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)

The expression of the nicotinic acetylcholine receptor (AChR) in vertebrate striated muscle is regulated both during development by nerve-evoked muscle activity and by local factors released or associated with the nerve ending. The expression pattern of AChR is achieved by coordinate regulation of four embryonic subunit mRNAs, alpha, beta, gamma and delta. We have taken the approach of identifying the similarities and differences among cis-acting regulatory elements of AChR genes to gain a better understanding of these mechanisms. Thus, to begin to define DNA sequences necessary for the transcriptional regulation of the mouse beta AChR gene, we have analyzed its 5'-flanking region. Primer extension and RNAase protection analyses showed that transcription initiates at one major and two minor sites, all of which are close to the translational initiation site. Using plasmids in which segments of the 5'-flanking region were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, we have demonstrated that 150 bp of the 5'-flanking region is active in C2 myotubes but not C2 myoblasts or NIH3T3 fibroblasts. This region contains a putative binding site for myoD, and when linked to CAT was transactivated by the muscle regulatory factors myoD, myogenin, and MRF4. Thus, a 150 bp sequence of the beta-subunit gene contains information necessary for developmental specificity and responsiveness to myogenic factors.
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PMID:The 5'-flanking region of the mouse muscle nicotinic acetylcholine receptor beta subunit gene promotes expression in cultured muscle cells and is activated by MRF4, myogenin and myoD. 131 51

The expression of the nicotinic acetylcholine receptor (AChR) in vertebrate striated muscle is regulated both during development and in response to nerve-evoked muscle activity. To define DNA sequences necessary for the transcriptional regulation of the mouse alpha-subunit AChR gene, we have isolated and analyzed the alpha-gene 5'-flanking region. Primer extension and RNase protection analysis showed that transcription initiates at 2 major and 12 minor sites close to the translational initiation site. Using a series of plasmids in which segments of the 5'-flanking region were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, we have defined an 86-base pair enhancer sequence that is active in C2 myotubes but not in C2 myoblasts or NIH3T3 fibroblasts. This enhancer contains three putative binding sites for myoD1, and the 5'-upstream regions linked to CAT were transactivated by the muscle regulatory factors, myoD1, and myogenin. Transactivation by MRF4 differed with the specific alpha-subunit construct tested. Whereas the alpha-subunit CAT constructs containing both the homologous as well as the heterologous myosin light chain 1 promoter were transactivated by myoD1 and myogenin, only the constructs containing their homologous promoter were transactivated by MRF4. Thus, an 86-base pair sequence of the alpha-subunit gene contains the information necessary for developmental specificity and responsiveness to myogenic factors.
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PMID:A developmental and tissue-specific enhancer in the mouse skeletal muscle acetylcholine receptor alpha-subunit gene regulated by myogenic factors. 165 1

Using the basic helix-loop-helix domain of the myogenic factor myogenin as a probe, we identified a clone from a sea urchin cDNA library with considerable sequence similarity to the vertebrate myogenic factors. This cDNA, sea urchin myogenic factor 1 (SUM-1), transactivated a muscle creatine kinase-chloramphenicol acetyltransferase reporter gene in 10T1/2 fibroblasts to a level comparable to that of the vertebrate myogenic factors. In addition, bacterially expressed beta-galactosidase-SUM-1 fusion protein interacted directly with the kappa E-2 site in the muscle creatine kinase enhancer core as assayed by electrophoretic mobility shift assays. Stably transfected SUM-1 activated the muscle differentiation program and converted 10T1/2 cells from fibroblasts to myotubes. In sea urchin embryos, SUM-1 RNA was not detected before gastrulation. It accumulated to its highest levels during the prism stage when myoblasts were first detected by myosin immunostaining and then diminished as myocytes differentiated. SUM-1 protein was localized in secondary mesenchyme cells when they could first be identified as muscle cells by myosin immunostaining. These results implicate SUM-1 as a regulatory factor involved in the early decision of a pluripotent secondary mesenchyme cell to convert to a myogenic fate. SUM-1 is an example of an invertebrate myogenic factor that is capable of functioning in mammalian cells.
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PMID:A myogenic factor from sea urchin embryos capable of programming muscle differentiation in mammalian cells. 206 3

The mouse myosin light-chain 1A (MLC1A) gene, expressed in the atria of the adult heart, is one of the first muscle genes to be activated when skeletal as well as cardiac muscles form in the embryo. It is also transcribed in skeletal muscle cell lines at the onset of differentiation. Transient transfection assays of mouse skeletal muscle cell lines with DNA constructs containing MLC1A promoter fragments fused to the chloramphenicol acetyltransferase (CAT) gene show that the first 630 bp of the promoter is sufficient to direct expression of the reporter gene during myotube formation. Two E boxes located at bp -76 and -519 are necessary for this regulation. MyoD and myogenin proteins bind to them as heterodimers with E12 protein and, moreover, transactivate them in cotransfection experiments with the MLC1A promoter in nonmuscle cells. Interestingly, the effect of mutating each E box is less striking in primary cultures than in the C2 or Sol8 muscle cell line. A DNA fragment from bp -36 to -597 confers tissue- and stage-specific activity to the herpes simplex virus thymidine kinase promoter in both orientations, showing that the skeletal muscle-specific regulation of the MLC1A gene is under the control of a muscle-specific enhancer which extends into the proximal promoter region. At bp -89 is a diverged CArG box, CC(A/T)6AG, which binds the serum response factor (SRF) in myotube nuclear extracts, as does the wild-type sequence, CC(A/T)6GG. Both types of CArG box also bind a novel myotube-enriched complex which has contact points with the AT-rich part of the CArG box and adjacent 3' nucleotides. Mutations within the CArG box distinguish between the binding of this complex and binding of SRF; only SRF binding is directly involved in the specific regulation of the MLC1A gene in skeletal muscle cell lines.
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PMID:A skeletal muscle-specific enhancer regulated by factors binding to E and CArG boxes is present in the promoter of the mouse myosin light-chain 1A gene. 762 50

We have isolated and analysed the 5' flanking region of the rat acetylcholine receptor (AChR) beta subunit gene and determined regulatory elements that confer muscle specificity. Deletion mapping revealed a minimal TATA-box-less promoter region containing an initiator motif. An 85-bp fragment has been shown to promote high muscle-specific expression of a chloramphenicol acetyltransferase (CAT) reporter construct upon transfection in primary muscle cells. This sequence can be functionally dissected in a basal muscle-specific promoter element carrying a M-CAT box that is flanked at the 5' end by an enhancer element with two binding sites for myogenic factors. Point mutations in the M-CAT box cause the loss of transcriptional activity of the basal promoter fragment. The enhancer activity depends on the presence of both E boxes that cooperate in a synergistic fashion. We therefore conclude that the control of muscle-specific and developmental expression of the rat AChR beta subunit gene requires both regulatory elements, the M-CAT box and two adjacent E boxes, located in close proximity to each other. Cotransfection experiments in NIH3T3 cells demonstrate that the rat AChR beta subunit gene can be transactivated by myogenic factors displaying a preference for myogenin, as well as MRF4 and myf5 compared to a clearly weaker responsiveness to MyoD1.
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PMID:Two adjacent E box elements and a M-CAT box are involved in the muscle-specific regulation of the rat acetylcholine receptor beta subunit gene. 791 88

The nicotinic acetylcholine receptor (AChR) in adult skeletal muscle is composed of alpha-, beta-, epsilon-, and delta-subunits and is localized at the neuromuscular junction; in contrast, the more diffusely distributed fetal form is composed of alpha-, beta-, gamma-, and delta-subunits. To define sequences necessary for the transcriptional regulation of the mouse epsilon-subunit gene, we sequenced and analyzed 1036 bp upstream of the transcription start site. Using deletion analysis of the 5'-flanking region linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and transfection of the resulting constructs into established cell lines, we demonstrate that a 151 bp fragment exhibits cell type- and differentiation-specific promoter activity. This activity was independent of a myogenic factor putative binding site (E-box). However, transactivation experiments with recombinant myoD, myogenin, or MRF4 showed that the E-box was functional and that MRF4 preferentially transactivates the epsilon-promoter. Thus, like other AChR promoters, the proximal region of the epsilon-promoter contains information for cell type-specific and developmental regulation of CAT and can be transactivated by myogenic factors in cultured cell lines. Unlike the other AChR promoters characterized to date, epsilon-promoter function can be partially independent of myogenic factors of the helix-loop-helix class.
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PMID:Cell type- and differentiation-dependent expression from the mouse acetylcholine receptor epsilon-subunit promoter. 826 73

Myogenin, as well as other MyoD-related skeletal muscle-specific transcription factors, regulate a large number of skeletal muscle genes during myogenic differentiation. During later development, innervation suppresses myogenin expression in the fetal hind limb musculature. Denervation of skeletal muscle reverses the effects of the nerve, and results in the reactivation of myogenin expression, as well as of other embryonic muscle proteins. Here we report that myogenin upstream sequences confer tissue- and developmental-specific expression in transgenic mice harboring a myogenin/chloramphenicol acetyltransferase (CAT) reporter construct. Using in situ hybridization to analyze serial sections of E12.5 embryos, we found colocalization of CAT and endogenous myogenin transcripts in the primordial muscle of the head and limbs, in the intercostal muscle masses, and in the most caudal somites. Later in development, we observed that the expression of the transgene and endogenous myogenin gene continued to be restricted to skeletal muscle but decreased shortly after birth; a period that coincides with the innervation of secondary myotubes. Furthermore, denervation of the mouse hind limbs induced a 10-fold accumulation of CAT and endogenous myogenin transcripts by 1 day after sciatic nerve resection; a 25-fold increase was observed by 4 days after denervation. Interestingly, we observed that the accumulation of CAT enzyme activity lagged considerably with respect to the increase in CAT transcripts. Our results indicate that the cis-acting elements that temporally and spatially confine transcription of the gene during embryonic development, and that mediate the responses to innervation and denervation of muscle, lie within the upstream sequences analyzed in these studies.
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PMID:Upstream sequences of the myogenin gene convey responsiveness to skeletal muscle denervation in transgenic mice. 828 16

Transcription of the genes coding for troponin I slow (TnIslow) and other contractile proteins is activated during skeletal muscle differentiation, and their expression is later restricted to specific fiber types during maturation. We have isolated and characterized the rat TnIslow gene in order to begin elucidating its regulation during myogenesis. Transcriptional regulatory regions were delineated by using constructs, containing TnIslow gene sequences driving the expression of the chloramphenicol acetyltransferase (CAT) reporter gene, that were transiently transfected into undifferentiated and differentiated C2C12 cells. TnIslow 5'-flanking sequences directed transcription specifically in differentiated cells. However, transcription rates were approximately 10-fold higher in myotubes transfected with constructs containing the 5'-flanking sequences plus the intragenic region residing upstream of the translation initiation site (introns 1 and 2), indicative of interactions between elements residing upstream and in the introns of the gene. Deletion analysis of the 5' region of the TnIslow gene showed that the 200 bp upstream of the transcription initiation site is sufficient to confer differentiation-specific transcription in C2C12 myocytes. MyoD consensus binding sites were found both in the upstream 200-bp region and in a region residing in the second intron that is highly homologous to the quail TnIfast enhancer. Transactivation experiments using transfected NIH 3T3 fibroblasts with TnI-CAT constructs containing intragenic and/or upstream sequences and with the myogenic factors MyoD, myogenin, and MRF4 showed different potentials of these factors to induce transcription. Transgenic mice harboring the rat TnI-CAT fusion gene expressed the reporter specifically in the skeletal muscle. Furthermore, CAT levels were approximately 50-fold higher in the soleus than in the extensor digitorum longus, gastrocnemius, or tibialis muscle, indicating that the regulatory elements that restrict TnI transcription to slow-twitch myofibers reside in the sequences we have analyzed.
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PMID:cis-acting sequences of the rat troponin I slow gene confer tissue- and development-specific transcription in cultured muscle cells as well as fiber type specificity in transgenic mice. 841 91

Doxorubicin (Dox, adriamycin), an antineoplastic agent that can cause dilated cardiomyopathy, selectively inhibits muscle-specific gene expression in rodent cardiac muscle cells. This study shows that Dox treatment of proliferating C2 myoblasts, an established cell line from mouse skeletal muscle, completely prevents both fusion and accumulation of muscle-specific gene transcripts without significantly altering non-muscle gene transcripts. When added to high density cultures, Dox only blocked myotube formation but did not inhibit induction of muscle-specific genes. Transient transfection into C2 myoblasts showed that the transcriptional expression of chloramphenicol acetyltransferase reporter plasmids regulated by either the cardiac alpha-actin promoter or the muscle creatine kinase enhancer, but not with a viral or beta-actin promoter, was significantly diminished by Dox in a dose-dependent manner. Moreover, exposure of C2 myoblasts to Dox had a profound effect on the expression of regulatory genes critical to the myogenic differentiation program; mRNAs for MyoD and myogenin were dramatically reduced and Id mRNA was concomitantly increased. In addition, there was diminished DNA binding activity of the muscle-specific transcription factor, MEF-2. These results suggest that Dox inhibits myogenesis by preventing muscle-specific gene expression, possibly through affecting the myogenic programs controlled by muscle-specific transcription factors.
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PMID:Antineoplastic agent doxorubicin inhibits myogenic differentiation of C2 myoblasts. 844 15

We have studied gene activation via CArG boxes in the context of myogenesis. The proximal CArG box of the human cardiac actin gene (HCA1) stimulates transcription from the herpes simplex virus thymidine kinase (TK) promoter in a tissue-specific fashion. Thus in transient transfection assays, when the expression of chloramphenicol acetyltransferase (CAT) from p(HCA1)4 TKCAT is compared to that derived from p(M1)4 TKCAT which contains an inactive mutated version (M1) of the HCA1 element, high levels of expression are seen in C2 mouse myoblasts and myotubes, and in the T4 myoblast cell line derived from the C3H10T1/2 cell line by 5-azacytidine treatment, whereas only low levels of expression are seen in the mouse L fibroblast cell line. The parental C3H10T1/2 cell line shows intermediate levels of expression. A similar situation is seen in stably transfected cell lines. Gene activation via CArG boxes was also analyzed in the course of myogenic conversion of C3H10T1/2 cells treated with 5-azacytidine. Our results indicate that activation of the CAT gene from the HCA1 element is slightly posterior to the appearance of the first MyoD1 and myogenin transcripts, concomitant with the appearance of cardiac alpha-actin transcripts, but clearly precedes the accumulation of myosin light-chain 1a transcripts and the appearance of troponin T-positive cells. These results further establish that CArG boxes can be seen as muscle-specific cis-acting regulatory element prior to terminal differentiation.
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PMID:Activation of gene expression via CArG boxes during myogenic differentiation. 845 94


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