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)

Gene transfer can be achieved in the adult rat heart in vivo by direct injection of plasmid DNA. In this report we define the spatial and temporal limits of reporter gene expression after a single intracardiac injection. pRSVCAT (100 micrograms), in which the Rous sarcoma virus long terminal repeat is fused to the chloramphenicol acetyltransferase reporter gene, and p alpha MHCluc (100 micrograms), in which the alpha-cardiac myosin heavy chain promoter is fused to the firefly luciferase gene, were injected into hearts, and reporter gene activities were assayed at various times. Both chloramphenicol acetyltransferase and luciferase were detectable in 100% of the rats from 1 to 7 days, in 60% of the rats from 17 to 23 days, and in 30% of the rats from 38 to 60 days after injection. Reporter gene activity was largely limited to a 1-2-mm region of the ventricle surrounding the injection site. Closed circular DNA was far more effective than linear DNA in transfecting cells in vivo. The relative strengths of three different promoters, Rous sarcoma virus long terminal repeat, alpha-myosin heavy chain, and alpha 1-antitrypsin, all fused to the luciferase reporter gene were determined. The constitutive viral promoter was approximately 20-fold more active than the cardiac-specific cellular promoter, and the liver-specific cellular promoter was not active at all in the cardiac environment. Thus, direct injection of genes into the heart offers a simple and powerful tool with which to assess the behavior of genes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Behavior of genes directly injected into the rat heart in vivo. 130 14

The effect of endothelin-1 on cardiac myosin heavy chain gene expression was examined using an isolated neonatal rat myocardial cell culture system. The effects of endothelin-1 on the expression of alpha- and beta- myosin heavy chain genes in the primary rat myocardial cell culture system were examined by S1 nuclease protection analysis. Endothelin-1 was found to stimulate both alpha- and beta- myosin heavy chain gene expression. The 5' flanking regions of both the alpha- and beta- myosin heavy chain gene promoters ligated to a reporter gene, chloramphenicol acetyltransferase, were used to study the effect of endothelin-1 on transcription. Myocardial cells treated with endothelin-1 increased the transcription rate of alpha- and beta- myosin heavy chain genes in a dose-dependent manner. Thus, the hypertrophic effect of endothelin-1 on cardiac myocytes involves augmentation of alpha- and beta- myosin heavy chain gene expression by increasing gene transcription.
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PMID:Endothelin stimulates cardiac alpha- and beta- myosin heavy chain gene expression. 156 2

The 5' flanking region of the rabbit myosin heavy chain (HC) beta gene extending 295 bp upstream from the cap site provides muscle-specific transcriptional activity. In this study, we have identified and functionally characterized cis-acting elements that regulate the muscle-specific expression within this region. By using linker-scanner (LS) mutants between -295 bp and a putative TATA box, we found five distinct positive cis-acting sequences necessary for transcription: element A, the sequences between -276 and -263, which contains a putative M-CAT motif in an inverted orientation; B, the sequences between -207 and -180; C, the sequences between -136 and -127; D, the sequences between -91 and -80; and E, a TATA consensus sequence at -28. The fragment containing both A and B elements dramatically enhanced the expression of the chloramphenicol acetyltransferase (CAT) gene driven by a heterologous promoter in differentiated muscle cells, whereas fragments containing either A or B elements alone had little or no effect in either muscle or nonmuscle cells. Therefore, these two elements appear to act cooperatively in determining a high level of muscle- and stage-specific expression. Unlike the typical enhancer element, this region functions in an orientation-dependent manner. In contrast, the fragment containing C and D elements activates the heterologous promoter in both muscle and nonmuscle cells in an orientation-independent manner.
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PMID:cis-acting elements responsible for muscle-specific expression of the myosin heavy chain beta gene. 157 72

The effects of thyroid hormone on expression of cardiac myosin heavy chain genes generally are thought to be mediated by nuclear 3,5,3'-triiodo-L-thyronine (T3) receptors that have been identified as the products of the protooncogene, c-erbA. This hypothesis has been tested by transfection of cardiomyocytes in primary culture with a plasmid, pRSVhEACAT-, expressing anti-sense c-erbA mRNA. Because only a low percentage of cells (20%) could be transfected in primary culture an alpha-myosin heavy chain-chloramphenicol acetyltransferase fusion construct was used as a reporter gene. The results indicate that the anti-sense plasmid almost completely blocks T3-induced activity of the reporter gene (less than 1% control) while transfection of a similar amount of the sense construct, pRSVhEACAT+, has no effect. When the c-erbA plasmids were cotransfected with constructs containing T3-independent promoters, no effects on expression were observed. The combined use of an anti-sense construct and a report gene provides a means of studying the role of c-erbA products in intracellular signal transduction even in differentiated, nondividing cells like those of the heart.
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PMID:An anti-sense c-erbA clone inhibits thyroid hormone-induced expression from the alpha-myosin heavy chain promoter. 169 Jul 29

The intergenic region between the mouse alpha-myosin heavy chain (MHC) and beta-MHC genes was analyzed in terms of its ability to drive gene expression in transgenic mice. Earlier, we reported that the entire intergenic region was sufficient to direct expression of the bacterial chloramphenicol acetyl transferase reporter gene in a tissue-specific and developmental stage-specific manner. Additional transgenic lines have been generated which include two deletions. The first deletion, alpha-3, which lacks the distal 2.5 kilobase pairs of the upstream region, is competent to direct tissue- and developmental-specific expression of the transgene. A larger deletion, in which only 138 base pairs upstream of the transcriptional start site remain, shows no chloramphenicol acetyltransferase activity in either muscle or non-muscle tissue. Tissue surveys of transgene expression indicated low levels of activity in the lung, and analyses via the polymerase chain reaction confirmed the presence of the endogenous alpha-MHC gene transcripts in this tissue. Subsequently, an alpha-MHC gene-specific riboprobe was used to detect the cognate transcripts in lung sections by in situ hybridization. The data show that, in the lung, the transcripts are localized to the thick intimal wall of the veins and venules.
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PMID:Tissue-specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice. 172 8

The mRNA encoding the sarcoplasmic reticulum (SR) Ca2+ ATPase is highly influenced by thyroid hormone (T3) in the hearts of intact animals. We show here that this effect of T3 can be mimicked in primary neonatal rat cardiocytes, both in serum-containing and in serum-free media; the expression of SR Ca2+ ATPase mRNA is myocyte-specific and is also modulated by retinoic acid (RA). RA also induces myosin heavy chain (MHC) alpha-mRNA in this system. The induction of Ca2+ ATPase mRNA is sensitive to T3 (EC50 approximately 30 pM) and less sensitive to RA (EC50 approximately 2 nM). Transient transfection experiments utilizing various segments of the Ca2+ATPase promoter fused to the reporter gene chloramphenicol acetyltransferase (CAT) indicate a minimal thyroid hormone response element (TRE) between nucleotides -262 and -322, while sequences between -322 and -559 are required for maximal trans-activation. RA is not able to regulate these constructs. Likewise, a clear effect of T3 but no effect of RA was observed when the CAT gene was driven by a TRE derived from the rat alpha-MHC gene. In contrast, CAT expression was induced by either hormone when placed under the control of a synthetic palindromic TRE. Taken together, these results indicate that T3 and RA induce gene expression in primary cardiac myocytes, but through distinct response elements and/or mechanisms.
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PMID:Influence of thyroid hormone and retinoic acid on slow sarcoplasmic reticulum Ca2+ ATPase and myosin heavy chain alpha gene expression in cardiac myocytes. Delineation of cis-active DNA elements that confer responsiveness to thyroid hormone but not to retinoic acid. 182 23

We report here for the first time direct injection of genes into fish muscle in vivo. Plasmids used contain either SV40 early promoter, rabbit beta-cardiac myosin heavy chain promoter, human MxA promoter or an artificial promoter, fused to a chloramphenicol acetyltransferase (CAT) or beta-galactosidase reporter gene. CAT assays revealed that most gene constructs were highly expressed. Histochemical analysis showed that beta-galactosidase was strongly expressed at the site of injection within muscle fibres. This method provides an excellent system for testing expression of gene constructs, including those of mammalian origin, in fish muscle in vivo and has the potential for fish vaccination.
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PMID:Strong expression of foreign genes following direct injection into fish muscle. 191 96

Two mouse genomic libraries were probed in order to isolate the murine cardiac myosin heavy chain (MHC) genes. Two overlapping cosmid clones that encode the cardiac genes were isolated. One of them encompasses the entire alpha-cardiac MHC gene, its 5'-flanking region and approximately 10 kilobase pairs (kb) of the 3'-end of the beta-cardiac MHC gene which we determined is located approximately 4 kb upstream of the alpha-cardiac MHC gene. Four clones isolated from a bacteriophage library were found to overlap with the 5'-region of the cosmid clones, and sequence analysis confirmed that the 5'-end of the beta-cardiac MHC gene was contained within one of the clones. Primer extension and polymerase chain reaction (PCR) analyses were used to define the transcriptional start site and the 5'-organization of the alpha-cardiac MHC gene. This region exhibits greater than 90% homology to the corresponding nucleotides of the rat alpha-cardiac MHC gene. To assess the importance of the intergenic region in directing expression of the alpha-cardiac MHC gene, a fragment containing the 3'-end of the beta-cardiac gene and the 5'-end of the alpha-cardiac gene was linked to a chloramphenicol acetyltransferase gene and used to generate transgenic mice. Analyses of the chloramphenicol acetyltransferase (CAT) activity in two lines indicate that the intergenic region is sufficient to properly direct expression in a tissue-specific manner.
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PMID:Isolation and characterization of the mouse cardiac myosin heavy chain genes. 202 17

Cultured neonatal rat cardiac myocytes express at least three isozymes of protein kinase C (PKC), and two PKC isozymes are translocated to different intracellular sites on activation with alpha 1-adrenergic agonists or phorbol myristate acetate. Differential intracellular localization upon activation was compatible with differential function, and we therefore asked whether PKC isozymes had distinct roles in regulating transcription of the cardiac myosin heavy chain (MHC) genes. Cardiac myocytes were transfected with chloramphenicol acetyltransferase reporter plasmids containing the promoters of the beta-MHC or alpha-MHC isogenes. An alpha 1-adrenergic agonist stimulated the beta-MHC promoter by 3-fold but had no effect on the alpha-MHC promoter. This pattern of MHC promoter regulation by an alpha 1 agonist was the same as that found previously for the endogenous MHC mRNAs in this model system. Myocytes were then co-transfected with the beta- or alpha-MHC-chloramphenicol acetyltransferase plasmids and expression plasmids encoding wild-type or constitutively activated mutants of the alpha- and beta-isozymes of PKC. Co-transfection with wild-type alpha-PKC or wild-type beta-PKC did not stimulate the beta-MHC promoter, and none of the expressed PKCs affected the alpha-MHC promoter. However, the constitutively activated mutant of beta-PKC stimulated the beta-MHC promoter by 8-fold, whereas stimulation by the activated alpha-PKC mutant was only 40% as great (3-fold). In contrast, the constitutively activated alpha-PKC and beta-PKC mutants were equally potent in stimulating a reporter plasmid containing AP-1 recognition sequences. All transfected PKCs were expressed equally in the myocytes, as judged by immunofluorescence. These data indicate that transcription of the beta-MHC isogene is stimulated preferentially by beta-PKC in cardiac myocytes and provide direct evidence for differential functions of alpa-PKC and beta-PKC in transcriptional regulation.
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PMID:Expression of a constitutively activated mutant of the beta-isozyme of protein kinase C in cardiac myocytes stimulates the promoter of the beta-myosin heavy chain isogene. 203 58

The organization of the cis-acting regulatory elements of a chick myosin heavy chain gene has been investigated. The data show that a gene which is transcribed in vivo in the fast white embryonic musculature is also the major transcript expressed during myotube differentiation of primary myoblasts derived from 12-day embryonic chick leg muscles. The upstream region of this gene consists of 7500 base pairs, and we have tested the ability of these sequences to drive expression of the chloramphenicol acetyltransferase gene in developing primary muscle cultures. Deletion analyses of the upstream region show that negative regulatory elements are present within 2000 base pairs of the basal promoter elements, the CCAAT and TAATA boxes. Removal of these elements reveals the presence of a strong positive element located near the start site of transcription. Sequence analysis showed that the region also contains a sequence characteristic of an enhancer found in the immunoglobulin heavy chain gene, ATGCAAAT, the "octa" element. Gel band-shift assays show that this octa sequence binds a transacting factor present in muscle nuclear extracts, although footprint analysis indicates a limited interaction. Transient assays carried out with a fragment in which the octa sequence has been mutated, with the subsequent abolition of protein binding, shows that the particular interaction probably plays a role in negatively modulating the action of the strong positive promoter element.
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PMID:Analysis of the upstream regulatory region of a chicken skeletal myosin heavy chain gene. 211 12


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