Frog diazepam-binding inhibitor: Peptide sequence, cDNA cloning, and expression in the brain

Three peptides derived from diazepam-binding inhibitor (DBI) were isolated in pure form from the brain of the frog Rana ridibunda. The primary structures of these peptides showed that they correspond to mammalian DBI- (1-39), DBI-(58-87), and DBI-(70-87). A set of degenerate primers, whose design was based on the amino acid sequence data, was used to screen a frog brain cDNA library. The cloned cDNA encodes an 87-amino acid polypeptide, which exhibits 68% similarity with porcine and bovine DBI. Frog DBI contains two paired basic amino acids (Lys-Lys) at positions 14-15 and 62-63 and a single cysteine within the biologically active region of the molecule. Northern blot analysis showed that DBI mRNA is expressed at a high level in the brain but is virtually absent in peripheral tissues. The distribution of DBI mRNA and DBI-like immunoreactivity in the frog brain was studied by in situ hybridization and immunocytochemistry. Both approaches revealed that the DBI gene is expressed in ependymal cells and circumventricular organs lining the ventricular cavity. Since amphibia diverged from mammals at least 250 million years ago, the data show that evolutionary pressure has acted to conserve the structure of DBI in the vertebrate phylum. The distribution of both DBI mRNA and DBI-like immunoreactivity indicates that DBI is selectively expressed in glial cells.