Open AccessA simplein vivofootprinting method to examine DNA-protein interactions over the yeastPYKUAS element
Author(s) -
loana Dumitru,
J B McNeil
Publication year - 1994
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/22.8.1450
Subject(s) - biology , footprinting , primer extension , dna , microbiology and biotechnology , saccharomyces cerevisiae , dna footprinting , biochemistry , guanine , yeast , activator (genetics) , genomic dna , gene , promoter , gene expression , nucleotide , base sequence
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 purine when Taq DNA polymerase encounters a modified adenine or guanine. The procedure was used to examine, in vivo DNA-protein contacts over the upstream activation site (UAS) of the Saccharomyces cerevisiae PYK gene. In vivo analysis, using isogenic strains of yeast and Escherichia coli transformed with plasmid DNAs, confirmed the binding of both the trans-acting factor RAP1 and the transcriptional activator GCR1 to cis-acting recognition sites located within the PYK UAS element.