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)

High levels of expression of cloned genes have been obtained in mammalian cells by using poxvirus-derived insertion/expression vectors. These vectors employ the cis-acting element (CAE I) that directs the transcription of one of the most strongly expressed genes of cowpox virus. This gene (the 160K gene) encodes the 160-kDa protein that is the major component of the A-type cytoplasmic inclusions. Its counterpart in vaccinia virus (VV) is the 94K gene contained in the HindIII A fragment of the viral DNA. Two insertion vectors have been constructed; each is designed to allow cloned genes to be placed immediately downstream of a modified version of CAE I within a poxvirus genome. One vector, p1200, enables the CAE I-cloned-gene constructs to be inserted into the thymidine kinase gene of VV. This vector was used to create a VV recombinant that directed expression of the chloramphenicol acetyltransferase (CAT) gene. The other vector, p2101, enables the CAE I-cloned-gene constructs to be inserted into the VV 94K gene. The prototype of this vector was used to create a VV recombinant that directed expression of a hybrid CAT-lacZ gene. Infection of cultured human cells with these recombinants led to high levels of synthesis of either the CAT gene product or the CAT-lacZ gene product. Each of these proteins was produced in quantities that were easily detected by Coomassie blue staining of total cell proteins resolved by polyacrylamide gel electrophoresis. We estimate that these vectors are capable of directing the synthesis of milligram amounts of gene product per 10(9) mammalian cells.
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PMID:A poxvirus-derived vector that directs high levels of expression of cloned genes in mammalian cells. 284 5

A series of mutations, including 5' and 3' deletions, as well as insertions were introduced into the 5' flanking nucleotide sequence of a vaccinia virus late gene. This DNA has been shown previously to contain all the necessary elements for correct regulation of the gene most probably transcribed by the viral RNA polymerase. To facilitate the assays, the mutated DNA was fused to the chloramphenicol acetyltransferase gene and inserted into the genome of live vaccinia virus. The effects of the mutations on expression of the chimeric gene were studied by both enzyme assays and nuclease S1 analysis. The results showed that 5' deletions up to about 15 bp from the putative initiation site of transcription still yielded high levels of gene expression. All mutations, however, that deleted the authentic late mRNA start site, abolished promoter activity.
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PMID:Functional analysis of the 5' flanking sequence of a vaccinia virus late gene. 301 74

DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system.
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PMID:Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. 309 28

Promoter elements responsible for directing the transcription of six tightly clustered vaccinia virus (VV) late genes (open reading frames [ORFs] D11, D12, D13, A1, A2, and A3) from the HindIII D/A region of the viral genome were identified within the upstream sequences proximal to each individual locus. These regions were identified as promoters by excising them from the VV genome, abutting them to the bacterial chloramphenicol acetyl transferase gene, and demonstrating their ability to drive expression of the reporter gene in transient-expression assays in an orientation-specific manner. To delineate the 5' boundary of the upstream elements, two of the VV late gene (A1 and D13) promoter: CAT constructs were subjected to deletion mutagenesis procedures. A series of 5' deletions of the ORF A1 promoter from -114 to -24 showed no reduction in promoter activity, whereas additional deletion of the sequences from -24 to +2 resulted in the complete loss of activity. Deletion of the ORF A1 fragment from -114 to -104 resulted in a 24% increase in activity, suggesting the presence of a negative regulatory region. In marked contrast to previous 5' deletion analyses which have identified VV late promoters as 20- to 30-base-pair cap-proximal sequences, 5' deletions to define the upstream boundary of the ORF D13 promoter identified two positive regulatory regions, the first between -235 and -170 and the second between -123 and -106. Background levels of chloramphenicol acetyltransferase expression were obtained with deletions past -88. Significantly, this places the ORF D13 regulatory regions within the upstream coding sequences of the ORF A1. A high-stringency computer search for homologies between VV late promoters that have been thus far characterized was carried out. Several potential consensus sequences were found just upstream from RNA start sites of temporally related promoter elements. Three major conclusions are drawn from these experiments. (i) The presence of promoters preceding each late ORF supports the hypothesis that each is expressed as an individual transcriptional unit. (ii) Promoter elements can be located within the coding portion of the upstream gene. (iii) Sequence homologies between temporally related promoter elements support the notion of kinetic subclasses of late genes.
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PMID:Molecular dissection of cis-acting regulatory elements from 5'-proximal regions of a vaccinia virus late gene cluster. 333 46

A vaccinia virus (VV) recombinant containing the DNA sequences encoding the bacterial chloramphenicol acetyltransferase (CAT) gene was constructed. The ability of the chimeric VV:CAT transcript to be translated in vitro into enzymatically active enzyme was assessed. Addition of mRNA isolated from the cytoplasm of VV:CAT infected cells to a mRNA-dependent reticulocyte lysate resulted in the synthesis of high levels of enzymatically active CAT. These results suggest that this assay may be used in concert with physical assays to study the expression and stability of chimeric transcripts in virus-infected cells.
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PMID:Cell-free translation of a chimeric eucaryotic-procaryotic message yields functional chloramphenicol acetyltransferase. 347 Oct 96

Nine recombinant vaccinia viruses that contain overlapping segments of the putative promoter region of the vaccinia virus thymidine kinase (TK) gene linked to DNA coding for the prokaryotic enzyme chloramphenicol acetyltransferase (CAT) were constructed. In each case, the RNA start site and 5 bp of DNA downstream were retained. No significant difference in CAT expression occurred as the deletion was extended from 352 to 32 bp before the RNA start site. Deletion of a further 10 bp, however, led to complete cessation of early promoter activity. Primer extension analysis of the 5' ends of the transcripts verified that the natural TK RNA start site was still used when only 32 bp of upstream DNA remained. Loss of early promoter activity was previously found when deletions were extended from 31 to 24 bp before the RNA start site of another vaccinia gene that is expressed constitutively throughout infection (M.A. Cochran, C. Puckett, and B. Moss, 1985, Proc. Natl. Acad. Sci. USA 82, 19-23). Sequence similarities in the promoter regions of these two genes were noted.
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PMID:Determination of the promoter region of an early vaccinia virus gene encoding thymidine kinase. 347 13

A putative promoter region, extending from 218 base pairs (bp) before (-218) to 10 bp after (+10) the RNA start site of a vaccinia virus gene encoding an Mr-28,000 precursor of a core polypeptide that is expressed only after the onset of DNA replication was linked to DNA coding for the procaryotic enzyme chloramphenicol acetyltransferase (CAT). When this chimeric gene was inserted into the genome of vaccinia virus, the infectious recombinant expressed CAT in a regulated fashion. A series of deletions starting upstream of the promoter region and extending toward the RNA start site were made. The effects of these mutations on CAT expression were examined in cells infected with recombinant viruses and confirmed in a helper virus-dependent transient assay system. A gradual reduction in CAT expression occurred as the deletions extended from -61 to -18. Mutants that retained 18 bp before and 10 bp after the RNA start site still expressed CAT as a late gene product, although at a submaximal level. A further 5'-to-3' deletion of 10 bp reduced CAT expression to background levels. To demonstrate that the effect on expression was not simply due to the bringing of upstream inhibitory sequences closer to the RNA start site, a point mutation substituting a G for the A at -12 was made. The sharp decrease in CAT expression indicated the importance of a run of eight A residues located between -15 and -7. Evidence that these mutations affected the level but not the site of transcriptional initiation was demonstrated by analysis of the 5' ends of the mRNAs from infected cells. The short DNA sequence required for accurate and temporally regulated transcription suggests that the same or overlapping signals are used for both aspects of this process.
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PMID:Determination of the transcriptional regulatory region of a vaccinia virus late gene. 378 25

A transient expression system in which chimeric genes are expressed in cells infected with vaccinia virus was developed. Recombinant plasmids containing the promoter regions of vaccinia virus genes ligated to the coding segment of the prokaryotic chloramphenicol acetyltransferase (CAT) gene were constructed. When the plasmids were introduced into vaccinia virus-infected cells by transfection, the chimeric gene was expressed and significant levels of CAT accumulated. CAT activity was not detected when the same recombinant plasmid was introduced into uninfected cells, nor was activity detected when the vaccinia virus promoter was absent from the plasmid or was replaced by simian virus 40 or Rous sarcoma virus promoters. This specificity indicated that expression is dependent on a cis-acting vaccinia virus promoter region within the recombinant plasmid and diffusible trans-acting transcription factors produced during virus infection. The lack of effect of a simian virus 40 enhancer element inserted upstream of the vaccinia virus promoter region also distinguished this system from systems dependent on RNA polymerase II. Although replication of the recombinant plasmid could not be detected in either uninfected or vaccinia virus-infected cells, an inhibitor of DNA synthesis significantly reduced CAT expression. This result, as well as the kinetics of CAT synthesis, suggests that replication of viral DNA templates can enhance transcription of chimeric genes in recombinant plasmids.
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PMID:Eukaryotic transient expression system dependent on transcription factors and regulatory DNA sequences of vaccinia virus. 385 41

A vaccinia virus gene that is expressed throughout the reproductive cycle was found to have two sets of RNA start sites approximately 55 nucleotides apart. The site nearest to the coding segment is used early in infection and the one further upstream is used after DNA replication. A series of 5' to 3' deletions were made in the promoter region, and the truncated DNA segments were then ligated to the coding portion of the procaryotic chloramphenicol acetyltransferase gene to measure expression. The effects of these mutations on chloramphenicol acetyltransferase synthesis were determined in a vaccinia virus helper-dependent transient expression system and by forming infectious vaccinia virus recombinants that contain the chimeric genes. Deletions extending up to 31 nucleotides before the late RNA start site had no effect on either early or late expression. Removal of an additional 15 nucleotides produced a dramatic decrease in late expression but had no effect on early expression. The latter was not diminished until the deletion was extended from 31 to 24 nucleotides before the early RNA start site. These results were confirmed by transcriptional analyses. We concluded that this vaccinia virus gene has two promoters and that the regulatory signals for each are located within 31 nucleotides of their sites of transcription.
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PMID:In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals. 397 82

A subset of vaccinia virus genes are expressed only after DNA replication. To investigate the regulation of such transcriptional units, a representative gene encoding a major late polypeptide (Mr, 28,000) was mapped and sequenced. Translatable mRNAs were heterogeneous in length and overlapped several early genes downstream. The 5' end of the message was located, and the DNA segment upstream was excised and ligated to the coding sequence of the easily assayable procaryotic chloramphenicol acetyltransferase gene. The resulting chimeric gene was recombined into the thymidine kinase locus of the vaccinia virus genome, and infectious recombinant virus was isolated. Both the time of chloramphenicol acetyltransferase synthesis in infected cells and the requirement for DNA replication indicate that the sequence upstream of the late gene contains cis-acting transcriptional regulatory signals.
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PMID:Regulation of expression and nucleotide sequence of a late vaccinia virus gene. 608 91


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