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)

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.
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PMID:Calcium phosphate transfection and cell-specific expression of heterologous genes in primary fetal rat hepatocytes. 867 28

The suitability of different purification methods for preparation of plasmid DNA for transfection into eukaryotic cells was systematically investigated. The reporter plasmid, pRSVcat, was prepared using several methods, and residual impurities in the preparations were quantitated. Transfection with these preparations was performed with several cell lines (HeLa, Huh7, COS7 and LMH) and two transfection methods: liposome-mediated and calcium phosphate transfection. Transfection efficiencies were determined by measuring chloramphenicol acetyltransferase expression. Higher transfection efficiencies were obtained with plasmid preparations of higher purity (those prepared by anion-exchange chromatography or two rounds of CsCl-gradient centrifugation) than with preparations of lower purity (those prepared using a silica-based DNA adsorption method or a single round of CsCl centrifugation). The results also demonstrated specifically that increasing concentrations of lipopolysaccharides in plasmid preparations directly correlate with decreasing transfection efficiencies.
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PMID:Short technical reports. Effects of lipopolysaccharide on transfection efficiency in eukaryotic cells. 874 59

Lipofection using the Lipofectin reagent was optimized to transiently transfect subcultured guinea pig endometrial stromal cells with a beta-galactosidase gene driven by a simian virus 40 promoter. Efficient transfection was obtained in the following conditions: a value of six for the ratio of lipofectin to DNA, a low cellular density (10(5) cells per 35-mm well) at the time of subculture (48 h before lipofection) and a lipofection duration of 12 hours. Lipofection was compared to calcium phosphate precipitation previously optimized in the same culture model. At a low cellular density, the lipofection method was found to be more efficient than the calcium phosphate precipitation. This result gives a great relevance to lipofection since the cultured cells available in an experiment are often limited. Then, using cells at low density and a plasmid containing the chloramphenicol acetyltransferase (cat) gene linked to an estrogen response element, it was shown that the lipofection procedure is a suitable tool for the evaluation of gene regulation by estrogen.
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PMID:Gene transfer into subcultured endometrial cells using lipofection. 877 Apr 11

A new method for coupling proteins to plasmid expression vectors is presented. Biotin was covalently attached to a plasmid expression vector containing a chloramphenicol acetyltransferase (CAT) gene. The specific label was one biotin per 100 bp. An electrophoretic mobility shift assay showed that the plasmid was capable of binding multiple streptavidin molecules. When transfected into mouse fibroblasts, the biotinylated plasmid retained 40% of the native plasmid's biological activity, as determined by CAT assay, and was not affected by the binding of streptavidin. The method allows for attachment of any protein to plasmid DNA expression vector while retaining biological function. Hybrid plasmids in which the transcription cassettes were kept free of biotin label were constructed by digesting biotinylated and unbiotinylated plasmids at sites outside the transcription cassette and re-ligating the digestion products. Electron microscopy studies show that the ligation products formed large tangled assemblages of plasmid DNA. When equimolar (with respect to gene number) amounts of these large hybrid biotinylated plasmids were transfected into mouse fibroblasts by means of calcium phosphate precipitation, an increase in CAT expression 25-fold greater than that of original biotinylated plasmid was observed. Slot-blot analysis of total DNA extracted from transfected cells shows that this enhanced activity was not due to increased transfection efficiency. Receptor-mediated delivery could not be shown when a complex comprising biotinylated asialoglycoprotein/streptavidin/biotinylated CAT expression vector was placed in media containing Hep G2 cells.
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PMID:Novel biotinylated plasmid expression vectors retain biological function and can bind streptavidin. 888 15

The genomic action of calcitriol is mediated through the interaction of the calcitriol receptor (VDR) with vitamin D response elements (VDREs) of the target genes. We have shown that the interaction of VDRs with VDREs is inhibited by uremic toxins. We hypothesize that uremic toxins form Schiff bases with the lysine residues of the VDR DNA binding domain and inhibit the VDR interaction with the VDRE. In this study, pyridoxal 5'-phosphate was used as a probe to test Schiff base formation as the inhibitory mechanism, since it forms Schiff bases with steroid receptors. Pyridoxal 5'-phosphate inhibited the VDR binding to the VDREs and chemically modified the DNA binding domain of the VDR in vitro. The inhibition was reversed when pyridoxal 5'-phosphate was preincubated with lysine. Further, this chemical agent also blocked the production of chloramphenicol acetyltransferase (CAT) enzyme induced by calcitriol in cells transfected with a constructed VDRE attached to a CAT reporter gene. This finding is consistent with the hypothesis that pyridoxal 5'-phosphate could interact with the VDR and impair its DNA binding within cells. Since induction of 24-hydroxylase synthesis is a receptor mediated process, we studied the effect of pyridoxal 5'-phosphate on the synthesis of renal 24-hydroxylase in rats. When pyridoxal 5'-phosphate was infused to rats, renal 24-hydroxylase activity was suppressed, consequently, degradation of calcitriol was also reduced in these animals. Thus, chemicals capable of Schiff base formation potentially could alter the physiological function of VDR and calcitriol.
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PMID:Effect of Schiff base formation on the function of the calcitriol receptor. 891 20

The promoter activity of an Acanthamoeba polyubiquitin gene was analyzed in its homologous system. A modified calcium phosphate transfection method using a neomycin marker vector was developed to achieve highly efficient transfection of the Acanthamoeba polyubiquitin gene into Acanthamoeba cells. In this transfection procedure, the calcium phosphate-DNA complex was formed gradually in the medium during incubation with cells and precipitated on the cells. The crucial factors for obtaining efficient transfection were the pH (6.95) of the transfection buffer used for the calcium phosphate precipitation and the amount (25 micrograms/96-well tissue culture plate) and form (circular) of transfecting DNA. Under these conditions, Acanthamoeba isolate 1B6 was transfected at an efficiency of about 40% with the constructed vector pOPSBU, a pOP13CAT-based polyubiquitin gene incorporated neomycin resistance vector. Acanthamoeba polyphaga was transfected at an efficiency of about 10% with this vector. Transfection of both Acanthamoeba strains appeared to result in low copy plasmid integration (about two copies per cell are suggested). The chloramphenicol acetyltransferase (CAT) assays showed that the promoter of the Acanthamoeba polyubiquitin gene in the constructed vector was especially strong in A. polyphaga, thus the pOPSBU-Acanthamoeba system may be useful for the construction of cDNA expression libraries, as well as for the expression of cloned genes.
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PMID:Stable transfection of Acanthamoeba. 909 Jan 13

The triose phosphate 3-phosphoglycerate phosphate translocator (TPT) is a chloroplast envelope inner membrane protein whose transit peptide has structural properties typical of a mitochondrial presequence. To study the TPT transit peptide in more detail, we constructed two chimeric genes encompassing the TPT transit peptide and either 5 or 23 amino-terminal residues of the mature TPT, both linked to the reporter chloramphenicol acetyltransferase (cat) gene. The precursors were synthesized in vitro and translocated to and processed in purified plant mitochondria. However, this import was not specific since both precursors were also imported into isolated chloroplasts. To extend this analysis in vivo, the chimeric genes were introduced into tobacco by genetic transformation. Analysis of CAT distribution in subcellular fractions of transgenic plants did not confirm the data obtained in vitro. With the construct retaining only 5 residues of the mature TPT, CAT was found in the cytosolic fraction. Extension of the TPT transit peptide to 23 residues of the mature TPT allowed specific import and processing of CAT into chloroplasts. These results indicate that, despite its unusual structure, the TPT transit peptide is able to target a passenger protein specifically into chloroplasts, provided that NH2-terminal residues of the mature TPT are still present. The discrepancy between the in vitro and in vivo data suggests that the translocation machinery is more stringent in the latter case and that sorting of proteins might not be addressed adequately by in vitro experiments.
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PMID:Different in vitro and in vivo targeting properties of the transit peptide of a chloroplast envelope inner membrane protein. 918 51

We have improved the productivity of an Escherichia coli cell-free protein synthesis system. First, creatine phosphate and creatine kinase were used as the energy source regeneration system, and the other components of the reaction mixture were optimized. Second, the E. coli S30 cell extract was condensed by dialysis against a polyethylene glycol solution to increase the rate of synthesis. Third, during the protein synthesis, the reaction mixture was dialyzed against a low-molecular-weight substrate solution to prolong the reaction. Thus, the yield of chloramphenicol acetyltransferase was raised to 6 mg/ml of reaction mixture. Stable-isotope labeling of a protein with 13C/15N-labeled amino acids for NMR spectroscopy was achieved by this method.
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PMID:Cell-free production and stable-isotope labeling of milligram quantities of proteins. 992 95

Cellular nuclease activity is a potential barrier to the successful delivery of foreign genes to mammalian cells. We tested the hypothesis that transfection in the presence of a specific DNase inhibitor can enhance the expression of foreign gene products. We have used DMI-2, a polyketide metabolite of Streptomyces sp. strain 560 to enhance the expression of bacterial chloramphenicol acetyltransferase (CAT) in the human lung adenocarcinoma cell line H441. DMI-2 has been shown previously to inhibit porcine DNase II, an acid pH nuclease contained in the endosomal/lysosomal compartment. Transfection of H441 cells in the presence of 0.1-1 microgram/ml DMI-2 caused: (1) 10-fold enhancement of CAT activity when the bacterial plasmid was complexed with either surfactant protein A-poly-lysine or transferrin-poly-lysine; (2) 1.5- to two-fold enhancement of CAT activity in cells exposed to lipofectin-DNA complexes: (3) no effect on transfection via calcium phosphate co-precipitation. DMI-2 alone showed no inherent transfection activity. In experiments using SP-A-poly-lysine and plasmid containing the beta-galactosidase reporter gene, DMI-2 increased the number of transfected cells. Methanolysis products of DMI-2 did not inhibit DNase II and did not enhance transfection efficiency. Taken together, the data support the hypothesis that nuclease action is a significant barrier to expression of foreign genes and inhibition of specific nucleases may facilitate transfection.
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PMID:Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor. 993 Mar 26

The product of the retinoblastoma susceptibility gene, pRB, is a nuclear phosphoprotein that controls cell growth by binding to and suppressing the activities of transcription factors such as the E2F family. Transactivation activity is inhibited when E2F is bound to hypophosphorylated pRB and released when pRB is phosphorylated by cyclin-dependent kinases (CDKs). To determine which of 16 potential CDK phosphorylation sites regulated the pRB-E2F interaction, mutant pRB proteins produced by site-directed mutagenesis were tested for the ability to suppress E2F-mediated transcription in a reporter chloramphenicol acetyltransferase assay. Surprisingly, no one CDK site regulated the interaction of pRB with E2F when E2F was bound to DNA. Instead, disruption of transcriptional repression resulted from accumulation of phosphate groups on the RB molecule.
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PMID:Cumulative effect of phosphorylation of pRB on regulation of E2F activity. 1020 50


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