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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A variety of DNA viruses are known to activate gene expression directed by the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). In light of the proposed use of recombinant vaccinia virus for HIV-1 vaccines, evaluation of the role of vaccinia virus in HIV-1 activation is warranted. To investigate whether vaccinia virus induces HIV LTR-directed gene expression, transient expression assays in Jurkat cells persistently infected with vaccinia virus (Jvac) using plasmid DNA containing the LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were performed. CAT activity in Jvac cells was always recorded, although the level appears to fluctuate independently of virus titers. Dual intracytoplasmic staining and fluorescence-activated cell sorter analysis showed that CAT activity was expressed in the infected cells. CAT expression was not due to plasmid replication, since plasmid DNA extracted from Jvac cells 48 h after transfection was restricted only by enzymes which recognize methylated sequences, indicating a prokaryotic source for the DNA. These findings suggest that a factor(s) present in vaccinia virus-infected cells is capable of activating the LTR of HIV-1.
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PMID:Activation of the human immunodeficiency virus type 1 long terminal repeat by vaccinia virus. 154 51

Activation of vaccinia virus late gene transcription is dependent on DNA replication and the expression of three genes: A1L, A2L, and G8R (J. G. Keck, C. J. Baldick, Jr., and B. Moss, Cell 61:801-809, 1990). To fully characterize the promoter elements of these trans-activator genes, we prepared more than 140 plasmid vectors containing natural and mutated DNA segments ligated to the Escherichia coli lacZ or chloramphenicol acetyltransferase reporter gene. Expression of the reporter genes occurred when the plasmids were transfected into vaccinia virus-infected cells and was enhanced when DNA replication was prevented, indicating that the A1L, A2L, and G8R promoters belong to the intermediate regulatory class. Deletional mutagenesis demonstrated that the regulatory elements of all three promoters extended between 20 and 30 nucleotides upstream of their RNA start sites. Single-base substitutions of the G8R promoter revealed two critical elements located from -26 to -13 (the core element) and -1 to +3 (the initiator element). Mutations in these regions drastically affected expression, as determined by beta-galactosidase and mRNA analyses. Additional mutations defined the TAAA sequence as the critical initiator element. The length, but not the nucleotide sequence, of the segment between the core and initiator regions was crucial. The requirement for the spacer to be 10 or 11 nucleotides was consistent with a single turn of a double helix. The A1L and A2L promoters resembled the G8R promoter, and mutations in the conserved bases had the predicted effects on expression. We concluded that the three intermediate promoters are composed of a 14-bp A+T-rich core sequence separated by one turn of the double helix from the TAAA initiator element.
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PMID:Mutational analysis of the core, spacer, and initiator regions of vaccinia virus intermediate-class promoters. 162 51

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

A vaccinia virus promoter was evaluated for regulation of a foreign gene in fowlpox virus by a transient expression assay. Fowlpox virus-infected quail cells, transfected with plasmid DNA containing chloramphenicol acetyltransferase (CAT) gene ligated to a vaccinia virus promoter, expressed CAT activity. No CAT activity was detected either in uninfected cells or fowlpox virus-infected cells. These results indicated that a heterologous vaccinia virus promoter can regulate expression of a foreign gene in fowlpox virus.
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PMID:Regulation of foreign gene in fowlpox virus by a vaccinia virus promoter. 215 94

A mouse cell line that constitutively synthesizes the bacteriophage T7 RNA polymerase was constructed. Fluorescence microscopy indicated that the T7 RNA polymerase was present in the cytoplasmic compartment. The system provided, therefore, a unique opportunity to study structural elements of mRNA that affect stability and translation. The in vivo activity of the bacteriophage polymerase was demonstrated by transfection of a plasmid containing the chloramphenicol acetyltransferase (CAT) gene flanked by T7 promoter and termination signals. Synthesis of CAT was dependent on the presence of a cDNA copy of the untranslated region of encephalomyocarditis virus (ECMV) RNA downstream of the T7 promoter, consistent with the absence of RNA-capping activity in the cytoplasm. CAT expression from a plasmid, pT7EMCAT, containing the T7 and EMCV regulatory elements was detected within 4 hr after transfection and increased during the next 20 hr, exceeding that obtained by transfection of a plasmid with the CAT gene attached to a retrovirus promoter and enhancer. Nevertheless, the presumably cap-independent transient expression of CAT from pT7EMCAT was increased more than 500-fold when the transfected cells also were infected with wild-type vaccinia virus. A protocol for high-level expression involved the infection of the T7 RNA polymerase cell line with a single recombinant vaccinia virus containing the target gene regulated by a T7 promoter and EMCV untranslated region.
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PMID:Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells. 220 64

Recombinant vaccinia viruses that express the bacteriophage T3 RNA polymerase (VV-T3pol) or the Escherichia coli lac repressor (VV-lacI) under control of the early-late vaccinia promoter P7.5 were constructed. To determine whether phage polymerase and lac repressor can function in the nucleus of mammalian cells, the bacterial chloramphenicol acetyltransferase (CAT) gene was cloned downstream of a T3 promoter (PT3-CAT) or downstream of a T3 promoter-lac operator fusion element (PT3Olac-CAT), and these reporter gene cassettes were introduced stably into NIH 3T3 or Ltk- cells. Infection of 3T3/PT3-CAT or Ltk-/PT3-CAT cells by VV-T3pol led to rapid expression of CAT (greater than 20 ng of CAT protein per 10(6) cells). The presence of hydroxyurea (which blocks virus DNA replication) did not prevent CAT production. When 3T3/PT3Olac-CAT cells were infected with both VV-T3pol and VV-lacI (multiplicities of infection of 2.5 and 10, respectively), greater than 30-fold repression of CAT gene activity by lac repressor was observed. This could be reversed to unrepressed levels by the presence of 10 mM o-nitrophenyl-beta-D-galactoside (IPTG) in the medium. Regulated expression of the target gene was observed with cell lines that had been maintained for over 1 year (greater than 50 passages in culture), and Southern blot analysis revealed the presence of the CAT gene only in the nuclear fraction in these cells, demonstrating the stability of the target gene. These results indicate that vaccinia virus-encoded proteins can function in the mammalian nucleus and provide the basis for a genetic system in which essential vaccinia virus genes, placed in the chromosome of a cell, can be used to complement defective virus particles. This approach may prove useful for other virus systems.
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PMID:Regulated expression of nuclear genes by T3 RNA polymerase and lac repressor, using recombinant vaccinia virus vectors. 220 24

The molecular mechanism of interferon action on vaccinia virus-specific immediate early protein synthesis was studied in interferon-treated chick cells. In line with previous observations, the synthesis of total vaccinia WR virus-specific mRNA, thymidine kinase (TK) mRNA, and several other early mRNAs was detectable by short [3H]uridine pulses. Under conditions of over 90% inhibition of poxvirus-specific TK induction, accumulation of TK mRNA was strongly inhibited. Northern blot analysis revealed strong degradation of residual TK mRNA prepared from interferon-treated chick embryo fibroblasts (CEF). Blot hybridization analysis using total vaccinia DNA and restriction fragment N as probes demonstrated a generally reduced steady-state amount of vaccinia virus-specific early mRNAs in interferon-treated CEF. When CEF were infected with a recombinant vaccinia virus strain into the TK gene of which the chloramphenicol acetyltransferase gene had been inserted, CAT activity was far lower in interferon-treated than in untreated CEF. We conclude that signals that specify rapid breakdown of viral TK mRNA in interferon-treated CEF are located in the regions flanking the coding sequences of the viral TK gene.
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PMID:Reduced steady-state levels of vaccinia virus-specific early mRNAs in interferon-treated chick embryo fibroblasts. 243 97

A recombinant vaccinia virus that directs the synthesis of bacteriophage T7 RNA polymerase provides the basis for the expression of genes that are regulated by T7 promoters in mammalian cells. The T7 transcripts, which account for as much as 30% of the total cytoplasmic RNA at 24 hr after infection, are largely uncapped. To improve the translatability of the uncapped RNA, the encephalomyocarditis virus (EMCV) untranslated region (UTR) was inserted between the T7 promoter and the chloramphenicol acetyltransferase (CAT) gene. Experiments with a reticulocyte extract demonstrated that the EMCV UTR conferred efficient and cap-independent translatability to CAT RNA synthesized in vitro by T7 RNA polymerase. In cells infected with recombinant vaccinia viruses containing the T7 promoter-regulated CAT gene, the EMCV UTR increased the amount of CAT RNA on polyribosomes. The polyribosome-derived CAT RNA, which contained the EMCV UTR, was translated in vitro in a cap-independent fashion as well. Use of the EMCV UTR significantly enhanced the vaccinia/T7 hybrid expression system as it resulted in a 4- to 7-fold increase in total CAT activity. A further approximately 2-fold improvement was achieved by incubating the cells in hypertonic medium, which favors the translation of uncapped picornavirus RNA over cellular mRNAs. With this newly modified expression system, CAT was the predominant protein synthesized by infected cells and within 24 hr accounted for greater than 10% of the total cell protein.
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PMID:Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. 254

A strong early promoter from the T1 open reading frame (ORF) within the terminal inverted repeat (TIR) of Shope fibroma virus (SFV) has been isolated and characterized. Promoter activity was determined by a transient gene expression assay in poxvirus-infected cells using the bacterial chloramphenicol acetyltransferase as a reporter gene. The sequences which constitute the boundaries of the promoter element were determined by 5' and 3' deletion analysis. The functional SFV T1 promoter domain comprises about 28 bp and includes, in addition to the transcriptional initiation site, a stretch of eight continuous A residues from position -18 to -11 which is critical for promoter function. Both the SFV T1 promoter and the vaccinia 7.5-kDa early/late promoter are active in the transient expression assay when the cells are infected with either the leporipoxvirus SFV or the orthopoxvirus vaccinia. To look more closely at the conservation of promoter function between poxvirus genera, a recombinant vaccinia virus containing the CAT gene driven by the SFV T1 promoter and a recombinant SFV containing the CAT gene driven by the vaccinia 7.5-kDa early/late promoters was constructed. The SFV T1 promoter behaves as an early promoter in the vaccinia genome, and both the T1 and the 7.5-kDa early/late promoters use transcriptional initiation sites in their heterologous genomic environment that are identical to the ones used in the native viral genome. The results from this work indicate that despite the relative lack of absolute sequence conservation, the transcriptional machinery, at least with respect to temporal regulation of early promoters and the position of transcript initiation, is conserved between these two poxvirus genera.
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PMID:Tumorigenic poxviruses: characterization of an early promoter from Shope fibroma virus. 254 12

A recombinant vaccinia virus that expresses the human immunodeficiency virus (HIV) trans-activator (tat) gene was constructed. The tat polypeptide migrated anomalously with an apparent molecular mass of 14 kDa on a sodium dodecyl sulfate-polyacrylamide gel and reacted with polyclonal anti-tat serum. The tat protein was localized predominantly in the cell nucleus despite the absence of other HIV proteins or intranuclear HIV DNA. Additional recombinant vaccinia viruses that contain the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under control of an early vaccinia promoter were constructed. Insertion of the HIV trans-activator-responsive (tar) sequence at the precise start of the CAT mRNA decreased CAT expression slightly. Trans-activation of vaccinia virus-encoded tarCAT failed to occur when CV-1 or HeLa cells were coinfected with the recombinant vaccinia virus expressing tat or when a HeLa cell line containing stably integrated copies of tat was used for infection, indicating the absence of transcriptional or translational effects under these conditions.
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PMID:Use of vaccinia virus vectors to study the synthesis, intracellular localization, and action of the human immunodeficiency virus trans-activator protein. 283 62


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