Identification of the G nucleotides in the repressor element that interact with NFY.

Labeled DNA probe (bottom strand labeled) corresponding to sequences +215 to +247 was partially methylated with dimethyl sulfate, incubated with nuclear extracts, and complexes formed were resolved on a 5% nondenaturing acrylamide gel. NFY protein bound probe (X), probe bound to nuclear proteins to form complex C3 (NX), and free probes (F) were eluted and cleaved at modified bases with piperidine and resolved on 20% acrylamide sequencing gel. Positions of the G residues where methylation interfered with protein interaction are shown by asterisks. The numbers represent the position of G residues in the bottom strand that correspond to each band. The analysis of the protein DNA complex formed with the probe that was labeled at the top strand is not shown since no difference was observed in the pattern compared with that of free probe. The figure shown is a representative of 3 independent experiments.