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)

Derivatives of Myxococcus xanthus FB(t) resistant to chloramphenicol (25 mug/ml) arose spontaneously with a frequency of approximately 10(-7). One of these organisms (FB(t)Cam(1) (r)) was characterized. FB(t)Cam(1) (r) showed a unique type of phenotypic instability. After transfer from medium containing chloramphenicol to medium lacking the drug, resistance was lost after approximately one generation. The loss resulted in a sharp drop in the total number of chloramphenicol-resistant organisms and was not due to segregation of chloramphenicol-susceptible organisms during growth. Cell-free extracts of strain FB(t)Cam(1) (r) converted chloramphenicol to acetyl chloramphenicols in a fashion implicating activity of chloramphenicol acetyltransferase. This activity was lost simultaneously with the loss of chloramphenicol resistance after removal of the drug from cultures. Organisms with a similar phenotype to FB(t)Cam(1) (r) could be produced at high frequencies when strain FB(t) was exposed to low concentrations of chloramphenicol (2 to 5 mug/ml), to 3-acetylchloramphenicol (25 mug/ml), or to 1,3-diacetylchloramphenicol (25 mug/ml). Since strain FB(t) is capable of deacetylating acetyl chloramphenicols, these effects are probably all due to low concentrations of chloramphenicol. In the presence of chloramphenicol, FB(t)Cam(1) (r) produced fruiting bodies and myxospores on fruiting agar; however, glycerol-induced myxospore formation was inhibited. In the absence of the antibiotic, chloramphenicol resistance was maintained by glycerol-induced myxospores.
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PMID:Chloramphenicol resistance in Myxococcus xanthus. 80 62

The oxygen-dependent promoter of the Vitreoscilla hemoglobin (VHb) gene has been shown to be functional in E. coli. Earlier studies established that the promoter is maximally induced under microaerobic conditions and that its activity is also influenced by the cAMP-CAP complex. We demonstrate here that the promoter can be used for regulated, high-level expression of recombinant proteins in two-stage fed-batch fermentations. The promoter is maximally induced at dissolved oxygen levels lower than 5% air saturation. Despite the influence of catabolite repression, glucose and glycerol-containing media give comparable product levels under carbon-limited conditions such as those encountered in typical fed-batch fermentations. The possibility of a third level of control of promoter activity is also indicated. This mode of induction can be repressed by addition of a complex nitrogen source such as yeast extract to the medium. The observed promoter activity can be modulated at least 30-fold over the course of high-cell density fermentations producing either cloned beta-galactosidase or cloned chloramphenicol acetyltransferase (CAT). Densitometer scanning of SDS-polyacrylamide gels revealed that beta-galactosidase was expressed to a level of approximately 10% of total cellular protein.
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PMID:Expression of recombinant proteins in Escherichia coli using an oxygen-responsive promoter. 136 36

The ugp operon of Escherichia coli includes genes involved in the uptake of sn-glycerol-3-phosphate and glycerophosphoryl diesters and belongs to the pho regulon which is induced by phosphate limitation. This operon has two transcriptional initiation sites, as determined by S1 nuclease mapping of the in vivo transcripts. The downstream promoter has multiple copies of the pho box, the consensus sequence shared by the pho promoters; the upstream promoter has a consensus sequence for the promoters regulated by cyclic AMP and its receptor protein, CRP. PhoB protein, which is the transcriptional activator for the pho regulon, protected the regulatory region with the pho boxes in DNase I footprinting experiments and activated transcription from the downstream promoter in vitro. Studies with transcriptional fusions between ugp and a promoterless gene for chloramphenicol acetyltransferase show that the upstream promoter is induced by carbon starvation in a manner that required the cya and crp genes. PhoB protein may act as a repressor for this upstream promoter, which also overlaps the upstream third pho box. The downstream promoter was induced by phosphate starvation and requires the PhoB protein for its activation as do the other pho regulon promoters. These results suggest that the two promoters function alternately in responding to phosphate or carbon starvation, thus providing the cell with a means to adapt to these physiological stresses.
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PMID:Dual regulation of the ugp operon by phosphate and carbon starvation at two interspaced promoters. 198 50

Several properties of a new oxygen-regulated promoter, OXYPRO, were tested in small-scale Escherichia coli cultures. Using OXYPRO, maximal activity of a reporter gene encoding chloramphenicol acetyltransferase (CAT) occurred in cultures that were tightly capped immediately after inoculation. This is probably a result of the reduced oxygen concentration attained in capped cultures, a condition known to be required for OXYPRO induction. CAT levels were significantly higher when the cells were grown in a glycerol-based medium. Similar levels of CAT expression were obtained when OXYPRO was compared to the trp-lac (tac) promoter. In addition, regulated expression of CAT occurred in a wild type strain of E. coli, suggesting that OXYPRO will be useful in most E. coli strains. Thus, OXYPRO provides a simple, inexpensive, and unobtrusive method to achieve high levels of cloned protein expression in most strains of E. coli. OXYPRO is available in a high copy plasmid with a convenient multiple cloning site for the insertion of genes for direct expression in E. coli.
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PMID:A new oxygen-regulated promoter for the expression of proteins in Escherichia coli. 269 65

The influence of cafestol, a lipid component found in boiled coffee, on low density lipoprotein (LDL) and lipid metabolism was investigated in CaCo-2 cells cultured on filter membranes. The rate of uptake and degradation of 125I-labeled tyramine cellobiose-LDL was increased 50% in CaCo-2 cells incubated with cafestol (20 micrograms/ml, 63 microM) for 24 h, whereas in cells incubated with 25-hydroxycholesterol (10 micrograms/ml, 25 microM) the rate of uptake and degradation showed a 30% decrease. A mixture of kahweol and cafestol, both natural components of coffee beans, modestly enhanced the rate of LDL uptake and degradation, as compared to pure cafestol. Incubation of cafestol with CaCo-2 cells induced a 3-fold up-regulation of LDL receptor mRNA, as compared to control cells. In contrast, incubation of the cells with 25-hydroxycholesterol produced a 30% decrease of LDL receptor expression. CaCo-2 cells were transfected with a promoter region containing the sterol regulatory element-1 (SRE-1) coupled to the reporter gene chloramphenicol acetyltransferase (CAT). When cells transfected with SRE-1 promoter were incubated with cafestol, there was a 20% up-regulation of CAT activity, whereas 25-hydroxycholesterol abolished this activity. Cafestol contributed to a significantly lowered secretion of cholesteryl ester and triacylglycerol, regardless of the radiolabeled precursor used ([2-14C]acetic acid, [1,2,3-3H]glycerol, [3H]water, and [1-14C]oleic acid). This reduction in secretion of lipids was accompanied by an increase in trichloroacetic acid-soluble activity when radiolabeled oleic acid was used as a tracer. We conclude that cafestol promotes an enhanced rate of uptake and degradation of LDL, probably due to an increase in transcription of LDL receptor mRNA and a reduced secretion of cholesteryl ester and triacylglycerol in CaCo-2 cells.
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PMID:Effect of a coffee lipid (cafestol) on regulation of lipid metabolism in CaCo-2 cells. 857 35

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 antibiotic-multiresistance IncF plasmid pRSB107 was isolated by a transformation-based approach from activated-sludge bacteria of a wastewater-treatment plant. It confers resistance to ampicillin, penicillin G, chloramphenicol, erythromycin, kanamycin, neomycin, streptomycin, sulfonamides, tetracycline and trimethoprim and against mercuric ions. Complete sequencing of this plasmid revealed that it is 120 592 bp in size and has a G+C content of 53.1 mol%. The plasmid backbone is composed of three replicons, RepFIA, RepFIB and RepFII, which are almost identical to corresponding regions located on the F-plasmid and on R100. The three replicons encode replication initiation (rep) and replication control, multimer resolution (mrs), post-segregational killing of plasmid-free cells (psk) and active plasmid partitioning (sopABC locus). Part of the F-leading region and remnants of the F-homologous DNA-transfer (tra) module complete the pRSB107 backbone. Plasmid pRSB107 contains a complex, highly mosaic 35 991 bp antibiotic-resistance region consisting of a Tn21- and a Tn10-derivative and a chloramphenicol-resistance module. The Tn21 derivative is composed of a mercury-resistance region (mer), a Tn4352B-like kanamycin/neomycin-resistance transposon, a streptomycin/sulfonamide-resistance module, remnants of the beta-lactam-resistance transposon Tn1, a macrolide-resistance module flanked by copies of IS26 and IS6100, remnants of Tn402 integrating a class 1 integron and the Tn21-specific transposition module. A truncated version of the tetracycline-resistance transposon Tn10 and the chloramphenicol acetyltransferase gene catA complete the pRSB107 resistance region. In addition to antibiotic resistance, pRSB107 encodes the following putative virulence-associated functions: (i) an aerobactin iron-acquisition siderophore system (iuc/iut); (ii) a putative high-affinity Fe(2+) uptake system which was previously identified on a pathogenicity island of Yersinia pestis and in the genome of the phytopathogen Erwinia carotovora subsp. atroseptica SCRI1043; (iii) an sn-glycerol-3-phosphate transport system (ugp); and (iv) the virulence-associated genes vagCD having a possible function in stable plasmid inheritance. All the accessory modules are framed by insertion sequences, indicating that pRSB107 was gradually assembled by integration of different horizontally acquired DNA segments via transposition or homologous recombination.
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PMID:The 120 592 bp IncF plasmid pRSB107 isolated from a sewage-treatment plant encodes nine different antibiotic-resistance determinants, two iron-acquisition systems and other putative virulence-associated functions. 1581 78

It has been suggested that the common (betaalpha)(8)-barrel enzyme fold has evolved by the duplication and fusion of identical (betaalpha)(4)-half barrels, followed by the optimisation of their interface. In our attempts to reconstruct these events in vitro we have previously linked in tandem two copies of the C-terminal half barrel HisF-C of imidazole glycerol phosphate synthase from Thermotoga maritima and subsequently reconstituted in the fusion construct HisF-CC a salt bridge cluster present in wild-type HisF. The resulting recombinant protein HisF-C*C, which was produced in an insoluble form and unfolded with low cooperativity at moderate urea concentrations has now been stabilised and solubilised by a combination of random mutagenesis and selection in vivo. For this purpose, Escherichia coli cells were transformed with a plasmid-based gene library encoding HisF-C*C variants fused to chloramphenicol acetyltransferase (CAT). Stable and soluble variants were identified by the survival of host cells on solid medium containing high concentrations of the antibiotic. The selected HisF-C*C proteins, which were characterised in vitro in the absence of CAT, contained eight different amino acid substitutions. One of the exchanges (Y143C) stabilised HisF-C*C by the formation of an intermolecular disulfide bond. Three of the substitutions (G245R, V248M, L250Q) were located in the long loop connecting the two HisF-C copies, whose subsequent truncation from 13 to 5 residues yielded the stabilised variant HisF-C*C Delta. From the remaining substitutions, Y143H and V234M were most beneficial, and molecular dynamics simulations suggest that they strengthen the interactions between the half barrels by establishing a hydrogen-bonding network and an extensive hydrophobic cluster, respectively. By combining the loop deletion of HisF-C*C Delta with the Y143H and V234M substitutions, the variant HisF-C**C was generated. Recombinant HisF-C**C is produced in soluble form, forms a pure monomer with its tryptophan residues shielded from solvent and unfolds with similar cooperativity as HisF. Our results show that, starting from two identical and fused half barrels, few amino acid exchanges are sufficient to generate a highly stable and compact (betaalpha)(8)-barrel protein with wild-type like structural properties.
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PMID:Stabilisation of a (betaalpha)8-barrel protein designed from identical half barrels. 1763 94