Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sindbis virus (SIN) is a small positive-strand enveloped RNA virus that infects a broad range of vertebrate and insect cells. A SIN vector (called dsSIN), designed for transient expression of heterologous RNAs and proteins, was engineered by inserting a second subgenomic mRNA promoter sequence into a nonessential region of the SIN genome. By using this vector, dsSIN recombinants have been constructed that express either bacterial chloramphenicol acetyltransferase, a truncated form of the influenza hemagglutinin (HA), or mini-genes encoding two distinct immunodominant cytotoxic T lymphocyte (CTL) HA epitopes. Infection of murine cell lines with these recombinants resulted in the expression of approximately 10(6)-10(7) chloramphenicol acetyltransferase polypeptides per cell and efficient sensitization of target cells for lysis by appropriate major histocompatibility complex-restricted HA-specific CTL clones in vitro. In addition, priming of an influenza-specific T-cell response was observed after immunizing mice with dsSIN recombinants expressing either a truncated form of HA or the immunodominant influenza CTL epitopes. This SIN expression system allows the generation of high-titered recombinant virus stocks in a matter of days and should facilitate mapping and mutational analysis of class I major histocompatibility complex-restricted T-cell epitopes expressed via the endogenous pathway of antigen processing and presentation.
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PMID:Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation. 137 87

An in vitro RNA synthesis system was established in which the influenza virus virion (minus-sense) RNA was made from the synthetic plus-sense RNA (cRNA) template by the purified viral polymerase complex. The cRNA promoter was studied by mutational analysis using the in vitro system, and on the basis of these experiments, the first 11 nucleotides of the 3' noncoding sequence were found to contain the minimum promoter required for virion RNA synthesis. The addition of extra nucleotides at the 3' end decreased the promoter activity of the templates, indicating that the viral polymerase does not recognize an internal promoter efficiently. The wild-type and mutated RNA templates were also tested in vivo by using the ribonucleoprotein transfection system. In contrast to the in vitro system, it was found that the majority of mutations at the 3'-terminal sequence significantly decreased or abolished chloramphenicol acetyltransferase (CAT) expression. These results suggest that the cRNA promoter overlaps other essential cis elements required for chloramphenicol acetyltransferase expression in vivo.
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PMID:Mutational analysis of the promoter required for influenza virus virion RNA synthesis. 160 47

A new transfection system for influenza virus was developed using the clone 76 cell line, in which the viral RNA polymerase and nucleoprotein (NP) genes can be expressed in response to dexamethasone. Ribonucleoprotein (RNP) complexes were reconstituted by expressing proteins from a chimeric NS-chloramphenicol acetyltransferase (CAT) RNA consisting of the full-length negative-strand RNA of the CAT gene positioned between the 5'- and 3'-terminal sequences of influenza virus RNA segment 8, and purifying NP from an NP gene-expressing Escherichia coli strain. When the reconstituted RNP was transfected into clone 76 cells, CAT was produced only when the synthesis of the three RNA polymerase subunits and NP was induced by treatment with dexamethasone.
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PMID:Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes. 160 55

We constructed A-type inclusion body (ATI) hybrid promoters, that is, late ATI promoters followed by tandemly repeated early regions of the promoter for the 7.5-kDa protein (the 7.5-kDa promoter). The repetition of the whole early promoter sequence of the 7.5-kDa gene, including the upstream consensus sequence and initiation region, efficiently increased the early expression of the bacterial chloramphenicol acetyltransferase gene in recombinant vaccinia virus. Recombinant vaccinia virus could express influenza virus hemagglutinin via the hybrid promoter more efficiently, induced higher levels of neutralizing antibody and cytotoxic T lymphocytes, and consequently protected mice more efficiently against challenge with influenza virus than did recombinant vaccinia virus containing the widely used 7.5-kDa promoter.
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PMID:Increased expression in vivo and in vitro of foreign genes directed by A-type inclusion body hybrid promoters in recombinant vaccinia viruses. 165 53

cDNAs for genome RNAs of influenza virus A/PR/8/34 were cloned and portions containing the ATG initiation codon for translation were inserted into the 5' leader sequence of the chloramphenicol acetyltransferase (CAT) gene in a pSV2cat vector. In cells that were transfected with a plasmid containing a cDNA segment for the early gene and then super-infected with influenza virus, the maximal CAT activity was obtained at the early stage of infection. In contrast, a plasmid containing a cDNA segment for the late gene directed the highest activity at the late stage of infection. These observations together with the previous observations [K. Yamanaka et al. (1988) Virus Genes 2: 19-30] indicate that the translational efficiency of influenza viral mRNA is subjected to temporal control following viral infection.
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PMID:Temporal control for translation of influenza virus mRNAs. 192 78

Appropriate RNAs are transcribed and amplified and proteins are expressed after transfection into cells of in vitro-reconstituted RNA-protein complexes and infection with influenza virus as the helper. This system permits us to study the signals involved in transcription of influenza virus RNAs. For the analysis we used a plasmid-derived RNA containing the reporter gene for chloramphenicol acetyltransferase (CAT) flanked by the noncoding sequences of the NS RNA segment of influenza A/WSN/33 virus. Mutations were then introduced into both the 5' and 3' ends, and the resulting RNAs were studied to determine their transcription in vitro and their CAT expression activity in the RNA-protein transfection system. The results reveal that a stretch of uninterrupted uridines at the 5' end of the negative-strand RNA is essential for mRNA synthesis. Also, a double-stranded RNA "panhandle" structure generated by the 5'- and 3'-terminal nucleotides appears to be required for polyadenylation, since opening up of these base pairs diminished mRNA synthesis and eliminated expression of CAT activity by the mutant RNAs. Finally, it was shown that this double-stranded RNA structural requirement is not sequence specific, since a synthetic GC clamp can replace the virus-coded RNA duplex. The data suggest that the viral RNA polymerase adds poly(A) by a slippage (stuttering) mechanism which occurs when it hits the double-stranded RNA barrier next to the stretch of uridines.
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PMID:The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure. 203 59

Influenza A and B viruses have not been shown to form reassortants. It had been assumed that the lack of genotypic mixing between influenza virus types reflected differences in polymerase and packaging specificity. In this study, we show that an influenza A virus polymerase transcribes and replicates a chloramphenicol acetyltransferase (CAT) gene flanked by the nontranslated sequences of an influenza B virus gene. Although the transcription level of this CAT gene was several times lower than that of a CAT gene flanked by the homologous nontranslated sequences of an influenza A virus, we proceeded to construct a chimeric type A/B influenza virus. Using recombinant DNA techniques, a chimeric neuraminidase gene was introduced into the genome of influenza A/WSN/33 virus. The hybrid influenza A/B virus gene contained the coding region of the A/WSN neuraminidase and the 3' and 5' nontranslated sequences of the nonstructural gene of influenza B/Lee virus. The resulting chimeric virus formed plaques in Madin-Darby bovine kidney cells but replicated more slowly and achieved lower titers than wild-type influenza A/WSN/33 virus. The chimeric virus was attenuated for mice as indicated by a 400-fold increase in its LD50. Interestingly, the virus was greatly restricted in replication in the upper respiratory tract and partially restricted in the lungs. Animals infected with the transfectant virus were highly resistant to influenza virus challenge. It appears that this chimeric virus has many of the properties desirable for a live attenuated virus vaccine.
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PMID:An influenza A virus containing influenza B virus 5' and 3' noncoding regions on the neuraminidase gene is attenuated in mice. 205 99

A system for the expression of a foreign gene derived from negative polarity RNA was developed using influenza virus, a negative-stranded RNA virus. From cDNA for the influenza virus RNA genome segment 8, the region coding for the nonstructural protein was deleted and replaced by the chloramphenicol acetyltransferase (CAT) gene. The resulting DNA sequence was placed under the control of the promoter of T7 RNA polymerase such that the antisense RNA to CAT mRNA was produced when transcribed by T7 RNA polymerase. Transfection of HeLa cells with this antisense CAT RNA in the presence of the helper ribonucleoprotein cores led to no significant production of the CAT. In contrast, when the RNA was covered with purified nucleoprotein prior to transfection, the CAT gene was efficiently expressed. This indicated that the viral RNA polymerase transcribed the RNA transfected as the RNA-nucleoprotein complexes. In addition, this system was used for analysis of the cis-acting region in transcription and the promoter structure of the viral RNA genome.
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PMID:In vivo analysis of the promoter structure of the influenza virus RNA genome using a transfection system with an engineered RNA. 205 14

A system is described that allows use of recombinant DNA technology to modify the genome of influenza virus, a negative-strand RNA virus, and to engineer vectors for the expression of foreign genes. Recombinant RNA is expressed from plasmid DNA in which the coding sequence of the influenza A virus NS gene is replaced with that of the chloramphenicol acetyltransferase gene. When transfected with purified influenza A virus polymerase proteins--in the presence of helper virus--the recombinant RNA is amplified, expressed, and packaged into virus particles, which can be passaged several times. The data indicate that the 22 5' terminal and the 26 3' terminal bases of the influenza A virus RNA are sufficient to provide the signals for RNA transcription, RNA replication, packaging of RNA into influenza virus particles.
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PMID:Amplification, expression, and packaging of foreign gene by influenza virus. 259 62

cDNAs for genome RNAs of influenza virus A/PR/8/34 were cloned, and portions containing the ATG for initiation codon of translation were inserted into the 5' leader sequence of the chloramphenicol acetyltransferase (CAT) gene in a pSV2cat vector. When transfected cells were super-infected with influenza virus, the CAT activity was found to vary in a time-dependent fashion: A construct containing a cDNA segment for the nonstructural (NS) protein directed the highest activity during the early stage of infection, while a construct containing a cDNA segment for the neuraminidase (NA) directed the highest activity during the late stage of infection. This time-dependent variation in the CAT activity is in good agreement with that of the synthesis rate of respective viral proteins in infected cells. We propose that the translational efficiency of viral mRNA is subjected to temporal control following viral infection, although viral protein synthesis itself is regulated primarily at the level of mRNA synthesis.
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PMID:Translational regulation of influenza virus mRNAs. 285 14


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