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 ribonucleoprotein core of reovirus is a multienzyme complex that transcribes messenger ribonucleic acid (mRNA) from double-stranded RNA templates. So far, the core has resisted attempts to disassemble it and identify the polypeptide species responsible for RNA polymerase activity. As an alternative approach, we tested pyridoxal 5-phosphate (PLP) as a potential affinity labeling reagent for reovirus transcriptase in vitro; PLP has been used as an affinity reagent for cellular and viral nucleic acid polymerases. We found that PLP inhibited reovirus transcriptase reversibly (apparent Ki = 0.2 mM), but the inhibition was noncompetitive with respect to each of the four ribonucleoside triphosphates. This interaction required both the aldehyde and phosphate moieties in PLP, since pyridoxamine and pyridoxal were relatively inactive. To identify the polypeptides involved, we labeled the PLP--core complex by reductive alkylation with [3H]borohydride. At PLP concentrations close to the apparent Ki, labeling was selective for the two largest virion polypeptides, lambda 1 and lambda 2. At saturation, there were only 10 high-affinity PLP binding sites per core in each of the lambda polypeptide species. These findings implicate either or both lambda polypeptide species in viral transcription and they indicate that a special population, representing no more than 10% of the total lambda molecules in each core, participates in RNA synthesis.
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PMID:Pyridoxal phosphate as a probe of reovirus transcriptase. 735 41

Hepatitis C virus is a positive single-strand RNA virus distantly related to flaviviruses. Therefore RNA replicase, an RNA-dependent RNA polymerase, may be essential for the replication of hepatitis C virus, as well as other RNA viruses. In this study we synthesized the recombinant polypeptide (HCV-NS5 antigen) with a 576 bp cDNA encoding a part of the NS5 region of the HCV genome that has the Gly-Asp-Asp motif. The antibody against this polypeptide was obtained from rabbit serum. In Western-blot analysis with NS5 IgG HCV antibody, an 84-kD protein was clearly detected as a single band in the microsomal fraction but not in the nuclear and mitochondrial fractions or in the cytosol fraction. Immunohistochemically, HCV-NS5 antigen was clearly stained in the cytoplasm of hepatocytes but not in the nucleus or cell membrane. Moreover, as determined on immunoelectron microscopy, HCV-NS5 antigen was demonstrated with fine granular distribution along the endoplasmic reticulum but not in other organelles, including the nucleus and mitochondria. Immunoreaction in other cell types was negative. These results indicate that replication of HCV may occur only in hepatocytes and that HCV-NS5 may be produced in the endoplasmic reticulum of these cells. HCV-NS5 antigen was stained only in the livers of hepatitis C virus-positive patients but not in sections from patients with chronic type B hepatitis or alcoholic fibrosis. In chronic type C liver disease, the overall detection rate of HCV-NS5 antigen was 56% (33% in chronic persistent hepatitis, 52% in chronic active hepatitis and 86% in cirrhosis).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Detection of antigens related to hepatitis C virus RNA encoding the NS5 region in the livers of patients with chronic type C hepatitis. 750 61

We have investigated the expression of diazepam binding inhibitor (DBI) (also called acyl-CoA-binding protein or endozepine) transcripts in different human tissues and tissue culture cell lines by reverse-transcriptase assisted PCR and RNase protection assay. Two different DBI transcripts capable of encoding polypeptides of 86 and 104 amino acids were detected in all the human tissues and cell lines studied. The transcript coding for the 86 amino acid DBI polypeptide was found to represent the majority of the total DBI transcript pool.
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PMID:The characterization of two diazepam binding inhibitor (DBI) transcripts in humans. 753 63

Mounting experimental evidence suggests that the TAT protein, released from human immunodeficiency virus-1 (HIV-1)-infected inflammatory cells, may genetically reprogram targeted cells within a localized environment to develop highly vascularized tumors of mesenchymal origin. The fibroblast growth factor (FGF) family of polypeptides has gained general acceptance as initiators of angiogenesis and functions as potent mitogens for mesoderm-derived cells. To evaluate a potential biological relationship between TAT and acidic FGF (FGF-1), primary murine embryonic fibroblasts either were transfected with the viral transactivator or were transduced (retrovirally mediated) with a secreted, chimeric form of the human polypeptide growth factor, human stomach tumor/Kaposi's sarcoma (hst/KS)FGF-1. Reverse transcriptase-polymerase chain reaction, Western blotting, in situ immunohistochemical, heparin affinity, DNA synthesis, and transient transfection techniques were used to confirm expression, localization, and functionality of the transgenes. Both transfected and transduced cells constitutively expressing either TAT or (hst/KS)FGF-1 adopted a transformed phenotype, maintained aggressive growth behavior, and demonstrated both induction of FGF-specific phosphotyrosyl proteins and nuclear association of FGF-1 and FGF-1 receptor. Increased levels of endogenous, murine FGF-1 mRNA (reverse transcriptase-polymerase chain reaction) and protein (immunoblot analysis) were apparent in both (hst/KS)FGF-1- and TAT-transformed cells. Medium conditioned by (hst/KS)FGF-1-transduced cells contained steady-state levels of biologically active FGF-1 which exhibited a representative molecular weight. Limited sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the conditioned medium from TAT-transformed cells demonstrated the appearance of FGF-1 as latent, high molecular weight complexes requiring reducing agents to activate full biological activity. Collectively, these results suggest that TAT induces the expression and secretion of FGF-1, which may be potentially relevant to the pathophysiological development of AIDS-Kaposi's sarcoma.
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PMID:The HIV-1 TAT protein induces the expression and extracellular appearance of acidic fibroblast growth factor. 754 39

The genome of cowpea mottle virus (CPMoV) is a positive ssRNA of 4029 nucleotides with six major open reading frames (ORFs). A non-coding region of 34 nucleotides precedes the first AUG. ORF1 encodes a 25 kDa polypeptide of unknown function and ORF2 encodes a 56 kDa putative RNA replicase. Like other members of carmoviruses, suppression of the amber termination codon of ORF1 would produce a readthrough polypeptide of 83 kDa. ORF3 and ORF4 encode two small proteins of 7.8 and 9.8 kDa, respectively. ORF5 encodes the 40 kDa capsid protein. ORF6 is located within ORF5 but is in a different frame and has no postulated function. CPMoV RNA is blocked at the 5' end and is not polyadenylated at the 3' end. Comparison of the physicochemical properties, genomic arrangement, and predicted amino acid sequences with those of other viruses justify the assignment of CPMoV to the genus Carmovirus, family Tombusviridae.
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PMID:The nucleotide sequence of cowpea mottle virus and its assignment to the genus Carmovirus. 759 92

An RNA-directed RNA polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue was purified to electrophoretic homogeneity. A terminal transferase activity that was found to cofractionate with RdRP from DEAE-Sepharose and DNA-cellulose columns was removed by chromatography on a Mono Q column. The highly purified RdRP exhibits a specific activity of 500 nmol x mg-1 x 30 min-1, which corresponds to a 100,000-fold enrichment of the enzyme. In buffer containing 50% glycerol, its activity decreased by about 15%/month. RdRP activity coincided with the silver staining intensity of a single 128-kDa polypeptide when the fractions eluted from the Mono Q column were analyzed by electrophoresis in a SDS-polyacrylamide gel. Its molecular mass and its sedimentation coefficient of 6.6 S indicate that RdRP is a nearly globular molecule. The catalytic activity of RdRP is resistant to alpha-amanitin and actinomycin D. In tomato leaves systemically infected with potato spindle tuber viroid, the activity of RdRP was found to be increased about 3-fold compared with RdRP isolated from healthy leaf tissue.
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PMID:RNA-directed RNA polymerase from tomato leaves. I. Purification and physical properties. 768 22

The nucleotide sequence of the 5'-end of feline calicivirus (FCV) Japanese F4 strain genome was determined. This region had 5311 bases and contained a large open reading frame (ORF1) encoding the non-structural proteins. The nucleotide sequence of the ORF1 region was highly conserved as compared with that of FCV F9 strain. When the deduced amino acid sequence of the ORF1 was compared with those of FCV F9 and CFI strains, the sequence was also highly conserved (88.9% and 88.8%, respectively). Functional motifs of the non-structural proteins were common to these strains. There were 2C polypeptide-, 3C cysteine protease- and 3D RNA-dependent RNA polymerase-like regions. The N-terminal region of 2C-like region continued upstream from the region identified by Neill [Virus Res. 17: 145-160]. Furthermore, the presence of 2B-like region was suggested in the upper stream of the 2C-like region, although the function of the region is unknown. When Kyte and Dolittle hydrophobicity profiles of the predicted amino acid sequences of the ORF1s of FCV F4 and F9 were computed and compared, both the profiles had striking similarities. In the region between residues 950-1000, there was a high rate of basic amino acid residues, suggesting that the polypeptide in this region of FCV may have a nucleic acid-binding function.
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PMID:The molecular cloning and sequence of an open reading frame encoding for non-structural proteins of feline calicivirus F4 strain isolated in Japan. 769 98

The double-stranded RNA genome of giardiavirus (GLV) has only two large open reading frame (ORFs). The 100-kDa capsid polypeptide (p100) is encoded by ORF1, whereas the only other viral polypeptide, the 190-kDa GLV RNA-dependent RNA polymerase (p190), is synthesized as an ORF1-ORF2 fusion protein by a (-1) ribosomal frameshifting. Edman degradation revealed that p100 was N-terminally blocked except for 2 to 5% of it that showed free N terminus starting from amino acid residue 33 of ORF1. Studies using antiserum targeted against amino acid residues 6 to 27 indicated that this region (NT) is absent from viral p100 and p190, while pulse-labelling experiments showed that NT is present in nascent p100 synthesized in GLV-infected Giardia lamblia but removed subsequently. In contrast, this region was retained in the two viral proteins synthesized in vitro, and it was not removed upon prolonged incubation or inclusion of microsomal fraction in the in vitro translation reaction mixtures. These results suggest that endoplasmic reticulum is not involved in the protein processing and that the precursors of p100 and p190 are incapable of cleaving themselves or each other. This specific cleavage was reproduced when lysates from GLV-infected G. lamblia were added, but not those from uninfected cells. The cleavage activity was relatively insensitive to phenylmethylsulfonyl fluoride, but it was inhibitable by leupeptin or E-64, two known specific inhibitors of cysteine protease. The possible origin of this processing activity is discussed.
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PMID:Maturation of giardiavirus capsid protein involves posttranslational proteolytic processing by a cysteine protease. 770 5

Appressorium formation in germinating Colletotrichum gloeosporioides is induced by the surface wax of its host. One of the genes expressed uniquely in C. gloeosporioides during appressorium formation induced by the host signal has been designated cap20, and this gene and its cDNA were cloned and sequenced. Nucleotide sequences of both revealed an open reading frame that could encode a 183-amino acid polypeptide that did not have significant homology with any known proteins. Reverse transcriptase-polymerase chain reaction detected cap20 gene transcripts at the infection front on the surface and within tomato fruits infected by C. gloeosporioides. Gene-disrupted mutants incapable of expressing cap20 showed a drastically decreased virulence on avocado and tomato fruits. These results suggest that cap20 plays a significant role in the infection of the host.
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PMID:Cloning of a gene expressed during appressorium formation by Colletotrichum gloeosporioides and a marked decrease in virulence by disruption of this gene. 775 29

Poliovirus (PV) 2C protein is a nonstructural polypeptide involved in viral RNA replication, whose biochemical activity(ies) in this process has not been defined. By using site-directed mutagenesis, it was shown previously that disruption of nucleotide-binding motifs present in this protein abolished viral RNA synthesis (C. Mirzayan and E. Wimmer, Virology 189:547-555, 1992; N. L. Teterina, K. M. Kean, E. Gorbalenya, V. I. Agol, and M. Girard, J. Gen. Virol. 73:1977-1986, 1992). We have tested whether PV 2C or 2BC protein provided in trans could rescue the replication of these mutated genomes. Rescuing proteins were provided either by cotransfection with helper chimeric PV-coxsackievirus genomes or by expression in cells with a vaccinia virus-T7 RNA polymerase transient-expression system. We report here that replication of mutated RNAs genomes was poorly supported in trans both by helper genomes and by expressed 2C or 2BC proteins. Similarly, very inefficient complementation was observed for two mutated genomes with lethal lesions in 3D polymerase coding sequence. Our results indicate that poliovirus RNA replication shows marked preference for proteins contributed in cis.
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PMID:Inefficient complementation activity of poliovirus 2C and 3D proteins for rescue of lethal mutations. 776 84


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