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).

In vitro transcription of zebrafish U6 wt or mutant promoter gene plasmids. A 100 μg aliquot of HeLa S100 extract was incubated with 1 μg of pGEM vector, hU6-1maxi, zU6-1maxi, TATATMUT promoter, or SPHMUT promoter plasmid DNAs, and included nucleoside triphosphates (with radiolabeled α-GTP) and α-amanitin at a final concentration of 2 μg/mL. Nucleic acids were purified and electrophoresed on a 10% polyacrylamide/8.3 M urea gel. “U6maxi” represents specific transcripts from U6 promoters. “tRNA^his” shows guanylyltransferase labeling of endogenous tRNA in the S100 extract and serves as a recovery and normalization control for quantification of each sample. Numbers in the row below the lane numbers are the relative percentage amounts of normalized zU6-1maxi transcript compared to the wt promoter (lane 3), and are the average of two independent reactions. Numbers in parentheses are the relative transcription levels from each experiment.

Relative expression of U6 maxigene transcripts from plasmids injected into zebrafish embryos. Equal masses of zU4-1maxigene and zU6-1maxigene plasmid DNAs were injected into zebrafish embryos, and total RNA was extracted at the 16-somite stage. Specific transcripts were amplified by RT-PCR, electrophoresed on 18% polyacrylamide gels, stained with ethidium bromide, and relative band intensities were determined. Band intensities from -RT samples (lanes 1, 3, 5, 7) were subtracted from +RT samples (lanes 2, 4, 6, 8), and U6-1maxi signals were normalized to U4-1maxi signals obtained from like RNA samples. Numbers in the row below the lane numbers are the relative percentage amounts of normalized zU6-1maxi RT-PCR product compared to the wt promoter set at 100% (lane 4), and are the average of two independent injection experiments for each plasmid DNA. Numbers in parentheses are the relative expression levels from each experiment.