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)

Efficient and controllable expression of a transgene usually requires the presence of intron sequences and much efforts have been made to produce retrovirus vectors that can transduce and integrate genes with introns. However, this has proven difficult because the viral RNA is spliced when it is synthesized in the nucleus of a producer cell. We describe a novel approach to avoid this problem. In our system the retroviral RNA is synthesized in the cytoplasm of the cell, not in the nucleus, in a reaction driven by the Semliki Forest virus (SFV) expression system. The approach was tested with a recombinant Moloney murine leukemia virus genome containing the chloramphenicol acetyltransferase (CAT) gene in association with an intron. This was inserted into a SFV transcription plasmid and the corresponding SFV vector RNA was transcribed in vitro. BHK-21 cells were then transfected with this vector RNA together with two additional SFV vectors that encode the Moloney murine leukemia virus packaging proteins. Retrovirus vectors containing intron-CAT sequences were produced at titers up to 1.3 x 10(6) infectious particles per ml during a 5-hr incubation period. The vectors faithfully transduced the intron-containing CAT gene into NIH 3T3 cells, where the intron-CAT RNA was subjected to efficient splicing and used for high level enzyme expression. Thus, the results show that intron containing genes can be efficiently packaged into retrovirus vectors by the SFV expression system.
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PMID:Packaging of intron-containing genes into retrovirus vectors by alphavirus vectors. 952 Apr 20

Cas-Br-E and Graffi are two murine viruses that induce myeloid leukemia in mice: while Cas-Br-E induces mostly non-T, non-B leukemia composed of very immature cells, Graffi causes exclusively a granulocytic leukemia (E. Rassart, J. Houde, C. Denicourt, M. Ru, C. Barat, E. Edouard, L. Poliquin, and D. Bergeron, Curr. Top. Microbiol. Immunol. 211:201-210, 1995). In an attempt to understand the basis of the myeloid specificity of these two retroviruses, we used DNase I footprinting analysis and gel mobility shift assays to identify a number of protein binding sites within the Cas-Br-E and Graffi U3 regions. Two protected regions include potential GATA binding sites. Methylation interference analysis with different hematopoietic nuclear extracts showed the importance of the G residues in these GATA sites, and supershift assays clearly identified the binding factors as GATA-1, GATA-2, and GATA-3. Transient assays with long terminal repeat (LTR)-chloramphenicol acetyltransferase constructs showed that these three GATA family members are indeed able to transactivate Cas-Br-E and Graffi LTRs. Thus, the availability and relative abundance of the various members of the GATA family of transcription factors in a given cell type could influence the transcriptional tissue specificity of murine leukemia viruses and hence their disease specificity.
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PMID:Members of the GATA family of transcription factors bind to the U3 region of Cas-Br-E and graffi retroviruses and transactivate their expression. 962 Oct 16

Bovine leukemia virus (BLV) is a member of the human T-cell leukemia virus (HTLV)/BLV group of retroviruses. These viruses regulate their own transcription by producing Tax, a protein which activates the virus promoter region, the long terminal repeat (LTR). To explore the molecular mechanisms involved in the transactivation, we identified protein binding elements by in vivo footprinting and analyzed their function by site- directed mutagenesis. We used in vivo dimethyl sulfate footprinting by ligation-mediated PCR to detect constitutive in vivo protein-DNA interactions in a BLV-producing cell line, Bat2Cl6. The U3 region and part of the R region of the LTR were footprinted. In addition to the cis-acting elements (three cyclic AMP-responsive elements [CREs] and two AP4 sites) reported by others to be important for Tax-mediated activation of the BLV LTR, we found footprints in regions flanking these elements and in the core promoter region. The importance of these sites for transcriptional activation was studied by site-directed mutagenesis followed by promoter function analysis of the mutants with a chloramphenicol acetyltransferase reporter system. Our data corroborate those of others showing that the CREs are necessary for transactivation of the LTR, and they identify two new functional sites not previously reported by others. We show that the middle region of the BLV U3 contains multiple dual-functioning cis-acting elements which act as either positive or negative regulatory elements depending on the cell type tested. This is the first report of a functional mapping of the cis-acting elements of a virus of the HTLV/BLV group.
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PMID:In vivo protein binding and functional analysis of cis-acting elements in the U3 region of the bovine leukemia virus long terminal repeat. 962 Oct 62

The human immunodeficiency virus type 1 (HIV-1) Vpr protein is a virion-associated protein that localizes in the nucleus of infected cells. Vpr has been shown to facilitate HIV infection of non-dividing cells such as macrophages by contributing to the nuclear translocation of the pre-integration complex. More recently, Vpr expression has been shown to induce an accumulation of cells at the G2 phase of the cell-cycle. We have previously reported that Vpr stimulates reporter gene expression directed from the HIV-1 long terminal repeat (LTR) as well as from heterologous viral promoters. However, the mode of action of Vpr-mediated transactivation remains to be precisely defined. We report here that, for a constant amount of transfected DNA, the level of chloramphenicol acetyltransferase (CAT) mRNA is increased in Vpr-expressing cells using either HIV-1 or a murine leukemia virus (MLV) SL3-3 LTR-CAT reporter construct. Moreover, this Vpr-mediated transactivation requires that promoters direct a minimal level of basal expression. Our mutagenic analysis indicates that the transactivation mediated by Vpr is not dependent on the ability of the protein to localize in the nucleus or to be packaged in the virions. Interestingly, all transactivation-competent Vpr mutants were still able to induce a cell-cycle arrest. Conversely, transactivation-defective mutants lost the ability to mediate cell-cycle arrest, implying a functional relationship between these two functions. Overall, our results indicate that the G2 cell-cycle arrest mediated by Vpr creates a cellular environment where the HIV-1 LTR is transcriptionally more active.
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PMID:Human immunodeficiency virus type 1 vpr protein transactivation function: mechanism and identification of domains involved. 983 15

Rous sarcoma virus (RSV) enhancer sequences in the long terminal repeat (LTR) have previously been shown to be sensitive to CpG methylation. We report further that the high density methylation of the RSV LTR-driven chloramphenicol acetyltransferase reporter is needed for full transcriptional inhibition in chicken embryo fibroblasts and for suppression of tumorigenicity of the RSV proviral DNA in chickens. In nonpermissive mammalian cells, however, the low density methylation is sufficient for full inhibition. The time course of inhibition differs strikingly in avian and mammalian cells: although immediately inhibited in mammalian cells, the methylated RSV LTR-driven reporter is fully inhibited with a significant delay after transfection in avian cells. Moreover, transcriptional inhibition can be overridden by transfection with a high dose of the methylated reporter plasmid in chicken cells but not in hamster cells. The LTR, v-src, LTR proviral DNA is easily capable of inducing sarcomas in chickens but not in hamsters. In contrast, Moloney murine leukemia virus LTR-driven v-src induces sarcomas in hamsters with high incidence. Therefore, the repression of integrated RSV proviruses in rodent cells is directed against the LTR.
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PMID:Inhibition of the rous sarcoma virus long terminal repeat-driven transcription by in vitro methylation: different sensitivity in permissive chicken cells versus mammalian cells. 1004 32

A number of studies have reported that human leukemia cells respond to exposure to power-line frequency electromagnetic fields (EMFs), providing evidence for an EMF-induced signaling pathway involving activation of protein tyrosine kinases (PTKs), phospholipase-Cy and protein kinase C (PKC). Because activation of PKC is also important in the signaling pathways that regulate the transcription factors NF-kappaB and AP-1, we evaluated the effect of exposure to a 60 Hz EMF on NF-kappaB or AP-1-dependent reporter gene expression in cells of the human promonocytic U937 leukemia cell line. Reporter genes were electroporated into U937 cells and activation of the NF-kappaB or AP-1 signaling pathway was evaluated by measuring chloramphenicol acetyltransferase (CAT) protein by CAT ELISA. In contrast to the effects of well-understood chemical or biological agents, the exposure to magnetic-field intensities of 0.08, 0.1, 1.0 or 1.3 mT had no effect on the NF-kappaB or AP-1 signaling pathways.
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PMID:NF-kappaB or AP-1-dependent reporter gene expression is not altered in human U937 cells exposed to power-line frequency magnetic fields. 1007 69

Using murine spermatogenic cell lines GC-1 spg and GC-2 spd(ts) as target cells, an attempt was made to design a retroviral vector that would transduce genes efficiently. Promoter activities of various retroviral long terminal repeats (LTRs) were examined by using chloramphenicol acetyltransferase (CAT) as a reporter. The U3 region of spleen focus-forming virus (SFFVp) showed higher enhancer activity than that of Moloney murine leukemia virus (Mo-MuLV) in both cell lines. The U3 region of myeloproliferative sarcoma virus (MPSV) showed higher activity only in GC-1 spg cells. Expression was suppressed by the repressor element of the primer-binding site (PBS) of the Moloney-related virus. The efficiency of transduction of the multidrug-resistance gene (mdr-1) by an Mo-MuLV-based vector was compared with hybrid vectors consisting of the murine embryonic stem cell virus (MESV) PBS and the LTR of either SFFVp or MPSV. Rhodamine efflux assays and colchicine-resistant colony-forming assays demonstrated higher gene expression by the hybrid vectors. Amphotropic and ecotropic receptors were found to be expressed and functional in both cell lines. Thus, these hybrid vectors represent a powerful tool by which to transfer genes into spermatogenic cells.
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PMID:Efficient gene transfer by hybrid retroviral vectors to murine spermatogenic cells. 1044 22

Alterations in gene expression are a hallmark of cardiac hypertrophy and heart failure. Among these, the decreased expression of the sarcoplasmic reticulum calcium ATPase (SERCA2) has been described. Elevated levels of cytokines in particular, Leukemia Inhibitory Factor (LIF) and Interleukin-6 (IL-6) have been shown to have the capacity to elicit hypertrophic responses in cultured cardiac myocytes. In this study, we investigated the effects of these cytokines (LIF & IL-6) on the regulation of SERCA2 levels in cardiac myocytes. Cultured neonatal rat ventricular myocytes were transfected with a 3.2 kb promoter plasmid construct containing the SERCA2 promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene, and subsequently treated with 10 ng/ml LIF or 10 ng/ml IL-6. LIF and IL-6 independently caused a significant (p < or = 0.05) 23-36% inhibition in SERCA2 promoter activity. LIF and IL-6 induced inhibition was also evident in SERCA2 mRNA levels as assessed by Northern analysis. Time course of inhibition of SERCA2 mRNA levels showed the most prominent decrease occurring after 48 hours of treatment, with both cytokines having a dose dependent effect on the inhibitory response. Western analysis using a polyclonal antibody to SERCA2 protein indicate a significant, 60% decrease in the amount of total SERCA2 protein in cultured myocytes treated with 10 ng/ml LIF or IL-6. In conclusion, the cytokines LIF and IL-6 downregulate SERCA2 gene expression and protein levels. The molecular mechanism responsible for cytokine induced downregulation of SERCA2 is at least partly transcriptional.
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PMID:Leukemia Inhibitory Factor and Interleukin-6 downregulate sarcoplasmic reticulum Ca2+ ATPase (SERCA2) in cardiac myocytes. 1075 45

The activation status of a recently identified STAT (signal transducers and activators of transcription) factor, LIL-Stat (lipopolysaccharide [LPS]/IL-1-inducible Stat) in adult T-cell leukemia (ATL) cells was investigated by electrophoretic mobility shift assays using nuclear extracts of leukemic cells from 7 patients with ATL and a GAS (gamma interferon activation site)-like element termed LILRE (LPS/IL-1-responsive element), which is found in the human prointerleukin 1beta (IL1B) gene. Spontaneous DNA binding of LIL-Stat was observed in all ATL cells examined. However, in normal human peripheral lymphocytes, DNA binding of LIL-Stat was detected only after stimulation with IL-1. These results demonstrated that LIL-Stat is constitutively activated in ATL cells. Furthermore, our transient transfection studies using LILRE chloramphenicol acetyltransferase (CAT) reporters argue that LIL-Stat in ATL cells functions as a transcriptional activator through binding to the LILRE in the IL1B gene. (Blood. 2000;95:2715-2718)
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PMID:Constitutive activation of LIL-Stat in adult T-cell leukemia cells. 1075 55

Specific structures found in the mRNA of picornavirus are known to allow a cap-independent translation. These structures, named internal ribosome entry sites (IRES), are also able to favor translation of the second cistron in bicistronic mRNAs. Their mechanism of action is not well understood. In the present study, two IRESs have been used: the IRES from poliovirus and a newly discovered IRES (SUR) composed of the 5' P untranslated sequence from SV40 early genes, the R structure, and a small part of the U5 region from the human leukemia virus-1 (HTLV-1). The bicistronic constructs containing the firefly luciferase gene as the first cistron and the chloramphenicol acetyltransferase (CAT) as the second cistron were driven by the Rous sarcoma virus (RSV) promoter and contained the early gene SV40 terminator. All the resulting plasmids were tested by transfection in HeLa and CHO cells. In the bicistronic mRNAs without IRES, the expression of the CAT gene was dependent on the distance between the two cistrons. The maximum efficiency in the expression of the second cistron was obtained when the intercalating RNA was composed of 30 to 90 nucleotides. This expression was deeply reduced when the intercalating fragment contained 8 or 300 nucleotides and was undetectable with 500 nucleotides. Unexpectedly, the luciferase mRNA was almost not expressed when the intercalating RNA was of 8 or 30 nucleotides. Expression of the luciferase gene occurred when the intercistronic RNA fragment was of 80 nucleotides and it became lower at 300 and 500 nucleotides. The same observations were done when the poliovirus or the SUR IRESs were added after the intercistronic spacers. However, expression of the CAT gene was amplified by both IRESs. When the CAT cistron preceded by the poliovirus or SUR IRES was introduced within luciferase cistron, 316 nucleotides before its termination codon, the IRESs were able to initiate translation of the following CAT gene irrespectively of the mRNA luciferase reading frame. Moreover, with all these constructs the highest expression level of the CAT cistron did not exceed 10% of that obtained with the same vector carrying only the CAT cistron. To identify a possible relation between the IRESs and the cap site, the CAT cistron preceded or not with an IRES was introduced 210 nucleotides downstream of the AUG codon of the luciferase gene (i.e., 258 nucleotides from the cap site) and 100 nucleotides after an added UAG termination codon. Expression of the CAT gene was not modified by the addition of the poliovirus IRES but it was strongly stimulated by the SUR IRES (the level of expression corresponded to 65% of that obtained with the same vector carrying only the CAT cistron). These results suggest that there is a cooperation between the cap and the SUR IRES and not the poliovirus IRES to stimulate translation. These data indicate that IRESs must be introduced in precise position to allow an efficient expression of the second cistron in bicistronic mRNAs.
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PMID:Effect of intercistronic length on internal ribosome entry site (IRES) efficiency in bicistronic mRNA. 1094 79


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