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 chloramphenicol resistance of Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis isolated from clinical materials was proved to be due to an inactivating enzyme produced by these bacteria. The inactivated products of chloramphenicol were identified as 1-acetoxy, 3-acetoxy and 1,3-diacetoxy derivatives by thin-layer chromatography and infrared spectroscopy. The responsible enzyme was thus confirmed to be chloramphenicol acetyltransferase. The enzyme was inducible. It was partially purified by ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. The enzymes obtained from S. haemolyticus, S. pneumoniae and S. faecalis have been compared with the conclusion that they are identical with respect to molecular weight (approximately 75,000-80,000), optimum pH and heat stability.
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PMID:Resistance mechanism of chloramphenicol in Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis. 1 97

We have expressed in the yeast Saccharomyces cerevisiae a full-length poliovirus cDNA clone under the control of the GAL10 promoter to better characterize the effect of poliovirus on host cell metabolism. We find that yeast cells are unable to translate poliovirus RNA in vivo and that this inhibition is mediated through the 5' untranslated region of the viral RNA. The in vivo inhibition of translation of poliovirus RNA and P2CAT RNA (which contains the 5' untranslated region fused upstream of the bacterial chloramphenicol transferase gene) can be mimicked in vitro in yeast translation lysates. In fact, a trans-acting inhibitor present in yeast lysates can inhibit translation of either poliovirus or P2CAT RNA in HeLa cell translation lysates. In contrast, when the inhibitor is added to translations programmed with chloramphenicol acetyltransferase RNA, yeast prepro-alpha-factor RNA, or an RNA containing the internal ribosome entry site of encephalomyocarditis virus, no inhibition is seen. The inhibitory activity has been partially purified by DEAE-Sephacel chromatography. The partially purified inhibitor is heat stable, escapes phenol extraction, is resistant to proteinase K and DNase I treatment, and is sensitive to RNase A digestion, suggesting that the inhibitor is an RNA. In an in vitro translation assay, the inhibitory activity can be overcome by increasing the concentration of HeLa cell lysate but not P2CAT RNA, suggesting that the inhibitor interacts (directly or indirectly) with one or more components of the HeLa cell translational machinery rather than with the viral RNA.
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PMID:Yeast cells are incapable of translating RNAs containing the poliovirus 5' untranslated region: evidence for a translational inhibitor. 130 48

The conditions and efficacy of transfection of vascular cells in primary culture using DEAE-dextran, calcium phosphate and lipofectin have been investigated using chloramphenicol acetyltransferase and luciferase as reporter genes. Subsequently factor VIII was expressed in endothelial and smooth muscle cells. Both reporter genes could be expressed after transfection of umbilical vein endothelial cells, umbilical artery smooth muscle cells and fibroblasts. The expression of both reporter genes in endothelial and smooth muscle cells was highest using lipofectin. After transfection of smooth muscle cells with both full-length and mutant factor VIII genes, factor VIII activity and antigen were secreted into the culture medium, the secretion remaining stable to serial cell passage. The secretion of factor VIII from transfected smooth muscle cells was confirmed by the immunoprecipitation of [35S]methionine labelled protein. Endothelial cells also were successfully transfected with the mutant factor VIII gene.
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PMID:The uptake and expression of the factor VIII and reporter genes by vascular cells. 160 16

A method for measuring nucleotide excision repair in response to UV irradiation and chemical-induced DNA damage has been developed, validated, and field tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiological studies seeking to investigate associations between DNA repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the suggestion that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum is manifested as a result of the reduced capacity of patients' cells to repair DNA damaged by UV-mimetic agents. For the assay, damaged, nonreplicating, recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (cat) reporter gene is introduced into lymphocytes by using a DEAE-dextran/DNA complex short-term transfection conditions. Excision repair of the damaged bacterial cat gene is monitored proportionately as a function of reactivated CAT enzyme activity following a 40-h repair/expression incubation period. The validity of the approach was indicated by the ability of the assay to discriminate xeroderma pigmentosum virus-transformed lymphocyte cell lines of both severe (complementation groups A and D) and moderate (complementation group C) excision repair deficiencies from repair-proficient cell lines. Similar results were observed when a mitogen-stimulated peripheral blood lymphocyte culture from an xeroderma pigmentosum A patient was assayed concurrently with mitogen-stimulated peripheral blood lymphocytes obtained from healthy individuals. Adaptation of this DNA repair assay as a field test in a pilot-tested select group of basal cell carcinoma patients and cancer-free controls led to the preliminary identification of a specific subset at risk for this disease as a consequence of significant reduction to the repair of photochemically (UV)-damaged plasmid DNA.
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PMID:Development and field-test validation of an assay for DNA repair in circulating human lymphocytes. 193 49

We describe a method for studying transient gene expression in primary avian erythroid cells that involves controlled osmotic shock, followed by DNA transfection using DEAE-dextran. Cells treated in this way reproducibly express high levels of chloramphenicol acetyltransferase (CAT) when transfected with a plasmid having the cat gene coupled to an appropriate viral promoter. An observed correlation between levels of CAT expression and extent of hemoglobin release during controlled shock makes it possible to choose optimum conditions for expression in erythroid cells at various stages of embryonic development. Using these techniques, we have investigated the effect on CAT expression of fusing to the cat gene various portions of the chicken adult beta-globin (beta A) gene. We show that in 9-day or 12-day embryonic erythrocytes, the promoter activity of the 5' flanking region of the beta A gene (in the absence of any viral promoters) is strongly stimulated by a downstream sequence, located in the region 110-588 base pairs on the 3' side of the poly(A) signal, that acts as an enhancer. Its activity is reduced in 5-day embryonic cells and absent in primary chicken fibroblasts and mouse L cells, suggesting that this transient expression system will be useful in studying developmentally regulated globin gene expression.
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PMID:Regulated gene expression in transfected primary chicken erythrocytes. 345 75

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.
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PMID:Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes. 346 23

We report the purification of chloramphenicol acetyltransferase (acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) by a two-step procecdure involving chromatography on a Sepharose 4B-reduced chloramphenicol matrix and DEAE-Sephadex A-50. This procedure resulted in a 120-fold purification with 50% recovery of the enzyme. Only one band of enzyme activity was present after electrophoresis on polyacrylamide gel. The enzyme is active over a broad pH range, maximal activity being observed near pH 7.6. Both chloramphenicol 1-acetate of chloramphenicol 3-acetate were found to be very stable in Tris-maleate buffer at pH 6.09 with negligible interconversion. The incubation at pH 6.0 of chloramphenicol 1-acetate with the purified chloramphenicol acetyltransferase yielded chloramphenicol 1,3-diacetate. These data indicate that the enzyme acetylates specifically at the 3-hydroxy position and the diacetylation is possible only because of non-enzymatic interconversion of chloramphenicol 3-acetate to chloramphenicol 1-acetate at higher pH values.
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PMID:A study of the enzymatic inactivation of chloramphenicol by highly purified chloramphenicol acetyltransferase. 699 33

Tissue factor (TF) is a cellular receptor and cofactor for factor VII/VIIa which initiates the blood coagulation cascade. We have investigated the role of 5'-flanking DNA sequences in regulating the expression of the human TF gene in human umbilical vein endothelial cells (HUVEC). Using a chloramphenicol acetyltransferase (CAT) reporter gene, we attempted to transfect primary cultured HUVEC (passage 3-4) with calcium phosphate coprecipitation, DEAE Dextran, lipopolyamine-coated DNA or electroporation. Electroporation in HEPES-buffered saline of 1 x 10(7) cells at 200V and 250 microF was found to be optimal. Using these conditions, varying lengths of TF 5'-flanking sequences coupled to the CAT reporter gene were tested in transient expression studies. CAT expression corrected for variation in transfection efficiency and cell viability revealed that the sequences between -111 and +14 base pairs are essential for minimal transcriptional activity. This region contains consensus sequences for a TATA box and three Sp1 binding sites. A domain from -382 to -111bp, which contains two AP-1 consensus elements, promoted high levels of gene expression. This transcriptional activity was repressed by 50% with constructs containing sequences between -550 and -382 bp. A further 2-fold drop in transcription activity was attributed to the region between -948 and -550 bp. These results suggest that the basal transcription of the human TF gene in HUVEC is mediated through at least two negative regulatory elements upstream of the proximal promoter domain. The proximal promoter region which contains two AP-1 sites is essential for efficient transcription.
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PMID:Efficient gene transfer into human umbilical vein endothelial cells allows functional analysis of the human tissue factor gene promoter. 780 34

NF-R2 is a DNA-binding protein that interacts with the MDR1 gene proximal promoter sequence. We previously reported that NF-R2 binds within the promoter's -126 and -102 regions, which contain the ATTCAGTCA motif. In the present study, we have purified NF-R2 from the nuclear extract of K562/ADM cells, a multidrug-resistant cell line derived from human myelogenous leukemia K562 cells, using sequential chromatography on Sephacryl S-300, DEAE-Sepharose, heparin-Sepharose and a DNA affinity column consisting of a repetitive synthetic ATTCAGTCA motif coupled to Sepharose. NF-R2 runs as a single protein of 75 kDa on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). CAT (chloramphenicol acetyltransferase) expression assay and gel mobility shift competition assay with mutated promoters revealed that the ATTCAGTCA motif is a positive regulatory element of MDR1 gene and that the motif is important for NF-R2 binding. These results suggest that NF-R2 may be involved in the positive regulation of the MDR1 gene transcription.
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PMID:Purification and characterization of NF-R2 that regulates the expression of the human multidrug resistance (MDR1) gene. 809 26

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


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