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 have developed and characterized a system for the transfer of plasmids encapsulated in large unilamellar vesicles (LUV) into Spiroplasma floricola BNR1 cells. The approach is based on the ability of S. floricola-derived LUV to fuse with S. floricola cells. The fusion was continuously monitored by an assay for lipid mixing based on the dequenching of the fluorescent probe octadecylrhodamine B (R18) that was incorporated into LUV at self-quenching concentrations. The fusion was also evaluated by fluorescence-activated cell sorter measurements and by sucrose density gradient analysis. LUV-cell fusion occurred only in the presence of low concentrations (5%) of polyethylene glycol (polyethylene glycol 8000) and depended on temperature, the LUV/cell ratio, and divalent cations in the incubation medium. Throughout the fusion process, spiroplasma cells remained intact and viable. Under optimal fusion conditions, the plasmid pACYC, encapsulated in LUV by reversed-phase evaporation, was transferred into live S. floricola cells and expressed chloramphenicol acetyltransferase activity. The expression was transient with maximal chloramphenicol acetyltransferase activity observed after 6 h of incubation of the transfected cells.
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PMID:Fusion-mediated transfer of plasmids into Spiroplasma floricola cells. 162 33

Plasmid DNA was transfected into tobacco mesophyll protoplasts by electroporation. Transfection efficiency was estimated, using a transient expression assay based on the measurement of chloramphenicol transacetylase activity or by scoring colonies expressing resistance to paromomycin, an aminoglycoside related to kanamycin. Under conditions of cell survival superior to 50% after electroporation, transient expression signals and transformation efficiencies were found to be proportional. Factors affecting the efficiency of transformation were studied. A clear-cut optimum voltage (250-300 V/cm) was detected. Among various salts tested, potassium chloride was the best electrolyte. No improvement of electroporation efficiency was obtained by a heat-shock (45 degrees C/5 min) treatment prior to electroporation or by the presence of polyethylene glycol in the electroporation medium. The physiological state of plants used as the protoplast source significantly affected the transfection ability of the resulting protoplasts. These results are discussed and compared to previously published procedures.
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PMID:Use of a transient expression assay for the optimization of direct gene transfer into tobacco mesophyll protoplasts by electroporation. 312 Jul 96

The ribonucleocapsids of many plant viruses are extremely stable. The protein coat protects the RNA genome against degradation during the accumulation and spread of progeny virions. Chimeric single-stranded RNA molecules were transcribed in vitro from recombinant plasmids and later encapsidated, in vitro, into ribonucleoprotein particles (pseudoviruses) 60 nanometers long that resembled tobacco mosaic virus. Transcripts encoding an assayable enzyme, chloramphenicol acetyltransferase (CAT), were packaged into pseudovirus particles to assess the utility of this single-stranded RNA delivery system in a wide range of cell types. In all cases, packaged CAT messenger RNA was uncoated and transiently expressed. Significantly higher levels of CAT activity were detected with packaged than with naked CAT messenger RNA after inoculation of plant protoplasts in the presence of polyethylene glycol or abrasive inoculation of intact leaf surfaces. Structural events that lead to the uncoating and expression of CAT messenger RNA showed no cell specificity. This observation may support the view that the comparatively restricted host range of a true plant virus results from events that occur later during the infection cycle.
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PMID:In vivo uncoating and efficient expression of foreign mRNAs packaged in TMV-like particles. 347 50

We have examined the usefulness of a protoplast fusion technique as a tool to transfer cloned genes into hematopoietic cells. Protoplasts carrying cloned plasmids, which would express specific markers when successfully transfected into human cells, were prepared and fused with human leukemic cell line K562 cells using polyethylene glycol as a fusogenic factor. As a result, K562 cells fused with protoplasts containing a plasmid pSV2-cat constructed to code for chloramphenicol acetyltransferase (CAT) expressed CAT activity efficiently. K562 cells were also readily transformed to geneticin-(G418) resistant cells following fusion with protoplasts carrying a plasmid pSV2-neo-SV-gpt, which confers the resistance of mammalian cells to G418 and mycophenolic acid. It was also demonstrated that the plasmid genome was stably integrated into the chromosomal DNA of G418-resistant K562 cells. Our results proved that protoplast fusion could be used to study the specific expression and the biologic activities of cloned genes in human hematopoietic cells.
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PMID:Protoplast-mediated gene transfer into human leukemia (K562) cells. 347 4

A method has been developed to transform plasmid deoxyribonucleic acid into protoplasts of the insect pathogen Bacillus thuringiensis. Protoplasts were formed by treatment of cells with lysozyme. The efficiency of formation of protoplasts was affected by the strain, the media, and the cell density. Deoxyribonucleic acid uptake was induced by polyethylene glycol. Deoxyribonucleic acid from the Staphylococcus aureus plasmid pC194 was used for transformation. Although this plasmid could not be isolated as a stable extrachromosomal element, its chloramphenicol resistance was transferred to the recipient protoplasts. This was confirmed by assay for the enzyme chloramphenicol acetyltransferase, which confers resistance to chloramphenicol. This suggested that pC194 acts as an insertion element in B. thuringiensis.
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PMID:Transformation of Bacillus thuringiensis protoplasts by plasmid deoxyribonucleic acid. 746 65

We modified a cell-free coupled transcription/translation system from Escherichia coli with the T7 phage RNA polymerase, and achieved a productivity as high as 0.4 mg protein/ml reaction mixture. First, we found that the optimal concentrations of phosphoenolpyruvate and poly(ethylene glycol) are interdependent; higher concentrations of the former should be used at higher concentrations of the latter. Second, the use of a condensed 30000 x g cell extract, in place of the conventional one, significantly increased the initial rate of protein synthesis. This phenomenon was demonstrated to be due to a reason other than elimination of inhibitory molecule(s) from the extract. For this system with the condensed extract, the phosphoenolpyruvate and poly(ethylene glycol) concentrations were again co-optimized, resulting in production of chloramphenicol acetyltransferase at a productivity of 0.3 mg/ml. Finally, the productivity was further increased up to 0.4 mg/ml, by supplementation of the pool of amino acids. This improved cell-free protein synthesis system is superior in productivity to any other cell-free systems reported so far, including the continuous-flow cell-free system.
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PMID:A highly efficient cell-free protein synthesis system from Escherichia coli. 877 39

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

Crystals of chloramphenicol acetyltransferase B2, an enzyme encoded by the transposon Tn2424 from Escherichia coli, have been obtained utilizing polyethylene glycol as a precipitant. The enzyme inactivates the antibiotic chloramphenicol and is a member of the xenobiotic acetyltransferase family. Two crystal forms were obtained and complete data sets have been collected at a synchrotron source: form I, which diffracted to 3.2 A, and form II, grown in the presence of NiCl(2), for which crystals of the apoenzyme and of the enzyme-chloramphenicol complex have been obtained. For the form II crystals, complete data sets have been collected at 2.7 and 3.2 A resolution, respectively. The space group of the above two crystal forms is P2(1)3, with unit-cell parameter a = 130 A.
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PMID:Crystallization and preliminary X-ray diffraction analysis of the chloramphenicol acetyltransferase from Tn2424. 1117 80

Alpha interferons (alpha-IFNs) are potent biologically active proteins synthesized and secreted by somatic cells during viral infection. Quantification of alpha-IFN concentrations in biological samples is used for diagnosis. More recently, recombinant IFNs have been used as antiviral, antiproliferative, and immunomodulatory therapeutic agents, and particularly for the treatment of chronic hepatitis C virus infection. For this purpose, IFN has recently been coupled to polyethylene glycol (PEG) to improve the pharmacokinetic properties. The measure of alpha-IFN in biological samples from treated patients could be useful to ensure compliance to therapy and the true IFN activity in relation to viral decay during follow-up. In particular, it could be used to monitor the PEG-IFN concentration in patients treated for hepatitis C virus infection. The most frequently used test is a bioassay based on the antiviral property of the IFN, but the assay is not highly reproducible. Here, we present a reporter test based on MxA promoter activation of chloramphenicol acetyltransferase expression (Mx-CAT). MxA is an antiviral protein induced and tightly regulated by alpha-IFN. The Mx-CAT assay showed good reproducibility of 15% and was suitable to quantify PEG-IFN and numerous other alpha-IFN subtypes as well, despite a differential MxA promoter activation in relation with the subtype. A good correlation was obtained with the reporter assay and a commercial enzyme-linked immunosorbent assay on samples from treated patients. This test could be useful for monitoring IFN therapy of chronically infected hepatitis C virus-infected patients treated with the standard IFN, PEG-IFN, and probably forthcoming recombinant IFNs.
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PMID:Quantification of different human alpha interferon subtypes and pegylated interferon activities by measuring MxA promoter activation. 1612 52

Protoplasts derived from cell suspensions of alfalfa (Medicago sativa L.) responded to treatment with fungal elicitor (FE) by an increase in endogenous chalcone synthase (CHS) activity but were unresponsive to reduced glutathione (GSH). Preexposure of protoplasts to polyethylene glycol and electroporation resulted in strong responsiveness to GSH but little change in responsiveness to FE. Protoplasts from suspension cultures which had been subcultured more than 12 times lost responsiveness to GSH, but not FE, as assessed by measuring expression of a chimeric gene containing a bean CHS promoter linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In protoplasts in which putative cis-acting CHS promoter sequences had been coelectroporated in trans with the intact CHS promoter-CAT construct, the extent of CAT expression depended upon the elicitor used (FE or GSH), the age (number of times subcultured) of the cells from which the protoplasts were isolated, and the nature of the coelectroporated CHS promoter sequence. For example, a region of the CHS promoter from -326 to -141 behaved as a trans-activator when coelectroporated with the CAT construct into unelicited protoplasts isolated from newly initiated cell suspensions, but the same region acted as a trans-silencer in the same protoplasts in the presence of FE. This silencer activity was much reduced in GSH-treated protoplasts. The results suggest that there are differences in the signal transduction pathways for elicitation of CHS transcription by FE and GSH, which involve previously indentified cis-elements in the CHS promoter.
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PMID:Stress Responses in Alfalfa (Medicago sativa L.): VI. Differential Responsiveness of Chalcone Synthase Induction to Fungal Elicitor or Glutathione in Electroporated Protoplasts. 1666 19


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