We had previously established cell culture conditions in which rat insulinoma cells can be maintained. The total amounts of insulin RNA in high and low glucose were identical. In conditions of low glucose, insulin mRNA was found to be shorter by approximately 100 bases. This difference was due to a significantly shorter poly(A) tract. To pursue these studies, we have utilized bovine papilloma virus (BPV) as an expression vector. In preliminary studies to optimize the system, we found that the orientation of the insulin insert in the BPV genome significantly influences gene expression. It may be that crucial promoter-enhancer interactions are affected by their relative orientations. Current studies to examine this point are in progress. In addition, flanking upstream sequences may contain inhibitory signals. Present studies will further map and characterize the potential inhibitory region, and define enhancer/promoter interactions.