Fig. 2

Binding of human SBF/Staf/ZNF143 to the SPH element of a zebrafish U6 gene promoter in vitro. A. EMSA. Approximately 3 fmol of radiolabeled zU6-3 DNA fragment containing the sequence from - 100 to + 20 (lanes 1–9) or hU6-1 DNA fragment from - 307 to - 190 (lanes 10–18) were incubated with 2 μL of human SBF/Staf/ZNF143-(88–638) expressed by in vitro transcription/translation (lanes 3–9 and 12–18) or with unprogrammed extract (lanes 2, 11), and electrophoresed on a 4% native polyacrylamide gel. In addition, in samples shown in lanes 4–6 or lanes 13–15 an unlabeled double-stranded oligonucleotide with the sequence including the human U6-1 SPH element were added with the labeled probe in amounts noted in the figure, or similarly, an unlabeled oligonucleotide containing the unrelated, OCT sequence was added to samples electrophoresed in lanes 7–9 or lanes 16–18. Sequences of the competitor oligonucleotides can be found in (Kunkel et al., 1996) where hU6-1 SPH is called NONOCT(long) and hU6-1 OCT is called OCTCON. B. DNase I footprinting demonstrates binding of human SBF/Staf/ZNF143 DBD to zebrafish U6 SPH element. Approximately 3 fmol of singly end labeled DNA fragment including the zU6-3 gene from - 100 to + 20 were incubated with amounts of purified human SBF/Staf/ZNF143 DBD protein as indicated in the figure. After limited DNase I treatment, nucleic acids were purified and electrophoresed on a 12% polyacrylamide/8.3 M urea gel, followed by autoradiography. The positions of the SPH and TATA elements and locations relative to the transcriptional start site were deduced by electrophoresis of G and T dideoxy sequencing reactions on the same gel (not shown).