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)

We examined the regulatory/promoter sequence of a calcium ionophore-inducible gene isolated from the rat genome. Whereas the promoter of this ubiquitously expressed gene is active under noninduced conditions, after induction by calcium ionophore A23187 this promoter is 10- to 25-fold more active than the simian virus 40 early promoter, as measured by chloramphenicol acetyltransferase activities. Within this regulatory/promoter region, we have identified a DNA fragment with enhancer-like properties immediately 5' to the TATA sequence. This 291-nucleotide fragment acts in cis to enhance expression of the neomycin phosphotransferase (neo) gene driven by the herpes simplex virus thymidine kinase promoter in an orientation-independent manner. In addition, this fragment can confer A23187 inducibility to the neo gene and effectively compete for positive regulatory factors involved in A23187 induction. Sequence analysis of this promoter reveals homology with viral core enhancer sequences, and the apparent organization of direct repeat domains is similar to those observed in viral enhancers.
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PMID:A calcium ionophore-inducible cellular promoter is highly active and has enhancerlike properties. 302 78

We have assayed the ability of segments of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer region to activate gene expression under the control of the SV40 early promoter and to compete for trans-acting enhancer-binding factors of limited availability in vivo in monkey CV-1 or human HeLa cells. The bacterial chloramphenicol acetyltransferase and the herpes simplex virus type 1 thymidine kinase genes were used as reporters in these assays. A 94-bp sequence located between SV40 nucleotides 179 and 272, including one copy of the 72-bp repeat, has been termed the minimal enhancer in previous studies. In the present study, we found that the 20-bp origin-proximal region located between nucleotides 179 and 198 was dispensable, since its removal caused only a slight reduction in enhancer activity. However, the deletion of another 4 bp up to nucleotide 202 abolished the enhancer activity. We propose that the minimal enhancer is a 74-bp sequence located between nucleotides 199 and 272, including 52 bp of one copy of the 72-bp repeat and a 22-bp adjacent sequence up to the PvuII site at 272. The nonamer 5'-AAGT/CATGCA-3', which we term the K core, occurred as a tandem duplication around the SphI site at nucleotide 200, and we found that this duplication was essential for enhancement and factor-binding activities. A heterologous core element (which we term the C core), 5'-GTGGA/TA/TA/TG-3', identified earlier (G. Khoury and P. Gruss, Cell 33:313-314, 1983; Weiher et al., Science 219:626-631, 1983) also occurred in duplicate, with one of the copies located within the 22-bp sequence near nucleotide 272 present outside the 72-bp repeat. We provide direct evidence that this 22-bp sequence augments enhancer activity considerably. We also found that in addition to the heterologous interaction occurring normally between the K and C cores within the minimal enhancer, certain homologous interactions were also permitted provided there was proper spacing between the elements.
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PMID:Minimal transcriptional enhancer of simian virus 40 is a 74-base-pair sequence that has interacting domains. 302 7

Two regions of the Epstein-Barr virus (EBV) genome together make up an element, oriP, which acts in cis to support plasmid replication in cells that express the EBV nuclear antigen 1 (EBNA-1). The two components of oriP are a region containing a 65-base-pair (bp) dyad symmetry and a region containing 20 copies of a 30-bp direct repeat. Here we show that the 30-bp family of repeats of oriP can function as a transcriptional enhancer that is activated in trans by the EBNA-1 gene product. In either EBV-genome-positive cells or in cells that express EBNA-1, the 30-bp family of repeats, when positioned in either orientation upstream or downstream, enhances expression of the chloramphenicol acetyltransferase (CAT) gene expressed from either the simian virus 40 early promoter or the herpes simplex virus type 1 thymidine kinase promoter. The extent of transcriptional enhancement varies with the promoter and cell type. This enhanced CAT expression reflects an increased level of CAT mRNA and does not result from amplification of the plasmids expressing CAT. In addition, plasmids carrying the gene for resistance to hygromycin B and the 30-bp family of repeats yielded 10 to 100 times more hygromycin B-resistant colonies than the vector lacking the 30-bp family of repeats in both EBV-genome-positive cells and cells that express EBNA-1. EBNA-1 is known to bind to sequences within the 30-bp family of repeats (D. R. Rawlins, G. Milman, S. D. Hayward, and G. S. Hayward, Cell 42:859-868, 1985), and these trans- and cis-acting elements together have at least two functional roles: (i) they are required for DNA replication dependent upon oriP, and (ii) they can enhance expression of genes linked to the 30-bp family of repeats of oriP.
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PMID:trans activation of an Epstein-Barr viral transcriptional enhancer by the Epstein-Barr viral nuclear antigen 1. 302 15

We have used a protoplast fusion protocol to introduce the genes encoding neomycin phosphotransferase (neo) and chloramphenicol acetyltransferase (CAT) into murine and human T-lymphocyte lines. Plasmid constructs containing the neo gene under the control of the promoters from the Rous sarcoma virus long terminal repeat (RSV LTR), the SV40 early region, or the herpes simplex virus thymidine kinase gene (HSV TK) can stably transform each of three T-cell lines to G-418 resistance. The characteristic frequencies for different cell lines can differ by at least two orders of magnitude, although initial DNA uptake and transient expression are similar. In the two murine cell lines, low numbers of gene copies are retained in long-term transformants. Prior to integration, transient expression assays for cat or neo gene products reveal that the differences in intrinsic promoter strength of different constructs are further influenced by the coding sequences being transcribed. Thus, while transient expression of the neo protein is similar from both the Rous LTR and the SV40 early promoter, the Rous LTR directs synthesis of CAT protein at levels two orders of magnitude higher than those from the SV40 early promoter.
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PMID:Differential transient and long-term expression of DNA sequences introduced into T-lymphocyte lines. 302 36

At early times after infection with herpes simplex virus, transcription from beta-promoters is initiated only in the presence of a functional 174,000 Mr phosphoprotein (ICP4), encoded by an immediate early (alpha) gene (IE4). A transient expression assay was used to analyze the requirement for two (ICP4 and ICP0) of the five alpha-gene products in the transcriptional regulation of model alpha and beta-gene promoters. These studies reveal that cells cotransfected with plasmids containing the alpha-gene sequences for infected cell proteins (ICPs) 4 and 0 and a thymidine kinase (TK, a beta-gene) gene or the thymidine kinase promoter fused to a chloramphenicol acetyltransferase (CAT) cassette accumulate 10 to 20-fold more RNA or exhibit 10 to 20-fold more CAT activity than cells cotransfected with a plasmid encoding either alpha-gene protein and a thymidine kinase indicator gene. Functional ICP4 is required for enhanced transcriptional activation in the transient expression assay system. It is also required for the uniform dispersal of ICP0 throughout the nucleus as shown by immunofluorescence staining analysis of transfected cells. Two alpha-promoter-CAT fusions were used as targets to study what effects ICP4, ICP0 and Vmw65 (the virion-associated alpha-gene transactivator) have on expression from alpha-promoters that contain all of the sequences that confer alpha-gene regulation, or only the core sequence governing basal level expression. We conclude that ICP4 can activate alpha-gene expression from the core sequence and, depending on its abundance, activate or repress expression from a promoter containing the sequences required for alpha-gene regulation. Independent of these alpha-regulatory sequences cotransfection with low levels of sequences encoding both ICP0 and ICP4 activate expression. At higher ratios of effector (both ICP4 and ICP0) the target accumulation of CAT activity decreases. Although a ts allele of IE4 (cloned from the mutant virus tsK) does not activate alpha-gene expression it can enhance the ability of ICP0 to activate a target containing alpha-regulatory sequences. Virus studies involving tsK support the conclusion that functional ICP4 is required to activate beta-promoters and to repress expression from alpha-promoters and help to explain the pleiotropic effects of the tsK mutation. These analyses have also revealed the presence of a novel RNA species that overlaps the sequences encoding ICP0. Our results suggest that co-ordinate regulation of HSV gene expression is mediated by the functional interaction of at least two alpha-gene products, ICP0 and ICP4.
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PMID:Co-ordinate regulation of herpes simplex virus gene expression is mediated by the functional interaction of two immediate early gene products. 302 83

The promoters for each of the immediate-early genes from herpes simplex virus type 1 were cloned and fused to a chloramphenicol acetyltransferase cassette. These chimeric genes were used as targets in a transient expression assay to determine how the immediate-early gene products ICP4 and ICP0 and the virion-associated stimulatory protein Vmw65 affected their expression in HeLa and Vero cells. The basal level of expression from these cassettes differed significantly depending on the extent of 5'-flanking sequence and the cell line that served as host. The promoters from IE-4 and IE-0 behaved in a qualitatively similar fashion independent of the host cell. However, the promoter for ICP27 had a unique response pattern: in Vero cells it acted as an alpha gene promoter, whereas in HeLa cells its response was more like that of a beta gene promoter. The promoter sequences for ICP22 and ICP47 behaved as the IE-4 and IE-0 promoters did in HeLa cells, but their response to the effector molecules in Vero cells was unlike that of other alpha gene promoters we have studied. Evidence is also presented for a role for ICP27 in autoregulation.
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PMID:Herpes simplex virus immediate-early promoters are responsive to virus and cell trans-acting factors. 303 26

To investigate the role of 5' noncoding leader sequence of herpes simplex virus type 1 (HSV-1) mRNA in infected cells, the promoter for the 65,000-dalton virion stimulatory protein (VSP), a beta-gamma polypeptide, was introduced into plasmids bearing the chloramphenicol acetyltransferase (cat) gene together with various lengths of adjacent viral leader sequences. Plasmids containing longer lengths of leader sequence gave rise to significantly higher levels of CAT enzyme in transfected cells superinfected with HSV-1. RNase T2 protection assays of CAT mRNA showed that transcription was initiated from an authentic viral cap site in all VSP-CAT constructs and that CAT mRNA levels corresponded to CAT enzyme levels. Use of cis-linked simian virus 40 enhancer sequences demonstrated that the effect was virus specific. Constructs containing 12 and 48 base pairs of the VSP mRNA leader gave HSV infection-induced CAT activities intermediate between those of the leaderless construct and the VSP-(+77)-CAT construct. Actinomycin D chase experiments demonstrated that the longest leader sequences increased hybrid CAT mRNA stability at least twofold in infected cells. Cotransfection experiments with a cosmid bearing four virus-specified transcription factors (ICP4, ICP0, ICP27, and VSP-65K) showed that sequences from -3 to +77, with respect to the viral mRNA cap site, also contained signals responsive to transcriptional activation.
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PMID:Herpes simplex virus virion stimulatory protein mRNA leader contains sequence elements which increase both virus-induced transcription and mRNA stability. 303 12

The chicken vitellogenin II gene is transcriptionally activated by estrogens. In transient transfection experiments in human T47D cells that contain receptors for various steroids, we showed estradiol, progestin, and androgen responses of a chimeric chicken vitellogenin II construct. This construct consists of DNA sequences from -626 to -590 upstream of the start of transcription of the chicken vitellogenin gene linked to the herpes simplex virus thymidine kinase promoter driving the transcription of the bacterial chloramphenicol acetyltransferase gene. Treatment of the transfected T47D cells with a combination of estradiol and the progestin R5020 led to a superinduction of chloramphenicol acetyltransferase activity, showing a synergistic action of these two steroids. This synergism was not observed upon treatment of the transfected cells with estradiol and the androgen dihydrotestosterone. Using point mutations in the vitellogenin gene fragment, we showed in functional and in in vitro DNase I footprinting assays with a purified progesterone receptor that, for the synergistic action of estradiol and R5020 to occur, the progesterone receptor must be bound to the vitellogenin gene fragment. The progesterone receptor-binding site was localized at -610 to -590, close to the consensus sequence (-626 to -613) for estrogen receptor binding and function. We therefore demonstrate here that two different steroid hormones can be functionally synergistic through the interaction of their corresponding receptors with two different binding sites adjacent to one another.
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PMID:Estrogen and progesterone receptor-binding sites on the chicken vitellogenin II gene: synergism of steroid hormone action. 324 57

Through substitution mutagenesis we identified the promoter elements responsible for basal expression and sterol-mediated repression of transcription of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-controlling enzyme of cholesterol biosynthesis. Mutant promoters containing 277 base pairs (bp) of reductase 5' flanking sequence were inserted into recombinant plasmids upstream of the coding region for bacterial chloramphenicol acetyltransferase. The plasmids were transfected into hamster fibroblasts, and transcription was measured in the presence and absence of sterols. Mutations in three regions that are known to bind nuclear proteins markedly reduced transcription. Mutation of another protein-binding region of 20 bp in length did not reduce transcription, but it did abolish sterol-mediated repression, producing an operator constitutive phenotype. This mutation also abolished protein binding to the corresponding 20-bp region of DNA as determined by footprinting assays. When a DNA fragment containing these 20 bp was inserted into the herpes simplex virus thymidine kinase promoter, sterol-mediated repression was observed. This sequence contains an octanucleotide that shows a 7/8-bp match with a previously identified regulatory sequence in repeat 2 of the low density lipoprotein receptor promoter, another sterol-repressible gene. We hypothesize that this octanucleotide, GTGGCGGTG, is the core binding site for a sterol-dependent protein that represses transcription.
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PMID:Operator constitutive mutation of 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter abolishes protein binding to sterol regulatory element. 334 49

A bioassay that is based on trans-activation has been developed for the detection and quantitation of the human immunodeficiency virus type 1 (HIV-1). Indicator cell lines were constructed that contain the HIV-1 long terminal repeat ligated to the chloramphenicol acetyltransferase (CAT) gene. Infection of these cells by HIV activates the expression of CAT protein. Isolates of HIV-1 with divergent nucleotide sequences activated the indicator cell lines to a similar extent, approximately 500- to 1000-fold. Human T cell lymphotropic viruses types 1 and 2, equine infectious anemia virus, and herpes simplex virus 1 did not activate the indicator cell lines. Isolates of simian immunodeficiency virus and human T cell lymphotropic virus type 4 activated these cells to a much lesser extent, which suggests that these viruses contain similar, but distinct, trans-activators. This assay can be used for the detection, quantitation, and typing of HIV and for studying the effect of drugs on the replication of HIV in different cellular backgrounds.
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PMID:A quantitative bioassay for HIV-1 based on trans-activation. 342 13


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