Fig. 3

Fig. 3 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.