Figure 3.

DNA-binding activity of the PLATZ1 protein. (A) Nucleotide sequence of the upstream region (–734 to –642) of the pea pra2 gene. The DE1 element is boxed. Arrows indicate the 68-bp DNA probe used in the EMSA experiment. The 31-bp DE1 probe is underlined. (B) The recombinant PLATZ1 protein binds to the 68-bp DNA fragment. Binding reactions were carried out with the recombinant PLATZ1 protein and the ³²P-labeled 68-bp DNA fragment (2.5 fmol) in the absence or presence of the unlabeled 68-bp DNA fragment as competitor. Lane 1, no protein; lane 2, 100 ng PLATZ1 protein; lanes 3–6, 200 ng PLATZ1 protein. As indicated, 100, 200 and 400-fold competitors were added (lanes 4, 5 and 6). Arrows indicate the free probe and the protein-bound complex. (C) Nucleotide sequences of various competitors used to define the PLATZ1-binding site. The positions of the mutated nucleotides are underlined. (D) Competition experiments using the 68-bp WT competitor and LS-mutated competitors. Binding reactions were carried out with 200 ng of the PLATZ1 protein and the ³²P-labeled 68-bp DNA fragment (2.5 fmol) in the absence or presence of the unlabeled competitor DNA. A 400-fold competitor was added in each reaction. The control does not contain added protein or competitor DNA.