Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacillus subtilis tryC2, thyA, thyB, lysogenic for the phage DNA polymerase negative mutant SPO2 susL244, was induced under conditions preventing phage and bacterial DNA synthesis. The biological activity of DNA from induced cells and from uninduced controls was assayed by transformation and transfection, respectively. About 50% of the phage DNA biological activity in DNA extracted from induced cells was resistant to exposure to pH 11.8 TO 11.9. This DNA was operationally defined as alkali-resistant phage DNA. Transforming bacterial DNA from uninduced or induced cells and transfecting DNA from uninduced cells were more than 95% inactivated after exposure to high pH. The alkali-resistant phage DNA was characterized by sucrose gradient centrifugation, by centrifugation in cesium chloride-propidium iodide, and by electron microscopy. It was found to consist of a majority of covalently closed circular DNA molecules. Length measurements of a few relaxed circular molecules indicate a molecular weight of these similar to that previously found for mature SPO2DNA. Attempts to isolate similar covalently closed circular phage DNA from induced bacteria lysogenic for SPO2 phage with a functional DNA polymerase gene were unsuccessful. The gene order in mature and prophage SPO2 was determined by rescue of single and double markers from the respective type of DNA. The data obtained show that prophage DNA is (genetically) permuted relative to mature DNA. The phage attachment site is suggested to be located between genes I and J.
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PMID:Induction of prophage SPO2 in Bacillus subtilis: isolation of excised prophage DNA as a covently closed circle. 0 67

Aspects of the Salmonella mutagenesis and Escherichia coli DNA polymerase deficient (pol A1) assay procedures for detecting environmental mutagens are discussed. The chief limitation of the pol A1-- assay involves substances that do not diffuse rapidly in agar. This problem can be overcome by performing the test in suspension. A simple procedure for accomplishing this is described. Although the Salmonella assay is more flexible, under routine conditions it does not respond to several classes of substances which give positive responses in the pol A1- system. For optimal testing, it is recommended that the two microbial assays be used in tandem. The DNA-modifying properties of povidone-iodine for eukaryotic and prokaryotic cells are described. Even though this substance does not display mutagenic properties in the standard Salmonella assay, it does so in suspension culture. The basis of the mutagenic and DNA-modifying properties of povidone-iodine appears to involve the iodination of the cystosine residue of DNA.
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PMID:Mutagenicity and DNA-modifying activity: a comparison of two microbial assays. 79 8

The sequence divergence of mitochondrial DNAs (mtDNA) from rat, mouse, guinea pig, monkey, and chicken has been examined by DNA-DNA hybridization. mtDNAs, isolated as closed circular molecules by propidium iodide-CsCl centrifugation, were labeled in vitro by use of Escherichia coli DNA polymerase I, and renatured (Tm-35 degrees) in the presence of a 2500-fold excess of heterologous mtDNA. Single-stranded and duples DNA were separated by hydroxylapatite chromatography. The thermal stability of heteroduplexes was compared to the homoduplex by thermal elution chromatography on hydroxylapatite columns. Heteroduplex fromation between the tritiated myDNAs and a 2500-fole excess of rar mtDNA were 70, 59, 37, and 22%, respectively, for mouse, guinea pig, monkey, and chicken. Similar results were obrained in reciprocal hybridizations where one of the other mtDNAs was present in excess. Considerable mismatching of sequences in all the heterohybrids was indicated by a 18-24 degrees depression in the te50 of the heteroduplexes compared with the homoduplex. There was no apparent change in heteroduplex formation when the concentration ratio of driving DNA in excess to [3H]mtDNA was varied between 1250 and 7500. Furthermore, a second renaturation with excess driving DNA after completion of the first reaction resulted in no detectable augmenting of heteroduplex formation. Similar sequences appear to be conserved preferentially in different organisms, since the presence of two of fouf different heterologous mtDNAs in excess resulted in only moderate and nonadditive increases in heteroduplex formation. Evolutionary divergence of mtDNA sequences appears to have occurred at rates similar to that for unique sequences nuclear DNA.
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PMID:Sequence homology between mitochondrial DNAs of different eukaryotes. 114 57

(Trimethylsilyl)acetylene was coupled with 1-(2,3,5-tri-O-acetyl-beta-D- arabinofuranosyl)-5-iodouracil to give 1- (2,3,5-tri-O-acetyl-beta-D-arabinofuranosyl)-5-[2-(trimethylsilyl)eth yny l] uracil. Lindlar hydrogenation of 4 gave 1-(2,3,4-tri-O-acetyl-beta-D-arabinofuranosyl)-5(Z)-[2- (trimethylsilyl)vinyl]uracil. Treatment of 5 with iodine monochloride (or sodium iodide/phenyliodine(III) dichloride) in benzene gave 1-(2,3,5-tri-O-acetyl-beta-D-arabinofuranosyl)-5(E)-(2-iodovinyl)uracil (7), whereas polar solvents favored the (Z)-iodovinyl isomer 8. Deacetylation of 7 gave 1-(beta-D-arabinofuranosyl)-5(E)-(2-iodovinyl)uracil (IVAraU, 9). A microscale in situ synthesis with Na*I gave [*I]IVAraU. Treatment of HSV-infected cells with [125I]IVAraU resulted in virus-dependent uptake associated with nucleoside phosphorylation by wild type or acyclovir-resistant DNA polymerase mutants (but not with TK-HSV-1 mutants). Uptake was virus-inoculum dependent and was detectable within 4 h postinfection. The process was not completely reversible. Virus-specified uptake of [125I]IVAraU may allow automated in vitro detection of HSV isolates.
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PMID:Nucleic acid related compounds. 65. New syntheses of 1-(beta-D-arabinofuranosyl)-5(E)-(2-iodovinyl)uracil (IVAraU) from vinylsilane precursors. Radioiodine uptake as a marker for thymidine kinase positive herpes viral infections. 206

A tumor-derived factor that inhibits cellular DNA synthesis was identified. The factor was extractable from a small-cell lung carcinoma cell line grown in either chemically defined medium or nu/nu mice and inhibited tritiated thymidine ([3H]dThd) incorporation by tumor cell lines of autologous, allogeneic, and xenogeneic origins. The viability of nonproliferating cells from normal tissue was not affected. Tumor extract inhibitory activity was trypsin labile but was resistant to other proteases, neuraminidase, lipase, DNase, RNase, glucosidase, extremes of pH-temperature, and reducing conditions. Inhibitory activity was reversibly bound to helix pomatia lectin but not to lentil, wheat germ, or concanavalin A lectins. Purification by size-exclusion high-performance liquid chromatography yielded a bioactive unimodal 12-kilodalton (kd) peak. The bioactive 12-kd moiety could be eluted from sodium dodecyl sulfate-polyacrylamide gels. Redosing of populations of the T-lymphoblastoid cell line CEM achieved an early (24 hr) sustained depression of pulse [3H]dThd incorporation and ultimately led to decreased population density of factor-treated populations. DNA histogram analysis demonstrated no change in cell cycle phase distribution after factor treatment. 5-Bromo-2'-deoxyuridine (BrdUrd) vs. propidium iodide with the two-parameter Fluorescence-Activated Cell Sorter analysis showed relative inhibition of non-S-phase BrdUrd uptake at 24 hours. A cell-free DNA polymerase assay demonstrated significant inhibition of non-alpha-polymerase-associated DNA synthesis in factor-treated cells. These studies suggest that this tumor-derived inhibitor of DNA synthesis represents a class of cellular products involved in the autoregulation of growth by regulation of DNA synthetic activity.
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PMID:Inhibition of DNA synthesis by a small-cell lung carcinoma-derived protein. 302 Mar 1

Terminal deoxynucleotidyl transferase (EC 2.7.7.31) is a eucaryotic DNA polymerase that does not require a template. The tryptophan environments in calf thymus terminal transferase were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was separated by time-resolved emission spectroscopy. Nanosecond fluorescence decays at 296-nm excitation and various emission wavelengths were deconvolved by global analysis, assuming that the lifetimes but not the relative weighting factors were independent of emission wavelength. The data were fit to three exponentials of lifetimes tau 1 = 1.4 ns, tau 2 = 4.5 ns, and tau 3 = 7.7 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 328, 335, and 345 nm. The accessibility of individual tryptophan environments to polar and nonpolar fluorescence quenchers was examined in steady-state and time-resolved experiments. In the presence of iodide and acrylamide, the steady-state emission spectra shift to the blue. However, at low quencher concentrations, the emission from the 7.7-ns component (maximum 345 nm) is hardly affected, suggesting that this hydrophilic tryptophan environment is buried within the protein. On the other hand, the red shift in the steady-state emission spectrum in the presence of trichloroethanol indicates that the 1.4-ns component (maximum 328 nm) is an exposed hydrophobic tryptophan environment. The results are consistent with an inside-out model for terminal transferase protein, with the more hydrophobic tryptophan(s) near the surface and the most hydrophilic tryptophan(s) in the core.
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PMID:Tryptophan fluorescence of terminal deoxynucleotidyl transferase: effects of quenchers on time-resolved emission spectra. 408 79

Bacteriophage T7-induced DNA polymerase is composed of a 1:1 complex of phage-induced gene 5 protein and Escherichia coli thioredoxin. Preparation of active subunits in the absence of sulfhydryl reagents indicates the reduced form of thioredoxin is sufficient for formation of the active holoenzyme. The oxidized form of thioredoxin, thioredoxin modified at one active site sulfhydryl by iodoacetate or methyl iodide, or thioredoxin modified at both active site sulfhydryls by N-ethylmaleimide, are all inactive, being defective in complex formation with gene 5 protein. Thioredoxin sulfhydryl groups present in native T7 DNA polymerase do not appear to be involved in an intersubunit disulfide bond; one and probably both sulfhydryls are available in the native holoenzyme for modification by N-ethylmaleimide. Furthermore, DNA substrates alter the reactivity of thioredoxin cysteines within the holoenzyme with respect to this reagent. Substrates for the single strand exonuclease enhance the reactivity of thioredoxin sulfhydryl groups while those for the polymerase or double strand exonuclease functions afford protection. It, therefore, seems likely that thioredoxin sulfhydryl groups are present in the reduced state within the native polymerase.
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PMID:T7-induced DNA polymerase. Requirement for thioredoxin sulfhydryl groups. 634 83

For the first time mosaic nucleic acids composed of 50% RNA and 50% DNA can be obtained as transcripts with T7 RNA polymerase. Two NTPs could be replaced simultaneously in a transcription reaction. This means more than 40 deoxynucleotides were inserted in one transcript. Previously, a maximum of two deoxynucleotides could be incorporated and 2'-O-methyl-NTPs were not substrates at all. We obtained reasonable transcript yields with a maximal level of 99% 2'-O-methyl-NTPs, and the products contained up to 58% 2'-O-methylnucleotides at more than 20 positions. Sequence-specific nucleotide incorporation was monitored by sequence ladders (partial alkali or iodine cleavage). No base misincorporations were detected with 100% dGTP, dCTP and dTTP, and with partial incorporation of dATP alpha S, 2'-O-methyl-GTP alpha S and 2'-O-methyl-CTP alpha S, whereas they were found with dATP, 2'-O-methyl-ATP alpha S and 2'-O-methyl-UTP alpha S. Quantitative data allow predetermined modification levels of partially modified transcripts. Highly modified transcripts can be used for structural and functional studies, in modification interference approaches and for in vitro evolution procedures. Modification interference studies revealed a small number of important phosphate and ribose moieties in RNase P substrates. The conversion of T7 RNA polymerase to a DNA polymerase extends the observation that there is no absolute distinction between RNA and DNA polymerases. Accordingly, an adapted concept of a primordial RNA world is presented.
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PMID:Enzymatic synthesis of 2'-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates. 754 Nov 30

We have investigated the cytopathic effects induced by the T-lymphotropic human herpesvirus 7 (HHV-7) on the CD4(+) T-lymphoblastoid SupT1 cell line and primary CD4(+) T lymphocytes. Acute in vitro HHV-7 infection induced (1) the formation of giant multinucleated syncytia, which eventually underwent necrotic lysis, and (2) single-cell apoptosis. Both cytopathic effects increased with the progression of infection and were blocked by phosphonoformic acid, a specific inhibitor of herpetic DNA polymerase. Using electron microscopy analysis of various samples, we found that all syncytia contained large amounts of virions and that most of them exhibited clear evidence of necrosis, whereas apoptosis was predominantly observed in single cells. Although empty viral capsids could be identified in the cytoplasm of approximately 25% of single cells exhibiting an apoptotic morphology, mature virions were hardly observed in these cells. In both coculture and cell-free HHV-7 infection experiments, a significant correlation was observed between the degree of single-cell apoptosis, evaluated by quantitative flow cytometry after propidium iodide staining, and the decrease in the total number of viable cells. Moreover, in cell-free infection experiments, apoptosis showed a positive correlation also with the viral load, monitored by quantitative HHV-7 DNA polymerase chain reaction. Thus, it appears that apoptosis occurred predominantly in uninfected bystander cells but not in productively HHV-7-infected cells.
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PMID:Human Herpesvirus 7 induces CD4(+) T-cell death by two distinct mechanisms: necrotic lysis in productively infected cells and apoptosis in uninfected or nonproductively infected cells. 937 61

DNA-targeted platinum phenanthridinium complexes were investigated in intact human cells and in tumour-bearing mice. The DNA sequence specificity of platinum phenanthridinium complexes was examined in intact human cells using a Taq DNA polymerase stop assay. It was found that the platinum phenanthridinium complexes had a similar sequence specificity to that of cisplatin. However, the rate at which DNA was damaged in intact human cells was 6-fold greater for the platinum phenanthridinium chloride complexes compared with cisplatin. These results are consistent with a DNA-targeting hypothesis where the attachment of an intercalating group to cisplatin places the platinum in close proximity to DNA and increases the rate of DNA platination. Platinum phenanthridinium iodide complexes were also tested, but damaged DNA at a rate similar to cisplatin. The platinum phenanthridinium complexes with shorter linker chain lengths damaged DNA more efficiently than the longer linker chain length complexes. The platinum phenanthridinium chloride complexes also showed significant anti-tumour activity in tumour-bearing (P388) mice.
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PMID:The interaction of DNA-targeted platinum phenanthridinium complexes with DNA in human cells. 1196 16


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