scholarly journals Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle.

1993 ◽  
Vol 13 (5) ◽  
pp. 2753-2764 ◽  
Author(s):  
S L Amacher ◽  
J N Buskin ◽  
S D Hauschka
Keyword(s):  
Skeletal Muscle ◽  
Cardiac Muscle ◽  
Striated Muscle ◽  
Regulatory Elements ◽  
High Level Expression ◽  
Muscle Creatine Kinase ◽  
Skeletal Myocytes ◽  
E Box ◽  
Nonmuscle Cells ◽  
High Level

We have used transient transfections in MM14 skeletal muscle cells, newborn rat primary ventricular myocardiocytes, and nonmuscle cells to characterize regulatory elements of the mouse muscle creatine kinase (MCK) gene. Deletion analysis of MCK 5'-flanking sequence reveals a striated muscle-specific, positive regulatory region between -1256 and -1020. A 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment confers high-level expression in skeletal myocytes but is inactive in myocardiocytes, indicating that skeletal and cardiac muscle MCK regulatory sites are distinguishable. To further delineate muscle regulatory sequences, we tested six sites within the MCK enhancer for their functional importance. Mutations at five sites decrease expression in skeletal muscle, cardiac muscle, and nonmuscle cells. Mutations at two of these sites, Left E box and MEF2, cause similar decreases in all three cell types. Mutations at three sites have larger effects in muscle than nonmuscle cells; an A/T-rich site mutation has a pronounced effect in both striated muscle types, mutations at the MEF1 (Right E-box) site are relatively specific to expression in skeletal muscle, and mutations at the CArG site are relatively specific to expression in cardiac muscle. Changes at the AP2 site tend to increase expression in muscle cells but decrease it in nonmuscle cells. In contrast to reports involving cotransfection of 10T1/2 cells with plasmids expressing the myogenic determination factor MyoD, we show that the skeletal myocyte activity of multimerized MEF1 sites is 30-fold lower than that of the 206-bp enhancer. Thus, MyoD binding sites alone are not sufficient for high-level expression in skeletal myocytes containing endogenous levels of MyoD and other myogenic determination factors.

Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle

1993 ◽  
Vol 13 (5) ◽  
pp. 2753-2764 ◽  
Author(s):  
S L Amacher ◽  
J N Buskin ◽  
S D Hauschka
Keyword(s):  
Skeletal Muscle ◽  
Cardiac Muscle ◽  
Striated Muscle ◽  
Regulatory Elements ◽  
High Level Expression ◽  
Muscle Creatine Kinase ◽  
Skeletal Myocytes ◽  
E Box ◽  
Nonmuscle Cells ◽  
High Level

We have used transient transfections in MM14 skeletal muscle cells, newborn rat primary ventricular myocardiocytes, and nonmuscle cells to characterize regulatory elements of the mouse muscle creatine kinase (MCK) gene. Deletion analysis of MCK 5'-flanking sequence reveals a striated muscle-specific, positive regulatory region between -1256 and -1020. A 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment confers high-level expression in skeletal myocytes but is inactive in myocardiocytes, indicating that skeletal and cardiac muscle MCK regulatory sites are distinguishable. To further delineate muscle regulatory sequences, we tested six sites within the MCK enhancer for their functional importance. Mutations at five sites decrease expression in skeletal muscle, cardiac muscle, and nonmuscle cells. Mutations at two of these sites, Left E box and MEF2, cause similar decreases in all three cell types. Mutations at three sites have larger effects in muscle than nonmuscle cells; an A/T-rich site mutation has a pronounced effect in both striated muscle types, mutations at the MEF1 (Right E-box) site are relatively specific to expression in skeletal muscle, and mutations at the CArG site are relatively specific to expression in cardiac muscle. Changes at the AP2 site tend to increase expression in muscle cells but decrease it in nonmuscle cells. In contrast to reports involving cotransfection of 10T1/2 cells with plasmids expressing the myogenic determination factor MyoD, we show that the skeletal myocyte activity of multimerized MEF1 sites is 30-fold lower than that of the 206-bp enhancer. Thus, MyoD binding sites alone are not sufficient for high-level expression in skeletal myocytes containing endogenous levels of MyoD and other myogenic determination factors.

scholarly journals An opportunistic promoter sharing regulatory sequences with either a muscle-specific or a ubiquitous promoter in the human aldolase A gene.

1993 ◽  
Vol 13 (1) ◽  
pp. 9-17 ◽  
Author(s):  
J P Concordet ◽  
M Salminen ◽  
J Demignon ◽  
C Moch ◽  
P Maire ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Integration Site ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Beta Globin ◽  
Fast Skeletal Muscle ◽  
High Level Expression ◽  
Adult Skeletal Muscle ◽  
High Level ◽  
Aldolase A

The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region.

A hormone-encoding gene identifies a pathway for cardiac but not skeletal muscle gene transcription

1994 ◽  
Vol 14 (5) ◽  
pp. 3115-3129
Author(s):  
C Grépin ◽  
L Dagnino ◽  
L Robitaille ◽  
L Haberstroh ◽  
T Antakly ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cardiac Muscle ◽  
Regulatory Elements ◽  
Cardiac Cells ◽  
Skeletal Myocytes ◽  
Encoding Gene ◽  
Muscle Gene ◽  
Encoding Genes ◽  
Dependent Pathway ◽  
Gata Gene

In contrast to skeletal muscle, the mechanisms responsible for activation and maintenance of tissue-specific transcription in cardiac muscle remain poorly understood. A family of hormone-encoding genes is expressed in a highly specific manner in cardiac but not skeletal myocytes. This includes the A- and B-type natriuretic peptide (ANP and BNP) genes, which encode peptide hormones with crucial roles in the regulation of blood volume and pressure. Since these genes are markers of cardiac cells, we have used them to probe the mechanisms for cardiac muscle-specific transcription. Cloning and functional analysis of the rat BNP upstream sequences revealed unexpected structural resemblance to erythroid but not to muscle-specific promoters and enhancers, including a requirement for regulatory elements containing GATA motifs. A cDNA clone corresponding to a member of the GATA family of transcription factors was isolated from a cardiomyocyte cDNA library. Transcription of this GATA gene is restricted mostly to the heart and is undetectable in skeletal muscle. Within the heart, GATA transcripts are localized in ANP- and BNP-expressing myocytes, and forced expression of the GATA protein in heterologous cells markedly activates transcription from the natural cardiac muscle-specific ANP and BNP promoters. This GATA-dependent pathway defines the first mechanism for cardiac muscle-specific transcription. Moreover, the present findings reveal striking similarities between the mechanisms controlling gene expression in hematopoietic and cardiac cells and may have important implications for studies of cardiogenesis.

scholarly journals A hormone-encoding gene identifies a pathway for cardiac but not skeletal muscle gene transcription.

1994 ◽  
Vol 14 (5) ◽  
pp. 3115-3129 ◽  
Author(s):  
C Grépin ◽  
L Dagnino ◽  
L Robitaille ◽  
L Haberstroh ◽  
T Antakly ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cardiac Muscle ◽  
Regulatory Elements ◽  
Cardiac Cells ◽  
Skeletal Myocytes ◽  
Encoding Gene ◽  
Muscle Gene ◽  
Encoding Genes ◽  
Dependent Pathway ◽  
Gata Gene

In contrast to skeletal muscle, the mechanisms responsible for activation and maintenance of tissue-specific transcription in cardiac muscle remain poorly understood. A family of hormone-encoding genes is expressed in a highly specific manner in cardiac but not skeletal myocytes. This includes the A- and B-type natriuretic peptide (ANP and BNP) genes, which encode peptide hormones with crucial roles in the regulation of blood volume and pressure. Since these genes are markers of cardiac cells, we have used them to probe the mechanisms for cardiac muscle-specific transcription. Cloning and functional analysis of the rat BNP upstream sequences revealed unexpected structural resemblance to erythroid but not to muscle-specific promoters and enhancers, including a requirement for regulatory elements containing GATA motifs. A cDNA clone corresponding to a member of the GATA family of transcription factors was isolated from a cardiomyocyte cDNA library. Transcription of this GATA gene is restricted mostly to the heart and is undetectable in skeletal muscle. Within the heart, GATA transcripts are localized in ANP- and BNP-expressing myocytes, and forced expression of the GATA protein in heterologous cells markedly activates transcription from the natural cardiac muscle-specific ANP and BNP promoters. This GATA-dependent pathway defines the first mechanism for cardiac muscle-specific transcription. Moreover, the present findings reveal striking similarities between the mechanisms controlling gene expression in hematopoietic and cardiac cells and may have important implications for studies of cardiogenesis.

scholarly journals Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

1996 ◽  
Vol 16 (4) ◽  
pp. 1649-1658 ◽  
Author(s):  
D B Donoviel ◽  
M A Shield ◽  
J N Buskin ◽  
H S Haugen ◽  
C H Clegg ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Transgenic Mice ◽  
Cardiac Muscle ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Muscle Creatine Kinase ◽  
Regulatory Regions ◽  
Mouse Muscle ◽  
Muscle Creatine

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt-1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5' region also had no effect on transgene expression in thigh skeletal muscle expression. All these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt - 1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.

An opportunistic promoter sharing regulatory sequences with either a muscle-specific or a ubiquitous promoter in the human aldolase A gene

1993 ◽  
Vol 13 (1) ◽  
pp. 9-17
Author(s):  
J P Concordet ◽  
M Salminen ◽  
J Demignon ◽  
C Moch ◽  
P Maire ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Integration Site ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Beta Globin ◽  
Fast Skeletal Muscle ◽  
High Level Expression ◽  
Adult Skeletal Muscle ◽  
High Level ◽  
Aldolase A

The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region.

Slow troponin C is present in both muscle and nonmuscle cells

1992 ◽  
Vol 70 (8) ◽  
pp. 691-697 ◽  
Author(s):  
Craig Berezowsky ◽  
Jnanankur Bag
Keyword(s):  
Skeletal Muscle ◽  
Polymerase Chain Reaction ◽  
Cardiac Muscle ◽  
Striated Muscle ◽  
Muscle Cells ◽  
Troponin C ◽  
Chain Reaction ◽  
Cardiac Muscle Cells ◽  
Nonmuscle Cells ◽  
Polymerase Chain

A common primer was used for synthesis of cDNAs from both chicken fast and slow troponin C (TnC) mRNAs. Synthesis of double-stranded cDNAs and their amplification by polymerase chain reaction gave specific products corresponding to these mRNAs. This method was used for determining the presence of TnC mRNAs in various tissues. Our results show that while the fast TnC mRNA is expressed only in the muscle cells, slow TnC mRNA is expressed in a number of nonmuscle cells. Not all nonmuscle cells, however, express slow TnC mRNA. Liver and brain tissues showed the presence of high levels of this mRNA, while it was absent in chicken smooth muscle and embryonic skin. The slow TnC mRNA was very stable in cardiac muscle cells. It degrades with a half-life of approximately 94 h. The same mRNA was less stable in skeletal muscle and liver cells. The half-lives were found to be only between 13 and 16 h in these cells. Our results suggest that slow TnC mRNA may function as the nonmuscle isoform of this contractile protein. Since slow TnC mRNA is the only TnC isoform present in cardiac muscle, liver and brain, it is possible that besides its role in regulating contraction of striated muscle slow TnC can also function in processes other than muscle contraction.Key words: troponin C mRNA, cardiac muscle, skeletal muscle, polymerase chain reaction, nonmuscle troponin C.

Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice

1993 ◽  
Vol 13 (9) ◽  
pp. 5266-5275
Author(s):  
R D Palmiter ◽  
E P Sandgren ◽  
D M Koeller ◽  
R L Brinster
Keyword(s):  
Transgenic Mice ◽  
Regulatory Elements ◽  
High Level Expression ◽  
Hypersensitive Sites ◽  
Enhanced Expression ◽  
Rat Growth ◽  
Flanking Sequences ◽  
High Level ◽  
Distal Regulatory Elements ◽  
Flanking Regions

DNA regions of 10 and 7 kb that flank the mouse metallothionein II (MT-II) and MT-I genes, respectively, were combined with a minimally marked MT-I (MT-I*) gene and tested in transgenic mice. This construct resulted in (i) position-independent expression of MT-I* mRNA and copy number-dependent expression, (ii) levels of hepatic MT-I mRNA per cell per transgene that were about half that derived from endogenous MT-I genes, (iii) appropriate regulation by metals and hormones, and (iv) tissue distribution of transgene mRNA that resembled that of endogenous MT-I mRNA. These features were not observed when MT-I* was tested without the flanking regions. These MT-I flanking sequences also improved the expression of rat growth hormone reporter genes, with or without introns, that were under the control of the MT-I promoter. Moreover, they enhanced expression from two of four heterologous promoters/enhancers that were tested. Deletion analysis indicated that regions known to have DNase I-hypersensitive sites were necessary but not sufficient for high-level expression. These data suggest that the DNA regions flanking the mouse MT-I and MT-II genes have functions like the locus control regions described for other genes.

scholarly journals The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle.

1991 ◽  
Vol 115 (2) ◽  
pp. 411-421 ◽  
Author(s):  
T J Byers ◽  
L M Kunkel ◽  
S C Watkins
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Cardiac Muscle ◽  
Striated Muscle ◽  
Adherens Junctions ◽  
Myotendinous Junction ◽  
Elevated Concentration ◽  
Western Blots ◽  
Functional Roles

We use a highly specific and sensitive antibody to further characterize the distribution of dystrophin in skeletal, cardiac, and smooth muscle. No evidence for localization other than at the cell surface is apparent in skeletal muscle and no 427-kD dystrophin labeling was detected in sciatic nerve. An elevated concentration of dystrophin appears at the myotendinous junction and the neuromuscular junction, labeling in the latter being more intense specifically in the troughs of the synaptic folds. In cardiac muscle the distribution of dystrophin is limited to the surface plasma membrane but is notably absent from the membrane that overlays adherens junctions of the intercalated disks. In smooth muscle, the plasma membrane labeling is considerably less abundant than in cardiac or skeletal muscle and is found in areas of membrane underlain by membranous vesicles. As in cardiac muscle, smooth muscle dystrophin seems to be excluded from membrane above densities that mark adherens junctions. Dystrophin appears as a doublet on Western blots of skeletal and cardiac muscle, and as a single band of lower abundance in smooth muscle that corresponds most closely in molecular weight to the upper band of the striated muscle doublet. The lower band of the doublet in striated muscle appears to lack a portion of the carboxyl terminus and may represent a dystrophin isoform. Isoform differences and the presence of dystrophin on different specialized membrane surfaces imply multiple functional roles for the dystrophin protein.

scholarly journals THE ULTRASTRUCTURE OF FROG VENTRICULAR CARDIAC MUSCLE AND ITS RELATIONSHIP TO MECHANISMS OF EXCITATION-CONTRACTION COUPLING

1968 ◽  
Vol 38 (1) ◽  
pp. 99-114 ◽  
Author(s):  
Nancy A. Staley ◽  
Ellis S. Benson
Keyword(s):  
Skeletal Muscle ◽  
Cardiac Muscle ◽  
Striated Muscle ◽  
Structural Features ◽  
Mammalian Skeletal Muscle ◽  
Striated Muscles ◽  
Thin Filaments ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Dense Granules

Frog ventricular cardiac muscle has structural features which set it apart from frog and mammalian skeletal muscle and mammalian cardiac muscle. In describing these differences, our attention focused chiefly on the distribution of cellular membranes. Abundant inter cellular clefts, the absence of tranverse tubules, and the paucity of sarcotubules, together with exceedingly small cell diameters (less than 5 µ), support the suggestion that the mechanism of excitation-contraction coupling differs in these muscle cells from that now thought to be characteristic of striated muscle such as skeletal muscle and mammalian cardiac muscle. These structural dissimilarities also imply that the mechanism of relaxation in frog ventricular muscle differs from that considered typical of other striated muscles. Additional ultrastructural features of frog ventricular heart muscle include spherical electron-opaque bodies on thin filaments, inconstantly present, forming a rank across the I band about 150 mµ from the Z line, and membrane-bounded dense granules resembling neurosecretory granules. The functional significance of these features is not yet clear.

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