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 cytoskeletal actins are abundant proteins in mammalian nonmuscle cells. We have previously reported that physiological concentrations of insulin induced beta-actin transcription in rat H4 hepatoma cells. To define whether one or more of the three CCArGG box elements or other elements within the beta-actin gene promoter is an insulin response element, we transfected H4 cells with regions of the human beta-actin gene promoter fused to the chloramphenicol acetyltransferase gene. A 350-basepair DNA fragment was isolated that mediates both insulin and serum effects. This fragment contains at least two up-stream elements, a CCAAT box and a CCArGG box, and accounts for more than 70% of the basal activity of the beta-actin promoter in H4 cells. There was a small, but significant, stimulatory effect of insulin over maximal serum induction, suggesting a difference in their mechanisms of action. Mutation of the CCAAT box drastically reduced basal expression, with no effect on insulin induction. In contrast, a mutation of the CCArGG element reduced basal expression and completely abolished insulin inducibility. Electrophoretic mobility shift assays suggested that insulin regulated the activity, but not the binding, of a factor(s) that associates with the CCArGG box. These data demonstrate that in H4 cells, insulin induction of beta-actin gene expression was mediated at least in part through one of the three beta-actin CCArGG elements.
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PMID:One of three CCArGG box/serum response elements of the beta-actin gene is an insulin-responsive element. 782 46

Previous studies have shown that the adipose tissue of young genetically obese Zucker rats was characterized by a coordinate overtranscription of lipogenic genes, suggesting that the fa mutation triggers transcription factor(s) acting in common on the promoters of these genes. To test this hypothesis, we developed a system of transient transfection of rat adipocytes with constructs containing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fatty acid synthetase (FAS) promoters fused to gene reporter CAT. Those transfected cells expressed high levels of promoter-driven chloramphenicol acetyltransferase (CAT) activity through correctly initiated transcription as shown by primer extension analysis. Using this system we found a direct effect of insulin on GAPDH and FAS gene expression in rat adipocytes. In transfected adipocytes from obese compared to lean rats, activity of GAPDH and FAS promoters fused to CAT, was 2.6- and 8-fold increased, respectively. In contrast when reporter gene activity was driven by either phosphoenolpyruvate carboxykinase or beta-actin promoter, no difference could be observed between lean and obese, pointing out the promoter specificity of genotype effect. 5' deletion analysis of GAPDH promoter allowed us to narrow down the fa responsive region to nucleotide -488-329. As assessed by gel retardation and DNase I footprinting analysis, adipocyte nuclear protein interactions to this 159-bp fragment were found to be identical and to footprint the same 20-bp sequence. This study pointed out that overexpression of GAPDH and FAS genes in adipose tissue of genetically obese rats relies on promoter activation, through a 159-bp cis-acting region within the GAPDH promoter. The effects of the fa mutation on trans-acting factors binding to this region remain to be identified.
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PMID:Evidence of increased glyceraldehyde-3-phosphate dehydrogenase and fatty acid synthetase promoter activities in transiently transfected adipocytes from genetically obese rats. 783 67

Expression of the human neutrophil elastase (NE) gene is limited to the early stage of myeloid cell differentiation in bone marrow cells. While NE gene expression is controlled mainly at the transcriptional level during bone marrow cell differentiation, the mechanism of transcriptional control is not fully understood. One motif of interest in the 5' flanking region of the gene is the six tandem repeats of a 53-bp nucleotide sequence (REP53) containing a potential binding site for a basic helix-loop-helix protein located at -1032 to -716. The REP53 sequence can function as a non-cell specific transcriptional enhancer which is capable of augmenting heterologous promoter activity. When the single REP53 element was inserted into the pAZ1037 chloramphenicol acetyltransferase (CAT) expression vector immediately upstream of the chicken beta-actin promoter in either normal or inverted orientation and used to transfect K-562 erythroleukemia or HeLa cervical carcinoma cells, these modified vectors achieved 2 to 3-fold higher CAT activity than the parental pAZ1037 vector irrespective of orientation of the REP53.
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PMID:Enhancer function of a 53-bp repetitive element in the 5' flanking region of the human neutrophil elastase gene. 794 85

Prolonged expression of activated ras mutants resulted in both neoplastic transformation and suppression of serum-induced c-fos expression in Rat1 fibroblasts. Expression of other serum-inducible genes, including c-jun and beta-actin, was not suppressed in ras-transformed Rat1 cells, indicating that these effects are specific for c-fos and that growth-factor signal transduction pathways remain essentially intact. Run-on transcription studies indicated that c-fos transcription was blocked at the level of initiation in these cells. Transient transfection studies using 360 bp from the wild-type c-fos promoter as well as a series of mutated c-fos promoter fragments linked to the chloramphenicol acetyltransferase gene indicated that repression of c-fos was mediated by approximately 49 bp immediately upstream of the dyad symmetry element (DSE). Deletion of this region, referred to as the upstream repressor region (URR), restored serum inducibility to the c-fos promoter in ras-transformed cells. In contrast, suppression of c-fos transcription was not affected by either deletion of 240 bp between the DSE and the TATA element or by base-substitution mutations that inactive the ternary complex factor and fos-AP-1-like binding sites. In addition, in vitro competition studies indicated that ras-transformed cells express one or more repressor factors that interact with as-yet-unidentified elements within the c-fos promoter (possibly the URR) and block serum induction of c-fos. These findings suggest that prolonged expression of activated ras results in the activation of one or more as-yet-unidentified proteins that suppress transcription of the c-fos gene by interacting with the URR.
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PMID:Mediation of suppression of c-fos transcription in rasT24-transformed rat cells by a cis-acting repressor element. 803 67

Transcription of the cytoskeletal beta-actin gene is rapidly induced by phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore, A23187, in cultured H4IIE hepatoma (H4) cells. PMA directly activates protein kinase C (PKC) and activation of PKC is necessary for the cellular actions of PMA, including induction of beta-actin gene transcription. In the present study, we determined the DNA sequence requirements for induction of the beta-actin gene by PMA and A23187. Constructs containing progressive deletions of normal and mutated human beta-actin 5' sequences fused to the reporter gene, bacterial chloramphenicol acetyltransferase, were analysed in transient transfections of H4 cells. We delineated the PMA response DNA element of the human beta-actin gene to the proximal CCArGG box (-62 to -53) in the 5' flanking region. In contrast, A23187 did not induce expression of transfected gene constructs containing this CCArGG box. Additionally, we demonstrated that CCArGG boxes from two other PMA-induced genes in H4 cells, c-fos and gamma-actin, could confer PMA inducibility to a heterologous promoter. This CCArGG box specifically interacts with one or more proteins present in nuclear extracts of H4 cells. These results indicate that in cultured cells, PMA-dependent induction of the beta-actin gene is mediated through the proximal CCArGG box. This suggests that the CCArGG box is a target for PKC action and may be involved in the control of other PKC regulated genes.
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PMID:Identification of beta-actin sequences necessary for induction by phorbol esters and calcium ionophores. 818 67

The transcriptional regulatory elements of the beta-actin gene of carp (Cyprinus carpio) have been examined in zebrafish and goldfish harbouring transgenes. The high sequence conservation of the putative regulatory elements in the beta-actin genes of animals suggested that their function would be conserved, so that transgenic constructs with the same transcriptional control elements would promote similar levels of transgene expression in different species of transgenic animals. To test this assumption, we analysed the temporal expression of a reporter gene under the control of transcriptional control sequences from the carp beta-actin gene in zebrafish (Brachydanio rerio) and goldfish (Carrasius auratus). Our results indicated that, contrary to expectations, combinations of different transcriptional control elements affected the level, duration, and onset of gene expression differently in developing zebrafish and goldfish. The major differences in expression of beta-actin/CAT (chloramphenicol acetyltransferase) constructs in zebrafish and goldfish were: (1) overall expression was almost 100-fold higher in goldfish than in zebrafish embryos, (2) the first intron had an enhancing effect on gene expression in zebrafish but not in goldfish, and (3) the serum-responsive/CArG-containing regulatory element in the proximal promoter was not always required for maximal CAT activity in goldfish, but was required in zebrafish. These results suggest that in the zebrafish, but not in the goldfish, there may be interactions between motifs in the proximal promoter and the first intron which appear to be required for maximal enhancement of transcription.
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PMID:Regulation of expression of transgenes in developing fish. 835 34

Doxorubicin (Dox, adriamycin), an antineoplastic agent that can cause dilated cardiomyopathy, selectively inhibits muscle-specific gene expression in rodent cardiac muscle cells. This study shows that Dox treatment of proliferating C2 myoblasts, an established cell line from mouse skeletal muscle, completely prevents both fusion and accumulation of muscle-specific gene transcripts without significantly altering non-muscle gene transcripts. When added to high density cultures, Dox only blocked myotube formation but did not inhibit induction of muscle-specific genes. Transient transfection into C2 myoblasts showed that the transcriptional expression of chloramphenicol acetyltransferase reporter plasmids regulated by either the cardiac alpha-actin promoter or the muscle creatine kinase enhancer, but not with a viral or beta-actin promoter, was significantly diminished by Dox in a dose-dependent manner. Moreover, exposure of C2 myoblasts to Dox had a profound effect on the expression of regulatory genes critical to the myogenic differentiation program; mRNAs for MyoD and myogenin were dramatically reduced and Id mRNA was concomitantly increased. In addition, there was diminished DNA binding activity of the muscle-specific transcription factor, MEF-2. These results suggest that Dox inhibits myogenesis by preventing muscle-specific gene expression, possibly through affecting the myogenic programs controlled by muscle-specific transcription factors.
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PMID:Antineoplastic agent doxorubicin inhibits myogenic differentiation of C2 myoblasts. 844 15

Transgenic mouse lines have been developed that express the tv-a receptor under the control of the chicken beta-actin promoter. These mice express the tv-a receptor in most or all tissues and in the early embryo. An avian leukosis virus (ALV)-based retroviral vector system was used for the efficient delivery of genes into preimplantation mouse embryos from these transgenic lines. Experimental animals could be generated quickly and easily by infecting susceptible blastocysts with ALV-based retroviral vectors. Expression of the delivered genes was controlled by either the constitutive viral promoter contained in the long terminal repeat or an internal nonviral tissue-specific promoter. Mating the infected founder chimeric animals produced animals that carry the ALV provirus as a transgene. A subset of the integrated proviruses expressed the chloramphenicol acetyltransferase reporter gene from either the promoter in the long terminal repeat or an internal promoter, which we believe indicates that many of the sites that are accessible to viral DNA insertion in preimplantation embryos are incompatible with expression in older animals. This approach should prove useful for studies on murine cell lineage and development, providing models for studying oncogenesis, and testing gene therapy strategies.
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PMID:Expression of transduced genes in mice generated by infecting blastocysts with avian leukosis virus-based retroviral vectors. 864 6

Membrane depolarization stimuli (high potassium concentration and veratridine) increased neuropeptide Y (NPY) mRNA abundance time-dependently, without a change in beta-actin mRNA level, in NG108-15 and PC12 cells. Although the induction by veratridine was blocked completely by tetrodotoxin, the induction by potassium was suppressed minimally. Voltage-dependent Ca channel blockers and calmodulin antagonists inhibited the increases by both depolarization stimuli completely, suggesting involvement of Ca2+/calmodulin-dependent kinases (CaM kinases). Transient assay using chloramphenicol acetyltransferase reporter genes containing the rat NPY gene promoter indicated that membrane depolarization and Ca entry stimulate transcription of the NPY gene. The depolarization-induced transactivation was also blocked by CaM kinase inhibitors. The 200-bp 5'-upstream region (-344/-145) was localized as a Ca2+/ calmodulin-responsive element (CaMRE), which confers depolarization-induced transactivation. It is interesting that this CaMRE did not contain the canonical Ca-responsive elements such as CRE, SRE, NF-AT, or the C/EBP beta-binding site and was separated from a 64-bp cyclic AMP/ phorbol 12-myristate 13-acetate-responsive element (-144/-81). These findings suggested that membrane depolarization regulates the NPY gene transcription positively through the unique CaMRE by activation of CaM kinases following Ca entry through L-type Ca channels.
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PMID:Ca2+/calmodulin-dependent transcriptional activation of neuropeptide Y gene induced by membrane depolarization: determination of Ca(2+)- and cyclic AMP/phorbol 12-myristate 13-acetate-responsive elements. 878 4

Peroxisome proliferators induce stearoyl-CoA desaturase activity (EC 1.14.99.5) in liver [Kawashima, Y., Hanioka, N., Matsumura, M. & Kozuka, H. (1983) Biochim. Biophys. Acta 752, 259-264]. We analyzed the changes in stearoyl-CoA desaturase 1 (SCD1) mRNA to further define the molecular mechanism for the induction of stearoyl-CoA desaturase by peroxisome proliferators. SCD1 mRNA was analyzed from the livers of BALB/c mice that had been fed diets supplemented with clofibrate or gemfibrozil. Clofibrate was found to induce liver SCD1 mRNA levels 3-fold within 6 hr to a maximum of 22-fold in 30 hr. Gemfibrozil administration resulted in a similar induction pattern. This induction is primarily due to an increase in transcription of the SCD1 gene, as shown by nuclear run-on transcription assays and DNA deletion analysis of transfected SCD1-chloramphenicol acetyltransferase fusion genes. The cis-linked response element for peroxisome proliferator-activated receptor (PPAR) was localized to an AGGTCA consensus sequence between base pairs -664 to -642 of the SCD1 promoter. Clofibrate-mediated induction of SCD1 mRNA was shown to be independent of polyunsaturated fatty acids, with peroxisome proliferators and arachidonic acid having opposite effects on SCD1 mRNA levels. Additionally, the activation of SCD1 mRNA by clofibrate was inhibited 77% by cycloheximide administration. Levels of liver beta-actin and albumin mRNAs were unchanged by these dietary manipulations. Our data show that hepatic SCD1 gene expression is regulated by PPARs and suggest that peroxisome proliferators and poly-unsaturated fatty acids act through distinct mechanisms.
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PMID:Peroxisome proliferators induce mouse liver stearoyl-CoA desaturase 1 gene expression. 879 Mar 49


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