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Diego Haro - One of the best experts on this subject based on the ideXlab platform.
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peroxisome proliferator activated receptor mediates induction of the mitochondrial 3 hydroxy 3 methylglutaryl coa synthase gene by fatty acids
Journal of Biological Chemistry, 1994Co-Authors: Joan C. Rodríguez, G Gilgomez, Fausto G Hegardt, Diego HaroAbstract:Abstract Fatty acids induce an increase in the transcription of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which encodes an enzyme that has been proposed as a control site of ketogenesis. We studied whether the peroxisome proliferator-activated receptor (PPAR) is involved in the mechanism of this transcriptional induction. We found that cotransfection of a rat mitochondrial HMG-CoA synthase promoter-Chloramphenicol Acetyltransferase reporter plasmid and a PPAR expression plasmid in the presence of the peroxisome proliferator clofibrate led to a more than 30-fold increase in Chloramphenicol Acetyltransferase activity, relative to the activity in the absence of both PPAR and inducer. Linoleic acid, a polyunsaturated fatty acid, increased this activity as potently as does clofibrate and more effectively than does monounsaturated oleic acid. We have identified, by deletional analysis, an element located 104 base pairs upstream of the mitochondrial HMG-CoA synthase gene, which confers PPAR responsiveness to homologous and heterologous promoters. This is the first example of a peroxisome proliferator-responsive element (PPRE) in a gene encoding a mitochondrial protein. This element contains an imperfect direct repeat that is similar to those described in the PPREs of other genes. Furthermore, gel retardation and cotransfection assays revealed that, as for other genes, PPAR heterodimerizes with retinoid X receptor and that both receptors cooperate for binding to the mitochondrial HMG-CoA synthase PPRE and subsequent activation of the gene. In conclusion, our data demonstrate that regulation of mitochondrial HMG-CoA synthase gene expression by fatty acids is mediated by PPAR, supporting the hypothesis that PPAR has an important role at the transcriptional level in the regulation of lipid metabolism.
Joan C. Rodríguez - One of the best experts on this subject based on the ideXlab platform.
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peroxisome proliferator activated receptor mediates induction of the mitochondrial 3 hydroxy 3 methylglutaryl coa synthase gene by fatty acids
Journal of Biological Chemistry, 1994Co-Authors: Joan C. Rodríguez, G Gilgomez, Fausto G Hegardt, Diego HaroAbstract:Abstract Fatty acids induce an increase in the transcription of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which encodes an enzyme that has been proposed as a control site of ketogenesis. We studied whether the peroxisome proliferator-activated receptor (PPAR) is involved in the mechanism of this transcriptional induction. We found that cotransfection of a rat mitochondrial HMG-CoA synthase promoter-Chloramphenicol Acetyltransferase reporter plasmid and a PPAR expression plasmid in the presence of the peroxisome proliferator clofibrate led to a more than 30-fold increase in Chloramphenicol Acetyltransferase activity, relative to the activity in the absence of both PPAR and inducer. Linoleic acid, a polyunsaturated fatty acid, increased this activity as potently as does clofibrate and more effectively than does monounsaturated oleic acid. We have identified, by deletional analysis, an element located 104 base pairs upstream of the mitochondrial HMG-CoA synthase gene, which confers PPAR responsiveness to homologous and heterologous promoters. This is the first example of a peroxisome proliferator-responsive element (PPRE) in a gene encoding a mitochondrial protein. This element contains an imperfect direct repeat that is similar to those described in the PPREs of other genes. Furthermore, gel retardation and cotransfection assays revealed that, as for other genes, PPAR heterodimerizes with retinoid X receptor and that both receptors cooperate for binding to the mitochondrial HMG-CoA synthase PPRE and subsequent activation of the gene. In conclusion, our data demonstrate that regulation of mitochondrial HMG-CoA synthase gene expression by fatty acids is mediated by PPAR, supporting the hypothesis that PPAR has an important role at the transcriptional level in the regulation of lipid metabolism.
Dennis E. Hruby - One of the best experts on this subject based on the ideXlab platform.
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8 use of fluorescent Chloramphenicol derivative as a substrate for Chloramphenicol Acetyltransferase assays
Recombinant DNA Methodology II, 1995Co-Authors: Dennis E. Hruby, Elizabeth M. WilsonAbstract:Publisher Summary This chapter discusses the use of fluorescent Chloramphenicol derivatives. The DNA sequences encoding bacterial Chloramphenicol Acetyltransferase (CAT) enzyme is fused to heterologous transcriptional regulatory signals and used as a “reporter gene” for measuring the rate of transcription of chimeric genes, as well as the translation and/or stability of the chimeric transcripts within the context of transformed bacterial cells, transfected tissue culture cells, or genetically engineered recombinant viruses. The chapter presents the two types of CAT assay procedures that are currently available. The first involves using Chloramphenicol as the substrate for acetylation. The acetylated derivatives are resolved from the substrate by thin-layer chromatography and quantitated by autoradiography and subsequent densitometric scanning or by eluting the acetylated derivatives from the plate followed by scintillation counting. The second method utilizes acetyl-Coenzyme A as the substrate. In this case, the acetylated reaction products are collected by organic extraction and quantitated directly by scintillation counting. The chapter additionally discusses the advantages of using a fluorescent Chloramphenicol substrate to assay CAT activity. The assay is rapid and easily quantified and the results can be evaluated immediately. No radioactive materials are required. This dispenses with the need for film, fluors, or scintillation counters and disposal of radioactive wastes.
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an nh2 terminal peptide from the vaccinia virus l1r protein directs the myristylation and virion envelope localization of a heterologous fusion protein
Journal of Biological Chemistry, 1993Co-Authors: M P Ravanello, Christine A. Franke, Dennis E. HrubyAbstract:Abstract The vaccinia virus L1R gene product is a late protein destined for insertion into the envelope of intracellular virus particles. Because this protein is co-translationally modified by the addition of myristic acid to the penultimate NH2-terminal glycine residue, it was of interest to identify the modification signal within the L1R protein and to assess the relevance of myristylation to protein localization. To this end, a family of chimeric reporter genes containing 0-13 codons from the NH2 terminus of the L1R open reading frame fused in-frame to the bacterial Chloramphenicol Acetyltransferase gene was constructed. The encoded proteins were tested as myristylation substrates in cell-free extracts and infected cells. The results obtained in vitro and in vivo were similar and suggested that although the NH2-terminal 5 amino acids of the L1R protein were the minimum signal required to observe modification by myristate, 12 amino acids were required to obtain wild type levels of myristylation with a modulating role played by the intervening amino acid residues. Furthermore, subcellular fractionation of infected cells expressing the fusion proteins indicated that the NH2 terminus of the L1R protein was capable of targeting the fusion proteins to membrane-containing fractions only if myristylated. In particular, the myristylated fusion protein containing the first 12 amino acids of the L1R protein abutted to the Chloramphenicol Acetyltransferase protein was found associated with the envelope of intracellular vaccinia virus particles.
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Use of fluorescent Chloramphenicol derivative as a substrate for Chloramphenicol Acetyltransferase assays.
Methods in Enzymology, 1992Co-Authors: Dennis E. Hruby, Elizabeth M. WilsonAbstract:Publisher Summary This chapter discusses the use of fluorescent Chloramphenicol derivative as a substrate for Chloramphenicol Acetyltransferase assays. The DNA sequences encoding bacterial Chloramphenicol Acetyltransferase (CAT) enzyme are commonly fused to heterologous transcriptional regulatory signals, and used as a “reporter gene” for measuring the rate of transcription of chimeric genes, as well as the translation and/or stability of the chimeric transcripts within the context of transformed bacterial cells, transfected tissue culture cells, or genetically engineered recombinant viruses. CAT activity has a number of advantages over the traditional methods: (1) No radioactive materials are required. This dispenses with the need for film, fluors, scintillation counters, and disposal of radioactive wastes; (2) the assay is rapid and easily quantified; (3) the results can be evaluated immediately. Taken together, these results would suggest that the use of a fluorescent Chloramphenicol substrate provides an attractive alternative method for measuring CAT activity in the extracts of cells, which express this enzyme. The development of a modified CAT enzyme assay that uses fluorescent Chloramphenicol substrates promises to enhance the utility of this system by eliminating the use of radioactive materials.
Fausto G Hegardt - One of the best experts on this subject based on the ideXlab platform.
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peroxisome proliferator activated receptor mediates induction of the mitochondrial 3 hydroxy 3 methylglutaryl coa synthase gene by fatty acids
Journal of Biological Chemistry, 1994Co-Authors: Joan C. Rodríguez, G Gilgomez, Fausto G Hegardt, Diego HaroAbstract:Abstract Fatty acids induce an increase in the transcription of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which encodes an enzyme that has been proposed as a control site of ketogenesis. We studied whether the peroxisome proliferator-activated receptor (PPAR) is involved in the mechanism of this transcriptional induction. We found that cotransfection of a rat mitochondrial HMG-CoA synthase promoter-Chloramphenicol Acetyltransferase reporter plasmid and a PPAR expression plasmid in the presence of the peroxisome proliferator clofibrate led to a more than 30-fold increase in Chloramphenicol Acetyltransferase activity, relative to the activity in the absence of both PPAR and inducer. Linoleic acid, a polyunsaturated fatty acid, increased this activity as potently as does clofibrate and more effectively than does monounsaturated oleic acid. We have identified, by deletional analysis, an element located 104 base pairs upstream of the mitochondrial HMG-CoA synthase gene, which confers PPAR responsiveness to homologous and heterologous promoters. This is the first example of a peroxisome proliferator-responsive element (PPRE) in a gene encoding a mitochondrial protein. This element contains an imperfect direct repeat that is similar to those described in the PPREs of other genes. Furthermore, gel retardation and cotransfection assays revealed that, as for other genes, PPAR heterodimerizes with retinoid X receptor and that both receptors cooperate for binding to the mitochondrial HMG-CoA synthase PPRE and subsequent activation of the gene. In conclusion, our data demonstrate that regulation of mitochondrial HMG-CoA synthase gene expression by fatty acids is mediated by PPAR, supporting the hypothesis that PPAR has an important role at the transcriptional level in the regulation of lipid metabolism.
G Gilgomez - One of the best experts on this subject based on the ideXlab platform.
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peroxisome proliferator activated receptor mediates induction of the mitochondrial 3 hydroxy 3 methylglutaryl coa synthase gene by fatty acids
Journal of Biological Chemistry, 1994Co-Authors: Joan C. Rodríguez, G Gilgomez, Fausto G Hegardt, Diego HaroAbstract:Abstract Fatty acids induce an increase in the transcription of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which encodes an enzyme that has been proposed as a control site of ketogenesis. We studied whether the peroxisome proliferator-activated receptor (PPAR) is involved in the mechanism of this transcriptional induction. We found that cotransfection of a rat mitochondrial HMG-CoA synthase promoter-Chloramphenicol Acetyltransferase reporter plasmid and a PPAR expression plasmid in the presence of the peroxisome proliferator clofibrate led to a more than 30-fold increase in Chloramphenicol Acetyltransferase activity, relative to the activity in the absence of both PPAR and inducer. Linoleic acid, a polyunsaturated fatty acid, increased this activity as potently as does clofibrate and more effectively than does monounsaturated oleic acid. We have identified, by deletional analysis, an element located 104 base pairs upstream of the mitochondrial HMG-CoA synthase gene, which confers PPAR responsiveness to homologous and heterologous promoters. This is the first example of a peroxisome proliferator-responsive element (PPRE) in a gene encoding a mitochondrial protein. This element contains an imperfect direct repeat that is similar to those described in the PPREs of other genes. Furthermore, gel retardation and cotransfection assays revealed that, as for other genes, PPAR heterodimerizes with retinoid X receptor and that both receptors cooperate for binding to the mitochondrial HMG-CoA synthase PPRE and subsequent activation of the gene. In conclusion, our data demonstrate that regulation of mitochondrial HMG-CoA synthase gene expression by fatty acids is mediated by PPAR, supporting the hypothesis that PPAR has an important role at the transcriptional level in the regulation of lipid metabolism.