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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The differential display (DD) technique, which is widely used almost exclusively for eukaryotic gene discovery, was optimized to detect differential mRNA transcription from both pure-culture and soil-derived bacterial RNA. A model system which included toluene induction of todC1 in Pseudomonas putida F1 was used to optimize the procedure. At 24-h tod induction was determined to be approximately 8 x 10(7) transcripts/microg or 0.08% of the total mRNA. The primer concentration, primer length, annealing temperature, and template, deoxynucleoside triphosphate, and MgCl2 concentrations were varied to optimize amplification of a todC1 fragment. The limit of detection of todC1 by DD was found to be 0.015 ng of total RNA template or approximately 10(3) transcripts. Once optimized, a todC1C2 gene fragment from P. putida F1 RNA was detected by using an arbitrary primer for the reverse transcriptase step in conjunction with the same arbitrary primer and a Shine-Dalgarno primer in the PCR. To verify the results, an arbitrary primer was used to detect recovery of a new salicylate-inducible naphthalene dioxygenase in Burkholderia cepacia JS150. The method was then used to detect mRNA induction in both inoculated and uninoculated toluene-induced soil microcosms. Several putative differentially expressed partial gene sequences obtained from the uninoculated microcosms were examined, and one novel fragment was found to be differentially expressed.
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PMID:Optimization of differential display of prokaryotic mRNA: application to pure culture and soil microcosms. 975 87

Competitive reverse transcriptase polymerase chain reaction (RT/PCR) was used to quantify the mRNA of the tcbC gene of Pseudomonas sp. strain P51. The tcbC gene encodes the enzyme chlorocatechol-1,2-dioxygenase involved in 1,2,4-trichlorobenzene (TCB) degradation. The mRNA content per cell was monitored in a batch culture growing on 1,2,4-TCB. No mRNA could be detected in the first 2 days of the lag phase. mRNA production became maximal with 20 molecules per cell in the early exponential growth phase but then decreased to less than 10 molecules per cell. When TCB was depleted and the cells entered the stationary phase, the mRNA content decreased slowly below the detection limit within 4 days. In order to compare detection of tcbC mRNA in pure culture and in river sediment, cells of strain P51 pregrown on TCB were added to sediment and RNAs extracted. In sediment samples containing 5 x 10(8) cells per gram the tcbC mRNA was quantifiable by RT/PCR. The mRNA recovery was about 3% as compared to the inoculum. The detection limit of the RT/PCR method was about 10(7) mRNA molecules per gram sediment or 10(6) copies per ml culture medium which corresponded in our case to 10(5) molecules per reaction vial.
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PMID:Quantification of bacterial mRNA involved in degradation of 1,2,4-trichlorobenzene by Pseudomonas sp. strain P51 from liquid culture and from river sediment by reverse transcriptase PCR (RT/PCR). 980 14

Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene by a partially reductive pathway in which the intermediate hydroxylaminobenzene is enzymatically rearranged to 2-aminophenol by hydroxylaminobenzene mutase (HAB mutase). The properties of the enzyme, the reaction mechanism, and the evolutionary origin of the gene(s) encoding the enzyme are unknown. In this study, two open reading frames (habA and habB), each encoding an HAB mutase enzyme, were cloned from a P. pseudoalcaligenes JS45 genomic library and sequenced. The open reading frames encoding HabA and HabB are separated by 2.5 kb and are divergently transcribed. The deduced amino acid sequences of HabA and HabB are 44% identical. The HAB mutase specific activities in crude extracts of Escherichia coli clones synthesizing either HabA or HabB were similar to the specific activities of extracts of strain JS45 grown on nitrobenzene. HAB mutase activity in E. coli extracts containing HabB withstood heating at 85 degrees C for 10 min, but extracts containing HabA were inactivated when they were heated at temperatures above 60 degrees C. HAB mutase activity in extracts of P. pseudoalcaligenes JS45 grown on nitrobenzene exhibited intermediate temperature stability. Although both the habA gene and the habB gene conferred HAB mutase activity when they were separately cloned and expressed in E. coli, reverse transcriptase PCR analysis indicated that only habA is transcribed in P. pseudoalcaligenes JS45. A mutant strain derived from strain JS45 in which the habA gene was disrupted was unable to grow on nitrobenzene, which provided physiological evidence that HabA is involved in the degradation of nitrobenzene. A strain in which habB was disrupted grew on nitrobenzene. Gene Rv3078 of Mycobacterium tuberculosis H37Rv encodes a protein whose deduced amino acid sequence is 52% identical to the HabB amino acid sequence. E. coli containing M. tuberculosis gene Rv3078 cloned into pUC18 exhibited low levels of HAB mutase activity. Sequences that exhibit similarity to transposable element sequences are present between habA and habB, as well as downstream of habB, which suggests that horizontal gene transfer resulted in acquisition of one or both of the hab genes.
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PMID:Sequence analysis and initial characterization of two isozymes of hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45. 1087 93

Amplification of DNA or RNA sequences using the polymerase chain reaction (PCR) or reverse transcriptase PCR (RT-PCR) requires primers of an appropriate length to be designed. Two hexamer restriction primers, denoted as E101 and H301, which correspond to sequences of EcoRI and HindIII recognition sites, respectively, were selected and used as primers in PCR and RT-PCR. We first applied the restriction primers to the plasmid DNA and bacterial (Pseudomonas) and plant (Cymbidium) genomic DNAs. We observed positive DNA amplifications with the recombinant plasmid DNA and bacterial and plant genomic DNAs. Purified viral RNA was used for template in the RT-PCR with the primers and successful DNA amplification was obtained. These results suggest that the 6-mer restriction primers can be useful for new applications in PCR.
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PMID:Restriction primers as short as 6-mers for PCR amplification of bacterial and plant genomic DNA and plant viral RNA. 1091 10

A novel single-chained antifungal protein with a molecular weight of 13 kDa displaying an N-terminal sequence with marked similarity to embryo-abundant protein from the white spruce was isolated from the seeds of Ginkgo biloba using ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose, and then gel filtration on Superdex 75. The protein, designated ginkbilobin, exerted potent antifungal activity against a variety of fungi, including Botrytis cinerea, Mycosphaerella arachidicola, Fusarium oxysporum, Rhizoctonia solani, and Coprinus comatus. Ginkbilobin exhibited a moderate antibacterial action against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. It suppressed the activity of HIV-1 reverse transcriptase and the proliferation of murine splenocytes.
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PMID:Ginkbilobin, a novel antifungal protein from Ginkgo biloba seeds with sequence similarity to embryo-abundant protein. 1111

The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal DNA (rDNA) and 16S rRNA, respectively, and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominantly present (PCR) and predominantly active (RT-PCR) bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere, such as Pseudomonas, Comamonas, Variovorax, and Acetobacter, or typical of root-free soil environments, such as Bacillus and Arthrobacter. The PCR-DGGE patterns observed for bulk soil were somewhat more complex than those obtained from rhizosphere samples, and the latter contained a subset of the bands present in bulk soil. DGGE analysis of RT-PCR products detected a subset of bands visible in the rDNA-based analysis, indicating that some dominantly detected bacterial populations did not have high levels of metabolic activity. The sequences detected by the RT-PCR approach were, however, derived from a wide taxonomic range, suggesting that activity in the rhizosphere was not determined at broad taxonomic levels but rather was a strain- or species-specific phenomenon. Comparative analysis of DGGE profiles grouped all DNA-derived root tip samples together in a cluster, and within this cluster the root tip samples from young plants formed a separate subcluster. Comparison of rRNA-derived bacterial profiles showed no grouping of root tip samples versus root base samples. Rather, all profiles derived from 2-week-old plant rhizosphere soils grouped together regardless of location along the root.
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PMID:Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA. 1113 42

Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsC and pmsB) showed homologies with chorismate-utilizing enzymes; a third ORF (pmsE) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and two predicted iron boxes were localized upstream of pmsC. We determined by reverse transcriptase-mediated PCR that the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminished SA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production.
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PMID:Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylic acid and the siderophore pseudomonine in the biocontrol strain Pseudomonas fluorescens WCS374. 1122 88

The endophytic diazotroph Azoarcus sp. strain BH72 is capable of infecting rice roots and of expressing the nitrogenase (nif) genes there. In order to study the genetic background for nitrogen fixation in strain BH72, the structural genes of nitrogenase (nifHDK) were cloned and sequenced. The sequence analysis revealed an unusual gene organization: downstream of nifHDK, a ferredoxin gene (fdxN; 59% amino acid sequence identity to R. capsulatus FdxN) and open reading frames showing 52 and 36% amino acid sequence identity to nifY of Pseudomonas stutzeri A15 and ORF1 of Azotobacter vinelandii were located. Northern blot analysis, reverse transcriptase PCR and primer extension analysis revealed that these six genes are located on one transcript transcribed from a sigma(54)-type promoter. Shorter transcripts sequentially missing genes of the 3' part of the full-length mRNA were more abundantly detected. Mutational analyses suggested that FdxN is an important but not the essential electron donor for dinitrogenase reductase. An in-frame deletion of fdxN resulted in reduced growth rates (59% +/- 9%) and nitrogenase activities (81%) in nitrogen-fixing pure cultures in comparison to the wild type. Nitrogenase activity was fully complemented in an fdxN mutant which carried a nifH promoter-driven fdxN gene in trans. Also, in coculture with the ascomycete Acremonium alternatum, where strain BH72 develops intracytoplasmic membrane stacks, the nitrogenase activity in the fdxN deletion mutant was decreased to 56% of the wild-type level. Surprisingly, the fdxN deletion also had an effect on the rapid "switch-off" of nitrogenase activity in response to ammonium. Wild-type strain BH72 and the deletion mutant complemented with fdxN in trans showed a rapid reversible inactivation of acetylene reduction, while the deletion mutant did not cease to reduce acetylene. In concordance with the hypothesis that changes in the redox state of NifH or electron flux towards nitrogenase may be involved in the mechanism of physiological nitrogenase switch-off, our results suggest that the ferredoxin may be a component involved in this process.
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PMID:Role of a ferredoxin gene cotranscribed with the nifHDK operon in N(2) fixation and nitrogenase "switch-off" of Azoarcus sp. strain BH72. 1137 40

The effectiveness of maintaining prokaryotic RNA in Synechococcus and Pseudomonas cells, fixed in 96% ethanol, 4% paraformaldehyde, or suspended in RNAlater, and held in cold storage for 3 months was compared. Fluorometric determination of the RNA extracted from Synechococcus and Pseudomonas cells indicated that the cell storage treatments tested were equally effective at maintaining their total RNA content. There was not any detectable decrease in the quantity of RNA isolated from the preserved samples during storage. Intact mRNA transcripts of the RuBisCO (rbcL) and nir genes were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) from preserved bacterial cells throughout 3 months of storage. In contrast, RT-PCR failed to amplify the mRNA of the rbcL and nitrite reductase genes in unfixed and/or unpreserved bacterial samples, suggesting that bacterial mRNA can be well maintained during a prolonged storage when cells are preserved properly. In addition, RNAlater is a useful reagent for the storage and maintenance of high quality RNA in unfrozen samples.
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PMID:RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples. 1147 Mar 50

In Pseudomonas syringae strains, the hrp-hrc pathogenicity island consists of an HrpL-dependent regulon that encodes a type III protein translocation complex and translocated effector proteins required for pathogenesis. HrpR and HrpS function as positive regulatory factors for the hrpL promoter, but their mechanism of action has not been established. Both HrpR and HrpS are structurally related to enhancer-binding proteins, but they lack receiver domains and do not appear to require a cognate protein kinase for activity. hrpR and hrpS were shown to be expressed as an operon: a promoter was identified 5' to hrpR, and reverse transcriptase PCR detected the presence of an hrpRS transcript. The hrpR promoter and coding sequence were conserved among P. syringae strains. The coding sequences for hrpR and hrpS were cloned into compatible expression vectors, and their activities were monitored in Escherichia coli transformants carrying an hrpL'-lacZ fusion. HrpS could function as a weak activator of the hrpL promoter, but the activity was only 2.5% of the activity detected when both HrpR and HrpS were expressed in the reporter strain. This finding is consistent with a requirement for both HrpR and HrpS in the activation of the hrpL promoter. By using a yeast two-hybrid assay, an interaction between HrpR and HrpS was detected, suggestive of the formation of a heteromeric complex. Physical interaction of HrpR and HrpS was confirmed by column-binding experiments. The results show that HrpR and HrpS physically interact to regulate the sigma(54)-dependent hrpL promoter in P. syringae strains.
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PMID:Enhancer-binding proteins HrpR and HrpS interact to regulate hrp-encoded type III protein secretion in Pseudomonas syringae strains. 1154 21


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