File:Os01g08415006.jpg

Figure 6. DNA binding affinity of OsMYB3R-2 protein. A, The alignment of MSA-like sequences shown in the promoters of type B cyclin genes: rice OsCycB1;1 and OsKNOLLE2, Arabidopsis cyc1bAt (Day et al., 1996) and cyc2aAt (Ferreira et al., 1994), and tobacco NtCYM (Ito et al., 1997), NACK1, and NACK2 (Ito et al., 1998) encoding kinesin-like proteins. The boxed 11-bp sequences share high homology with each other. The nucleotide positions are numbered from the transcription start sites. The motifs of binding sites of c-Myb (Howe and Watson, 1991) and v-Myb (Grotewold et al., 1994) are also shown. B, Probes and competitors used in EMSA. BP, A 378-bp fragment of OsCycB1;1 promoter upstream of the transcription start site ATG; RT1, an MSA cis-acting element of BP; RT2, an MSA trans-acting element of BP; RT3, an MSA cis-acting element of OsKNOLLE2 promoter upstream of the transcription start site ATG; RT1mut, mutant of the RT1 motif; BPWTand RT1WT, competitors of biotinlabeled probes of BP and RT1, respectively. C to E, EMSA assays of OsMYB3R-2 protein. Abbreviations are the same as in B. Binding reaction mixtures were incubated with the probes and mock-translated product (mock = probe + no protein) or in vitrosynthesized OsMYB3R-2, with GST as a control, in the presence or absence of a 200-fold molar excess of unlabeled oligonucleotide competitors. DNA binding affinity of OsMYB3R-2 was confirmed experimentally twice. AL1 and AL4, OsMYB3R-2-antisense lines; OL5 and OL7, OsMYB3R-2-overexpressing lines.