show that 8-azido-ATP acted as a competitive inhibitor of dUTP with a Ki of approximately 173 microM. Also, protection studies demonstrated that nucleotide competitors inhibit photoincorporation of the photoaffinity analogues [gamma-32P]5-azido-dUTP and [gamma-32P]8-azido-ATP. This suggests that while catalytic activity is limited to dUTP, other nucleotides can bind the active site.
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PMID:Purification and characterization of the vaccinia virus deoxyuridine triphosphatase expressed in Escherichia coli. 879 59

Oligodendrocytes in multiple sclerosis brain may be under a direct attack by proinflammatory cytokines, particularly tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma). In this study, we have examined the in vitro cytotoxic effects of the two cytokines, individually and in combination, on oligodendrocyte lineage cells using morphological criteria, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay (MTT), terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL), and agarose-gel electrophoretic analysis of fragmented DNA. IFNgamma exerted a dose-dependent cytotoxic effect on cultured CG4 cells, an oligodendrocyte progenitor cell line, and in primary cultures of purified oligodendrocyte progenitors. TNFalpha, while by itself being only mildly toxic, greatly potentiated the cytotoxicity of IFNgamma. The cytokine effects were developmentally modified in that their cytotoxic and cooperative effects became less evident in more differentiated cells. A cell-permeable peptide inhibitor (i.e., z-VAD.fmk) of caspases partially suppressed apoptotic changes elicited by the cytokine combination in CG4 cells but not in primary oligodendrocytes. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of mRNA prepared from cytokine-treated cultures revealed an increased expression of the death receptor, Fas. The results suggest particular vulnerability of oligodendrocyte progenitors to a combination of TNFalpha and IFNgamma involving an activation of the cell death program.
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PMID:TNFalpha potentiates IFNgamma-induced cell death in oligodendrocyte progenitors. 984 48

Although fetal breathing movements are required for normal lung development, there is uncertainty concerning the specific effect of absent fetal breathing movements on pulmonary cell maturation. We set out to evaluate pulmonary development in a genetically defined mouse model, the myogenin null mouse, in which there is a lack of normal skeletal muscle fibers and thus skeletal muscle movements are absent in utero. Significant decreases were observed in lung:body weight ratio and lung total DNA at embryonic days (E)14, E17, and E20. Reverse transcriptase/polymerase chain reaction, in situ immunofluorescence, and electron microscopy revealed early lung cell differentiation in both null and wild-type lungs as early as E14. However at E14, myogenin null lungs had decreased 5'-bromo-2-deoxyuridine incorporation compared with that of wild-type littermates, whereas at E17 and E20, increased Bax immunolabeling and terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick-end labeling staining were detected in the myogenin null mice but not in the wild-type littermates. These observations highlight the importance of skeletal muscle contractile activity in utero for normal lung organogenesis. Null mice lacking the muscle-specific transcription factor myogenin exhibit a secondary effect on lung development such that decreased lung cell proliferation and increased programmed cell death are associated with lung hypoplasia.
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PMID:Pulmonary hypoplasia in the myogenin null mouse embryo. 1069 67

The objective of this study was to examine placentas after delivery from normal, healthy patients at term gestation. The placentas were from elective cesarean sections (n = 10, prior to the onset of labor) and spontaneous vaginal delivery (n = 10, after labor). We found that deoxyribonucleic acid laddering was present in all placentas and consistent with the pattern found in tissues that undergo apoptosis. Paraffin-embedded sections of placental villi stained by the in situ terminal deoxynucleotidyl transferase mediated biotinylated dUTP nick end labeling method revealed positive apoptotic nuclei in the placental villi. Reverse-transcriptase polymerase chain reaction demonstrated expression of messenger RNA for testosterone-repressed prostate message 2 and B cell lymphoma/leukemia-2 in the placenta. Our data demonstrate that apoptosis occurs in human term placenta.
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PMID:Apoptosis in human term placenta. A morphological and gene expression study. 1096 89


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