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)

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.
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PMID:Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids. 132 38

A plasmid expression vector was constructed to direct the synthesis of foreign proteins in Escherichia coli as fusions with cyclomaltodextrin glucanotransferase (CGT) with cytoplasmic location (delta ssCGT). The ability of CGT to bind to covalently immobilized cyclodextrins was utilized in purifying fused target proteins. A large proportion of the cytoplasmically synthesized delta ssCGT formed inclusion bodies which adopted the active conformation at considerably high refolding concentration (67 microM delta ssCGT solution). By lowering the cultivation temperature the proportion of the soluble delta ssCGT was slightly increased. Intracellularly expressed delta ssCGT provides a potential affinity handle which forms easily refoldable inclusion bodies increasing the yield and stability, and possibly allows the expression of lethal target proteins. Interestingly, the interaction between one model fusion protein delta ssCGT-CAT (CAT, chloramphenicol acetyltransferase) and the E. coli heat shock protein GroEL was observed.
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PMID:Expression in E. coli and purification of intracellular proteins by fusion to cyclomaltodextrin glucanotransferase. 136 55

We established basic conditions for transient gene expression and selection of antibiotics in the cultured cell line of silkworm, Bombyx mori, by use of the promoter of the heat shock protein (hsp70) gene of Drosophila melanogaster. The control promoter (hsp70) promoted the expression of chloramphenicol acetyltransferase (CAT) gene ligated at the downstream, dependent on the orientation of the promoter in the silkworm cell. The cell line is able to be supplied for the promoter assay of the silkworm genes. The concentration for the drug selection was determined as 0.75 mg/ml on neomycin analog, G418 (geneticin).
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PMID:Basic conditions for the drug selection and transient gene expression in the cultured cell line of Bombyx mori. 148 68

The promoter sequences involved in the basal expression of a human 70-kilodalton heat shock protein (HSP70) gene during Xenopus embryogenesis were analyzed by microinjection of mutant promoters of a HSP70--chloramphenicol acetyltransferase fusion gene into fertilized eggs and following their expression during early development. While deletion of the HSP70 gene promoter to--100 base pairs (bp) did not affect basal transcription in postmidblastula stage embryos, linker-scanner mutations in the CCAAT and purine box elements blocked expression. However, extension of the 5' boundary to--188 bp restored full wild-type expression to these mutants. These results suggest that multiple redundant cis-acting regulatory elements present in the human HSP70 gene promoter can function during Xenopus embryogenesis.
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PMID:Compensatory effect of distal promoter sequences on the basal expression of a microinjected 70-kilodalton heat shock protein gene after the midblastula transition of Xenopus laevis embryogenesis. 149 61

The function of the hormone-binding domain of the human progesterone receptor was examined in a yeast cell system and in mammalian HeLa cells using mutant receptors, progesterone, the progesterone agonist R502, and the antagonists RU-486, Org31806, and Org31376. The hormone-binding domain, located on the carboxy terminal of the peptide, is known to initiate a conformational change in the receptor upon binding an agonist or antagonist, then shedding of associated proteins including the heat shock protein, dimerization of the receptor, and finally, binding to DNA, leading to transcription. Binding of a progesterone antagonist such as RU-486 elicits all these events except transcription. First the progesterone receptor was inserted in yeast with a plasmid, and a set of mutants were generated, using beta galactosidase as an indicator. A mutant progesterone receptor, U-P1, was selected for mechanistic studies, that binds and was activated by antagonists, but was inactive with progesterone. This receptor had a deletion at base 2636, resulting in a shift of reading frame so that a stop codon 36 nucleotides downstream caused truncation of 54 amino acids at the C-terminus and addition of 12 novel amino acids. Western blot analysis confirmed the expected molecular weight. The mutant receptor was active with RU-486, suggesting that the C-terminus may be responsible for poor transcription with RU-486, suggesting in normal receptors. 2 other truncated mutants were inactive with progesterone. These data suggested that the terminal 42 amino acids of the progesterone receptor are needed to bind progesterone, and that the antagonist is contacting different amino acids than the native receptor, possibly inducing a different conformational change. The activity of the UP-1 mutant was also confirmed in HeLa cells, with the chloramphenicol acetyltransferase reporter system. The results were interpreted to mean that progesterone agonists and antagonists contact at least some different amino acids in the hormone binding domain of the receptor, and that the conformational changes resulting from binding these agents are different. It appears that the C-terminus of the receptor contains an inhibitory domain which, when removed, turns antagonists into agonists.
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PMID:The mechanism of RU486 antagonism is dependent on the conformation of the carboxy-terminal tail of the human progesterone receptor. 158 49

We have introduced hsp-cat plasmid DNA into Spodoptera frugiperda (Lepidoptera: Noctuidae) cells by transfection with purified DNA (1 to 48 micrograms/ml) mixed with the polycation polybrene (100 micrograms/ml) in serum-free Grace's medium. The hsp-cat construct contains a gene coding for the bacterial enzyme chloramphenicol acetyltransferase (CAT), whose expression is controlled by a promoter derived from a Drosophila heat shock (hsp) gene. Expression of CAT activity in transfected Spodoptera cells was induced by a 2-h heat shock at 43 degrees C. The temperature of the heat shock was based on conditions that maximized the expression of endogenous heat shock protein genes in these cells. CAT activity was maximal in cells that were exposed to the heat shock 2 d after transfection; by 4 d, activity was diminished, and little activity was detectable after 6 d. Transfection frequencies, which varied with DNA concentration and ranged as high as 6000 per million cells, were determined using a histochemical staining procedure.
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PMID:Polybrene-mediated transfection of cultured lepidopteran cells: induction of a Drosophila heat shock promoter. 238 51

We examined the effects of cellular aging on the regulation of heat shock gene expression in IMR-90 human diploid fibroblasts. Heat shock (42-43 degrees C) and canavanine (200-400 micrograms/ml) were used to evoke the heat shock response in these cells. We showed that heat shock induced the synthesis of proteins with apparent molecular weights of 98,000, 89,000, 78,000, 72,000, 64,000, 50,000 and 25,000, with heat shock protein (HSP) 89 and 72 being most prominent. Canavanine induced the synthesis of the four high molecular weight HSPs, particularly HSP 89 and HSP 78, without noticeably enhancing synthesis of the low molecular weight HSPs. We found that, while a similar series of HSPs were induced in the young and old cells, there was a marked decrease in the magnitude of this induction in the old cells. Using cells with defined population doubling levels, we observed a direct correlation of the inducibility of HSP synthesis and the replicative potential of the cells used. Analysis of the amount of translatable and hybridizable mRNA, by the methods of in vitro translation and Northern blot hybridization, demonstrated that the induction of HSPs synthesis can be accounted for by increases in their mRNA. Nuclear runoff transcription provided evidence that the decrease in inducible expression of the HSPs in aging IMR-90 cells was attributable to a transcriptional mechanism. This conclusion was substantiated by analysis of the hsp 70 promoter activity in transient expression assay of the hsp 70 promoter-chloramphenicol acetyltransferase construct. We propose that there is an age-associated dysfunction in the signaling mechanism of the heat shock response.
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PMID:Attenuated induction of heat shock gene expression in aging diploid fibroblasts. 274 27

Hemin-induced differentiation of the human erythroleukemia cell line K562 results in the expression and accumulation of erythroid-specific gene products such as embryonic and fetal hemoglobins and the elevated synthesis of the major heat shock protein HSP70. This activity was suggested to represent activation of a heat shock gene during erythroid maturation independent of stress induction. In this study, we demonstrate that hemin induces the transcription of two members of the human HSP70 gene family, HSP70 and GRP78 (BiP). However, the induction of HSP70 by hemin showed characteristics consistent with the molecular events associated with a heat shock or stress response. The increase in HSP70 gene transcription was accompanied by induction of the stress-induced form of the heat shock transcription factor. Moreover, a heat shock element was required for the hemin responsiveness of chimeric heat shock promoter-chloramphenicol acetyltransferase genes transiently expressed in transfected K562 cells.
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PMID:Hemin-induced transcriptional activation of the HSP70 gene during erythroid maturation in K562 cells is due to a heat shock factor-mediated stress response. 279 86

We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43 degrees C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to the HSP70 synthesized in vivo. From the analysis of S1 nuclease-resistant mRNA-DNA hybrids, the HSP70 gene appears to be transcribed as an uninterrupted mRNA of 2.3 kilobases. We show that the cloned HSP70 gene contains the sequences necessary for heat shock-induced expression by two criteria. First, hamster cells transfected with a subclone containing the HSP70 gene and flanking sequences synthesized a HSP70-like protein upon heat shock. Second, human cells transfected with a chimeric gene containing the 5' flanking sequences of the HSP70 gene and the coding sequences of the bacterial chloramphenicol acetyltransferase gene transcribed the chimeric gene upon heat shock. We show that the HSP70 mRNA transcribed in an adenovirus 5 transformed human cell line (293 cells) is identical to the HSP70 mRNA induced by heat shock.
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PMID:Structure and expression of the human gene encoding major heat shock protein HSP70. 285 50

The polycation 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) is superior to calcium phosphate for the introduction of purified DNA into cultured Aedes albopictus (mosquito) cells. Adsorption of the polybrene-DNA complex to mosquito cells was essentially linear for 6 h. However, the rate of adsorption of DNA increased when the DNA-polybrene mixture was preincubated for several hours prior to addition to cells. A recombinant plasmid carrying an inducible chloramphenicol acetyltransferase gene under the control of a Drosophila heat shock protein (hsp) promoter was used to show that expression of transfected DNA was highest when cells were treated with a freshly prepared polybrene-DNA mixture. Optimal expression was observed in cells transfected with 4-13 micrograms of DNA per 10(6) cells; transfection with 24 micrograms of DNA resulted in reduced CAT expression. Variation in the polybrene-DNA ratio improved transfection with high levels of DNA. In mosquito cells, CAT expression was independent of DNA methylation.
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PMID:Factors affecting polybrene-mediated transfection of cultured Aedes albopictus (mosquito) cells. 346 5


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