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)

Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms.
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PMID:Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice. 766 7

The beta-myosin heavy chain (beta-MyHC) gene is expressed in cardiac and slow skeletal muscles. To examine the regulatory sequences that are required for the gene's expression in the two compartments in vivo, we analyzed the expression pattern of a transgene consisting of the beta-MyHC gene 5' upstream region linked to the chloramphenicol acetyltransferase reporter gene. By using 5600 bp of 5' upstream region, the transgene was expressed at high levels in the slow skeletal muscles. Decreased levels of thyroid hormone led to the up-regulation of the transgene in both cardiac and skeletal muscles, mimicking the behavior of the endogenous beta-MyHC gene. After deleting the distal 5000 bp, the level of reporter gene expression was strongly reduced. However, decreased levels of thyroid hormone led to an 80-fold skeletal muscle-specific increase in transgene expression, even upon the ablation of a conserved cis-regulatory element termed MCAT, which under normal (euthyroid) conditions abolishes muscle-specific expression. In contrast, cardiac-specific induction was not detected with the deletion construct. These observations indicate that the cardiac and skeletal muscle regulatory elements can be functionally segregated on the beta-MyHC gene promoter.
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PMID:Segregation of cardiac and skeletal muscle-specific regulatory elements of the beta-myosin heavy chain gene. 787 16

The interactions of trans-acting factors with their respective cis-acting elements in the 5' upstream region of the beta myosin heavy chain gene (MyHC) regulate its tissue- and developmental stage-specific expression. The role of three conserved elements, an MCAT or TEF-1 binding site, a C-rich region, and a beta e3 region, in muscle-specific gene expression was analyzed in vivo. Each cis-acting site was ablated in the context of the beta MyHC promoter, fused to the chloramphenicol acetyltransferase reporter gene, and used to generate transgenic mice. In contrast to results obtained in vitro, the data demonstrate that mutating any one of these cis-acting elements does not affect the level or tissue specificity of transgene expression. Sequences upstream of -600 can functionally substitute for any one of these regulatory cassettes and are important both for high levels of expression as well as for controlled muscle specificity. Mutation of any two of the cis-acting elements also does not affect transgene expression. However, simultaneous mutation of the three sites significantly reduces expression, indicating that these conserved sequences do play an important role and that combinatorial interactions underlie the beta MyHC's regulation.
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PMID:In vivo regulation of the mouse beta myosin heavy chain gene. 798 72

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

We have investigated the regulated expression of genes injected into the heart of large mammals in situ. Reporter constructs using the chloramphenicol acetyltransferase gene under the control of muscle-specific beta-myosin heavy chain (beta-MHC) or promiscuous (mouse sarcoma virus) promoters were injected into the canine myocardium. There was a linear dose-response relation between the level of gene expression and the quantity of plasmid DNA injected between 10 and 200 micrograms per injection site. The level of reporter gene expression did not correlate with the amount of injury imposed on the cardiac tissue. There was no regional variation in expression of injected reporter genes throughout the left ventricular wall. By use of both the mouse sarcoma virus and a muscle-specific beta-MHC promoter, reporter gene expression was one to two orders of magnitude greater in the heart than in skeletal muscle. Expression in the left ventricle was threefold higher than in the right ventricle. Chloramphenicol acetyltransferase activity was detected at 3, 7, 14, and 21 days after injection, with maximal expression at 7 days after injection. Statistical analysis of coinjection experiments revealed that coinjection of a second gene construct (Rous sarcoma virus-luciferase) is useful in the control of transfection efficiency in vivo. Furthermore, using reporter constructs containing serial deletions of the 5' flanking region of the beta-MHC gene, we performed a series of experiments that demonstrate the utility of this model in mapping promoter regions and identifying important regulatory gene sequences in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Gene injection into canine myocardium as a useful model for studying gene expression in the heart of large mammals. 843 91

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

We recently reported that interleukin-1beta (IL-1beta) induces a novel form of cardiac myocyte hypertrophy characterized by an increase in protein content but an absence of the fetal program of skeletal alpha-actin or beta-myosin heavy chain (beta-MHC) gene expression (Palmer, J. N., Hartogensis, W. E., Patten, M., Fortuin, F. D., and Long, C. S. (1995) J. Clin. Invest. 95, 2555-2564). Because of the apparent disparity between this myocardial phenotype and that seen with other hypertrophic agents in culture, such as catecholamines, we investigated the effect of IL-1beta on alpha1-induced cardiomyocyte hypertrophy. Although there was no augmentation in total protein when IL-1beta and phenylephrine were given simultaneously, IL-1beta attenuated the increase in contractile protein mRNAs (skeletal alpha-actin and beta-MHC) in response to phenylephrine. Transient transfection studies with skeletal alpha-actin and beta-MHC promoter constructs linked to the chloramphenicol acetyltransferase (CAT)-reporter gene indicate that repression occurred at the level of gene transcription. In view of the previously reported activity of the zinc finger protein YY1 in the negative regulation of the skeletal alpha-actin promoter in cardiomyocytes (MacLellan, W. R., Lee, T. C., Schwartz, R. J., and Schneider, M. D. (1994) J. Biol. Chem. 269, 16754-16760), we investigated the potential role of this factor in the IL-1beta-mediated effects. Using transient transfection, we found that a mutation in the YY1 binding site of the skeletal alpha-actin promoter abolished the inhibitory effect of IL-1beta. We further found that the 127-base pair fragment of the skeletal alpha-actin promoter required for the IL-1beta effect is also required for inhibition by the overexpression of YY1 in the myocytes. Furthermore, increased levels of YY1 protein are found in IL-1beta treated myocytes. Taken together these results suggest that the repression of contractile protein gene transcription by IL-1beta may be due, at least in part, to activation of the negative transcription factor YY1.
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PMID:Interleukin-1beta is a negative transcriptional regulator of alpha1-adrenergic induced gene expression in cultured cardiac myocytes. 870 83

The molecular mechanisms underlying heart and skeletal muscle-specific gene expression during development and in response to physioloic stimuli are largely unknown. Using a novel immunohistochemical procedure to detect chloramphenicol acetyltransferase (CAT), we have investigated, in vivo at high resolution, the ability of cis-acting DNA sequences within the 5' flanking region of the mouse beta myosin heavy chain (MyHC) gene (beta-MyHC) to direct appropriate gene expression throughout development. A 5.6-kb fragment 5' to the beta-MyHC's transcriptional start site was linked to the reporter gene encoding CAT (cat) and used to generate transgenic mice. The anti-CAT in situ assay described in this report allowed us to define the ability of the promoter fragment to direct appropriate temporal, tissue- and muscle fiber type-specific gene expression throughout early development. In skeletal muscles, the transgene expression profile mimics the endogenous beta-myHC's at all developmental stages and is appropriately restricted to slow (type I) skeletal fibers in the adult. Surprisingly, transgene expression was detected in both the atria and ventricles during embryonic and fetal development, indicating that ventricular specification involves elements outside the 5.6-kb fragment. In contrast, in the adult, hypothyroid conditions led to transgene induction specifically in the ventricles, suggesting that distinct regulatory mechanisms control fetal versus adult beta-MyHC expression in the cardiac compartment.
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PMID:Developmental Modulation of a beta myosin heavy chain promoter-driven transgene. 872 85


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