Characterization of the mouse insulin receptor gene promoter

The 5' flanking region of the mouse insulin proreceptor gene was isolated, and the 5' boundary of the minimal promoter was mapped. Genomic clones encompassing greater than 30 kilobases of the gene contain the promoter and exons 1 and 2 interrupted by an approximately 20-kilobase intron at the codon for amino acid 7 of the alpha subunit. The nucleotide sequence of a 1.3-kilobase fragment containing 766 base pairs of the 5' flanking region and the entire first exon was determined. Two major transcription start sites were mapped by S1 nuclease analysis to sites located 469 and 424 nucleotides upstream from the initiation codon for translation. The 5' terminus of an insulin proreceptor cDNA, isolated from a mouse 3T3-L1 adipocyte cDNA library, corresponds to the 3'-most major start site of transcription. The 5' deletion mutants of the 5' flanking region of the proreceptor gene, linked upstream of the bacterial chloramphenicol acetyltransferase reporter gene, were transfected into 3T3-L1 preadipocytes and assayed for promoter activity. The 5' boundary of the minimal promoter, which directs unexpectedly high levels of reporter gene expression, maps to a region 22 base pairs upstream from the 3'-most major transcription start site.