Fig. 5

Identification of a tuba1a promoter enhancer. (A) 5′ promoter deletions identify tuba1a regulatory elements. PC12 cells were co-transfected with the indicated tuba1a (α1T)-luciferase constructs along with CMV-CAT for normalization. Two days post-transfection cells were assayed for luciferase and CAT activity. Reported are relative light units (RLU) representing luciferase activity normalized to CAT activity. (_*p < 0.05, t-test). (B) The - 182/-104 tuba1a sequence confers activation to a heterologous minimal enkephalin (MEK) promoter in an orientation independent manner. PC12 cells were transfected and assayed, as described in (A), with the indicated constructs. (_*p < 0.01, t-test). (C) The - 182/-104 tuba1a enhancer binds proteins present in PC12 nuclear extracts. Radiolabelled - 182/-104 tuba1a enhancer was mixed with and without PC12 nuclear extract (NE) and DNA:protein complexes resolved on native acrylamide gels (EMSA). Shown is a representative autoradiogram indicating free probe (F) and 3 DNA:protein complexes (arrows). Specificity of DNA:protein binding is indicated by competition with 50x unlabeled tuba1a enhancer DNA (competitor). (D) Sequence comparison of the proximal promoter of tuba1a from goldfish (gf) and zebrafish (zf). The conserved protein binding site identified in Fig. 6 is underlined. The CAAT and TATA boxes are identified by dashed underline. An arrow identifies the transcription start site, identified in goldfish, and carrots above and below the sequence identifies the end of exon 1 and beginning of intron 1. Values in (A) and (B) are means +/- SEM.