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Nucleic Acids Research, 1994, Vol. 22, No. 8 1450-1455
© 1994

MOLECULAR BIOLOGY

A simple in vivo footprinting method to examine DNA-protein interactions over the yeast PYK UAS element

loana Dumitru and J.Bryan McNeil*

Department of Microbiology, University of Toronto Toronto, Ontario M5S 1A8, Canada

*To whom correspondence should be addressed at: Department of Biology, York University, 4700 Keele Street. North York M3J 1P3, Canada

Received December 16, 1993. Accepted March 6, 1994.

In this report a modification to the in vivo footprinting assay is described. The method includes dimethyl sulfate treatment of whole yeast cells, followed by reiterative primer extension of the methylated genomic DNA using Taq DNA polymerase. Under appropriate reaction conditions chain extension terminates opposite a methylated purlne when Taq DNA polymerase encounters a modified adenlne or guanine. The procedure was used to examine, in vivo DNA-protein contacts over the upstream activation site (UAS) of the Saccharomyces cerevlslae PYK gene. in vivo analysis, using isogenic strains of yeast and Escherichla coli transformed with plasmid DNAs, confirmed the binding of both the frens-actlng factor RAP1 and the transcriptlonal activator GCR1 to cis-acting recognition sites located within the PYK UAS element.


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