Figure 3

The majority of the 41 bp PRE-1 sequence is required for binding of nuclear factors in vitro. Large deletions and 3 bp mutations of PRE-1 eliminate trans-factor(s) binding. A. Electrophoretic mobility shift assay demonstrates that PRE-1 deletion probes P4.1-3 are unable to form a complex with eye nuclear protein but are sufficient to compete away factors from binding to full length P4 at the indicated molar excess of unlabelled probe. C. Electrophoretic mobility shift assay demonstrates that mutant PRE-1 probes P4MA-D are unable to bind eye nuclear protein but P4ME is, indicating that 3 bp sequences mutated in A-D are necessary for trans-factor(s) binding to PRE-1. B, D. Sequence of PRE-1 deletion and mutant probes. Nucleotides required for binding are indicated by boxes and 3 bp mutated sequences are in lowercase and underlined. Nuclear protein (NP), non-related (NR), probe 4 (P4 or PRE-1), free probe (FP). Asterisks indicate radiolabelled probe and arrow indicates the DNA-protein complex of interest.