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)

We have examined the transcriptional regulation of the rabbit myosin heavy chain (HC) beta gene by using DNA-mediated transfection experiments. To analyze the activity of the myosin HC beta promoter in a myogenic background, cultured myoblasts from 12-day-old chick embryonic breast muscle were transfected with a chimeric gene containing 781 base pairs of the promoter region fused to the gene for chloramphenicol acetyltransferase (CAT). As indicated by the transient expression of chloramphenicol acetyltransferase, the activity of the promoter in myoblast cultures increased at least 32-fold following differentiation and was selectively inhibited when myogenesis was blocked with 5-bromodeoxyuridine. Furthermore, RNase protection experiments showed that the in vivo myosin HC beta transcriptional initiation (or cap) site was utilized in the transfected skeletal muscle cells and also that the regulation of the exogenous promoter was similar to the induction of the endogenous skeletal alpha-actin gene. The results indicated that the exogenous promoter is regulated in a tissue- and stage-specific manner. By creating progressive 5' deletions of the promoter, we showed that only the region extending -294 base pairs upstream from the cap site is necessary for the muscle-specific expression. Linker-scanner mutagenesis of this region indicated that the positive regulation in differentiated skeletal muscle is mediated by at least two distinct elements within the 5'-flanking region of the myosin HC beta gene.
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PMID:Muscle-specific regulation of a transfected rabbit myosin heavy chain beta gene promoter. 256 93

Members of the myosin heavy chain (MyHC) gene family show developmental stage- and spatial-specificity of expression. We report on the characterization and identification of a porcine skeletal fast MyHC gene, including its corresponding 5' end cDNA and 5' regulatory region. This MyHC isoform was found exclusively in skeletal muscles from about the last quarter of gestation through to adulthood. Expression of this isoform was higher postnatally and its spatial distribution resembled a rosette cluster; each with a ring of fast fibres surrounding a central slow fibre. This rosette pattern was absent in the adult diaphragm but about 20% of the fibres continued to express this MyHC isoform. Further in vivo expression studies, in a variety of morphologically and functionally diverse muscles, showed that this particular skeletal MyHC isoform was expressed in fast oxidative-glycolytic fibres, suggesting that it was the equivalent of the fast IIA isoform. Two domains in the upstream regulatory region were found to confer differentiation-specific expression on C2 myotubes (-1007 to -828 and -455 to -101), based on in vitro transient expression assays using the chloramphenicol acetyltransferase (CAT) reporter gene. Interestingly, for high levels of CAT expression to occur, a 3' region, extending from the transcriptional start site to part. of intron 2, must be present in all the DNA constructs used.
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PMID:Molecular characterization of a developmentally regulated porcine skeletal myosin heavy chain gene and its 5' regulatory region. 761 92

We examined the effect of cyclical mechanical stretch on the regulation of cardiac myosin heavy chain genes using an isolated neonatal rat cardiocyte culture system. Cultured cardiocytes grown on a flexible membrane were deformed by vacuum to 20% of maximum elongation, at 60 cycles/min in a serum-free medium. Cyclical stretch did not cause myocyte damage as assessed by supernatant LDH measurement and trypan blue exclusion test. The levels of myosin heavy chain (MyHC) mRNA increased as early as 1 h after stretch, reaching 12-fold over the control in 24 h, as shown by Northern blot analysis. However, the proximal 5'-flanking regions of the alpha- and beta-MyHC gene which were linked to chloramphenicol acetyltransferase (CAT) reporter gene did not exhibit enhanced CAT activity following cyclical stretch. Deletion of the chimeric constructs to shorten the 5'-flanking regions of the MyHC genes generated by polymerase chain reaction amplification did not enhance the CAT activity under cyclical stretch. This finding suggests that the stretch-response element of the alpha- and beta-MyHC gene promoter is probably not present in the proximal region of either the alpha- or beta-MyHC genes.
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PMID:Regulation of human cardiac myosin heavy chain genes by cyclical mechanical stretch in cultured cardiocytes. 775 35

Despite the importance of smooth muscle cell proliferation in vascular pathophysiological states, the mechanisms regulating smooth muscle cell growth and differentiation are poorly understood. Previous studies have shown that adult rabbit smooth muscles express two types of myosin heavy chain (MHC) isoforms, SM1 and SM2, which are generated through alternative RNA splicing from a single smooth muscle MHC (SMHC) gene. In the present study, we isolated and characterized the rabbit SMHC gene promoter. DNA sequence analysis of the upstream region of the SMHC gene revealed several putative cis-DNA regulatory elements proximal to the transcription start site. Most notably, cis-acting regulatory elements that closely resemble CC(A/T)6GG (CArG box) and myocyte enhancer binding factor 2 (MEF-2)-type sequence motifs were found in the SMHC 5'-flanking region. In addition, six E-box motifs were found in the 5'-flanking region of the SMHC gene between -374 and -2109 base pairs from the transcription start site. A series of transient transfection assays using SMHC promoter deletion constructs indicated that a promoter fragment extending to 2266 base pairs upstream of the transcription start site has the highest reporter activity in cultured rat aortic smooth muscle cells. Gel mobility shift analyses using the MEF-2-like sequence located at -1540 revealed a specific DNA protein complex, whereas the CArG-like element located at -1275 did not show protein binding. The SMHC promoter construct, p509-CAT, which included neither the CArG- nor MEF-2-type motifs, conferred 32% of chloramphenicol acetyltransferase activity in the same cells, whereas the construct p188-CAT, which contained the minimal promoter elements (TATA box), was significantly less active (7%; 2.0-fold over background). This is the first report describing the promoter elements of a gene whose expression is restricted to smooth muscle cells.
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PMID:Identification of functional promoter elements in the rabbit smooth muscle myosin heavy chain gene. 798 72

Hypertrophy of the myocardium in response to pressure or volume overload elicits a change in myofibrillar protein content as a result of changes in both transcriptional and translational regulation of gene expression. Hemodynamic overload caused by aortic constriction produced changes in the expression of the two isoforms of myosin heavy chain (MHC) with a 319% increase in beta-MHC mRNA and a 54% decrease in alpha-MHC mRNA (P < 0.01). Cardiac unloading as a result of heterotopic transplantation resulted in a decrease in cardiac mass and a similar shift in MHC isoform expression. In this study. We investigated cardiac gene transcription to understand how different hemodynamic stimuli produce similar cardiac phenotypes. We studied the in vivo activity of the alpha-MHC promoter (-2564 to +421 bp of the transcriptional start site) by directly injecting a recombinant expression plasmid (pAM3LUC) into the ventricular tissue of coarctated animals as well as into the unloaded heterotopic transplanted heart. When expressed as a function of the activity of a constitutively active viral promoter (pSVCAT), pAM3LUC activities were 18.4 +/- 2.9, 24.6 +/- 2.6, and 25.0 +/- 4.5 (x10(4)) luciferase/chloramphenicol acetyltransferase units in the hypertrophied ventricles of 2-, 3-, and 7-day coarctated animals, respectively. These values were not statistically different from pAM3LUC activity in control hearts of sham operated animals even though alpha-MHC mRNA content was decreased by 54% in the hypertrophied myocardium. This disparity between transcriptional activity and mRNA content suggests that alpha-MHC expression in the hypertrophic ventricle is in part regulated by a posttranscriptional mechanism. In contrast, alpha-MHC promoter activity in the unloaded transplanted hearts decreased significantly by 37% compared to control working hearts and suggests that a transcriptional mechanism of regulation of the alpha-MHC gene may account for the phenotypic expression observed in the unloaded myocardium.
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PMID:Posttranscriptional modification of myosin heavy-chain gene expression in the hypertrophied rat myocardium. 815 71

Serial deletion constructs derived from the 5'-flanking regions of the human cardiac alpha- and beta-myosin heavy chain genes were generated by polymerase chain reaction (PCR) amplifications. Generation of different length chimeric constructs were based on the complete sequence of the human cardiac myosin heavy chain genes. The primers were synthesized with HindIII and BamH1 sites and were linked to any designed nucleotide of the 5' flanking sequence of the myosin heavy chain gene(s). Following the PCR amplification and the site-directed mutagenesis, the PCR products were verified by DNA sequencing and subsequently ligated to the chloramphenicol acetyltransferase (pBLCAT3) reporter gene which was restricted with Hind III and BamH1. Neonatal rat cardiocytes were used to assay the promotor activity (i.e. CAT activity) of different lengths of the chimeric constructs of the gene.
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PMID:Serial deletion constructs of human cardiac myosin heavy chain genes generated by PCR amplification. 823 79

The role of two putative, cis-acting thyroid hormone-responsive elements, TRE1 and TRE2, located at -129 to -149 and -102 to -120, respectively, on the murine alpha-myosin heavy chain (MHC) gene, has been investigated in transgenic mice. These motifs are present in a 4.5-kilobase fragment lying upstream of the transcriptional start site of the mouse alpha-MHC gene: this fragment directs appropriate expression of a reporter gene in transgenic mice (Subramaniam, A., Jones, W. K., Gulick, J., Wert, S., Neumann, J., and Robbins, J. (1991) J. Biol. Chem. 266, 24613-24620). Here, we independently mutate the TRE1 and TRE2 elements by base substitution. The mice were analyzed for transgene expression in different muscle and non-muscle tissues including the atria and ventricles. Normal levels of transgene expression were observed in euthyroid mice carrying a mutation in TRE1. In contrast to these results, mice in which TRE2 was mutated showed reduced levels of CAT activity in both the atria and ventricles, suggesting a previously undefined role for this element in the constitutive up-regulation of the alpha-MHC gene. In hypothyroid mice carrying either of these mutations, the complete cessation of ventricular expression of the chloramphenicol acetyltransferase transcripts that takes place in the alpha-5.5 (wild type) animals did not occur.
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PMID:Transgenic analysis of the thyroid-responsive elements in the alpha-cardiac myosin heavy chain gene promoter. 844 Jul 18

The 5' upstream region of the murine beta-myosin heavy chain (MHC) gene has been isolated and tested for its ability to drive gene expression in transgenic mice. Three classes of transgenic mice were generated. The constructs contained approximately 5000, 2500, and 600 base pairs of beta-MHC upstream sequence fused to the chloramphenicol acetyltransferase gene and were termed beta 5, beta 2.5, and beta .6, respectively. Muscle-specific expression was observed with all three constructs. However, only the beta 5 lines directed high levels of muscle-specific transgene expression in both pre- and postbirth mice. Expression driven by the two shorter constructs was two to three orders of magnitude lower when the chloramphenicol acetyltransferase specific activities were compared. These data suggest that a distal-positive element directs high levels of gene expression in the ventricle and in slow skeletal muscles. Analyses of transgene expression during heart maturation revealed that some of the beta 5 lines were not able to respond in an appropriate manner to developmental transcriptional cues. Unlike the endogenous beta-MHC gene, which is down regulated in the ventricles around the time of birth, reporter gene expression in the majority of the lines generated was not shut off in the ventricles of the adult animals. These data indicate that high levels of muscle-specific beta-MHC gene expression are dependent upon the combinatorial interactions of a number of sequence elements that are distributed over a large region of the gene's upstream sequence.
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PMID:In vivo analysis of the murine beta-myosin heavy chain gene promoter. 844 7

The intergenic region between the mouse alpha-cardiac myosin heavy chain and beta-myosin heavy chain genes has previously been shown to direct expression of the bacterial chloramphenicol acetyltransferase reporter gene in transgenic mice in a tissue-specific manner. Sequence analyses located a putative myocyte-specific enhancer-binding factor (MEF-2) site situated in the regulatory region of this gene proximal to the start site of transcription. The role of this element in directing the cardiac compartment-specific expression of the transgene was assessed. The polymerase chain reaction was used to perform substitution mutagenesis of the MEF-2 binding site, and lack of MEF-2 binding was confirmed by gel retardation assays. The resultant construct was used to generate transgenic mice. Surprisingly, transgene expression was not down-regulated, but was significantly increased in the hearts of the MEF-2 mutant mice. In addition, cardiac-specific expression of the transgene was perturbed with significant levels of ectopic expression occurring in the aorta.
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PMID:Role of myocyte-specific enhancer-binding factor (MEF-2) in transcriptional regulation of the alpha-cardiac myosin heavy chain gene. 844 97

Thyroid hormone exerts marked effects on cardiovascular function. Expression of cardiac alpha- and beta-myosin heavy chain (MHC) isoforms can be altered in response to thyroid hormone as well as by hemodynamic changes imposed on the heart. The molecular mechanisms that mediate these changes are not completely known. We studied the contractile and thyroid hormone responsiveness of the betaMHC promoter in both cultured cardiac myocytes and in vivo by direct DNA transfer. Using transient transfection of neonatal rat cardiomyocytes, the activities of recombinant reporter plasmids containing betaMHC 5'-flanking sequences terminating at positions -2250, -1145, -670, and -354 were decreased significantly in cultures containing L-T3 (50 nM). Similar deletion analysis showed that 5'-flanking regions terminating within -2250 to -151 bp were contractility responsive; however, deletion to position -126 attenuated this response. In vivo betaMHC promoter activity, determined by injecting the recombinant plasmid into the myocardium, was significantly higher by 2-fold in hyperthyroid than in euthyroid ventricles (2.47 +/- 0.41 vs. 1.33 +/- 0.25 luciferase/ chloramphenicol acetyltransferase; P<0.05). Increased ventricular workload, produced by aortic coarctation for 5 days, resulted in ventricular hypertrophy (heart/body weight, 4.05 +/- 0.19 vs. 3.42 +/- 0.16 mg/g; P < 0.02) and a 3.4-fold increase in betaMHC messenger RNA content. However, betaMHC promoter activity in vivo was not significantly different between rats experiencing aortic coarctation and sham-operated rats (1.49 +/- 0.41 vs. 0.96 +/- 0.27 luciferase chloramphenicol acetyltransferase, respectively) and was similar to that in euthyroid animals. These results show that betaMHC promoter activity is T3 responsive in cultured myocytes and in vivo, but that the increase in betaMHC messenger RNA observed in the in vivo pressure overloaded myocardium cannot be explained entirely by transcription control mechanisms.
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PMID:Thyroid hormone and hemodynamic regulation of beta-myosin heavy chain promoter in the heart. 860 87


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