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)

The influenza virus NS1 protein was shown to stimulate translation of the M1 protein. M-CAT RNA, which contains the chloramphenicol acetyltransferase (CAT) reporter gene and the terminal noncoding sequence of segment 7 (coding for the M1 and M2 proteins), was ribonucleoprotein transfected into clone 76 cells expressing the influenza virus RNA polymerase and NP proteins required for the transcription and replication of influenza virus ribonucleoproteins. When the cells were superinfected with a recombinant vaccinia virus which expresses the NS1 protein, CAT expression from the M-CAT RNA was significantly stimulated but transcription was not altered. The expression of NS-CAT RNA, which contains noncoding sequences of segment 8 (coding for the NS1 and NS2 proteins), was not altered by the NS1 protein. Site-directed mutagenesis showed that the sequence GGUAGAUA upstream of the initiation codon on segment 7 was required for stimulation.
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PMID:Influenza virus NS1 protein stimulates translation of the M1 protein. 750 95

A new in vivo replication system for influenza virus was developed by using the clone 76 cell line, in which the viral RNA polymerase and nucleoprotein genes can be expressed in response to dexamethasone. The chimeric NS-chloramphenicol acetyltransferase (CAT) RNAs in the sense and antisense orientations positioned between the 5'- and 3'-terminal sequences of the influenza virus RNA segment 8 can be replicated [both genomic RNA (vRNA) and complementary RNA (cRNA) were transcribed] in the clone 76 cells treated with dexamethasone. These data indicate that three RNA polymerase proteins (PB1, PB2, and PA) and nucleoprotein are sufficient for replication of the influenza virus genome. Analysis of mutant cRNAs containing a base-substitution or a deletion in the 3'-conserved terminal 13 nucleotides revealed that important cis elements in the cRNA for vRNA synthesis reside at positions 2, 3, and 7 to 13 nucleotides from the 3'-end.
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PMID:An in vivo study of the replication origin in the influenza virus complementary RNA. 768 Oct 56

We have succeeded in engineering changes into the genome of influenza B virus. First, model RNAs containing the chloramphenicol acetyltransferase gene flanked by the noncoding sequences of the HA or NS genes of influenza B virus were transfected into cells which were previously infected with an influenza B helper virus. Like those of the influenza A viruses, the termini of influenza B virus genes contain cis-acting signals which are sufficient to direct replication, expression, and packaging of the RNA. Next, a full-length copy of the HA gene from influenza B/Maryland/59 virus was cloned. Following transfection of this RNA, we rescued transfectant influenza B viruses which contain a point mutation introduced into the original cDNA. A series of mutants which bear deletions or changes in the 5' noncoding region of the influenza B/Maryland/59 virus HA gene were constructed. We were able to rescue viruses which contained deletions of 10 or 33 nucleotides at the 5' noncoding region of the HA gene. The viability of these viruses implies that this region of the genome is flexible in sequence and length.
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PMID:Influenza B viruses with site-specific mutations introduced into the HA gene. 781 5

An established cell line, clone 64, in which the expression of the RNA polymerase PB1 and PA subunit genes and the nucleoprotein (NP) gene but not the PB2 subunit gene of influenza virus can be induced by the addition of dexamethasone, was used to analyze the replication and transcription machineries of the influenza virus. Both NS-CATc and NS-CATv, the chimeric nonstructural protein chloramphenicol acetyltransferase (NS-CAT) RNAs in the sense and antisense orientations positioned between the 5'- and 3'-terminal sequences of influenza virus RNA segment 8 (the NS gene), respectively, can be transcribed into the corresponding complementary-strand RNA in clone 64 cells only when treated with dexamethasone. Although sense-strand poly(A)+ CAT RNA was detected in the dexamethasone-treated clone 64 cells transfected with NS-CATv RNA, CAT activity was not detected in these cells and the isolated poly(A)+ CAT RNA was inert in an in vitro translation system. However, when the poly(A)+ CAT RNA was capped by using a purified yeast mRNA capping enzyme (mRNA guanylyltransferase), the capped poly(A)+ CAT RNA became translatable in the in vitro translation system. These results indicated that PB1, PA, and NP can support the replication of the influenza virus genome as well as the transcription to yield uncapped poly(A)+ RNA and that PB2 is specifically required for the synthesis of capped RNA.
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PMID:The RNA polymerase PB2 subunit is not required for replication of the influenza virus genome but is involved in capped mRNA synthesis. 781 36

A program (HOOK) is described for generating potential ligands that satisfy the chemical and steric requirements of the binding region of a macromolecule. Functional group sites with defined positions and orientations are derived from known ligand structures or the multicopy simulation search (MCSS) method (Miranker, A., Karplus, M. Proteins 11:29-34, 1991). HOOK places molecular "skeletons" from a database into the protein binding region by making bonds between sites ("hooks") on the skeleton and functional groups. The nonpolar interactions with the binding region of candidate molecules are assessed by use of a simplified van der Waals potential. The method is illustrated by constructing ligands for the sialic acid binding site of the hemagglutinin from the influenza A virus and the active site of chloramphenicol acetyltransferase. Aspects of the HOOK program that lead to a highly efficient search of 10(5) or more skeletons for binding to 10(2) or more functional group minima are outlined.
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PMID:HOOK: a program for finding novel molecular architectures that satisfy the chemical and steric requirements of a macromolecule binding site. 793 34

RNA polymerase I transcription has been used for expression of influenza vRNA molecules, with influenza hemagglutinin or other cDNAs precisely inserted between mouse rDNA promoter and terminator sequences. In in vitro studies generation of HA vRNA transcripts in high rates and correct formation of their 5' ends as well as their 3' ends has been achieved for such hybrid DNA templates. For in vivo expression studies, the HA coding region was replaced by chloramphenicol acetyltransferase (CAT), also in vRNA antisense orientation, with both influenza terminal sequences beyond start and stop codons being retained on the resulting transcript. Following transfection with precisely constructed hybrid DNA templates and depending on infection with influenza virus, CAT activity could be demonstrated. Templates resulting in 3' extended vRNA molecules did not give this result. vRNA-CAT molecules were not only recognized by influenza viral RNA polymerase for synthesis of plus strand mRNAs, but also were packaged into progeny virus particles, as shown by CAT activity in infected cells after passaging of virus containing supernatants.
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PMID:RNA polymerase I-mediated expression of influenza viral RNA molecules. 800 59

An in vivo system in which expression of a synthetic influenza virus-like chloramphenicol acetyltransferase (CAT) RNA is driven by influenza virus proteins synthesized from cloned cDNAs has been developed. Expression of the four influenza virus core proteins (nucleoprotein, PA, PB1 and PB2) was performed by transfection of four pGEM recombinant plasmids, each containing one of the four viral genes, into cell cultures previously infected with a vaccinia virus recombinant encoding the T7 RNA polymerase (vTF7-3). When a naked negative-sense influenza virus-like CAT RNA was transfected into cells expressing the four influenza virus proteins, CAT activity was detected in the cell extracts, demonstrating that the expressed proteins had RNA-synthesizing activity. In this system, CAT RNA templates containing additional nucleotides at the 3' end were also expressed, resulting in CAT activity. This showed that the influenza virus polymerase can recognize its promoter when located internally on an RNA template. In influenza virus-infected cells however, CAT activity was detected only when the CAT RNA contained the viral promoter at the exact 3' end and was transfected as in vitro assembled ribonucleoprotein. These results are discussed in terms of the different requirements of the two helper systems for expression of an exogenously added RNA.
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PMID:Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system. 804 17

Human androgen receptor (hAR) is a ligand-dependent transcription factor that mediates androgen-induced actions on target tissues. Transfection studies in the human prostate cancer cell line LNCaP examine the ability of dihydrotestosterone (DHT), hydroxyflutamide (HO-FLU), cyproterone acetate (Cypro.A), and RU 23908-10 to stimulate or to inhibit the transcription activation of mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase (MMTV-CAT). DHT stimulated transcription activation of MMTV-CAT gene in LNCaP cells in a dose-dependent manner. HO-FLU, Cypro.A, and RU 23908-10, though only partially, also stimulated the transcription activation of MMTV-CAT. Despite this, 100- to 1,000-fold molar excess of all antiandrogens inhibited the agonistic activity of 10 nM DHT in this system. Receptor binding assays confirmed that HO-FLU, Cypro.A, and RU 23908-10 competed with DHT for AR binding in LNCaP cells. Western blot analysis using AR antipeptide antibodies raised in rabbits revealed the presence of two AR protein bands in LNCaP cells, following treatment with antiandrogens. Increasing doses of HO-FLU stimulated the expression of the 114-kDa AR by 2.5-fold, but did not affect the 108-kDa AR. Increasing doses of Cypro.A and RU 23908-10 decreased the levels of both the 114-kDa and the 108-kDa AR. Although the exact nature of 108-kDa and 114-kDa AR in LNCaP cells is still unknown, these data suggest that the regulatory actions of each individual antiandrogen on AR expression in LNCaP cells may be different.
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PMID:Antiandrogens inhibit human androgen receptor-dependent gene transcription activation in the human prostate cancer cells LNCaP. 814 66

cDNAs containing the coding sequences of influenza type A virus polymerase proteins (PB1, PB2 and PA) and nucleoprotein (NP) have been expressed in mammalian cells by T7 polymerase provided by a recombinant vaccinia virus. The resulting proteins are able to form a complex that can copy a negative sense influenza-like RNA, transcribed from input DNA by the T7 polymerase, into a positive sense RNA that is translated into active chloramphenicol acetyltransferase (CAT). In this system there is no requirement for helper virus or purified viral core proteins, thus it will allow manipulation of all proteins as well as template for studies of replication in influenza virus.
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PMID:Expression of functional influenza virus A polymerase proteins and template from cloned cDNAS in recombinant vaccinia virus infected cells. 816 49

In this report we describe the rescue of a transfectant influenza A virus which stably expresses a heterologous protein, bacterial chloramphenicol acetyltransferase (CAT). The foreign sequences encoding CAT are expressed as part of an essential influenza virus segment, that coding for the neuraminidase (NA) protein. The novel way by which this was achieved involved inserting in frame the 16-amino-acid self-cleaving 2A protease of foot-and-mouth disease virus between the CAT and the NA coding sequences. The resultant gene produces a polyprotein which is proteolytically cleaved to release both CAT and NA. The intramolecular cleavage occurs at the C terminus of the 2A sequence between a glycine-proline dipeptide motif such that the released NA protein has an additional N-terminal proline residue. The transfectant virus is stable upon passage in tissue culture. CAT activity is expressed at high levels in cell culture supernatants and in the allantoic fluid of infected eggs. Since the chimeric segment must maintain the heterologous reading frame to retain viability, the virus stability is dependent upon concomitant synthesis of the heterologous protein. This design may be particularly appropriate for utilization of influenza virus as a mammalian expression vector.
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PMID:Expression of a foreign protein by influenza A virus. 820 22


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