Specific protection of nucleotides in the lac operator from DMS methylation and DNase I nicking by crude bacterial cell extracts |
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Authors: | G B Ruvkun V Sundaresan F M Ausubel |
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Affiliation: | Cellular and Developmental Group, Department of Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138 U.S.A. |
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Abstract: | Crude bacterial cell extracts prepared from an Escherichia coli lacIq strain were shown to protect specific nucleotides in the lac operator from methylation by dimethyl sulfate (DMS) or digestion by DNase I, whereas no protection was observed using extracts prepared from a nearly isogenic lacI- strain. These experiments show that it is not necessary to use purified regulatory proteins in experiments designed to localize sequences on DNA which interact with proteins. Therefore, crude cell extracts should be useful in DNA "footprinting" experiments to define regions of DNA which bind to unknown regulatory proteins. |
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Keywords: | DNA footprinting regulatory proteins bp base pairs CAP catabolic activation protein distilled water DMS dimethyl sulfate IPTG βME β-mercaptoethanol UREB universal restriction enzyme buffer |
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