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)

Terminal deoxynucleotidyl transferase (TdT) was used to prepare copolymers of dA and 1,N6-ethenodeoxyadenosine (epsilon dA). When used as templates for Escherichia coli DNA polymerase I (Pol I) and compared with poly (dA), normal dTTP incorporation was not significantly affected by the presence of 7% epsilon dA. dGTP misincorporation was only slightly increased and occurred about once for every 500 epsilon dA residues. The error-prone polymerase from avian myeloblastosis virus (AMV reverse transcriptase) increased this error rate 5- to 20-fold to a maximum of 1 dG/25 epsilon dA. No dCTP misincorporation was detected with either polymerase. In transcription with E. coli DNA-dependent RNA polymerase, no errors were revealed by nearest neighbor analysis. Poly (dA) treated with chloroacetaldehyde under conditions producing the same proportion of epsilon dA (without the hydrated form) as the synthesized template behaved in the same manner with a similar low level of misincorporation of dG. Such treatment of alternating poly d(A-T) caused structural changes indicative of crosslinks but did not alter its template properties. Increasing the amount of epsilon dA in either synthesized or modified polymers greatly decreased the template activity without increasing the error rate. It is suggested that epsilon dA generally does not prevent dT incorporation but behaves as a bulky lesion which is bypassed. In contrast to the low mutagenic efficiency of epsilon dA, O4-methyldeoxythymidine (m4dT), in copolymers with dA, directed the misincorporation of 1 dG/12 m4dT with Pol I and 1 dG/3 m4dT with reverse transcriptase. Nearest neighbor analysis of transcripts showed the incorporation of 1 dG/12 m4dT. These data are in agreement with the previous reported mutagenicity of m4dT in alternating poly d(A-T, m4T).
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PMID:Assessment of mutagenic efficiency of two carcinogen-modified nucleosides, 1,N6-ethenodeoxyadenosine and O4-methyldeoxythymidine, using polymerases of varying fidelity. 620 83

To examine the possible role of the vaccinia virus glutaredoxin as a cofactor for viral ribonucleotide reductase, viral growth, DNA synthesis, and dNTP pools were measured in infections of B-SC-40 monkey kidney cells with wild type vaccinia virus and with mutants of vaccinia that lacked a functional reductase or glutaredoxin. In infections of untreated host cells, the lack of viral ribonucleotide reductase or glutaredoxin had only small effects upon virus growth. When host cells were pretreated with alpha-amanitin, which blocks host RNA polymerase II but not viral transcription, viral DNA synthesis was markedly reduced in infections with either of the mutants when compared with wild type infections. Relative to dNTP levels in wild type infections, pools of dCTP, but not of the other dNTPs, were significantly reduced in infections of amanitin-treated cells with either mutant. The parallel depletion of dCTP in the two mutant suggests that the role of glutaredoxin may be to function as a cofactor for viral ribonucleotide reductase. The data suggest that both viral proteins become essential for DNA replication only when levels of the corresponding host cell proteins are depleted.
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PMID:Roles of vaccinia virus ribonucleotide reductase and glutaredoxin in DNA precursor biosynthesis. 749 96

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 incorporation of 2'-deoxynucleoside-5'-O-(1-thiotriphosphates) into RNA transcripts using T7 RNA polymerase. With the exception of [alpha-S]dGTP, we obtained full-length transcripts of pre-tRNA(Phe) and pre-tRNA(Tyr) using an appropriate mixture of 2'-deoxynucleoside 5'-O-(1-thiotriphosphate) and the corresponding normal nucleoside triphosphate. The yields of the transcripts were comparable to those obtained with unmodified NTPs. Both substrates, [alpha-S]dTTP and [alpha-S]dATP, were inserted specifically. However, [alpha-S]dCTP was excluded at specific sites. We could not obtain transcripts using the deoxyguanosine derivative.
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PMID:Enzymatic RNA synthesis with deoxynucleoside 5'-O-(1-thiotriphosphates). 767 87

We have developed a multiplex, competitive, reverse-transcriptase polymerase chain reaction (RT-PCR) method which measures absolute levels of renin, angiotensinogen, and the housekeeping transcript elongation factor-1 alpha (EF-1 alpha) mRNA. Sample RNA was simultaneously titrated with serial dilutions of renin, angiotensinogen, and EF-1 alpha competitor RNAs which flanked the endogenous concentrations of target transcripts. The samples were coreverse transcribed in the presence of random primers and resulting first-strand cDNA was coamplified for 10-15 cycles with [32P]-dCTP and primers for renin angiotensinogen, after which EF-1 alpha primers were added. Amplified DNA was separated by electrophoresis on polyacrylamide gel and radioactivity in the bands was quantified by direct radioanalytical scanning. Three conditions were necessary to obtain absolute quantification of renin and angiotensinogen mRNA levels: (a) exogenous competitor RNA was used to control for tube-to-tube variability in the efficiencies of reverse transcription and amplification; (b) Sample RNA was titrated with flanking concentrations of competitor RNA to correct for intraassay differences in the efficiency of amplification due to concentration differences between competitor and target templates; and (c) a housekeeping transcript EF-1 alpha was used to control for tube-to-tube differences in RNA loading and/or degradation. We show that the multiplex RT-PCR method is precise and accurate over approximately three logs of transcript concentration and sensitive to less than 5 and 0.5 fg for renin and angiotensinogen mRNA, respectively. This method will be useful for absolute quantification of target mRNAs, especially when the amount of sample RNA is limited or unknown and/or the gene expression is low.
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PMID:An improved method for absolute quantification of mRNA using multiplex polymerase chain reaction: determination of renin and angiotensinogen mRNA levels in various tissues. 788 70

The effects of single base pair substitutions at the initiation sites of lacUV5 promoter on the transcription start site selection by E. coli RNA polymerase were systematically studied. Transcription start sites were mapped by sizing the cytosine-specifically terminated transcripts produced in vitro by using a chain terminator 3'-deoxycytidine 5'-triphosphate (3'-dCTP) in transcription reactions. Transcription of a prototype lacUV5 promoter initiated with three purines (-1G, +1A and +2A; +1 representing the predominant start site) located 6-8 bp downstream from the Pribnow box. All the substitutions affected the start site selection, resulting in a change in the number of start sites (from 3 to 2 or 1) and/or a shift of the major start site (to -1 or +2). None of the variants started outside the 3-bp region and at the positions substituted by a pyrimidine. Purine-to-pyrimidine changes suppressed not only initiation at the substituted position but also, in some cases, at the other purine position. Purine-to-purine changes also shifted the major start site or suppressed the initiation at other sites. Changes at -2 and +5 also affected the start site selection. Thus, the sequence context around the initiation sites of lacUV5 promoter strongly influences the selection of initiating nucleotides by E. coli RNA polymerase.
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PMID:Start site selection at lacUV5 promoter affected by the sequence context around the initiation sites. 798 16

A novel photoreactive deoxycytidine analog, 4-[N-(p-azidobenzoyl)-2-aminoethyl]-dCTP (ABdCTP), has been synthesized and incorporated at specific sites within the SUP4 tRNA(Tyr) gene. Immobilized single-stranded DNA was annealed to specific oligonucleotides and AB-dCMP incorporated into DNA by primer extension. DNA photoaffinity labeling with AB-dCMP was used to survey protein-DNA contacts in initiation and elongation complexes of RNA polymerase III (Pol III), and compared to DNA photoaffinity labeling using the previously described photoreactive deoxyuridine analog, 5-[N-(pazidobenzoyl)-3-aminoallyl]-dUMP (AB-dUMP) [Bartholomew et al. (1993) Mol. Cell.Biol. 13,942-952]. In contrast to previous studies, we have used a crude protein fraction rather than highly purified preparations of Pol III and transcription factors TFIIIC and TFIIIB to examine if some component of the transcription complex is lost upon purification. Eleven nucleotide positions from bp-17 to bp +17 (+1 being the start site of transcription) on the nontranscribed strand were modified and shown to have little or no effect on transcription complex formation, initiation, or elongation as determined by multiple-round transcription assays. Efficient photoaffinity labeling by DNA containing AB-dCMP gave results comparable to that with AB-dUMP at proximal nucleotide positions and provided new evidence for the placement of the 160 and 31 kDa subunits of Pol III near the 5' end of the transcriptional bubble in an elongation complex. A novel 40 kDa protein was cross-linked at bps -17, -9, and -8 in a TFIIIC-dependent manner that had not been previously detected.
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PMID:Probing the protein-DNA contacts of a yeast RNA polymerase III transcription complex in a crude extract: solid phase synthesis of DNA photoaffinity probes containing a novel photoreactive deoxycytidine analog. 870 56

Hyperoxia-exposure results in neutrophil accumulation and edema in the exposed lung. Intercellular adhesion molecule-1 (ICAM-1), a ligand for neutrophil beta 2 integrins, is upregulated in hyperoxia-exposed lungs and enhances neutrophil-mediated injury. Because tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) are potent inducers of ICAM-1, we investigated whether TNF-alpha and IL-1 beta mRNA increase prior to the increase in ICAM-1 mRNA in hyperoxia-exposed mouse lungs. We exposed mice to > 95% oxygen for up to 96 h, isolated lung RNA, and assessed ICAM-1, TNF-alpha, and IL-1 beta mRNA by Northern blotting. We found that neither, TNF-alpha nor IL-1 beta mRNA was detectable prior to 96 h, while ICAM-1 mRNA increased by 48 h. To further assess TNF-alpha and IL-1 beta mRNA, we employed quantitative reverse-transcriptase polymerase chain reaction (RTPCR) using a mimic DNA (mimic) species as an internal control for PCR. Mimic DNA was identical to reverse-transcribed cDNA (wild type), except for 147 bp of irrelevant DNA ligated into the original cDNA. For each lung RNA sample we reverse transcribed total lung RNA and coamplified the resulting wild-type cDNA with serial dilutions of mimic DNA in a PCR containing [32P] dCTP. After PCR, we electrophoresed the samples and determined the concentration of TNF-alpha and IL-1 beta wild-type cDNAs by the ratios of wild type to mimic counts. We found no increase in TNF-alpha or IL-1 beta mRNA through 72 h of hyperoxia exposure, while there was an approximately 10-fold increase in TNF-alpha mRNA and a 35-fold increase in IL-1 beta mRNA within 2 h in the lungs of animals exposed to endotoxin. In conclusion, our data suggest that TNF-alpha and IL-1 beta are not responsible for the upregulation of ICAM-1 in hyperoxia-exposed mouse lungs.
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PMID:Hyperoxic increases in lung ICAM-1 mRNA are independent of TNF-alpha and IL-1 beta mRNA. 937 69

In this study, human oropharyngeal epidermoid carcinoma KB cells that were resistant to 2,2-difluorodeoxycytidine (dFdCyd) were selected and designated the KB-Gem clone. The KB parental cell line IC50 was 0.3 microM dFdCyd, as compared with the KB-Gem clone IC50 of 32 microM dFdCyd. The KB-Gem clone demonstrated overexpression of ribonucleotide reductase (RR) M2 subunit mRNA (9-fold) and overexpression of M2 protein (2-fold); RR activity was 2.3-fold higher than the KB parental cell line. Both the dATP and dCTP pools of the KB-Gem clone increased 2-fold over the parental cell line, with no change in the dGTP and dTTP pools. Reverse transcriptase-PCR was used to clone the cDNA of deoxycytidine kinase (DCK). Resulting sequences revealed two silent mutations in the KB-Gem clone. The amino acid sequence of the DCK protein and mRNA expression remained unchanged. The KB-Gem clone's DCK enzyme activity was 56% of that of the parental cell line. After the endogenous dNTPs were removed with a G-25 column, no difference was evident between the enzyme activities of the KB-Gem clone and parental cells. Thus, contrary to previous hypotheses, DCK deficiency does not play the primary role in the resistance mechanism of dFdCyd, accepting a secondary role to the overexpression of the target gene, RR, and pool expansion.
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PMID:Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line. 1048 55

"Mesenchymal-Epithelial Transition in the Developing Metanephric Kidney: Gene Expression Study by Differential Display," by Sergei Y. Plisov, Sergey V. Ivanov, Kiyoshi Yoshino, Lee F. Dove, Tatiana M. Plisova, Kathleen G. Higinbotham, Irina Karavanova, Michael Lerman, and Alan O. Perantoni The above article originally appeared in Volume 27, Number 1, the May 2000 issue, of genesis on pp. 22-31. The wrong affiliations were listed for two of the co-authors: Sergey V. Ivanov is affiliated with Intramural Research Support Program, SAIC-Frederick, Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland. Michael Lerman is affiliated with Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland. On page 24, left column, under "In situ mRNA Hybridization" in the "Results" section, the sentence, "To verify the results of DD and to determine in which cells of the developing kidney the differentially displayed genes were expressed we applied mRNA hybridization (ISH)," should read: "To verify the results of DD and to determine in which cells of the developing kidney the differentially displayed genes were expressed we applied in situ mRNA hybridization (ISH)." On page 27, the legend for Figure 2, should read: "In situ RNA hybridization with thin sections of 19 dpc fetal kidney. Labeled antisense RNA was in vitro transcribed from cloned cDNA fragments obtained after differential display." On page 30, right column, under "In situ Hybridization" in the "Methods" section, the sentence, "To generate sense or antisense probes, 5 &mgr;g of plasmids with cloned cDNA fragments were linearized either with NcoI or SpeI (Promega) restriction enzymes and transcribed with T7 or SP6 RNA polymerase in the presence of alpha-35S-dCTP," should read: "To generate sense or antisense probes, 5 &mgr;g of plasmids with cloned cDNA fragments were linearized either with NcoI or SpeI (Promega) restriction enzymes and transcribed with T7 or SP6 RNA polymerase in the presence of alpha-35S-CTP." The authors regret these errors.
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PMID:Erratum 1095 6


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