In vivo Footprinting of a Muscle Specific Enhancer by Ligation Mediated PCR
- Creators
- Mueller, Paul R.
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Wold, Barbara
Abstract
In vivo protein-DNA interactions at the developmentally regulated enhancer of the mouse muscle creatine kinase (MCK) gene were examined by a newly developed polymerase chain reaction (PCR) footprinting procedure. This ligation mediated, single-sided PCR technique permits the exponential amplification of an entire sequence ladder. Several footprints were detected in terminally differentiated muscle cells where the MCK gene is actively transcribed. None were observed in myogenic cells prior to differentiation or in nonmuscle cells. Two footprints appear to correspond to sites that can bind the myogenic regulator MyoD1 in vitro, whereas two others represent muscle specific use of apparently general factors. Because MyoD1 is synthesized by undifferentiated myoblasts, these data imply that additional regulatory mechanisms must restrict the interaction between this protein and its target site prior to differentiation.
Additional Information
© 1989 American Association for the Advancement of Science. Received 9 July 1989; accepted 20 September 1989. We thank J. Miner for providing template plasmids used in the RNase protection assays and aza-myoblast RNA's; S. Hauschka for the MM14 and DD1 cell lines; S. Sharp for BC_3H1 RNA's; P. Garrity, J. Johnson, U. Landegren, H. Weinhard, and P. Mathers for helpful discussions; S. Hauschka, J. Buskin, H. Arnold, E. Olsen, A. Lassar, and H. Weintraub for supplying information before publication; and N. Davidson, D. Anderson, K. Zinn, A. Riggs, G. Pfeifer, S. Steigerwald, and members of the Wold group for critical reading of this manuscript. Supported by NIH (General Medicine) grants RR07003 and GM35526 (B.W.). B.W. would like to dedicate this work to D.E.W.Errata
In the research artide "In vivo footprinting of a muscle specific enhancer by ligation mediated PCR" by P. R. Mueller and B. Wold (10 Nov. 1989, p. 780), an error was introduced after the galley proofs were approved by the authors. The ninth sentence of the legend for figure 2 contained an error in the concentration of deoxynudeoside triphosphate. It should have read, "Hybridization was stopped by transferring to ice; a solution of 7.5 µL of 20 mM MgCl2, 20 mM dithiothreitol (DTT), and 0.2 mM of each deoxynudeoside triphosphate (dNTP) was added...." The printed version read, "... 0.02 mM of each deoxynudeoside triphosphate (dNTP).. . ." This erroneous tenfold reduction in dNTP concentration may have a significant negative impact on the efficiency of the first strand synthesis reaction and therefore on the ultimate success of the ligation mediated PCR procedure.Additional details
- Eprint ID
- 52319
- Resolver ID
- CaltechAUTHORS:20141203-101540445
- NIH
- RR07003
- NIH
- GM35526
- Created
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2014-12-03Created from EPrint's datestamp field
- Updated
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2023-06-01Created from EPrint's last_modified field