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 identified an Epstein-Barr virus (EBV) gene product which functions in transient-expression assays as a nonspecific trans activator. In Vero cells, cotransfection of the BglII J DNA fragment of EBV together with recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene gave up to a 100-fold increased expression of CAT activity over that in cells transfected with the recombinant CAT constructs alone. The BglII J fragment acted promiscuously, in that increased CAT synthesis was observed regardless of whether the promoter sequences driving the CAT gene were of EBV, simian virus 40, adenovirus, or herpes simplex virus origin. Cleavage of cloned BglII-J plasmid DNA before transfection revealed that activation was dependent upon the presence of an intact BMLF1 open reading frame. This was confirmed with subclones of BglII-J and with hybrid promoter-open reading frame constructs. This region of the genome is also present in the rearranged P3HR-1-defective DNA species, and defective DNA clones containing these sequences produced a similar activation of CAT expression in cotransfection experiments. The heterogeneous 45-60-kilodalton polypeptide product of BMLF1 may play an important regulatory role in expression of lytic-cycle proteins in EBV-infected lymphocytes.
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PMID:Promiscuous trans activation of gene expression by an Epstein-Barr virus-encoded early nuclear protein. 301 81

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

The BamHI M DNA fragment of Epstein-Barr virus was shown to activate transcription of the cotransfected chloramphenicol acetyltransferase gene under the control of the simian virus 40 early promoter. Both the BamHI-BglII and the HindIII-BamHI subfragments of the BamHI M fragment, corresponding to the rightward reading frame BMRF1 and the leftward reading frame BMLF1, respectively, had the ability to activate transcription from the simian virus 40 promoter. The trans-activating function was well correlated with the expression of nuclear early antigens, which suggests that early antigens encoded by BMRF1 and BMLF1 are responsible for trans-activation and possibly play a role in regulated expression of virus genomes.
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PMID:Both the rightward and the leftward open reading frames within the BamHI M DNA fragment of Epstein-Barr virus act as trans-activators of gene expression. 304 Oct 51

Sequence variations of the 5'-upstream region of latent membrane protein 1 (LMP-1) in two Epstein-Barr virus (EBV) strains have been reported before (Chen et al., 1992). To investigate the effect of these variations on gene expression, we constructed a series of deletion plasmids encompassing positions -950 to +20 of the LMP-1 promoter region and tested for the ability to drive chloramphenicol acetyltransferase (CAT) gene expression in C33A cells. Results showed that the promoter activities of constructs from NPC strain were 3-fold lower than the corresponding constructs from the B95-8 strain. In addition, the region between -54 and +20 contained the basic, constitutive promoter activity for both strains. Sequence analysis of this region indicated that an activating transcription factor (ATF) binding site, TGACGTAG, which is present in B95-8 strain was changed to TCTCGTAG in NPC strain. A chimeric plasmid study suggested that these sequence variations in the ATF binding site may contribute to the 3-fold increase of CAT activity observed for B95-8 strain. Furthermore, the activity of the promoter constructs was not activated by EBV-encoded nuclear antigen 2 (EBNA-2) in C33A cells. However, the promoter activities were upregulated in B-lymphocyte cells such as CG3 and CA46 cells. The biological significance of this difference in promoter activity of LMP-1 gene between two strains and the involvement of the cellular factors were discussed.
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PMID:Characterization of 5'-upstream sequence of the latent membrane protein 1 (LMP-1) gene of an Epstein-Barr virus identified in nasopharyngeal carcinoma tissues. 748 24

Epstein-Barr virus (EBV)-inducing activity was previously demonstrated to occur in various foodstuffs, including dried salted fish in southern China and 'harissa', a homemade spice mixture in Tunisia, whose consumption is epidemiologically associated with an increased risk for developing nasopharyngeal carcinoma (NPC). For the isolation and the characterization of active ingredients in harissa, we used as a rapid screening assay the induction of the chloramphenicol acetyltransferase (CAT) activity through the EBV-DR promoter in DR-CAT Raji cells. After fractionation of harissa and column chromatography on Sepharose-CL4B, the major inducing activity was associated with a macromolecular fraction which was chemically characterized as liginin-containing complexes. The active material enhanced EBV-DR induction with an activity comparable to the tumor promoter and strong EBV inducer, 12-O-tetradecanoylphorbol-13-acetate. Experiments with inhibitors of protein kinase C-related pathways suggested that the EBV-inducing activity of lignin fractions operates through a different pathway. Our results on the presence of specific lignin fractions in high-risk food items that can induce important cellular functions linked to tumor promotion are discussed in relation to NPC genesis and etiology.
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PMID:Characterization of macromolecular lignins as Epstein-Barr virus inducer in foodstuff associated with nasopharyngeal carcinoma risk. 763 18

EBNA 1 is the only antigen expressed in both Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma cells. Previous studies showed that the mRNA of EBNA 1 in these two tumor tissues was initiated from a promoter located in the Bam HI F fragment (Fp) on the viral genome. Two regulatory elements located in the downstream Bam HI Q region include an EBNA 1 binding site and a positive regulatory region between the Fp and the EBNA1 binding site. This data strongly suggested that a cellular factor(s) may modulate the usage of the Fp. To locate the shortest responsible viral sequence, we constructed a series of luciferase gene and chloramphenicol acetyltransferase (CAT) gene plasmids that contained various portions of the Bam HI F/Q region. Plasmid DNA was then introduced into cells to examine the promoter activity of each construct. By this method, we identified a 186-bp fragment within the Bam HI Q region that possessed the highest activity. This promoter was designated as Qp and found to be orientation-dependent and down-regulated by EBNA 1 in both the type I BL cells and human epithelial cells. Furthermore, RNase protection assay showed that a transcription initiation site was located at nucleotide 62,416 of the EBV genome. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis further confirmed that the transcript was initiated from the Qp, and not the Fp. Therefore, our data suggested that a novel promoter, Qp, located within the Bam HI Q existed for the EBNA 1 expression in the latently infected type 1 BL cells. The biological significance of the selection of the Qp needs further investigation.
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PMID:Identification of a novel promoter located within the Bam HI Q region of the Epstein-Barr virus genome for the EBNA 1 gene. 766 54

We have developed a simple and highly sensitive tissue culture-based assay for the biological activity of steroids and synthetic steroidal compounds. A DNA cassette, containing a synthetic steroid-inducible promoter controlling the expression of a bacterial chloramphenicol acetyltransferase gene (GRE5-CAT), was inserted into an Epstein-Barr virus (EBV) episomal vector which replicates autonomously in primate and human cells. We then used this promoter/reporter system to generate two stably transfected human cell lines. In the cervical carcinoma cell line HeLa, which expresses high levels of glucocorticoid receptor, the GRE5 promoter is inducible over 100-fold by the synthetic glucocorticoid dexamethasone. In the breast carcinoma cell line T47D, which expresses progesterone and androgen receptors, the GRE5 promoter is inducible over 100-fold by either progesterone or dihydrotestosterone. In both cell lines basal expression of CAT activity is strictly dependent on the presence of steroid, so that very low levels of induction can be detected. Thus, the cell lines can be used to test for low levels of agonist activity in steroid antagonists. These cell lines can be used to screen compounds for steroid agonist or antagonist activity by testing extracts of cells grown in microtiter wells directly using a colorimetric CAT assay. This system should provide a sensitive and efficient method for screening and analysis of the activity of large numbers of natural or synthetic steroid agonists or antagonists.
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PMID:A simple and sensitive high-throughput assay for steroid agonists and antagonists. 776 3

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) has been shown to transactivate both cellular and viral gene promoters including the promoter for the viral terminal protein 1 gene (TP-1). We investigated whether three other EBV nuclear antigens EBNA-3A, -3B, and -3C (which themselves share a degree of primary sequence homology) could also play a role in TP-1 gene regulation. The TP-1 promoter sequence was linked to the chloramphenicol acetyltransferase (CAT) gene and used in cotransfection experiments in an EBV negative cell line with various combinations of vectors expressing individual EBNA-3s. In the absence of other EBV proteins, the EBNA-3s did not stimulate TP-1 promoter activity. In the presence of EBNA-2, the EBNA-3s were shown to be capable of reducing the level of TP-1 promoter-driven CAT activity. The EBNA-3s had no effect on a panel of heterologous promoters, indicating that EBNA-2 and/or transcription elements specific to the TP-1 promoter are essential for the observed activity of the EBNA-3s. The functional antagonism between the EBNA-2 and EBNA-3 proteins may be important in the overall viral strategy.
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PMID:The Epstein-Barr virus determined nuclear antigens EBNA-3A, -3B, and -3C repress EBNA-2-mediated transactivation of the viral terminal protein 1 gene promoter. 797 64

The regulatory mechanisms which control latency and reactivation of the Epstein-Barr virus (EBV) are not fully understood. To determine whether DNA methylation is involved, we examined the BamHI-H divergent promoter, which also encompasses the origin for lytic replication (oriLyt) of EBV. The divergent promoter was highly methylated in the stringently latent HH514cl16 cell line and largely unmethylated in the semipermissive FF41-1 cell line. Expression vectors in which the divergent promoter directed transcription of the chloramphenicol acetyltransferase gene were made. Using in vitro methylation and transient transfections, we found an inverse correlation between the number of sites methylated and level of gene expression. Similar patterns of inhibition were observed when the methylated promoter was activated by BZLF1 or BRLF1 and in lymphoid or epithelial cells. The role of two CpG dinucleotides in the BRLF1 binding sites of the divergent promoter was determined by site-directed mutagenesis. The results indicated that site-specific methylation of these CpGs was not solely responsible for inhibition of expression by methylation. DNA methylation also reduced DNA replication mediated by oriLyt. These results suggest that hypermethylation of the divergent promoter and oriLyt may suppress transcription and lytic replication of EBV.
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PMID:Regulation of Epstein-Barr virus BamHI-H divergent promoter by DNA methylation. 821 55

Epstein-Barr virus (EBV) is a herpesvirus that transforms B-cells (B-LCL) and has undergone intense scrutiny owing to its association with Burkitt's lymphoma, nasopharyngeal carcinoma, and immunoblastic lymphomas. B-LCL have also proven useful in the study of human immunology. We describe a novel system for inducing efficient foreign gene expression in B-LCL using biotinylated adenovirus as an endosome-disrupting agent. Plasmid DNA is coupled to the exterior of viral particles by streptavidin-polylysine chimeric proteins. Up to 67% of B-LCL may be induced to express foreign genes in vitro in transient expression systems, and gene expression lasts for at least 17 days. We have expressed firefly luciferase, beta-galactosidase (beta-gal), chloramphenicol acetyltransferase, HIV gag, and env genes, as well as infectious HIV, and the EBV-specific BZLF gene in B-LCL with this system. In vivo delivery of a beta-gal reporter gene to B-LCL was documented in a SCID mouse model. Potential applications include study of genetic regulation of EBV infection and transformation events, study of potential gene therapies for EBV-related B-cell tumors, and production of antigen-presenting cells for use in immunologic assays. Because of the high percentage of cells transformed and the length of foreign gene expression, the possibility of examining foreign gene expression in transient assays, without selection for clonal populations, exists.
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PMID:Efficient foreign gene expression in Epstein-Barr virus-transformed human B-cells. 829 Dec 40


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