Journal: Nucleic Acids Research
Article Title: Different roles for Abf1p and a T-rich promoter element in nucleosome organization of the yeast RPS28A gene
Figure Lengend Snippet: Comparison of in vivo nucleosome positions on the wild-type RPS28A promoter and a promoter containing a mutated T-rich element. Nystatin-permeabilized spheroplasts prepared from wild-type (ERS3) and mutant cells (ETS6) were treated with either Mnase ( A ) or DNaseI ( B ). Distances are from the translational start codon ATG. Protected regions of 160–170 bp are interpreted as positioned nucleosomes (closed elipses). The 130 bp Mnase-protected region that covers the Abf1p site ( Xba I) in the wild-type promoter (ERS3) has decreased to 85 bp when the T-rich element is mutated (ERS6). Concomitantly, downstream nucleosomes are shifted towards the Abf1p binding site.
Article Snippet: For chromatin samples, after the 0 min time sample, either 250 U/4 ml Mnase (micrococcal endonuclease) or 180 U/4 ml DNaseI (both from Boehringer-Mannheim) was added to the nystatin-permeabilized spheroplasts, and 500 µl time samples (1, 2.5, 5, 9, 15 and 25 min) were added to 50 µl StopMnase (10% SDS, 0.1 M EDTA and 50 µg proteinase K).
Techniques: In Vivo, Mutagenesis, Binding Assay