Figure 8.

Gel mobility-shift assays with purified recombinant Myb1 protein. (A) DNA-binding activity of Myb1 to several PR-1a promoter fragments. The locations of the fragments in the PR-1a promoter relative to the transcriptional start site are given in parenthesis. (B) The Myb1 protein binds preferentially to MBSII (GTTTGGT) found in PR-1a promoter fragment P2. The binding reaction fractionated in the first lane did not contain Myb1 while those in other lanes contained 200 ng of Myb1. The following double-strand oligonucleotides were used as probes: MBSI (5′-CGGAATTCCCTAACTGACGCATCGATGGGA-3′), MBSIm (5′-CGGAATTCCCTCCCTGACGCATCGATGGGA-3′), MBSII (5′-CGGAATTCTGTTTGGTATGCATCGATGGGA-3′), and MBSIIm (5′-CGGAATTCTGTTGCCTATGCATCGATGGGA-3′). (C) Competition for binding of Myb1 to the MBSII probe. The binding assay was performed by preincubating unlabeled competitor oligonucleotides for 15 min with Myb1 before addition of 1 ng of ³²P-labeled MBSII probe. Reactions fractionated in lanes 1, 4, 7, and 10 contained no competitors, while those in lanes 2, 5, 8, and 11 contained 20 ng competitors and those in lanes 3, 6, 9, and 12 contained 200 ng competitors.