Mutagenesis of the Rat Insulin II 5′-Flanking Region Defines Sequences Important for Expression in HIT Cells

Abstract

To define the cis-acting elements important for rat insulin II gene expression, we analyzed the effects of 5′ deletions and linker-scanning mutations on the expression of a rat insulin II reporter gene in an insulinoma cell line (HIT). The reporter gene contained 448 base pairs of 5′-flanking sequence joined to the bacterial chloramphenicol acetyltransferase gene. Expression of the 5′ deletion mutations indicated that the minimal sequence requirement for efficient expression was 218 base pairs of 5′-flanking sequence, and at least three regions downstream from −218 were important for transcription. A more precise localization of these elements and the cis-acting sequences in the promoter was achieved by analysis of the expression of 18 linker-scanning mutations. In these studies at least four other regions important for expression of the rat insulin II gene were identified. These findings suggest that the sequences important for rat insulin II and rat insulin I expression may differ significantly despite the high degree of sequence similarity in their 5′-flanking regions.