FIG. 5.

Oligonucleotide B is bound by a cellular factor(s) but functions poorly in heterologous contexts. (A) Oligonucleotide B forms specific complexes with 293T cell nuclear extracts. Labeled oligonucleotide B (lane 1) was incubated with 293T cell nuclear extracts (lanes 2 to 8) in the absence of competitor oligonucleotide (lane 2) or in the presence of the following unlabeled competitor oligonucleotides: WT oligonucleotide B (20- and 200-fold) (lanes 3 and 4, respectively), mutant oligonucleotide 1 (20- and 200-fold) (lanes 5 and 6, respectively), and mutant oligonucleotide 2 (20- and 200-fold) (lanes 7 and 8, respectively). The specific shifted bands are shown by arrows. WT-B, WT oligonucleotide B; mut-1, mutant oligonucleotide 1; mut-2, mutant oligonucleotide 2. (B) Repeats of oligonucleotide B mediate RTA activation poorly in a heterogeneous promoter. Oligonucleotide B or a long oligonucleotide spanning site B (oligonucleotide BL) (both illustrated in Fig. 3A) was inserted in tandem repeats (with the number of repeats as shown) into the SV40 minimal promoter in the pGL3 promoter, which drives the expression of luciferase. Individual reporter constructs were cotransfected into 293T cells with either empty vector or RTA expression vector, and the fold induction by RTA was calculated; the error bars represent standard deviations of the results from at least two independent experiments.