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

---

Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to study transcriptional regulation of hepatic genes during development, a method for transfer of fusion genes to primary cultures of fetal hepatocytes was required. The aim of this study was to assess currently available transfection methods and optimize the best method for use with cultured fetal hepatocytes. The Rous sarcoma virus 5' long terminal repeat controlling transcription of the beta-galactosidase reporter gene (pRSV lac Z II) was used to assess electroporation, lipofection, DEAE-dextran and calcium phosphate transfection in cultured primary fetal hepatocytes. The success of transfection was determined by histochemical detection and quantitation of beta-galactosidase activity. Results showed that calcium phosphate transfection was optimal for fetal hepatocytes with respect to beta-galactosidase activity and cell survival. For maximum transfection of cells, 10 micrograms/ml DNA, HEPES buffered saline transfection buffer at pH 7.05 and a 24 hr expression period for the reporter gene were employed. Glycerol shock did not increase transfection efficiency significantly. The method was simplified by adding calcium chloride solution to DNA diluted in transfection buffer and the resulting co-precipitate added directly to the medium covering the cells. Transfection 24 hr after initial culture and a precipitate incubation time of 20 hr were optimal. The suitability of this method was confirmed with a liver-specific promoter controlling beta-galactosidase and chloramphenicol acetyltransferase expression. In conclusion this study shows that a modified calcium phosphate transfection method is most effective for transferring DNA to primary cultured fetal hepatocytes. It is concluded that this method is appropriate for use with fetal hepatocytes and will facilitate studies of gene regulation during liver development.
...
PMID:Calcium phosphate transfection and cell-specific expression of heterologous genes in primary fetal rat hepatocytes. 867 28

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.
...
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

We constructed plasmids expressing mRNA7 coding the nucleocapsid (N) protein of JHM strain of MHV (JHMV) under the control of Rous sarcoma virus (RSV) LTR or human elongation factor (EF) 1a promoter, referred to as pRSV-mRNA7 and pEF-mRNA7, respectively. Although only a slight level of cytolysis was observed by the spleen cells from C57BL/6 mice injected intramuscularly with pEF-mRNA7, the spleen cells from the mice administered with pRSV-mRNA7 showed a significant level of cytolytic activities against the cells expressing the viral N protein. The difference in the level of specific cytolysis might have been mainly due to a difference in the expression levels of the N protein in the muscles between the mice injected with pEF-mRNA7 and pRSV-mRNA7, since the specific activity of chloramphenicol acetyltransferase (CAT) in muscles from the mice injected with plasmid DNA expressing CAT gene directed by RSV LTR was significantly higher than that in those administered by the plasmid DNA directed by EF-1a promoter.
...
PMID:Cytolytic activity induced by intramuscular injection of plasmid DNA expressing the nucleocapsid protein of the JHM strain of mouse hepatitis virus into C57BL/6 mice. 887 70

Previously, our laboratory showed that human cytomegalovirus (HCMV) activates human immunodeficiency virus type 1 (HIV-1) in brain-derived cells with limited HIV-1 gene expression but inhibits HIV-1 in cells fully permissive for replication of both viruses (F. M. Jault, S. A. Spector, and D. H. Spector, J. Virol. 68:959-973, 1994). To investigate these effects further, we developed a model system that uncouples HIV-1 gene expression from long terminal repeat (LTR) activity. Two monoclonal U373-MG astrocytoma/glioblastoma cell lines (LTRIG and LIGHIVDC) were generated, each containing an integrated copy of an LTR-chloramphenicol acetyltransferase (CAT) construct and the Escherichia coli lacI gene. LIGHIVDC also has an inducible HIV-1 genome controlled by a Rous sarcoma virus promoter with lac operator sequences. Basal LTR-mediated CAT activity is 65-fold higher in LIGHIVDC than in LTRIG, and this activity is further increased (20-fold) following incubation of LIGHIVDC with isopropyl-beta-D-thiogalactopyranoside (IPTG). Tat protein can be detected by immunostaining in LIGHIVDC. However, Rev-mediated transport and subsequent translation of the singly spliced and unspliced HIV-1 mRNAs is inefficient. In the absence of Tat, HCMV stimulated CAT activity approximately 20-fold, and this activation required HCMV gene expression but not viral DNA replication. LTR-directed transcription was unaffected by HCMV infection in LIGHIVDC but was inhibited in these cells when they contained increased Tat levels following IPTG induction. These results support the hypothesis that HCMV can induce the HIV-1 LTR when HIV-1 gene expression is minimal and that a threshold level of HIV-1 gene products is necessary for HCMV to inhibit this promoter.
...
PMID:A model system for human cytomegalovirus-mediated modulation of human immunodeficiency virus type 1 long terminal repeat activity in brain cells. 909 43

Intramuscular injection of plasmid DNA is an efficient method to introduce a foreign gene into a live animal. We investigated several factors affecting the gene transfer efficiency and the following immune response by intramuscular injection of plasmid DNA. When the strength of several highly efficient viral promoters was compared in muscle by using the chloramphenicol acetyltransferase (CAT) gene as an indicator, cytomegalovirus (CMV) immediate early promoter was found to be stronger than any other viral promoters including Rous sarcoma virus (RSV), murine leukemia virus (SL3-3) and simian virus 40 (SV40) early promoters. Inclusion of adenovirus tripartite leader (TPL) sequences and a synthetic intron in the 5' untranslated region of mRNA moderately stimulated the CAT expression. On the other hand, the expression of encephalomyocarditis virus (EMCV) VP1 gene was greatly enhanced by the TPL sequences and an intron. The level of humoral immune response by intramuscular injection of various VP1 expression plasmids was compared. The seroconversion rate was highly dependent on the strength of the expression vector. However, the ratio of IgG1 and IgG2a immune response was not significantly variable depending on the strength of the expression vector. Also, the efficiency of the sindbis virus-based DNA vector was examined for the gene expression and immune response. Although a high level of CAT expression was obtained in muscle by using this system, VP1 was not produced as much as the conventional expression vectors. Furthermore, little humoral immune response was elicited by intramuscular injection of VP1-expressing sindbis vector, suggesting that this system was not superior to the conventional vector for DNA immunization.
...
PMID:Comparison of various expression plasmids for the induction of immune response by DNA immunization. 933 93

In order to synthesize human erythropoietin protein in the oviduct of laying hens, localized in vivo gene transfer was attempted by using electroporation. In Experiment 1, transcriptional activities were compared by using four viral and cellular promoters, i.e., the 1.35-kbp long ovalbumin promoter, SV40 early promoter, Rous sarcoma virus long terminal repeat (RSV LTR), and the miw promoter, which is a hybrid of RSV LTR and chicken beta-actin promoter. These promoters were fused immediately upstream to the chloramphenicol acetyltransferase reporter gene. The results of chloramphenicol acetyltransferase activity showed that the miw promoter was the strongest, followed by SV40, RSV LTR, and the ovalbumin promoter in decreasing order. The intensity of the miw promoter was 250 times as high as that of the ovalbumin promoter. In Experiment 2, plasmid DNA containing the human erythropoietin gene, driven either by the ovalbumin promoter or the miw promoter, was transfected in vivo, and the production of human erythropoietin protein was detected by ELISA. The results indicated that the synthesis of human erythropoietin protein was attained in the chicken oviduct, and its concentration was higher when driven by the miw promoter than the ovalbumin promoter.
...
PMID:Synthesis of human erythropoietin in vivo in the oviduct of laying hens by localized in vivo gene transfer using electroporation. 949 97

For many in vivo gene therapy clinical applications, it is desirable to control the expression of the transferred transgene using pharmacologic agents. To evaluate the feasibility of accomplishing this using corticosteroids, pharmacologic agents widely used in clinical medicine, we constructed replication deficient adenoviral (Ad) vectors containing an expression cassette with a chimeric promoter comprised of five glucocorticoid response elements (GRE) and the chloramphenicol acetyltransferase reporter gene (AdGRE.CAT) or the murine thrombopoietin cDNA (AdGRE.mTPO). In vitro studies showed the vectors functioned as expected, with marked glucocorticoid-induced upregulation of the CAT or mTPO transgenes. To evaluate the inducibility of the GRE promoter in vivo, the AdGRE. CAT vector was administered intravenously to C57B1/6 mice, and CAT activity was quantified in liver before and after intraperitoneal administration of dexamethasone. The GRE promoter activity was dependent on the dexamethasone dose, with a 100-fold increase in CAT expression with 50 microg dexamethasone, similar to the levels observed in vivo with the Rous sarcoma virus long terminal repeat constitutive promoter. After dexamethasone administration, maximum CAT activity was observed at day 2, with a slow decline to baseline levels by 2 weeks. Based on these observations, we hypothesized that a single administration of an Ad vector-mediated transfer of the chimeric GRE inducible promoter driving the mTPO cDNA would enable repetitive administration of corticosteroids to repetitively upregulate platelet levels for 1 to 2 weeks. The data show that this occurs, with dexamethasone administration every 3 weeks associated with 1-week elevations (at each 3-week interval) of serum mTPO levels, megakaryocyte numbers in bone marrow, and platelet levels fourfold to sixfold over baseline. Thus, with the appropriate promoter, it is possible to use a commonly used pharmacologic agent to upregulate the expression of a newly transferred gene on demand.
...
PMID:Intermittent, repetitive corticosteroid-induced upregulation of platelet levels after adenovirus-mediated transfer to the liver of a chimeric glucocorticoid-responsive promoter controlling the thrombopoietin cDNA. 968 Mar 50

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.
...
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

Production of the glycoprotein hormone alpha-subunit (GPH alpha) was enhanced by sodium butyrate (Btr) in HeLa cells. Induction of the HeLa alpha-subunit gene by Btr was inhibited by the simultaneous addition of cycloheximide (CHX), indicating a requirement for continued protein synthesis. Transient expression assays using plasmids containing the GPH alpha gene promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene demonstrated that the GPH alpha promoter is inducible by Btr in HeLa cells, and this induction could be prevented by 2-deoxyglucose (dGlc). CAT production driven by the SV-40 early promoter, the cytochrome P-450-IA1 promoter, and the Rous sarcoma virus long terminal repeat was also enhanced by Btr, but the augmented synthesis was not inhibited by the addition of dGlc, demonstrating that the effect is restricted only to some promoters. CAT synthesis could be induced by Btr when the GPH alpha promoter extended upstream to position -169 (relative to the transcription start site at +1) but not when the promoter terminated at -150, classifying the DNA between these termini as a Btr-responsive element (BRE). This region overlaps the composite trophoblast-specific enhancer. Inactivation of enhancer subdomains by site-directed mutagenesis confirmed the deletion analysis and ranked their response to Btr as CRE < TSE < URE < alpha ACT. Electrophoretic mobility shift analysis failed to detect any significant difference among several enhancer binding proteins in nuclear extracts from untreated and Btr-treated cells. Together, these results suggest that Btr-mediated induction of the alpha-subunit gene in HeLa cells is manifest either through the synthesis of a new transcription factor(s), which is inhibited by CHX but required for increased transcription from the GPH alpha gene promoter, or through the activity of existing factors that may require glycosylation or phosphorylation by a modification system that is inducible by Btr and inhibited by dGlc and CHX. These results further suggest that the factor is not an enhancer-binding protein or that Btr increases its transactivation potential without altering its DNA-binding activity.
...
PMID:Sodium butyrate-mediated induction of the glycoprotein hormone alpha-subunit gene: requirement for continued protein synthesis, identification of a butyrate-responsive element, and inhibition of promoter activation by 2-deoxyglucose. 1040 94

In order to study the transactivational property of the X gene in the methylotropic yeast Pichia pastoris, a Rous sarcoma virus-chloramphenicol acetyltransferase (RSV-CAT) cassette was co-transformed and integrated into the host yeast strain as a reporter which showed an overwhelming CAT activity. Immunoprecipitation of the yeast cell extracts with an X-specific monoclonal antibody, however, showed a low level expression of the X gene. Therefore besides a trans-effect of the X protein, the enhanced reporter activity could be a manifestation of a cis-effect of the X gene sequences also. Therefore, unlike the transactivation studies with X gene in animal cells where limited functional activity is observed, P. pastoris appears to be an excellent system to study cis- and trans-aspects of gene regulation by the X gene.
...
PMID:The X gene of hepatitis B virus shows a high level stimulation of the Rous sarcoma virus long terminal repeat in the methylotropic yeast, Pichia pastoris. 1045 40


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---