Figure 2

Identification of PRE-1 in TαCP. A. Annotated zebrafish TαC proximal promoter sequence. The sequence of probes 1-4 used in electrophoretic mobility shift assays are highlighted by rounded boxes. The PRE-1 (probe 4) sequence is highlighted in pink. The most 5'-end of zebrafish TαC ESTs (accession numbers DY550630, BG307391) are indicated by asterisks and underlined. The translation start codon is indicated by a box and methionine (Met). Putative trans-factor binding sites for Ret 3, NRE, PCE-1, PCE-II, Otx, CBE, Nr2e3 and E-boxes, based on homology to known cis-elements are indicated by boxes [26–29, 33, 36, 51, 52]. NRE, Nrl Response Element. PCE, photoreceptor conserved element. CBE, Crx-binding element. B. PRE-1 specifically binds nuclear factors expressed in ocular tissue. Electrophoretic mobility shift assay of radiolabelled PRE-1 (TαC probe 4) incubated with increasing concentrations of zebrafish eye or body nuclear protein. PRE-1 binds nuclear protein isolated from adult zebrafish eyes but not body nuclear protein. C. Competition analyses indicate that binding of trans-factor(s) is specific because unlabelled PRE-1 at 100 or 200-fold molar excess is able to compete away binding of eye nuclear protein to labelled PRE-1, whereas an unlabelled non-related competitor at 200-fold molar excess is not. 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.