We have examined the DNA sequences responsible for regulation of insulin expression in the B-cells of pancreas. A DNA transfection procedure for introducing DNA molecules into established lines of transformed hamster B-cells (HIT) has been established. Using this protocol, a sequence upstream of the rat insulin I gene has been identified which can activate expression from a heterologous promoter to high levels in the HIT cell line. This element shares several properties with viral enhancer elements and is presently being chararcterized in detail. Current studies also focus on the identification of pancreatic cell-specific factors that bind upstream regulatory regions of the rat insulin gene and to identify the particular insulin regulatory sequences that interact with these proteins. Sequences located 1.5kb upstream of the rat insulin I cap site have been identified as memebers of a major rat long interspersed repetitive (LINE) sequence. These sequences have a down regulatory or negative effect on enhancer function as shown in transient assay system using the SV40 enhancer and a heterologous indicator gene. They reduce enhancer-mediated transcriptional activity 10-fold when positioned either 5' or 3' to the indicator gene. The mechanism of inhibition is currently under study.