Mutational Analysis of the Mouse Somatostatin Receptor Type 5 Gene Promoter

Abstract

We have previously characterized the structure of the murine somatostatin receptor type 5 gene (sst5). Initial transient transfection studies in pituitary somatolactotropes (GH(3)) mapped the promoter activity of this gene to a region 290 bp upstream of the transcription start site. The current study identifies the sst5 promoter region critical for basal activity. A series of deletions was generated, and promoter activity was localized to a region between -83 and -19. Similar promoter deletion patterns were evident in five pituitary cell types. Seven 10-bp transversion mutations encompassing the region between -83 and -19 were generated, and functional activity was assessed. Promoter activity was reduced by the mutations spanning -67 to -47 compared with the wild-type construct. Another mutation between -26 and -17 resulted in promoter activity reduction in GH(3) cells, but not TtT-97 thyrotropes. Deoxyribonuclease I protection analysis of the sst5 promoter region between -208/+47 was performed using GH(3) and TtT-97 nuclear extracts. The most striking protected regions, located between -61 and -41 and -25 and -3, correlated with functionally important regions identified by transfection studies. In summary, the mouse sst5 gene promoter has been characterized, and functional activity and nuclear factor interactions were mapped to two specific promoter regions. The region between -67 and -47 appears to contain a nucleotide sequence critical for basal transcriptional regulation of the mouse sst5 gene in pituitary cells.