Cloning, sequencing and characterization of the tilapia insulin gene

Using degenerate primers based on insulin sequences from other organisms, we report the cloning of the complete tilapia (Oreochromis niloticus) insulin gene. Using nested primers and a cassette ligation strategy we have also cloned 932 base pairs (bp) of 5' flanking and 1152 bp of 3' flanking sequence. The tilapia insulin gene has the similar three exon (one untranslated), two intron distribution found in all insulin genes sequenced to date. However, intron 1 is unique in having a smaller size (73 bp) than found in other organisms. 5' RNA extension revealed the presence of two potential transcriptional start sites. A perfect TATA box is located at -30 bp from the first transcriptional start site. Interestingly, the 5' upstream region contains a microsatellite close to the same position of a unique minisatellite found only in humans and primates. The upstream region also contains several potential control elements to regulate insulin expression that are found in mammalian insulin genes.