Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

High levels of nonfused chloramphenicol acetyltransferase, beta-galactosidase, and beta-glucuronidase expressed under the control of new vector constructs of the polyhedrin promoter in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus were investigated by SDS-PAGE and RNA dot blot analysis of total cytoplasmic RNA. When the polyhedrin ATG start codon was converted to ATT by site-directed mutagenesis, translation initiated at downstream ATG codons resulting in high yields of nonfused foreign proteins. When a stop codon was inserted downstream from and in phase with the polyhedrin ATG codon but upstream from the ATG of a foreign gene, nonfused proteins were also produced, but at lower levels. The level of steady-state polyhedrin gene-promoted mRNA was not affected by the mutation from ATG to ATT or the insertion of in phase stop codons downstream from the polyhedrin ATG.
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PMID:High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors. 249 80

The transcriptional and translational signals required for efficient expression of the chloramphenicol acetyltransferase, beta-galactosidase, and tissue plasminogen activator genes, under the control of the polyhedrin promoter in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus, were investigated by SDS-PAGE and RNA dot blot analysis. The recombinant baculoviruses all contained alterations in the leader sequence or 5' proximal coding region of the polyhedrin gene. Highest levels of foreign proteins and polyhedrin-linked mRNAs were observed when portions of the coding sequence of the polyhedrin gene were fused in phase with the foreign gene. Recombinant viruses in which the foreign gene was inserted upstream from the polyhedrin ATG start codon expressed nonfused products but at lower levels than contructs which produced fusion proteins. A corresponding decrease in the levels of mRNAs produced by such constructs was also observed. Some constructs in which the foreign gene was inserted out of phase downstream from the polyhedrin start codon expressed nonfused protein products at low levels but produced polyhedrin-linked mRNA at levels comparable to vectors which produced protein fusions. These data suggest that reinitiation of translation can take place at AUG start codons a short distance downstream from the primary polyhedrin start codon. These results indicate that sequences immediately upstream from the polyhedrin start codon are important for regulation of transcription and that additional sequences near the AUG start codon can have a dramatic influence on the levels of translation observed.
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PMID:Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors. 314 47

Vibrio anguillarum strains were isolated from chloramphenicol-resistant bacteria in diseased fish. Plasmid Rms418, which confers chloramphenicol resistance, was transferred from V. anguillarum GN11379 to Escherichia coli K12 by conjugation. The Rms418-encoded chloramphenicol acetyltransferase (CAT) [EC 2.3.1.99] was isolated and purified to homogeneity using affinity chromatography on immobilized p-amino-chloramphenicol or ATP. The general CAT could be adsorbed by a matrix with a chloramphenicol base ligand (Zaidenzaig, Y. & Shaw, W.V. (1976) FEBS Lett. 62,266-271), but the Rms418-encoded CAT was not bound under these conditions. The specific activity of the enzyme, when measured by the spectrophotometric assay, was 71.4 units/mg protein at 37 degrees C. The molecular weight of the enzyme treated with SDS and 2-mercaptoethanol was shown to be approximately 22,000. The molecular weight of the native enzyme, as determined by gel filtration, was approximately 69,000, and the optimal pH was 7.8. The Km values for chloramphenicol and CoASAc were 34.5 and 150 microM, respectively. Enzyme activity was inhibited by HgCl2, p-chloromercuribenzoate (p-CMB), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and ethylendiaminotetraacetic acid (EDTA). The half life at 53 degrees C was approximately 100 min.
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PMID:Purification and some properties of a chloramphenicol acetyltransferase mediated by plasmids from Vibrio anguillarum. 314 69

The primary biological effect of the estrogen estradiol-17 beta (17 beta E2) on bone is to decrease bone resorption. However, whether 17 beta E2 affects osteoclast differentiation or function directly or through its action on osteoblasts is unclear. To investigate this question we examined the human preosteoclastic cell line FLG 29.1 for evidence of functional estrogen receptors (ERs). Southern blotting of reverse transcription-PCR amplification products with a 32P-labeled cDNA probe for the human ER mRNA demonstrated that FLG 29.1 cells express ER mRNA. Binding of [3H]17 beta E2 to nuclear ERs was steroid specific with approximately 400 saturable, high affinity (Kd approximately 1 nM) binding sites per cell nucleus. Nuclear ERs covalently labeled with [3H]tamoxifen aziridine showed an apparent molecular weight of 65,000 by SDS/PAGE and Western blotting with the D75 monoclonal antibody to human ER. Pretreatment of cells with 0.1, 1.0, or 10 nM 17 beta E2 induced a dose- and time-dependent specific binding of progesterone to FGL 29.1 cells, and stimulation of the cells with 10 nM and 100 nM 17 beta E2 significantly (P < 0.05) reduced cell proliferation. Transcriptional activity of the ER gene was detected by transient transfection of cells with the pERE-BLCAT plasmid containing the estrogen response element for the vitellogenin A2 gene and the bacterial chloramphenicol acetyltransferase reporter gene. Treatment of FLG 29.1 cells with 10 nM 17 beta E2 increased chloroamphenicol acetyltransferase expression from 5- to 29-fold compared to controls. These observations suggest a potential role for estrogen in osteoclastogenesis.
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PMID:Functional estrogen receptors in a human preosteoclastic cell line. 770 3

In the late stages of an entomopoxvirus infection, virions become embedded within a crystalline occlusion body or spheroid. Spheroids are composed primarily of a single polypeptide, spheroidin. We describe the construction of a genetically modified Amsacta moorei entomopoxvirus (AmEPV) in which the spheroidin gene coding sequences are deleted and replaced with those of a heterologous reporter gene encoding chloramphenicol acetyltransferase (CAT). A transfer vector, pAmCP1, was prepared containing a unique BamHI site in lieu of the spheroidin gene coding region, together with 1 kbp of upstream and downstream DNA sequence that flanks the spheroidin gene. The flanking sequences provide the transcriptional control signals and also guide homologous recombination so that the spheroidin gene coding region can be replaced with that of the foreign gene. The transfer vector was designed so that the translational start codon of the introduced foreign gene would be utilized. A recombinant virus, AmEPV.CAT, was produced by transfecting AmEPV-infected cells with the transfer vector encoding the CAT gene. The recombinant virus was isolated from wild-type virus by identifying plaques with a spheroidin-negative phenotype. Light microscopy and SDS-PAGE analysis demonstrated that no spheroids or spheroidin protein were produced in the recombinant virus-infected cells. The recombinant virus was able to replicate to high titres (10(7) p.f.u./ml) in insect cells indicating that the spheroidin gene is non-essential for AmEPV replication in vitro. Moderate levels of CAT were synthesized in recombinant virus-infected cells and temporal analyses indicated that CAT synthesis followed the pattern of spheroidin production suggesting that the spheroidin gene promoter was functioning under normal regulatory control in the genetically modified virus.
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PMID:Genetic modification of an entomopoxvirus: deletion of the spheroidin gene does not affect virus replication in vitro. 784 25

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

We have investigated a mechanism of the regulation of mucin core polypeptide (MUC1) gene expression, which is induced by a soluble stimulatory factor, in KM12C human colon carcinoma cells. Conditioned media from normal human colon tissues elevated the level of expression of MUC1 mRNA. Transcriptional activation of the MUC1 gene was analyzed by transient expression of MUC1-CAT reporter plasmids containing the 5'-flanking sequence of the MUC1 gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. A region between base pairs -531 and -520 of the 5'-flanking sequence of the MUC1 gene was sufficient for the induction of CAT activity by normal colon conditioned medium (NCCM). Mutagenesis of 3 base pairs within the region corresponding to sequence -531 to -517 from ACAGGGAGCGGTTAG to ACAGGGAGATTTTAG substantially decreased the induction of CAT activity by NCCM. Nuclear extracts from untreated or NCCM-treated KM12C cells were tested for their interaction with 32P-labeled oligonucleotides corresponding to this sequence. A specifically retarded band was identified after electrophoretic analysis. The quantity or mobility of this band was not changed by NCCM treatment. When an oligonucleotide with three point mutations was used as a competitor, the retarded band remained at the same position. This element (positions -531 to -520), which we call the responsive mucin element, does not contain any sequence that corresponds to previously described cis-acting elements. A protein component complexed with this sequence was identified with a molecular mass of approximately 70 kDa by SDS-polyacrylamide gel electrophoresis.
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PMID:Transcriptional regulation of the MUC1 mucin gene in colon carcinoma cells by a soluble factor. Identification of a regulatory element. 819 40

The chymosin-sensitive sequence of bovine k-casein A (kappa-CN A) was investigated as a cleavable linker site between the two domains of a streptavidin-chloramphenicol acetyltransferase fusion protein. Two DNA sequences were synthesized which encode the amino acids from 101 to 107 and from 97 to 113 of bovine kappa-CN A. These sequences were separately cloned in-frame to a streptavidin expression vector used for fusion protein construction. The gene for chloramphenicol acetyltransferase (CAT) was then cloned in-frame to a streptavidin-chymosin-sensitive linker vector forming plasmids pStCL1CAT and pStCL2CAT. The fusion protein was expressed in Escherichia coli and SDS-PAGE and Western blot analysis of chymosin-treated cell lysates showed a pH-dependent cleavage of the fusion proteins. Fusion proteins were also bioselectively immobilized onto biotinylated controlled-pore glass beads and treated with chymosin. CAT was specifically released by chymosin treatment and was identified by SDS-PAGE.
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PMID:Investigating the use of the chymosin-sensitive sequence of kappa-casein as a cleavable linker site in fusion proteins. 872 7

The L-II element (-149 to -126 bp) in the enhancer unit of the rat pyruvate kinase L (PKL) gene is required for cell-type-specific transcription and induction by carbohydrates. This element was found to bind multiple nuclear proteins with different heat stabilities. A heat-labile factor was shown to be hepatocyte nuclear factor (HNF) 4 by the electrophoretic mobility-shift assay (EMSA) using various competitor DNAs and anti-HNF4 serum. A heat-stable factor was purified from rat liver nuclear extract and was resolved as two protein bands migrating at about 33 kDa on SDS/polyacrylamide gels. Peptide sequence analysis revealed that these proteins were nuclear factor (NF) 1-L and NF1/Red1. The heat-stable factor was also identified as a member of the NF1 family by using various competitor DNAs and anti-NF1 serum in an EMSA. In addition, we found that a factor bound to the accessory site of the rat S14 gene, which is necessary for carbohydrate responsiveness of this gene, was also a member of the NF1 family, raising the possibility that the NF1 family is involved in the carbohydrate regulation of gene transcription by interactions with other proteins. The NF1 family members and HNF4 interacted with overlapping sequences of the L-II element, wherein the 5' half-site was more critical for NF1 binding, and the 3' site was more important for HNF4 binding. Co-transfection of a vector expressing either NF1-L or NF1/Red1 repressed the transcription of the PKL enhancer unit-chloramphenicol acetyltransferase (CAT) fusion gene in HepG2 cells, whereas co-transfection of a vector expressing HNF4 activated the transcription of the same reporter gene. Furthermore NF1 family members antagonized the effect of HNF4 on PKL enhancer unit-CAT fusion gene expression when both expression plasmids were co-transfected. We conclude that NF1 family members and HNF4 regulate transcription of the PKL gene in an opposing manner by binding overlapping sequences of the L-II element.
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PMID:Members of the nuclear factor 1 family and hepatocyte nuclear factor 4 bind to overlapping sequences of the L-II element on the rat pyruvate kinase L gene promoter and regulate its expression. 921 Apr 17


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