Non-interfering adsorbents in alkaloidal analysis**An abstract of a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, University of Washington, Seattle, Washington.

Johnson, Estelle Koozin; Rising, L. Wait

doi:10.1002/jps.3030290610

Cited by 3 publications

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“…Sequences were confirmed by dideoxynucleotide sequencing. Correct initiation of transcription was previously confirmed for the 1256MCK-CAT construct and a construct in which the 206-bp enhancer was linked to the MCK basal promoter (bp Ϫ80 to ϩ7) (34).…”

Section: Methodsmentioning

confidence: 73%

E-Box Sites and a Proximal Regulatory Region of the Muscle Creatine Kinase Gene Differentially Regulate Expression in Diverse Skeletal Muscles and Cardiac Muscle of Transgenic Mice

Shield

Haugen

Clegg

et al. 1996

Molecular and Cellular Biology

106

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Previous analysis of the muscle creatine kinase (MCK) gene indicated that control elements required for transcription in adult mouse muscle differed from those required in cell culture, suggesting that distinct modes of muscle gene regulation occur in vivo. To examine this further, we measured the activity of MCK transgenes containing E-box and promoter deletions in a variety of striated muscles. Simultaneous mutation of three E boxes in the 1,256-bp MCK 5 region, which abolished transcription in muscle cultures, had strikingly different effects in mice. The mutations abolished transgene expression in cardiac and tongue muscle and caused a reduction in expression in the soleus muscle (a muscle with many slow fibers) but did not affect expression in predominantly fast muscles: quadriceps, abdominals, and extensor digitorum longus. Other regulatory sequences with muscle-type-specific activities were found within the 358-bp 5-flanking region. This proximal region conferred relatively strong expression in limb and abdominal skeletal muscles but was inactive in cardiac and tongue muscles. However, when the 206-bp 5 enhancer was ligated to the 358-bp region, high levels of tissue-specific expression were restored in all muscle types. These results indicate that E boxes and a proximal regulatory region are differentially required for maximal MCK transgene expression in different striated muscles. The overall results also imply that within skeletal muscles, the steady-state expression of the MCK gene and possibly other muscle genes depends on transcriptional mechanisms that differ between fast and slow fibers as well as between the anatomical and physiological attributes of each specific muscle.Creatine kinase catalyzes the regeneration of ATP from creatine phosphate and provides energy for contraction in all striated muscle types. The muscle creatine kinase (MCK) gene is transcriptionally activated during differentiation of myoblasts to myocytes (8, 31) and encodes the predominant creatine kinase isoform expressed in mammalian skeletal and cardiac muscle. While it seems likely that intrinsic differences in striated muscle anatomy and physiology would affect MCK gene expression, little is known concerning the steady-state mechanisms of MCK transcription in diverse muscle types. This transgenic study seeks to identify regulatory regions and elements within the MCK gene that are involved in controlling muscle-type-specific expression.Several regions within the mouse MCK gene 5Ј-flanking sequence are required for muscle-specific expression in cultured myocytes and cardiomyocytes (1, 32, 64). Of particular interest is a 206-bp enhancer located approximately 1 kb upstream of the transcription start site. This region functions in an orientation-independent manner and drives the expression of heterologous promoters in skeletal myocytes (32). The 5Ј MCK enhancer contains a number of sequence motifs-including the E box, CArG, and AT-rich sites-which are recognized by tissue-specific and ubiquitous transcription factors. Mutation...

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Section: Methodsmentioning

confidence: 73%

E-Box Sites and a Proximal Regulatory Region of the Muscle Creatine Kinase Gene Differentially Regulate Expression in Diverse Skeletal Muscles and Cardiac Muscle of Transgenic Mice

Shield

Haugen

Clegg

et al. 1996

Molecular and Cellular Biology

106

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“…The enh[right E mut]117MCK-CAT and enh[right E mut]80MCK-CAT constructs, containing the 206-bp enhancer harboring a mutation in the right E box linked to the MCK promoter, either bp Ϫ117 to Ϫ1 (mice 50 to 57) or bp Ϫ80 to ϩ7 (mice 45 to 49), were formed from ligation of a blunt-ended SphIBamHI fragment of the mutated pUC-E vector into SalI-cut, blunt-ended 117MCK-CAT or ligation of a blunt-ended HindIII-BamHI fragment of the right E box-mutated pUC-E vector (9) into SalI cut, blunt-ended 80MCK-CAT, respectively. Correct initiation of transcription for the 1256MCK-CAT and enh117MCK-CAT constructs were confirmed by RNase protection analysis (31).…”

Section: Methodsmentioning

confidence: 85%

Analysis of Muscle Creatine Kinase Gene Regulatory Elements in Skeletal and Cardiac Muscles of Transgenic Mice

Donoviel

Shield

Buskin

et al. 1996

Molecular and Cellular Biology

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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 of 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 muscle cells and in whole adult transgenic muscle. This suggests that there are alternative mechanisms of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.The muscle creatine kinase (MCK) gene is transcriptionally activated during striated muscle differentiation and is expressed at high levels in adult heart and skeletal muscles (11,39). Previous cell culture analyses of MCK gene regulation have implicated both a 5Ј muscle-specific enhancer (bp Ϫ1256 to Ϫ1050) and the adjacent 1-kb region of DNA (bp Ϫ1049 to ϩ7) as playing important roles in expression of the MCK gene in skeletal and cardiac muscle (24,29,63). The MCK enhancer contains a number of conserved DNA motifs, which are also found in the regulatory regions of many other muscle-specific genes (reviewed in references 12, 22, and 49). These motifs bind trans-acting factors in vitro and are critical control elements in cultured muscle cells (2,3,8,9,24). The MCK enhancer sites include E boxes, which contain the core consensus binding sequence CANNTG for the myogenic basic helix-loop-helix (bHLH) proteins (MyoD, Myf-5, myogenin, and MRF-4) (for reviews, see references 17, 22, 49, and 72); a CArG element, containing the consensus serum response factor-binding sequence CC(A/T) 6 GG (68); and an AT-rich site, which has been shown in gel shift assays to bind ubiquitously expressed factors (1, 15a, 25), as well as MHox and MEF-2 (14). The 1-kb region of DNA between the enhancer and the basal promoter exhibits low-level activity in cultured skeletal myocytes and cardiomyocytes (2).Mutation of eac...

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Datura innoxia— a potential commercial source of scopolamine

Gerlach¹

1948

Econ Bot

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Non-interfering adsorbents in alkaloidal analysis**An abstract of a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, University of Washington, Seattle, Washington.

Cited by 3 publications

References 0 publications

E-Box Sites and a Proximal Regulatory Region of the Muscle Creatine Kinase Gene Differentially Regulate Expression in Diverse Skeletal Muscles and Cardiac Muscle of Transgenic Mice

E-Box Sites and a Proximal Regulatory Region of the Muscle Creatine Kinase Gene Differentially Regulate Expression in Diverse Skeletal Muscles and Cardiac Muscle of Transgenic Mice

Analysis of Muscle Creatine Kinase Gene Regulatory Elements in Skeletal and Cardiac Muscles of Transgenic Mice

Datura innoxia— a potential commercial source of scopolamine

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