Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The tertiary structure in the 3'-untranslated region (3'-UTR) of Bamboo mosaic virus (BaMV) RNA is known to be involved in minus-strand RNA synthesis. Proteins found in the RNA-dependent RNA polymerase (RdRp) fraction of BaMV-infected leaves interact with the radio labeled 3'-UTR probe in electrophoretic mobility shift assays (EMSA). Results derived from the ultraviolet (UV) cross-linking competition assays suggested that two cellular factors, p43 and p51, interact specifically with the 3'-UTR of BaMV RNA. p43 and p51 associate with the poly(A) tail and the pseudoknot of the BaMV 3'-UTR, respectively. p51-containing extracts specifically down-regulated minus-strand RNA synthesis when added to in vitro RdRp assays. LC/MS/MS sequencing indicates that p43 is a chloroplast phosphoglycerate kinase (PGK). When the chloroplast PKG levels were knocked down in plants, using virus-induced gene silencing system, the accumulation level of BaMV coat protein was also reduced.
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PMID:Chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of Bamboo mosaic virus. 1716 94

Eukaryotic mRNAs that prematurely terminate translation are recognized and degraded by nonsense mediated decay (NMD). This degradation pathway is well studied in animal and yeast cells. The data available imply that NMD also takes place in plants. However, the molecular mechanism of recognition and degradation of plant RNAs containing premature terminator codon (PTC) is not known. Here we report that in plant cells this mechanism involves the recognition of the sizes of the 3'-untranslated regions (3'UTR). Plant 3'UTRs longer than 300 nucleotides induce mRNA instability. Contrary to mammalian and yeast cells, this destabilization does not depend on the presence of any specific sequences downstream of the terminator codon. Unlike nuclear-produced mRNAs, plant virus vector long 3'UTR-containing RNAs, which are synthesized directly in the cytoplasm, are stable and translated efficiently. This shows that RNAs produced in the cytoplasm by viral RNA-dependent RNA polymerase are able to avoid the proposed mechanism.
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PMID:Stability of plant mRNAs depends on the length of the 3'-untranslated region. 1722 92

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.
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PMID:Multiple enzyme activities of flavivirus proteins. 1731 55

The Toxocara canis "abundant novel transcripts" (ant) are four highly expressed products, constituting >18% of ESTs from the infective stage of this widely prevalent nematode parasite. Using 5' RACE, we determined full-length sequences for each ant gene, between 1.8 and 2.8kb. The four genes (termed ant-3, -5, -30 and -34), share no coding sequence similarity, although their 3'UTRs (untranslated regions) are homologous. Predicted ANT-5 and ANT-30 proteins show distant similarity to RNA regulatory proteins, RNA-dependent RNA polymerase and DEAH-box helicase, respectively. Surprisingly, ant-3 appears to be bi-cistronic, encoding two ORFs (ANT-3.1 and -3.2), each with a predicted N-terminal signal sequence. Antibodies raised to recombinant proteins did not react with native parasite products, indicating that protein expression did not accord with transcript abundance. However, antibody reactivity to two gene products (ANT-3.1 and ANT-34) was present in patient sera, suggesting that these proteins are synthesized later in infection. To test whether 3'UTRs may regulate expression, the ant-34 3'UTR sequence was inserted adjacent to enhanced green fluorescent protein (EGFP) for transformation of Caenorhabditis elegans. The ant-34 3'UTR greatly reduced EGFP expression, inhibiting both transcription and translation. We identified a tract in this UTR with significant sequence complementarity to the C. elegans micro-RNA lin-4. While infective stage parasites stockpile high levels of the ant transcripts, we suggest that translation is repressed, possibly by a mechanism involving 3' UTR motifs shared by the four genes.
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PMID:Four abundant novel transcript genes from Toxocara canis with unrelated coding sequences share untranslated region tracts implicated in the control of gene expression. 1870 93

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3'-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (K(app)(m) approximately 100-400 mum) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 10(3)- to 10(4)-fold, revealing a much reduced nucleotide requirement for elongation (K(app)(m) approximately 0.03-0.09 microm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 microm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3'-end of the HCV(-)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.
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PMID:Hepatitis C virus NS5B polymerase exhibits distinct nucleotide requirements for initiation and elongation. 1884 Jun 5

A cotton (Gossypium hirsutum L.) genomic clone encompassing a 17.9-kb DNA fragment was found to contain a delta-12 fatty acid desaturase gene (designated FAD2-4). The FAD2-4 open reading frame has 1,155bp and is uninterrupted, encoding a conceptual FAD2-4 polypeptide of 384 amino acids that has 98% identity with the cotton FAD2-3 polypeptide. The FAD2-4 gene has a single intron of 2,780 bp in its 5'-untranslated region (5'-UTR). The 3'-flanking regions and 5'-UTR introns in the FAD2-4 and FAD2-3 genes are quite different, indicating that the genes might be paralogs in the cotton genome. Reverse transcriptase (RT)-PCR analysis indicated that the FAD2-4 and FAD2-3 genes were expressed in all tissues examined, including seeds, seedling tissues, young and mature leaves, roots, stems, developing flower buds, and ovule fibers. These constitutive patterns of expression were notably different from that of the FAD2-1 gene, which was restricted to seeds and developing flower buds, or to the expression of a newly-identified FAD2-2 gene isoform, which was barely detectable in roots, hypocotyls, stems, and fibers, but was expressed in all other tissues. The FAD2-4 coding region was expressed in yeast and shown to encode a functional delta-12 desaturase, converting oleic acid (C18:1) to linoleic acid (C18:2) in recombinant yeast cells. In addition, both the FAD2-4 and the FAD2-3 genes were transferred into the Arabidopsis thaliana fad2-1 mutant background where they effectively restored wild type fatty acid composition and growth characteristics. Finally, the cotton FAD2-4 green fluorescent protein (GFP) fusion polypeptide appeared to be localized in the endomembrane system of transgenic Arabidopsis plants in the complemented fad2-1 mutant background, supporting a functional ER location for the cotton FAD2-4 polypeptide in this heterologous plant system. Thus, a new functional member of the FAD2 gene family in cotton has been characterized, indicating a complex regulation of membrane lipid desaturation in this important fiber/oilseed crop.
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PMID:Identification and expression of a new delta-12 fatty acid desaturase (FAD2-4) gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana plants. 1921 93

RNA elements within the flavivirus genome may play essential regulatory roles during virus replication. Here, recombinant West Nile virus (WNV) NS5 protein was used in combination with WNV subgenomic RNA templates to establish in vitro RNA-dependent RNA polymerase and RNA-binding assays. These assays identified mutations in the stem-loop A (SLA) region of the 5' untranslated region (5'UTR) altering NS5 RNA synthesis and RNA-binding capability. These mutations were then introduced into the full-length WNV genome by reverse genetics. Further analysis of the mutant viruses showed that deletion of nt 46-60, which disrupted the stem and side loop of SLA, greatly compromised virus replication, whereas mutations that destroyed the top loop of SLA required for RNA synthesis in vitro did not significantly alter virus replication. These results suggest that SLA present in the 5'UTR of WNV is essential for RNA synthesis in vitro and for virus replication.
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PMID:RNA elements within the 5' untranslated region of the West Nile virus genome are critical for RNA synthesis and virus replication. 2001 34

Sesbania mosaic virus (SeMV), a single-strand positive-sense RNA plant virus, belongs to the genus Sobemoviruses. Mechanism of replication in Sobemoviruses is poorly understood. In the present study, SeMV RNA-dependent RNA polymerase (RdRp) was overexpressed and purified as a thioredoxin-tagged protein. The recombinant SeMV RdRp could synthesize RNA from genomic or subgenomic RNA templates, even in the absence of the protein primer, VPg. Analysis of the product indicated that it was double-stranded and that the mode of initiation was de novo. Mutational analysis of the 3' UTR of subgenomic RNA revealed that a stem-loop structure at the 3' end was important. Further, analysis of this stem-loop showed that the SeMV RdRp was capable of recognizing stem-loop structures of various lengths and forms. These results demonstrate that the SeMV RdRp is capable of primer-independent RNA synthesis in vitro.
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PMID:Primer-independent initiation of RNA synthesis by SeMV recombinant RNA-dependent RNA polymerase. 2033 53

To identify hepatitis C virus (HCV) transmission routes among injection drug users in Northern Vietnam, plasma samples were collected from 486 drug users in Hai Phong. Plasma viral RNA was extracted from 323 (66.5%) samples that were positive for anti-HCV antibodies. Portions of the HCV 5'-untranslated (5'UTR)-Core and NS5B genes were amplified by reverse-transcriptase polymerase chain reaction, sequenced directly, and genotyped in 194 and 195 specimens, respectively. Both regions were genotyped in 137 specimens. In the 5'UTR-Core region, genotype 6a was predominant (32.5%), followed by genotype 1a (23.7%), genotype 1b (20.6%), and genotype 6e (14.4%). In the NS5B region, genotype 1a was predominant (42.6%), followed by genotype 1b (24.1%), genotype 6a (14.4%), genotype 3b (7.2%), and genotype 6e (5.1%). Of the 137 specimens with both regions genotyped, 23 (16.8%) showed discordant genotyping results between the two regions, suggesting possible recombination and/or dual infection. Phylogenetic analysis revealed close associations between Hai Phong strains and strains from Southern China: the Yunnan province for genotype 3b; the Guangxi province for genotype 6e; the USA for genotype 1a; and Southern Vietnam for genotypes 1a and 6e. The human immunodeficiency virus (HIV) infection rate among HCV-infected injection drug users was 52.6-55.4% and did not differ significantly by HCV genotype. Most drug users infected with HIV-1 [98.8% (171/173)] were co-infected with HCV. These results suggest multiple routes of HCV transmission among injection drug users in Northern Vietnam that may also be HIV transmission routes.
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PMID:Multiple routes of hepatitis C virus transmission among injection drug users in Hai Phong, Northern Vietnam. 2057 71

In plants, small interfering RNAs (siRNAs) with sequence homology to transcribed regions of genes can guide the sequence-specific degradation of corresponding mRNAs, leading to posttranscriptional gene silencing (PTGS). The current consensus is that siRNA-mediated PTGS occurs primarily in the cytoplasm where target mRNAs are localized and translated into proteins. However, expression of an inverted-repeat double-stranded RNA corresponding to the soybean FAD2-1A desaturase intron is sufficient to silence FAD2-1, implicating nuclear precursor mRNA (pre-mRNA) rather than cytosolic mRNA as the target of PTGS. Silencing FAD2-1 using intronic or 3'-UTR sequences does not affect transcription rates of the target genes but results in the strong reduction of target transcript levels in the nucleus. Moreover, siRNAs corresponding to pre-mRNA-specific sequences accumulate in the nucleus. In Arabidopsis, we find that two enzymes involved in PTGS, Dicer-like 4 and RNA-dependent RNA polymerase 6, are localized in the nucleus. Collectively, these results demonstrate that siRNA-directed RNA degradation can take place in the nucleus, suggesting the need for a more complex view of the subcellular compartmentation of PTGS in plants.
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PMID:Posttranscriptional gene silencing in nuclei. 2117 64


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