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

Use of the translation-inhibiting drug cycloheximide has indicated that the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene, the sole EHV-1 IE gene, encodes a major viral regulatory protein since IE mRNA translation is a prerequisite for all further viral gene expression (W.L. Gray, R. P. Baumann, A. T. Robertson, G. B. Caughman, D. J. O'Callaghan, and J. Staczek, Virology 158:79-87, 1987). An EHV-1 IE gene expression vector (pSVIE) in combination with chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs was used in transient transfection assays to characterize the regulatory functions of the IE gene product. These experiments demonstrated that (i) the EHV-1 IE gene product is a bifunctional protein capable of both positive and negative modulation of gene expression; (ii) the IE gene product possesses an autoregulatory function which represses the IE promoter; (iii) IE autoregulation is dependent on IE promoter sequences mapping within positions -288 to +73 relative to the transcription initiation site (+1) of the IE gene; (iv) the IE gene product can independently activate the EHV-1 tk promoter (an early promoter) by as much as 60-fold; (v) two EHV-1 beta-gamma (leaky late) promoters, those of IR5 (gene 5 in the inverted repeat) and the glycoprotein D gene, demonstrate a requirement for both the IE gene product as well as a gene product encoded within the EHV-1 XbaI G fragment for significant activation; and (vi) the IE gene product is capable of activating heterologous viral promoters.
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PMID:Characterization of the regulatory functions of the equine herpesvirus 1 immediate-early gene product. 130 21

The herpes simplex virus type 1 (HSV-1) alpha proteins ICP4, ICP0, and ICP27 are trans-acting proteins which affect HSV-1 gene expression. To investigate potential interactions between these alpha products and to determine the specificity of action of the alpha proteins in combination with each other compared with their activities individually, we performed a series of transient-expression assays. In these assays we used plasmids containing the alpha genes encoding ICP4, ICP0, and ICP27 either singly or in combination as effectors and HSV-1 genes of different kinetic classes and heterologous genes as targets. The HSV-1 targets consisted of promoter-regulatory domains from alpha (ICP0 and ICP27), beta (thymidine kinase and alkaline exonuclease), beta-gamma (glycoprotein D, glycoprotein B, and VP5), and gamma (glycoprotein C) genes, each fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target genes consisted of the simian virus 40 early promoter with enhancer and the Rous sarcoma virus long terminal repeat promoter and enhancer each fused to the CAT gene. Target promoter activity was measured by the assay of CAT activity in extracts of transfected cells and by Northern (RNA) blot hybridization of CAT mRNA. The results of these experiments showed that ICP4 activated only HSV-1 target genes, whereas ICP0 activated all of the targets and ICP27 had little effect on any of the targets. ICP4 and ICP0 had a synergistic effect when inducing HSV-1 targets, but they did not have this effect on the heterologous targets pSV2-CAT or pRSV-CAT. In fact, lower levels of CAT activity and CAT mRNA were found in the presence of both effectors than with ICP0 alone. Most interestingly, although the effector plasmid containing the ICP27 gene had little effect on its own, two different and marked effects depending on the target were observed when ICP27 was combined with ICP4 or ICP0 or both. A trans-repression of the induction seen with ICP4 and ICP0 was found when ICP27 was present in the transfections with pSV2-CAT, pRSV-CAT, pICP0-CAT, pICP27-CAT, pTK-CAT, pgD-CAT, pgB-CAT, and pgC-CAT. This resulted in CAT activity levels which were similar to or lower than the basal level of expression of the target genes in the absence of effector plasmids. This trans-repression occurred over a wide range of concentrations of input ICP27 plasmid. In contrast to this repressive effect of ICP27, a trans-activation was seen when ICP4, ICP0, and ICP27 plasmids were combined in transfections with pAE-CAT and pVP5-CAT as targets. This trans-activation also occurred over a 10-fold range of input ICP27 plasmid. These results suggest that ICP27 can facilitate both down
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PMID:The herpes simplex virus type 1 alpha protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0. 284 67

On the basis of experiments with mutant virus and transfection with isolated genes, the herpes simplex virus immediate-early gene product ICP4 is known to positively regulate the transcription of viral early and late genes and negatively regulate expression from its own promoter. Binding of ICP4 to DNA sequences in several viral genes has been reported, yet the significance of ICP4-DNA interaction in transcriptional activation remains unclear. We have studied this problem by using the early glycoprotein D (gD) gene, which possesses a binding site at approximately -100 relative to the RNA initiation site. We linked this promoter and various mutant constructs to the chloramphenicol acetyltransferase gene in order to measure promoter activity in transient transfections both in the presence and in the absence of an ICP4-encoding plasmid. The natural promoter was activated 3.3-fold, and a deletion construct lacking the binding site was activated minimally (1.7-fold). Constructs containing multiple tandem repeats of the binding site (three or five inserts) demonstrated higher expression in the presence of ICP4 than did the natural promoter while retaining low levels of expression when unstimulated. Gel mobility shift assays and DNase I footprinting analyses indicated that ICP4 associated with multiple binding sites. In vitro transcription from a gD promoter construct containing multiple binding sites showed increased RNA synthesis in the presence of partially purified ICP4. These data provide the first direct evidence that binding of ICP4 to a specific DNA sequence in the gD gene contributes to activation of transcription.
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PMID:Role for DNA-protein interaction in activation of the herpes simplex virus glycoprotein D gene. 284 78

We have previously identified a novel first exon of Duffy gene and two inverse GATA motifs in the 600 bp 5' flanking region. The proximal GATA is positioned downstream from the start position of endothelium and upstream from that of erythroid. One base substitution (-365T --> C) was found in the proximal GATA motif from three black Fy(a-b-) individuals, and was regarded as a common polymorphic mutation in black Fy(a-b-) individuals. The upstream sequence of the novel first exon was inserted in the upstream of chloramphenicol acetyltransferase (CAT) gene and transfected in human erythroleukemia cell line (HEL) and human microvascular endothelial cells (HMvEC). The black type mutation abolished the CAT transcription in HEL cells but not in HMvEC. Deletion mutagenesis study revealed that the proximal GATA motif represent the erythroid regulatory core region for Duffy gene. Gel shift assay showed that the proximal GATA motif is the target sequence of GATA-1. These studies indicate that the black type mutation abolishes Duffy gene expression in erythroid but not in postcapillary venule endothelium, which is compatible with the Northern blot and immunohistochemical observation in black Fy(a-b-) individuals.
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PMID:Characterization of the Duffy gene promoter: evidence for tissue-specific abolishment of expression in Fy(a-b-) of black individuals. 865 34

Porcine adenovirus-3 (PAV-3) was developed as an expression vector using homologous recombination in Escherichia coli BJ 5183. As a prerequisite, the complete genome of PAV-3 was first introduced as a PacI restriction fragment into a bacterial plasmid. The plasmid, when PacI restricted and transfected into swine testicular cells, produces an infectious virus. The potential of this procedure was demonstrated by the construction of several PAV-3 recombinants. Part of the E3 region, which is nonessential for virus replication under cell culture conditions, was identified and deleted from the virus genome. The gene for glycoprotein D (gD) of pseudorabies virus (PRV), which elicits PRV-neutralizing antibodies in pigs, was cloned and expressed from the E3 region of PAV-3. A 50 kDa polypeptide was identified in recombinant PAV-3-infected cell lysates by immunoprecipitation assays using gD-specific monoclonal antibodies. In another experiment, a region between the right inverted terminal repeat and the promoter of the E4 region was used to clone and express the chloramphenicol acetyltransferase (CAT) gene under the control of SV40 immediate early promoter. CAT gene expression was observed irrespective of the orientation of the CAT gene. These results indicate that the helper-independent recombinant PAV-3 could be used as an expression vector and has potential as a recombinant vaccine vector in pigs.
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PMID:Development of porcine adenovirus-3 as an expression vector. 1009 94