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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 previously used mice bearing a myosin light chain-chloramphenicol acetyltransferase (MLC1-CAT) transgene to show that adult muscle cells bear a heritable, cell autonomous memory of their rostrocaudal position. CAT mRNA and protein are expressed in a > 100-fold rostrocaudal gradient in skeletal muscles of developing and adult MLC1-CAT mice (Donoghue, M. J., Merlie, J. P., Rosenthal, N. and Sanes, J. R. (1991). Proc. Natl. Acad. Sci. USA 88, 5847-5851; Donoghue, M. J., Alvarez, J. D., Merlie, J. P. and Sanes, J. R. (1991). J. Cell Biol. 115, 423-434). Moreover, both in primary cultures and in myogenic cell lines prepared from individual muscles of these mice, CAT levels reflect the body position from which the myoblasts were derived (Donoghue, M.J., Morris-Valero, R., Johnson, Y.R., Merlie, J.P. and Sanes, J. R. (1992). Cell 69, 67-77). Here, we show that the methylation state of the MLC1-CAT transgene in skeletal muscles is also graded along the rostrocaudal axis: methylation levels decrease and expression levels increase in the order, jaw-->neck-->chest and forelimb-->hindlimb. Methylation levels are also approx. 10-fold higher in rostrally derived than in caudally derived myogenic cell lines, which express low and high levels of CAT, respectively. Within each cell line, undifferentiated cells (myoblasts), which do not express the transgene, and differentiated cells (myotubes), which do, are indistinguishable in methylation state. Thus, differentiation-related changes in transgene expression do not affect position-related levels of transgene methylation. On the other hand, treatment of rostrally derived lines with the demethylating agent, 5-azacytidine, decreases methylation and increases expression of the transgene. Thus, perturbation of methylation affects expression. Taken together, these results suggest that methylation provides a genomic imprint of rostrocaudal body position that may serve as a component of the positional memory that mammalian cells retain into adulthood.
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PMID:An axial gradient of transgene methylation in murine skeletal muscle: genomic imprint of rostrocaudal position. 129 32

We previously documented a greater than 100-fold rostrocaudal gradient of chloramphenicol acetyltransferase (CAT) expression in the muscles of adult mice that bear a myosin light chain-CAT transgene: successively more caudal muscles express successively higher levels of CAT. Here we studied the development and maintenance of this positional information in vitro. CAT levels reflect the rostrocaudal positions of the muscles from which the cells are derived in cultures established from adult muscles, in clones derived from individual adult myogenic (satellite) cells, in cultures prepared from embryonic myoblasts, and in cell lines derived by retrovirus-mediated transfer of an oncogene to satellite cells. Our results suggest that myoblasts bear a positional memory that is established in embryos, retained in adults, cell autonomous, heritable, stable to transformation, and accessible to study in clonal cell lines.
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PMID:Mammalian muscle cells bear a cell-autonomous, heritable memory of their rostrocaudal position. 155 34

We recently generated and characterized transgenic mice in which regulatory sequences from a myosin light chain gene (MLC1f/3f) are linked to the chloramphenicol acetyltransferase (CAT) gene. Transgene expression in these mice is specific to skeletal muscle and graded along the rostrocaudal axis: adult muscles derived from successively more caudal somites express successively higher levels of CAT. To investigate the cellular basis of these patterns of expression, we developed and used a histochemical stain that allows detection of CAT in individual cells. Our main results are as follows: (a) Within muscles, CAT is detected only in muscle fibers and not in associated connective tissue, blood vessels, or nerves. Thus, the tissue specificity of transgene expression observed by biochemical assay reflects a cell-type specificity demonstrable histochemically. (b) Within individual muscles, CAT levels vary with fiber type. Like the endogenous MLC1f/3f gene, the transgene is expressed at higher levels in fast-twitch (type II) than in slow-twitch (type I) muscle fibers. In addition, CAT levels vary among type II fiber subtypes, in the order IIB greater than IIX greater than IIA. (c) Among muscles that are similar in fiber type composition, the average level of CAT per fiber varies with rostrocaudal position. This position-dependent variation in CAT level is apparent even when fibers of a single type are compared. From these results, we conclude that fiber type and position affect CAT expression independently. We therefore infer the existence of separate fiber type-specific and positionally graded transcriptional regulators that act together to determine levels of transgene expression.
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PMID:Fiber type- and position-dependent expression of a myosin light chain-CAT transgene detected with a novel histochemical stain for CAT. 171 85

The myosin light chain (MLC) 1/3 enhancer (MLC enhancer), identified at the 3' end of the skeletal MLC1/3 locus, contains a sequence motif that is homologous to a protein-binding site of the skeletal muscle alpha-actin promoter. Gel shift, competition, and footprint assays demonstrated that a CArG motif in the MLC enhancer binds the proteins MAPF1 and MAPF2, previously identified as factors interacting with the muscle regulatory element of the skeletal alpha-actin promoter. Transient transfection assays with constructs containing the chloramphenicol acetyltransferase reporter gene demonstrated that a 115-bp subfragment of the MLC enhancer is able to exert promoter activity when provided with a silent nonmuscle TATA box. A point mutation at the MAPF1/2-binding site interferes with factor binding and abolishes the promoter activity of the 115-bp fragment. The observation that an oligonucleotide encompassing the MAPF1/2 site of the MLC enhancer alone cannot serve as a promoter element suggests that additional factor-binding sites are necessary for this function. The finding that MAPF1 and MAPF2 recognize similar sequence motifs in two muscle genes, simultaneously activated during muscle differentiation, implies that these factors may have a role in coordinating the activation of contractile protein gene expression during myogenesis.
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PMID:The myosin light chain enhancer and the skeletal actin promoter share a binding site for factors involved in muscle-specific gene expression. 204 75

Transgenic mice were produced in which expression of the reporter gene chloramphenicol acetyltransferase (CAT) is controlled by regulatory elements of a rodent myosin light chain gene. CAT activity was readily detectable in muscles of these mice but negligible in a variety of nonmuscle tissues. Unexpectedly, levels of CAT expression varied greater than 100-fold from muscle to muscle, forming a gradient in which a muscle's position in the rostrocaudal axis was correlated with its level of CAT enzyme activity and abundance of CAT mRNA. Thus, rostral muscles (innervated by cranial nerves) had the lowest levels of CAT, thoracic muscles had intermediate levels, and caudal muscles (innervated through lumbar and sacral roots) had the highest levels. We established that myosin light chain sequences are responsible for the gradient of CAT expression but observed no strong gradient of endogenous myosin light chain expression. We argue that elements that are silent or masked by other sequences in their native context are revealed in the transgene and that the rostrocaudal gradient of gene expression they produce reveals the existence of a positionally graded endogenous regulator of gene expression. These transgenic mice provide evidence that cells in adult mammals retain "positional information" of a sort hitherto studied largely in embryos. The transgene they express may provide a means for determining how such positional values are generated and maintained.
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PMID:Rostrocaudal gradient of transgene expression in adult skeletal muscle. 206 62

The mRNAs for myogenic functions are coordinately transcribed with polyomavirus (Py) early mRNA during in vitro differentiation of mouse C2 myoblast cells. Sequence analysis shows that the A domain of the Py enhancer includes an E1A-like consensus sequence that is also found in the 5' upstream region of two genes expressed during myoblast differentiation: alpha-actin and myosin light chain. Therefore, the coordinate expression of such genes with Py early mRNA may be activated by a common cellular regulatory factor. In the present work, we report that C2 cells surviving Py infection are unable to differentiate and do not express alpha-actin and myosin light-chain mRNAs. Hybrids between such Py-resistant myoblast cells and the parental cells exhibited dominance of the permissibility to Py growth and of the expression of myogenic mRNAs. In C2 cells transiently transfected with a chimeric plasmid (pSVPy12CAT) harboring the bacterial chloramphenicol acetyltransferase (CAT) gene driven by the Py enhancer-promoter region, the CAT gene was expressed irrespective of their stage of differentiation. Moreover, undifferentiated stably transfected cells expressing the CAT gene restricted viral growth. Py-resistant C2 myoblasts transiently transfected with pSVPy12CAT also expressed the CAT gene driven by the Py enhancer. This contradictory finding is similar to results previously obtained by other investigators with cloned genes specific for myogenic functions, and it may be explained by a structural difference between the pSVPy12CAT and the Py genomic organizations in which the viral enhancer operates.
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PMID:Polyomavirus genome and polyomavirus enhancer-driven gene expression during myogenesis. 255 61

The rat myosin light chain (MLC)1/3 gene locus contains a potent muscle-specific enhancer, located downstream of the coding region, greater than 24 kilobases away from the MLC1 transcription start site. To assess the role of this enhancer in the activation of MLC expression during development, transgenic mice were generated carrying multiple copies of a MLC1 promoter-chloramphenicol acetyltransferase (CAT) transcription unit linked to a genomic fragment including the enhancer. CAT expression was detected in four mouse lines, up to 1000-fold higher in skeletal muscles than in other tissues. Activation of endogenous MLC1 transcription in these animals 4 days before birth was reflected in the onset of CAT transgene expression. This study identifies the transcriptional control elements necessary to activate the 21-kilobase MLC1/3 locus at the appropriate fetal stage and indicates that the MLC enhancer is sufficient to induce developmentally regulated expression from the MLC1 promoter exclusively in skeletal muscle cells.
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PMID:Myosin light chain enhancer activates muscle-specific, developmentally regulated gene expression in transgenic mice. 281 57

The expression of myogenin is suppressed during innervation and has been implicated in determining properties of skeletal muscle which are regulated by electrical activity. We previously reported that transcription driven by 3700 bp of the mouse myogenin upstream sequence (MYG3700) is activated by denervation in transgenic mice (Nucleic Acids Res. 21, 5684-5693, 1993). To extend our investigation of the activity dependence of the myogenin promoter, we have utilized myoblast implantation as a novel approach to in vivo reporter analysis. Myoblasts for hindlimb injections were generated by stable transfection of chloramphenicol acetyltransferase (CAT) reporters into a beta-galactosidase-expressing line of C2 cells. In vitro characterization of stable myoblast clones carrying myogenin-CAT deletion constructs revealed that while the proximal myogenin 5'-flanking sequence confers myotube specificity, high-level expression requires a region upstream (-335 to -1102) which depends on chromosomal integration for its function. For analysis by implantation, incorporation of injected myoblasts into existing myofibers was confirmed by histochemical staining. Using clonal myoblasts harboring nicotinic receptor alpha-subunit (alpha 800) and myosin light chain receptors as positive and negative controls, respectively, for denervation responsiveness, we determined that the nuclei of injected myoblasts are susceptible to regulatory signals imposed by nerve-induced electrical activity of the myofiber into which they incorporate. In in vivo analysis of myogenin upstream sequence by implantation, CAT activities of MYG3700 and MYG1565 reporters in injected limbs increased up to fourfold within 4 days after denervation, whereas the activities of MYG1102 and MYG335 were unchanged. By 10 days after denervation, all myogenin reporters displayed denervation responsiveness. These implantation data suggest an early phase of denervation activation, one that is mediated by control elements residing within -1102 to -1565 of the myogenin upstream sequence. Thus, the combined analyses of stable reporter myoblast lines in vitro and in vivo by implantation provide an efficient means of evaluating regulatory regions for high-level expression and neural modulation of muscle gene transcription.
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PMID:Analysis of neural-responsive myogenin upstream sequences by myoblast implantation. 861 76

Intramuscular injection of plasmid constructs promises to be an effective way of carrying out gene therapy for muscle disorders as well as using muscle as an in vivo expression system for disorders that involve the gene product being secreted into the bloodstream. The effectiveness of this method depends on the design of the cassette used for the expression of the cDNA of the introduced gene. We tested the levels of expression achieved by a number of muscle-specific promoters and a myosin light chain enhancer when spliced to the reporter gene chloramphenicol acetyltransferase (CAT), in vitro and in vivo by injection into fast and slow muscles of the mouse. The results show that the highest levels of expression are achieved by a combination of a truncated myosin heavy chain promoter and the enhancer, and that a whole range of expression levels is obtained with the other combinations tested. The data show that a cassette based on these elements should provide efficient vectors for the introduction and expression of genes following intramuscular injection of naked DNA.
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PMID:Myosin regulatory elements as vectors for gene transfer by intramuscular injection. 961 76