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)

Pyrrolizidine alkaloids (PAs) are constitutive plant defense compounds with a sporadic taxonomic occurrence. The first committed step in PA biosynthesis is catalyzed by homospermidine synthase (HSS). Recent evidence confirmed that HSS evolved by gene duplication from deoxyhypusine synthase (DHS), an enzyme involved in the posttranslational activation of the eukaryotic translation initiation factor 5A. To better understand the evolutionary relationship between these two enzymes, which are involved in completely different biological processes, we studied their tissue-specific expression. RNA-blot analysis, reverse transcriptase-PCR, and immunolocalization techniques demonstrated that DHS is constitutively expressed in shoots and roots of Senecio vernalis (Asteraceae), whereas HSS expression is root specific and restricted to distinct groups of endodermis and neighboring cortex cells located opposite to the phloem. All efforts to detect DHS by immunolocalization failed, but studies with promoter-beta-glucuronidase fusions confirmed a general expression pattern, at least in young seedlings of tobacco (Nicotiana tabacum). The expression pattern for HSS differs completely from its ancestor DHS due to the adaptation of HSS to the specific requirements of PA biosynthesis.
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PMID:Cell-specific expression of homospermidine synthase, the entry enzyme of the pyrrolizidine alkaloid pathway in Senecio vernalis, in comparison with its ancestor, deoxyhypusine synthase. 1222 85

Three genes encoding phosphoenolpyruvate carboxylase were isolated from Nicotiana sylvestris and designated Nsppc1-3. Sequencing of nucleotides showed that the coding sequence and deduced amino acid sequences were highly conserved among the genes, but sequences for noncoding regions including introns and 5'-flanking regions were not conserved. Analysis of the transcript level of the genes by a combination of reverse transcriptase-PCR and restriction fragment polymorphism showed mostNsppc1 in the leaves, stems, roots, and cultured cells of N. sylvestris. beta-Glucuronidase activity was detected histochemically in mesophyll cells in leaves, lateral buds, and vascular bundles in roots of transgenic tobacco harboring a chimeric construct of the Nsppc1 promoter and the gene for beta-glucuronidase. Deletion analysis indicated the presence of a silencer-like element for basal expression in the promoter region of Nsppc1.
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PMID:Genomic structure and promoter analysis of phosphoenolpyruvate carboxylase in a C3 plant, Nicotiana sylvestris. 1235 29

NifA, the transcriptional activator of nitrogenase (nif) genes, has up to now been described to be regulated in its activity via the sensor NifL only for members of the gamma-subgroup of the PROTEOBACTERIA: This paper reports a functionally similar NifL-like protein outside this group in Azoarcus sp. strain BH72, a diazotrophic grass endophyte belonging to the beta-subgroup of the PROTEOBACTERIA: Its structural genes for nitrogenase (nifHDK) are regulated in response to combined nitrogen and O(2) and expressed endophytically inside rice roots. In order to characterize nitrogen-regulatory genes, an Azoarcus sp. BH72 genomic library was used to select cosmids that complemented a nifA mutation in Azotobacter vinelandii. Sequence analysis of the 3.4 kb genomic region complementing nifA showed two ORFs with sequence identities of 44% to NifL and 61% to NifA of Azotobacter vinelandii. According to Northern blot and reverse transcriptase PCR analysis, the nifLA transcript was more abundant at low combined nitrogen and O(2) levels, results which were corroborated by GUS (beta-glucuronidase) assays using a transcriptional nifL::gusA fusion. N(2) fixation was abolished in a NifLA(-) and a NifA(-) mutant, wild-type fixation being restored by nifLA in trans. The NifLA(-) mutant also failed to activate nifH::gus expression, indicating that NifA is the obligate transcriptional activator for nifHDK. A nifL mutant was diazotrophic and did not show repression of nifH::gusA by ammonium or O(2), suggesting that NifL of Azoarcus sp. strain BH72 has a similar role in inactivating NifA in response to O(2) and combined nitrogen as NifL in bacteria of the gamma-PROTEOBACTERIA:
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PMID:Identification of a NifL-like protein in a diazotroph of the beta-subgroup of the Proteobacteria, Azoarcus sp. strain BH72. 1236 54

Each fiber of cotton (Gossypium hirsutum) is a single epidermal cell that rapidly elongates to 2.5 to 3.0 cm from the ovule surface within about 16 d after anthesis. A large number of genes are required for fiber differentiation and development, but so far, little is known about how these genes control and regulate the process of fiber development. To investigate gene expression patterns in fiber, a cDNA, GhTUB1, encoding beta-tubulin was isolated from a cotton fiber cDNA library. The analyses of RNA northern-blot hybridization and reverse transcriptase-polymerase chain reaction demonstrated that GhTUB1 transcripts preferentially accumulated at high levels in fiber, at low levels in ovules at the early stage of cotton boll development, and at very low levels in other tissues of cotton. The corresponding GhTUB1 gene including the promoter region was isolated by screening a cotton genomic DNA library. To demonstrate the specificity of the GhTUB1 promoter, the 5'-flanking region including the promoter and 5'-untranslated region was fused with the beta-glucuronidase reporter gene. The expression of the reporter chimera was examined in a large number of transgenic cotton plants. Histochemical assays demonstrated that GhTUB1::beta-glucuronidase fusion genes were expressed preferentially at high levels in fiber and primary root tip of 1- to 3-d-old seedlings and at low levels in other tissues such as ovule, pollen, seedling cotyledon, and root basal portion. The results suggested that the GhTUB1 gene may play a distinct and required role in fiber development. In addition, the GhTUB1 promoter may have great potential for cotton improvement by genetic engineering.
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PMID:Molecular characterization of the cotton GhTUB1 gene that is preferentially expressed in fiber. 1237 34

Cyst nematodes induce a metabolically highly active syncytial cell complex in host roots. The syncytia are symplastically isolated. Because they form a strong sink, assimilates must be imported via the apoplast, thus suggesting that specific membrane-bound sugar transport proteins are expressed and activated. To identify possible candidate genes, transgenic Arabidopsis plants expressing different reporter genes under the control of different promoters from Arabidopsis sugar transporter genes were infected with the beet cyst nematode (Heterodera schachtii). With polymerase chain reaction, 13 additional sugar transporters were tested for their presence in the syncytia through the use of a syncytium-specific cDNA library. Analysis of the infected roots showed that the promoter of the sucrose (Suc) transporter AtSUC2 gene that codes for a companion cell-specific Suc transporter in noninfected plants was found to be expressed in syncytia. Its expression patterns in beta-glucuronidase and green fluorescent protein plants were monitored. Syncytium-specific gene expression was confirmed by reverse transcriptase-polymerase chain reaction. Results support the idea that AtSUC2 mediates the transmembrane transfer of Suc. AtSUC2 is the first disaccharide carrier described to be activated by pathogens.
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PMID:The companion cell-specific Arabidopsis disaccharide carrier AtSUC2 is expressed in nematode-induced syncytia. 1252 15

Following adeno-associated virus (AAV)-mediated transduction, cellular RNA preparations can be contaminated with AAV single-stranded DNA. The single-stranded DNA genome of recombinant AAV vectors can serve as an efficient, but undesirable, template for traditional reverse transcriptase-polymerase chain reaction (RT-PCR) methods. Consequently, recombinant AAV gene therapy presents a unique challenge to the design of sensitive and reliable methods to detect vector-derived mRNA. Several methods have been proposed to reduce the presence of single- and double-stranded vector DNA without compromising RNA specificity. For example, DNase I, although widely used, can be ineffective at completely removing the AAV single-stranded DNA genome. We have developed a sensitive real-time RNA-Specific reverse transcriptase PCR (RS-PCR) method that is independent of DNase I treatment. The RS-PCR method relies on the generation of a first-strand cDNA template using a primer with a linker sequence, X, at the 5'- end such that synthesis of second-strand cDNA incorporates the X-linker sequence into the cDNA template. The RS-PCR then utilizes forward and reverse primers targeting AAV vector sequence and the X-primer site, respectively, while a vector-specific Taqman probe makes sensitive real-time detection possible. We present data to validate the sensitivity and RNA specificity of the RS-PCR method and propose two unique endogenous control strategies by monitoring expression of both beta-glucuronidase and endogenous cystic fibrosis transmembrane conductance regulator (CFTR). Finally, we demonstrate the utility of this new RS-PCR method in detecting recombinant AAV-CFTR expression, including, an in vitro transduction assay and methods to support both preclinical and clinical trials.
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PMID:A sensitive, real-time, RNA-specific PCR method for the detection of recombinant AAV-CFTR vector expression. 1293 41

p-Chlorophenoxyisobutyric acid (PCIB) is known as a putative antiauxin and is widely used to inhibit auxin action, although the mechanism of PCIB-mediated inhibition of auxin action is not characterized very well at the molecular level. In the present work, we showed that PCIB inhibited BA::beta-glucuronidase (GUS) expression induced by indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid, and 1-naphthaleneacetic acid. PCIB also inhibited auxin-dependent DR5::GUS expression. RNA hybridization and quantitative reverse transcriptase-polymerase chain reaction analyses suggested that PCIB reduced auxin-induced accumulation of transcripts of Aux/IAA genes. In addition, PCIB relieved the reduction of GUS activity in HS::AXR3NT-GUS transgenic line in which auxin inhibits GUS activity by promoting degradation of the AXR3NT-GUS fusion protein. Physiological analysis revealed that PCIB inhibited lateral root production, gravitropic response of roots, and growth of primary roots. These results suggest that PCIB impairs auxin-signaling pathway by regulating Aux/IAA protein stability and thereby affects the auxin-regulated Arabidopsis root physiology.
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PMID:p-Chlorophenoxyisobutyric acid impairs auxin response in Arabidopsis root. 1452 8

A screening process was applied to extracts made from Sutherlandia frutescens (L.) R. Br (Fabaceae) and Lobostemon trigonus (Boraginaceae) as identified by the Botany Department, University of Port Elizabeth to detect if any of the extracts inhibited the human immunodeficiency virus (HIV). For purposes of dereplication, sulphated polysaccharides were removed and bovine serum albumin (BSA) was included in the assays to adsorb non-specific tannins potentially present. In the reverse transcriptase (RT) assay, an aqueous extract of the Lobostemon leaves inhibited HIV-1 RT with an IC50 value of 49 microg/ml, while in the protease assay no inhibition was seen. In the alpha- and beta-glucosidase assays, no significant inhibition was seen with the inclusion of BSA, indicating tannin-based inhibitory effects on these two enzymes. The beta-glucuronidase inhibitory activity, however, was retained in the presence of BSA. The study shows that Sutherlandia extracts contain inhibitory compounds active against HIV target enzymes, while aqueous Lobostemon leaf extracts contain a potent HIV-1 RT inhibitor, thus showing a potential mechanistic action of these plants in aiding HIV-positive patients.
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PMID:Anti-HIV activities of organic and aqueous extracts of Sutherlandia frutescens and Lobostemon trigonus. 1558 58

Drought treatment induces the accumulation of dcTLP, which is similar in structure to the thaumatin-like proteins (TLPs) found in the embryogenic calli, seedlings, and mature plants of carrot (Daucus carota). We isolated a full-length dcTLP cDNA clone from carrot and characterized the 5' upstream sequences. The coding region of dcTLP consisted of 645 nucleotides; the theoretical pI value was 4.9, and its molecular weight was approximately 22 kDa. The production of dcTLP transcripts in the seedlings increased dramatically with dehydration treatment but was not affected by abscisic acid (ABA), salicylic acid, or jasmonic acid. The expression patterns of dcTLP mRNA at different developmental stages and in response to a variety of signal molecules was analyzed using reverse transcriptase-PCR and promoter analysis with fused genes of 0.5-kb 5' upstream sequences in which beta-glucuronidase (GUS) reporter genes (gus) were established. The induction of dcTLP was found to be highly specific to drought stress in the embryogenic calli, seedlings, and mature plants. Our results suggest that this new isoform of TLP that has been isolated from carrot is a drought-specific, ABA-independent, non-organ-specific, and non-developmental-stage-specific protein.
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PMID:Drought-inducible-but ABA-independent-thaumatin-like protein from carrot (Daucus carota L.). 1578 5

Previous gene transfer studies of the herpes simplex virus type 1 (HSV-1) using the latency-associated transcript (LAT) promoter have reported a decrease in transgene expression in the brain over time, but the extent of this decrease has not been measured and it is unknown if expression eventually stabilizes. We examined LAT promoter-mediated transgene expression in the mouse brain for 1 year following intracranial injection with a HSV-1 vector expressing human beta-glucuronidase (GUSB). The vector genome copy number remained stable from 2 to 52 weeks. Quantitative reverse transcriptase PCR detected a peak of LAT intron expression at 2 weeks (corresponding to the end of the acute phase of viral infection), followed by stable expression during latency (13-52 weeks). The number of GUSB-positive cells also had a peak in the acute phase and then was stable during latency (13-52 weeks). GUSB enzymatic activity was maintained at 11% of normal at 6 and 12 months, indicating that the LAT promoter is capable of driving stable transgene expression in the brain.
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PMID:Stable levels of long-term transgene expression driven by the latency-associated transcript promoter in a herpes simplex virus type 1 vector. 1612 87


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