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)

Using the polymerase chain reaction and standard recombinant DNA techniques, a series of new multipurpose low-copy-number (lcn) vectors, pWSK29, pWKS30, pWKS129 and pWKS130, have been constructed. Plasmids pWSK29 and pWKS30 carry the ampicillin-resistance marker (ApR), 20 unique cloning sites flanked by T7 and T3 RNA polymerase promoters, the lacZ alpha gene and the bacteriophage f1 origin of replication (ori) for production of single-stranded (ss) DNA in the presence of a helper phage. Plasmids pWSK129 and pWKS130 carry the kanamycin-resistance marker (KmR) and have 16 unique cloning sites flanked by T7 and T3 RNA polymerase promoters positioned within the lacZ alpha gene. Plasmids pWSK129 and pWKS130 also contain the f1 ori for the generation of ss DNA in the presence of a helper phage. All of the plasmids have an lcn of six to eight per cell. Each vector can be used for: (i) complementation analysis, (ii) generating unidirectional deletions with exonuclease III/S1 nuclease, (iii) DNA sequencing, (iv) high-level gene expression using T7 RNA polymerase, and (v) run-off transcription. They are very useful for analyzing genes encoding proteins which are toxic in Escherichia coli in high copy number.
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PMID:Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. 205 70

Transposition of the ampicillin-resistant transposon Tn3 was reproduced in vitro using the Escherichia coli cell extract. In this cell-free system, we used plasmid DNA carrying mini-Tn3 as donor and phage lambda DNA as target and assayed for ampicillin-resistance transducing phages formed by cointegration of these DNA molecules. Ampicillin-resistance transducing phages, which were obtained by in vitro packaging of lambda DNA after the in vitro transposition reaction, were formed only in the presence of Tn3 transposase. The reaction required mini-Tn3 with the proper sequence and orientation of the terminal inverted repeats of Tn3. The reaction also required DNA synthesis but not RNA synthesis by E. coli RNA polymerase.
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PMID:In vitro transposition of transposon Tn3. 217 35

A new class of rifamycin-resistant mutants of Escherichia coli was obtained by lysogenic insertions of bacteriophage Mu Amp DNA. Rifamycin resistance is closely linked to the ampicillin resistance conferred by the prophage. Mapping by conjugation with auxotrophic markers revealed that the rifamycin-resistant mutations are located between 28 and 37 min on the E. coli chromosome standard map, some distance from the rpoB gene at 89.5 min. The DNA-dependent RNA polymerase of these mutants is highly sensitive to rifampicin.
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PMID:Mu-induced rifamycin-resistant mutations not located in the rpoB gene of Escherichia coli. 352 57

The binding of Escherichia coli RNA polymerase to antibiotic-resistance promoters was examined using the nitrocellulose filter assay. Four filter-retainable HaeIII fragments were observed with pBR322 and the promoter-probe plasmids, pBRH1, pBRH2 and pBRH4. Of the three fragments studied, two were shown to carry promoters for the ampicillin (Ap) and tetracycline (Tc) resistance genes, while the third present in pBRH1 appears to be the promoter for colicin E1 immunity (Colimm). Although the formation of filter-retainable complexes involving the Tcr promoter was sensitive to high salt, Apr promoter complexes were not. It was also shown that plasmids containing only the "firm-binding" portion of the Tcr promoter could still bind RNA polymerase in vitro despite the fact that these plasmids confer no in vivo Tcr. Additional filter-binding experiments performed with AluI-digested pBR322 DNA revealed the presence of a fifth RNA polymerase binding site on pBR322. This site is probably the promoter for the 100 bp transcript thought to be involved in the initiation of plasmid replication. An analysis of the recombinant plasmid (pKTR25) which carries the Kan-B portion of the EcoRI kanamycin (Kn) resistance fragment revealed that this fragment contains two RNA polymerase binding sites. We believe that these sites are responsible for the insertional activation of the Tcr gene and may be the promoters for the Knr and fusidic acid (Fa) resistance genes.
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PMID:Construction and characterization of E. coli promoter-probe plasmid vectors. II. RNA polymerase binding studies on antibiotic-resistance promoters. 624 25

The transposition-replication reaction of phage Mu has been reproduced in a cell-free reaction system. Two assay methods were used for the detection of transposition products. The first method uses lambda DNA as the target of transposition and a plasmid containing the ends of Mu DNA and an ampicillin-resistance gene as the donor; after the reaction, in vitro lambda packaging allows the scoring of ampr transducing phages generated by transposition. In the second method, the products made in the presence of a radioactive precursor for DNA synthesis are directly analyzed by gel electrophoresis and unique product species are identified. The reaction requires a donor DNA carrying the two Mu ends in their proper relative orientation, extracts containing the A and B gene products of Mu, and host factor(s). RNA synthesis by E. coli RNA polymerase is not required for the reaction. The products include both cointegrates and simple inserts. Both types of products show incorporation of radioactive DNA precursors; however, simple inserts do not seem to undergo a full round of DNA replication.
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PMID:In vitro transposition of bacteriophage Mu: a biochemical approach to a novel replication reaction. 631 1

We have developed plasmid-based expression systems that encode modified forms of T7 RNA polymerase (RNAP) having 6-12 histidine residues fused to the amino terminus. The histidine-tagged RNAPs (His-T7 RNAPS) are indistinguishable from the wild-type (WT) enzyme in nearly all biochemical assays. Similar plasmids that encode His-tagged T3 and SP6 RNAPs have also been constructed. To facilitate site-directed mutagenesis of the RNAP gene, the size of the target plasmid was minimized by using T7 RNAP itself as a selectable marker. BL21 (DCAT4) cells (which carry a chromosomal copy of the chloramphenicol acetyltransferase cat gene under control of a T7 promoter) are resistant to chloramphenicol when functional T7 RNAP is expressed, thus allowing the selection and maintenance of the target plasmid in these cells. Mutagenesis is accomplished by denaturing the plasmid, annealing mutagenic DNA primers, and repairing the plasmid with T4 DNA polymerase. Two DNA primers are used: one corrects a defect in the bla gene, the other introduces the desired mutation into the RNAP gene; 30-85% of the ampicillin-resistant transformants carry the desired mutation in the RNAP gene. By using BL21 (DCAT4) cells as a recipient for transformation the functional integrity of the RNAP gene may conveniently be monitored by assessing the level of chloramphenicol resistance in vivo. Methods for rapid, simultaneous purification of multiple samples of modified (His-tagged) and conventional RNAPs are described. Together, these developments greatly enhance our ability to characterize this important class of enzymes.
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PMID:Rapid mutagenesis and purification of phage RNA polymerases. 911 96

Strong transcription of phage promoters often renders the host E. coli cells containing the phage RNA polymerase inviable. When expression of the phage SP6 RNA polymerase gene in one plasmid was induced in the E. coli JM109 cells, cells that bear an active SP6 promoter were inviable. When it was not induced (the polymerase was still produced in low levels), viability of the host cells and stability of the promoter-bearing plasmids depended on the orientation of the promoter with respect to that of the replication origin and on the sequence of the origin. A group of SP6 promoter-bearing plasmids (group I plasmids) that had the promoter directed towards the ColE1 replication origin, rendered the polymerase-containing host cells inviable in selective media. When the sequence of the origin was different (group II plasmids), this adverse effect was not observed. When the promoter direction was same as the replication origin and the ampicillin-resistant gene (group III plasmids), many satellites formed around the colonies on ampicillin-containing agar plates. These effects were caused by strong transcription of the phage SP6 promoter by its RNA polymerase, since they were reduced or eliminated by inserting an active terminator just downstream of the promoter. The viability of host cells and copy number of the promoter/terminator-bearing plasmids appear to be quantitatively related with efficiency of initiation and termination of the phage transcription. These systems may be useful for in vivo screening for mutant variants of the phage promoter, polymerase and terminator that are affected in their efficiency.
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PMID:Viability of E. coli cells containing phage RNA polymerase and promoter: interference of plasmid replication by transcription. 983 56

We found that the presence of plasmids expressing tetracycline resistance or chloramphenicol resistance genes, but not those expressing ampicillin resistance or kanamycin resistance genes, in Escherichia coli led to the retardation of the process of removal of the heat-aggregated proteins (i.e. the S fraction) from the bacterial cells. The presence of chloramphenicol acetyltransferase in the S fraction is demonstrated. Moreover, we observed that the expression of T7 RNA polymerase gene had an influence on S fraction removal. These results suggest that high level production of some heterologous proteins which are accumulated in the cytoplasm, but not proteins exported through the cell membranes, may cause overloading of the S fraction and delay in the removal of heat-aggregated proteins from bacterial cells.
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PMID:The effect of some antibiotic-resistance-conferring plasmids on the removal of the heat-aggregated proteins from Escherichia coli cells. 1042 10

Cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) was developed to recognize individual genes in a single bacterial cell. In CPRINS, the amplicon was long single-stranded DNA and thus retained within the permeabilized microbial cells. FISH with a multiply labeled fluorescent probe set enabled significant reduction in nonspecific background while maintaining high fluorescence signals of target bacteria. The ampicillin resistance gene in Escherichia coli, chloramphenicol acetyltransferase gene in different gram-negative strains, and RNA polymerase sigma factor (rpoD) gene in Aeromonas spp. could be detected under identical permeabilization conditions. After concentration of environmental freshwater samples onto polycarbonate filters and subsequent coating of filters in gelatin, no decrease in bacterial cell numbers was observed with extensive permeabilization. The detection rates of bacterioplankton in river and pond water samples by CPRINS-FISH with a universal 16S rRNA gene primer and probe set ranged from 65 to 76% of total cell counts (mean, 71%). The concentrations of cells detected by CPRINS-FISH targeting of the rpoD genes of Aeromonas sobria and A. hydrophila in the water samples varied between 2.1 x 10(3) and 9.0 x 10(3) cells ml(-1) and between undetectable and 5.1 x 10(2) cells ml(-1), respectively. These results demonstrate that CPRINS-FISH provides a high sensitivity for microscopic detection of bacteria carrying a specific gene in natural aquatic samples.
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PMID:Recognition of individual genes in diverse microorganisms by cycling primed in situ amplification. 1626 64

It was demonstrated that bifidobacteria and lactic acid bacteria B. adolescentis and Lactobacillus sp. synthesized extracellular enzymes cleaving glycoside bonds in the molecules of dextran, pectic acid, and soluble starch. The maximal production of extracellular beta-galactosidase by B. adolescentis 91-BIM and 94-BIM at a rate of 0.08 and 0.03 U/mg h was observed during the exponential growth phase at 5 and 12 h of cultivation, respectively. The cultures of bifidobacteria retained 60-70% of beta-galactosidase and alpha-amylase activities after six months of storage. The bifidobacterium strains studied were resistant to amphotericin and aminoglycosides (gentamicin, kanamycin, and netromycin). The lactam antibiotics (ampicillin, benzylpenicillin, bicillin 3, bicillin 5, and carbenicillin), the preparations inhibiting protein synthesis at the level of ribosomes (lincomycin), RNA polymerase inhibitors (rifampin), cephalosporin, and Maxipime inhibited the growth of bifidobacteria. Rifampin, erythromycin, amphotericin, Maxipime, Fortum, doxycycline, levomycetin, streptomycin, and the aminoglycosides netromycin, gentamicin, and kanamycin did not have an effect on the growth of Lactobacillus sp., whereas semisynthetic derivatives of penicillin, carbenicillin and ampicillin, inhibited its growth as well as Oxamp and lincomycin. The lactam antibiotics benzylpenicillin, bicillin 3, and bicillin 5 inhibited the growth of lactic acid bacilli by 30-90%.
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PMID:[Production of hydrolases by lactic acid bacteria and bifidobacteria and their antibiotic resistance]. 1747 4


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