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)

Functional analyses of the p10 gene promoter from the Autographa californica nuclear polyhedrosis virus (AcNPV) were performed by progressively deleting the 230 nucleotides upstream from the p10 coding sequences towards the ATG codon. Truncated promoter sequences retaining the full 5' non-coding leader of p10 were inserted in front of the chloramphenicol acetyltransferase (CAT) gene, and promoter activity in transfected AcNPV-infected cells was measured using the transient CAT expression assay. The removal of sequences to a position 101 nucleotides upstream from the p10 ATG did not affect the level of CAT expression. Deletion of a further 13 nucleotides reduced CAT expression by three- to fourfold, but the removal of three more nucleotides, which deleted most of the baculovirus very late gene transcription consensus sequence, almost completely abolished activity. The removal of the TATA motif had no effect on the level of transient expression. We conclude that a sequence of about 101 nucleotides upstream from the ATG codon of p10 is sufficient for high level promoter activity in this transient system.
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PMID:Analysis of the promoter of the Autographa californica nuclear polyhedrosis virus p10 gene. 265 27

5' deletion mutants of the Autographa californica nuclear polyhedrosis virus very late p10 gene promoter have been prepared and subjected to a transient expression assay in infected Spodoptera frugiperda cells. The control plasmid contained the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the p10 promoter, which was included in a 230 bp sequence upstream from the p10 translation initiation codon. The control plasmid also contained a segment of the hr5 enhancer downstream from the CAT gene. Promoter activity was unaffected by 5' deletion to position -77, which lies about 11 bp upstream from the p10 cap site. However, deletion of 12 more bp completely eliminated p10 promoter activity. Thus, the 5' border of the p10 promoter lies downstream from position -77, and the region between positions -77 and -65 contains an element that is important to promoter activity. This is the region that is conserved near the cap sites of late baculovirus genes. Our studies also show that transient expression of CAT under the control of the p10 promoter and hr5 enhancer is higher when transfection occurs prior to infection by virus.
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PMID:Studies on the control region of the p10 gene of the Autographa californica nuclear polyhedrosis virus. 265 31

Deletions were made in the 5' non-coding (leader) sequence of the Autographa californica nuclear polyhedrosis virus (AcNPV) p10 gene which progressively removed nucleotides upstream from the ATG translation initiation codon. The effect of these deletions on p10 gene expression was studied using a transient expression assay. Fragments containing the putative promoter and the entire or partly deleted 5' leader sequence of the p10 gene were inserted in front of the chloramphenicol acetyltransferase (CAT) gene in the pSVO-CAT construct. Transfection of AcNPV-infected Spodoptera frugiperda cells with these plasmids resulted in higher CAT expression with increasing representation of the 5' leader sequence. The lowest level of CAT expression was found with a construct containing only 10% of the 5' leader sequence, but this was enhanced on average by 50-fold if the entire 5' leader sequence was retained. The results indicate that the entire 5' leader sequence of the p10 gene is necessary for the high level of expression. The normal transcription initiation site was utilized in the transient expression of CAT. The data are discussed in relation to the strong promoter of the baculovirus polyhedrin gene.
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PMID:Functional analysis of the p10 gene 5' leader sequence of the Autographa californica nuclear polyhedrosis virus. 283 97

The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) replicates in insect cell lines in culture. In mammalian cells, however, the virus cannot be propagated. AcNPV DNA does not replicate or persist and is not transcribed in mammalian cells (Tjia et al., 1983). In insect cells productively infected with AcNPV, at least two major late viral gene products have been recognized, the polyhedrin, which makes up the bulk of the polyhedral inclusion bodies in infected cell nuclei, and a 10,000 Da protein (p10) of unknown function. The p10 promoter has been fused to the prokaryotic gene for chloramphenicol acetyltransferase (CAT) as a reporter gene (Knebel et al., 1985). Activity of this construct can be elicited in AcNPV-infected insect cells but not in uninfected insect cells or in mammalian cells. Presumably, the late p10 promoter requires other AcNPV gene products for activity. When the pAcp10-CAT construct is transfected into BHK21 hamster cells at about 18 h after infection with human adenovirus type 5 (Ad5), the insect AcNPV promoter is transactivated in cells of the heterologous mammalian species. The results of S1 protection analyses on the RNA from Ad5-infected and pAcp10-CAT transfected cells reveal that the p10 promoter is used for initiation of transcription. Similarly, the p10 insect virus promoter is activated in BHK21 hamster cells cotransfected with the HindIII-G fragment of adenovirus type 2 (Ad2) DNA which contains the E1A and parts of the E1B region in the left terminal 7.8% of the Ad2 genome. Moreover, in human 293 cells or in BHK297-C131 hamster cells, which both carry and constitutively express the E1 region of Ad5 DNA, the pAcp10-CAT construct is also expressed, and similarly in HE7 hamster cells which carry appreciable portions of the Ad2 genome (Klimkait and Doerfler, 1985). It is concluded that adenovirus functions are capable of transactivating a heterologous insect virus promoter in mammalian cells.
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PMID:Activation of an insect baculovirus promoter in mammalian cells by adenovirus functions. 296 53

The p10 gene of Autographa californica nucleopolyhedrovirus has two putative AATAAA polyadenylation signals. The downstream signal is used predominantly, as was determined by analysing 3' cDNA ends. This downstream motif is followed by a GT-rich sequence, known to be important for efficient polyadenylation in mammalian systems. To analyse the importance of polyadenylation for p10 gene expression, recombinant viruses with altered 3' untranslated regions (UTRs) were tested using chloramphenicol acetyltransferase (CAT) as a reporter. Surprisingly, after inactivation of the downstream AATAAA motif, CAT expression remained at the same high level as observed with a wild-type 3' UTR. Polyadenylation occurred 24-28 nucleotides further downstream, probably due to an ATTAAA sequence motif. Replacing the p 10 3' UTR with the SV40 early terminator sequence as part of an hsp70-lacZ-SV40 gene cassette, which is commonly used in baculovirus expression vectors, resulted in a reduction in reporter gene expression. Polyadenylation occurred far more efficiently in the original p10 3' UTR than in the SV40 terminator sequence, as was shown by testing the SV40 terminator separately. These results indicate that in order to obtain high levels of foreign gene expression, vectors that provide a wild-type p10 3' UTR are to be preferred over those containing the hsp70-lacZ-SV40 gene cassette. Comparison of the p10 genes of various baculoviruses showed the presence of at least one AATAAA or ATTAAA motif in combination with a GT-rich sequence in the 3' UTR, suggesting an evolutionary conservation of these two elements, thereby maintaining the high level of p10 gene expression.
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PMID:Role of the 3' untranslated region of baculovirus p10 mRNA in high-level expression of foreign genes. 1046 25

In this report, we describe a novel recombinant Semliki Forest virus (SFV) expressing T7 RNA polymerase (T7-RP), which was shown to drive transient expression of the chloramphenicol acetyltransferase (cat) gene in mammalian and mosquito cells after transfection of plasmids carrying the reporter gene under the control of the T7 promoter. To our knowledge, this is the first description of a T7-RP-based expression in mosquito cells. Expression of the cat gene was significantly enhanced in mammalian cells by inserting the sequence of the encephalomyocarditis virus internal ribosome entry site between the T7 promoter and the 5' end of the cat gene. In mosquito cells, the level of chloramphenicol acetyltransferase activity was increased by flanking the cat gene with the Autographa californica baculovirus p10 gene 5' and 3' untranslated regions. SFV expressing T7-RP appears, therefore, to be an alternative to other virus-based gene-expression systems in mammalian cells and a powerful tool for protein expression in mosquito cells.
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PMID:Transient gene expression in mammalian and mosquito cells using a recombinant Semliki Forest virus expressing T7 RNA polymerase. 1064 25