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 isolated an avian muscle cell line (QM) which has the essential features of established mammalian muscle cell lines. The experiments reported here were undertaken to determine the suitability of QM cells for the introduction and analysis of cloned transgenes. The promoter of the cardiac troponin T (cTNT) gene has been previously shown to contain sequence elements which govern muscle-specific expression of the chloramphenicol acetyltransferase (CAT) gene in transiently transfected primary cell cultures. We show here that QM cells stably harboring cTNT promoter-CAT fusion genes up-regulate CAT expression in concert with myogenic differentiation, and that as few as 110 upstream nucleotides are sufficient for such differentiation-dependent regulation. In addition, both transient and stable transfection experiments demonstrate that differentiated QM cells possess trans-acting factors necessary for the expression of the skeletal alpha-actin promoter, despite the absence of mRNA or protein product from the endogenous sarcomeric actin genes in these cells. Finally, to follow the developmental potential of QM cells in vivo, we created a clone, QM2ADH, which constitutively expresses the histochemical marker transgene encoding Drosophila alcohol dehydrogenase. When surgically inserted into the limb buds of developing chick embryos, QM2ADH cells are incorporated into endogenous developing muscles, indicating that QM cells are capable of recognizing and responding to host cues governing muscle morphogenesis. Thus, QM cells are versatile as recipients of transgenes for the in vitro and in vivo analysis of molecular events in muscle development.
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PMID:Transgene expression in the QM myogenic cell line. 198 14

The chicken gene encoding cardiac troponin T (cTNT) is expressed in both cardiac and skeletal muscle during early embryonic development, but is specifically repressed in skeletal muscle during fetal development. To determine if the cis-acting sequences governing transcription of a single gene in these two related cell types are the same, we have transfected promoter/upstream segments of the cTNT gene coupled to the bacterial chloramphenicol acetyltransferase gene into primary cultures of early embryonic cardiac and skeletal muscle cells. Using this assay system, chloramphenicol acetyltransferase activity directed by the cTNT promoter/upstream region was between two and three orders of magnitude higher in cardiac or skeletal muscle cells than in fibroblast cells, indicating that cis elements responsible for cell-specific expression reside in this region of the cTNT gene. Deletion experiments showed that a 67-nucleotide DNA segment residing between 268 and 201 nucleotides upstream of the cTNT transcription initiation site is required for cTNT promoter activity in embryonic cardiac cells. This region is not required in embryonic skeletal muscle cells because a cTNT promoter construction containing only 129 upstream nucleotides is transcriptionally active in these cells. These results demonstrate that different cis-acting sequences are required for cTNT expression in early embryonic cardiac and skeletal muscle cells. Nonessential regions residing farther upstream, on the other hand, affected the level of expression of these minimum regions in a similar manner in both cell types. The data from these experiments indicate, therefore, that transcription of the cTNT promoter in early embryonic cardiac and skeletal muscle cells is governed both by common and divergent regulatory elements in cis and in trans.
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PMID:Analysis of the upstream regions governing expression of the chicken cardiac troponin T gene in embryonic cardiac and skeletal muscle cells. 304 42

To study the transcriptional regulation of the rat cardiac troponin T (cTnT) gene, chimeric genes composed of the upstream region (-757 to +193 base pairs (bp) relative to the transcription initiation site) of the cTnT gene and the bacterial chloramphenicol acetyltransferase (CAT) gene were constructed and transfected into primary cultures of neonatal cardiomyocytes and cardiac fibroblasts. Deletion analysis showed that a 41-bp fragment (-249 to -209 bp) containing the MEF-2-like motif is an essential element for minimal cardiac-specific expression of the rat cTnT gene. The proximal promoter (-208 to -1 bp) contains two consensus CArG boxes, one M-CAT motif, one AP2 site, and one TATA box. The construct (cTNT-208) composed of the CAT reporter gene driven by this proximal promoter did not show cardiac muscle-specific expression. Ligation of consensus MEF-2-like sequence into the upstream of this chimera only partially increased its ability to express in cardiomyocytes. These results suggest that the spacing among MEF-2-like motif and proximal promoter and/or the flanking sequences of the MEF-2-like motif are important in determining cardiac muscle-specific expression. By footprint analysis with a DNA fragment (-303 to +6 bp), we identified three novel regions (called A, B, and C) protected by protein extract from rat hearts, in addition to the known motifs such as MEF-2, M-CAT, and CArG. Gel retardation with the probe (-235 to -141 bp), containing the MEF-2-like motif, one of the CArG boxes, and the C region, or the 41-bp probe (-249 to -209 bp), containing the MEF-2-like motif, revealed different DNA-protein complexes formed by heart, skeletal muscle, and liver extracts. By using DNA affinity purification, DNA-binding proteins with apparent molecular masses of 22-26 kDa were identified from rat heart extract but not from skeletal and liver extracts, suggesting the involvement of cardiac-specific proteins in regulating the cTnT gene expression.
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PMID:Characterization of cis-regulating elements and trans-activating factors of the rat cardiac troponin T gene. 798 78

The M-CAT binding factor transcription enhancer factor 1 (TEF-1) has been implicated in the regulation of several cardiac and skeletal muscle genes. Previously, we identified an E-box-M-CAT hybrid (EM) motif that is responsible for the basal and cyclic AMP-inducible expression of the rat cardiac alpha-myosin heavy chain (alpha-MHC) gene in cardiac myocytes. In this study, we report that two factors, TEF-1 and a basic helix-loop-helix leucine zipper protein, Max, bind to the alpha-MHC EM motif. We also found that Max was a part of the cardiac troponin T M-CAT-TEF-1 complex even when the DNA template did not contain an apparent E-box binding site. In the protein-protein interaction assay, a stable association of Max with TEF-1 was observed when glutathione S-transferase (GST)-TEF-1 or GST-Max was used to pull down in vitro-translated Max or TEF-1, respectively. In addition, Max was coimmunoprecipitated with TEF-1, thus documenting an in vivo TEF-1-Max interaction. In the transient transcription assay, overexpression of either Max or TEF-1 resulted a mild activation of the alpha-MHC-chloramphenicol acetyltransferase (CAT) reporter gene at lower concentrations and repression of this gene at higher concentrations. However, when Max and TEF-1 expression plasmids were transfected together, the repression mediated by a single expression plasmid was alleviated and a three- to fourfold transactivation of the alpha-MHC-CAT reporter gene was observed. This effect was abolished once the EM motif in the promoter-reporter construct was mutated, thus suggesting that the synergistic transactivation function of the TEF-1-Max heterotypic complex is mediated through binding of the complex to the EM motif. These results demonstrate a novel association between Max and TEF-1 and indicate a positive cooperation between these two factors in alpha-MHC gene regulation.
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PMID:Transcription enhancer factor 1 interacts with a basic helix-loop-helix zipper protein, Max, for positive regulation of cardiac alpha-myosin heavy-chain gene expression. 919 27

To investigate the underlying mechanism regulating cardiac gene expression, transgenic mice carrying the rat cardiac troponin T proximal promoter (-497 bp from the transcriptional start site) fused to a LacZ or chloramphenicol acetyltransferase (CAT) reporter gene were analyzed. The LacZ expression pattern throughout development was very similar to that of the endogenous cardiac troponin T gene. Within this promoter, a high degree of sequence homology was found at 2 sites, modules D (-335 to -289 bp) and F (-249 to -209 bp). Both regions contain at least a TCTG(G/C) direct repeat and an A/T-rich site, whereas only the F module has a muscle enhancer factor 2 (MEF2)-like motif. No significant decrease in CAT transgene expression was observed when only the MEF2 core sequence was mutated. However, when the MEF2 core sequence and its flanking TCTGG site were mutated (Mut5), CAT transgene expression was significantly decreased in the heart, and ectopic expression of the transgene was also observed. When mutations were introduced into this promoter to destroy all upstream TCTG(G/C) direct repeats in the D module (MutD), CAT expression remained cardiac specific, but the expression level was dramatically decreased. Relaxation of cardiac-specific transgene expression became even more severe in transgenic mice carrying double mutations (Mut[D+5]). In addition, CAT activity in the heart was nearly abolished. These results suggest that D and F modules have an additive function in determining the level of expression in the heart and only the F module confers cardiac-specific expression.
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PMID:Identification of cis elements in the cardiac troponin T gene conferring specific expression in cardiac muscle of transgenic mice. 1070 Apr 54