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)

The P3 promoter activity of Bovine papillomavirus (BPV) and cis-acting DNA element of P3 promoter required for transcription were examined using chloramphenicol acetyltransferase (CAT) assay. The results show that P3 promoter is a very weak promoter compared to P2 promoter in BPV and E2 transactivator is required for the maximal transcription of P3 promoter in a BPV upstream regulatory region (URR)-dependent manner. Deletion experiments by nuclease Bal-31 were carried out to define P3 promoter element. The DNA sequences between nt 712 and nt 802 of BPV are required for efficient transcription of the P3 promoter. This 90 bp region contains SV40 enhancer core sequences and an ATF binding site.
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PMID:P3 promoter element of bovine papillomavirus. 839 Mar 22

A new Escherichia coli expression vector with increased stability was developed based on bacteriophage Mu. Unlike traditional expression vectors, the vector described herein is chromosome based rather than existing as an autonomously replicating plasmid. The chromosomal location resulted in extreme stability of the vector even in the absence of selective pressure. Both replication and heterologous protein synthesis could be induced by temperature shift. Expression of the heterologous gene was controlled by the Mu middle promoter and was dependent on the presence of the transactivator, Mor, of the Mu middle promoter. Four proteins, beta-galactosidase, chloramphenicol acetyltransferase, porcine somatotropin and human growth hormone, were made from this vector at levels ranging from 5 to 20% of total cell protein. Expression from the middle promoter was highest when inductions were done in rich media. The expression of some genes varied in different strains.
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PMID:A chromosomal expression vector for Escherichia coli based on the bacteriophage Mu. 847 59

Transcription from the HIV-1 long terminal repeat (LTR) was shown to be inhibited by DNA CpG methylation both in vivo and in vitro. Enzymatic methylation of CpG sites localized within the LTR decreased the transcription of the CAT reporter gene, chloramphenicol acetyltransferase, as assayed by the transient expression of this gene in tissue culture. The inhibitory effect could be initially overcome, in trans, by the transactivator tat. As a function of time, the presence of tat had no observable effect on transcription, within the limits of detection sensitivity, suggesting that the level of basal transcription was reduced to very low levels. This effect is suggestive of the involvement of cellular CpG methylation-dependent inhibitory factors which have been characterized by other laboratories. These data imply that transactivation is reduced to low levels after longer periods of time when the DNA template is sparsely methylated. The transcriptional inhibitory process may involve proteins such as MeCP which may interact with methylated DNA more slowly and/or weakly. Conversely, densely methylated DNA was transcriptionally repressed immediately which suggests the rapid/strong association of the cellular inhibitory factor(s). The transcriptional inhibitory effect was also observed in an in vitro transcription run-off system. These data suggest that the methylation-mediated inhibition of transcription is directly affected by CpG methylation density and may involve other factors.
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PMID:Transcription of the HIV-1 LTR is regulated by the density of DNA CpG methylation. 849 86

The transgenic mouse system is a powerful tool for the study of gene function. However, when the analysis involves genes that are critical for the normal developmental process, the usefulness of transgenic mouse systems is limited (for review see Hanahan, 1989; Westphal and Gruss, 1989; Byrne et al., 1991). This is due to potential transgene interference with development in case of ectopic or high level expression. As a result, establishing permanent transgenic mouse lines expressing these types of genes has proven difficult. To circumvent these difficulties, a binary transgenic mouse system has been established, termed the Multiplex System (Byrne and Ruddle, 1989). This is a two-tiered gene activation system in which expression of the gene of interest occurs only in offspring carrying transgenes encoding both components: transactivator and transresponder. Transactivator lines contain the gene encoding the VP16 protein of herpes simplex virus. Transresponder lines harbour the gene of interest linked to the IE promoter which includes recognition sequences for the VP16 transactivator. Previously, the inducibility of a chloramphenicol acetyltransferase reporter gene in newborn offspring that carried both a transactivator and transresponder transgene (Byrne and Ruddle, 1989) has been shown. Moreover, it has been demonstrated that expression of the VP16 protein was not detrimental to development and that transactivation appeared to be tissue specific. Here, the potential of the system for the expression of transgenes in early mouse embryogenesis was examined, using the Escherichia coli beta-galactosidase gene as a reporter in the transresponder mouse strain. To direct expression of VP16, the murine Hoxc-8 promoter, which is known to be active during early development, was used. Embryos from crosses of transactivators to transresponders were isolated at different stages of development and stained for beta-galactosidase activity. Transactivation, as demonstrated by strong beta-galactosidase staining, could be detected as early as eight days of development. At all stages examined, the pattern of lacZ transresponder gene expression accurately reflected the activity of the Hoxc-8 promoter controlling VP16 expression. It is demonstrated that the Multiplex System can be used to express transresponder transgenes in a spatially and temporally defined manner in multiple cell types early during mouse embryogenesis.
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PMID:Spatial and temporal regulation of a lacZ reporter transgene in a binary transgenic mouse system. 858 38

Notch is a transmembrane receptor that plays a critical role in cell fate determination. In Drosophila, Notch binds to and signals through Suppressor of Hairless. A mammalian homologue of Suppressor of Hairless, named CBF1 (or RBPJk), is a ubiquitous transcription factor whose function in mammalian Notch signaling is unknown. To determine whether mammalian Notch can stimulate transcription through a CBF1-responsive element (RE), we cotransfected a CBF1-RE-containing chloramphenicol acetyltransferase reporter and N1(deltaEC), a constitutively active form of human Notch1 lacking the extracellular domain, into DG75, COS-1, HeLa, and 293T cells, which all contain endogenous CBF1. N1(deltaEC) dramatically increased chloramphenicol acetyltransferase activity in these cells, indicating functional coupling of Notch1 and CBF1. The activity was comparable to that produced by the Epstein-Barr virus protein EBNA2, a well-characterized, potent transactivator of CBF1. To test whether CBF1 and Notch1 interact physically, we tagged CBF1 with an epitope from the influenza virus hemagglutinin or with the N-terminal domain of gal4, and transfected the tagged CBF1 plus N1(deltaEC) into COS-1 cells. Cell lysates were immunoprecipitated and immunoblotted with several anti-Notch1 antibodies [to detect N1(deltaEC)] or with antibodies to hemagglutinin or gal4 (to detect CBF1). Each immunoprecipitate contained a complex of N1(deltaEC) and CBF1. In summary, we find that the truncated, active form of human Notch1, N1(deltaEC), binds CBF1 and activates transcription through a CBF1-RE-containing promoter. We conclude that CBF1 is a critical downstream protein in the human Notch1 signaling pathway.
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PMID:Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element. 864 33

The experiments presented herein were designed to understand the molecular mechanism(s) by which membrane Ig (mIg)-dependent signals are integrated at the level of the junB promoter to induce gene transcription. Functional studies using chloramphenicol acetyltransferase reporter gene constructs that contained deleted 5' flanking region junB sequences identified a region located between -194 and -87 that contains an Ets binding site and a putative cAMP response element binding site (CRE-like). Point mutagenesis of the CRE-like site blocked junB promoter activation in response to mIg cross-linking in mature Bal17 B cells. Nuclear extract binding activity to a synthetic oligonucleotide containing the junB CRE-like site was detected in unstimulated B cells and was increased in response to mIg cross-linking. Binding activity was competed with unlabeled oligonucleotides that contained the junB CRE-like site or the somatostatin CRE consensus motif, the latter observation suggests that members of the activating transcription factor/CRE binding protein (CREB) family may mediate mIg-dependent junB transcription. Consistent with this interpretation, recombinant CREB and activating transcription factor proteins bound the junB CRE-like site, but did not interact with a mutant CRE-like site. Expression of a dominant negative CREB protein blocked mIg-mediated transcription from a junB CRE-like site-chloramphenicol acetyltransferase reporter gene. CRE-like nucleoprotein complexes from Bal17 B cells contained constitutively bound CREB-1, which was phosphorylated on serine 133 in response to mIg cross-linking. Activating transcription factor-1 protein was also constitutively expressed in CRE-like nucleoprotein complexes. Collectively, these results suggest that components of the protein kinase A signaling pathway are recruited by mIg to induce junB transcription.
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PMID:Transcriptional regulation of the junB promoter in mature B lymphocytes. Activation through a cyclic adenosine 3',5'-monophosphate-like binding site. 868 8

Gene therapy approaches for human immunodeficiency virus type 1 (HIV-1) infections focus on the transfer of critical genetic elements into CD4+ T lymphocytes and CD34+ stem cells. Ideally, expression of the anti-HIV-1 gene constructs should be induced during early stages of infection to combat high turnover of the replicating virus. In this study, we investigated the activity of two promoters, HIV-1 long terminal repeat (HIV-1-LTR) and Rous sarcoma virus (RSV) LTR fused with the transactivation response element (TAR) from the HIV-1-LTR (ie RSV-TAR) in presence of Tat, the major HIV-1 transcriptional transactivator and an early gene product in HIV-1 infection. Comparative expression from both of these plasmids was analyzed by measuring expression of a reporter gene, chloramphenicol acetyltransferase (CAT), after transfection of the promoter-CAT constructs and a Tat-expressing plasmid into CEM T lymphocytic cells and peripheral blood mononuclear cells (PBMC). The HIV-1-LTR could be transactivated by Tat in both unstimulated and stimulated cells. Although the RSV-TAR had a relatively high basal level of expression, Tat transactivation of this chimeric promoter occurred only in unstimulated cells. These results suggest that the HIV-1-LTR may be a better promoter for therapeutic gene expression in anti-HIV-1 intracellular immunization approaches.
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PMID:Evaluation of relative promoter strengths of the HIV-1-LTR and a chimeric RSV-LTR in T lymphocytic cells and peripheral blood mononuclear cells: promoters for anti-HIV-1 gene therapies. 885 98

The Zta transactivator is crucial for both Epstein-Barr virus (EBV) lytic gene expression and lytic DNA replication. We have used a cotransfection-replication assay to examine the effect of mutations in the Zta activation domain (amino acids [aa] 1 to 167) on Zta replication activity. Deletion of Zta aa 25 to 86, which are critical for transcriptional activation of ori-Lyt, or aa 93 to 141 did not adversely affect replication of an ori-Lyt-containing target plasmid. However, removal of aa 2 to 25 (delta2-25) abolished replication activity. Within this subdomain, deletion of aa 2 to 10 (delta2-10) or mutation of codons 18 and 19 (m18/19) or 22 and 26 (m22/26) did not affect replication competency, while deletion of codons 13 to 19 (delta13-19) or mutation at codons 12 and 13 (m12/13) impaired Zta replication function. Each of the replication-negative Zta variants was capable of transactivating expression from both BHLF1 promoter-chloramphenicol acetyltransferase constructions and the BMRF1 promoter on endogenous EBV genomes in Raji cells with efficiency comparable to that of the wild-type polypeptide. Thus, a replication contribution of Zta was functionally separable from its transactivation activity and was supplied by the N-terminal region encompassing aa 11 to 25. Replication by a subset of the impaired Zta mutants was partially rescued upon the addition of Rta to the replication assay. The contribution of Rta mapped to domain II of the Rta activation domain and was specific for this region. A chimeric Rta-EBNA-2 transactivation domain fusion, which retains the DNA-binding and transactivation properties associated with wild-type Rta, failed to rescue replication-deficient Zta. Our data suggest that Rta may act as an ancillary replication factor in EBV ori-Lyt DNA synthesis by stabilizing Zta-replisome interactions.
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PMID:A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator. 897 Sep 53

Caffeic acid phenethyl ester (CAPE) is a phenolic antioxidant derived from the propolis of honeybee hives. CAPE was shown to inhibit the formation of intracellular hydrogen peroxide and oxidized bases in DNA of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated HeLa cells and was also found to induce a redox change that correlated with differential growth effects in transformed cells but not the nontumorigenic parental ones. Mediated via the electrophile or human antioxidant response element (hARE), induction of the expression of NAD(P)H quinone oxidoreductase (NQO1) and glutathione S-transferase Ya subunit genes by certain phenolic antioxidants has been correlated with the chemopreventive properties of these agents. Here, we determined by Northern analysis that CAPE treatment of hepatoma cells stimulates NQO1 gene expression in cultured human hepatoma cells (HepG2), and we characterized the effects of CAPE treatment on the expression of a reporter gene either containing or lacking the hARE or carrying a mutant version of this element in rodent hepatoma (Hepa-1) transfectants. A dose-dependent transactivation of human hARE-mediated chloramphenicol acetyltransferase (cat) gene expression was observed upon treatments of the Hepa-1 transfectants with TPA, a known inducer, as well as with CAPE. The combined treatments resulted in an apparent additive stimulation of the reporter expression. To learn whether this activation of cat gene expression was effected by protein kinase C in CAPE-treated cells, a comparison was made of cat gene activity after addition of calphostin, a protein kinase C inhibitor. Calphostin reduced the cat gene induction by TPA but not by CAPE, suggesting that stimulation of gene expression in this system by these agents proceeds via distinct mechanisms. Band-shift experiments to examine binding of transactivator proteins from nuclear extracts of treated and untreated cells to a hARE DNA probe showed that TPA exposure increased the binding level. In contrast, binding of factors to this probe was inhibited after either in vivo treatment of cells with CAPE or in vitro addition of this compound to the nuclear extract. In view of the clear stimulation by CAPE of gene expression mediated by hARE, possible explanations of this result are discussed.
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PMID:Caffeic acid phenethyl ester stimulates human antioxidant response element-mediated expression of the NAD(P)H:quinone oxidoreductase (NQO1) gene. 901 71

Hepatitis B virus (HBV) preS1 fused to the GAL4 DNA-binding domain functioned as a transcriptional activation domain in yeast and mammalian cells. The GAL4-preS1 fusion proteins derived from the preS1 of all three tested HBV subtypes (adr, adw and ayw) specifically activated the transcription of a lacZ or chloramphenicol acetyltransferase reporter gene linked to a GAL4-responsive promoter in transient transfection assays using yeast or HepG2 cells, respectively. Deletion analyses showed that the segments of preS1 from residues 21 to 90 and from residues 21 to 56 are sufficient and essential for the activity, respectively. Stable expression of GAL4-preS1 in Chinese hamster ovary cells also produced transactivator activity. These results suggest that preS1 fused to any DNA-binding domain of transcription factors would have transactivation potential.
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PMID:Hepatitis B virus preS1 functions as a transcriptional activation domain. 915 26


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