Meeting Abstract

34.2  Jan. 5  Defining the promoter region of a second insulin gene in zebrafish PAPASANI, MR*; HALTERMAN, K; HILL, RA; University of Idaho; University of Idaho; University of Idaho papasani@uidaho.edu

A second insulin gene, insb has been identified and shown to be expressed in the pancreas, head and blastomeres during embryonic development of zebrafish. Towards understanding gene regulatory networks underlying insb mRNA expression, we have isolated and characterized the proximal promoter region of insb. We have conducted luciferase reporter assays to investigate the promoter activity of three different fragments of the 5�flanking region of insb, -668 (-668/+3), -373 (-373/+3), and -180 (-180/+3) relative to the transcription start site (+1). We have transfected two different cell lines: 1) zebrafish embryonic cell line (ZF4) that expresses insb mRNA, and 2) mouse pancreatic islet &beta-cell line (&betaTC3) containing the mammalian &beta-cell specific regulatory network. In transfected &betaTC3, the regions -668/+3 and -373 /+3 were strongly activated showing relative luciferase activity levels of 20.6581, 18.4248 respectively compared to baseline levels of the promoterless luciferase vector. In contrast, the activity levels of -180/+3 region were not above baseline levels. Data from transfected ZF4 cells were consistent with the &betaTC3 model as -668/+3 and -373/+3 showed relative luciferase activity levels of 2.9076, 2.1234 respectively and -180/+3 activity levels were below baseline. In &betaTC3, activity levels of -668/+3 and -373/+3 were similar (P>0.05). However in ZF4, -668/+3 showed an increase in promoter activity compared to -373/+3 (P<0.05). These data suggest that the proximal promoter regions -668/+3, -373/+3 retain promoter activity and the -373 to -180 region is critical for promoter activity of insb.