FROG DIAZEPAM-BINDING INHIBITOR - PEPTIDE SEQUENCE, CDNA CLONING, ANDEXPRESSION IN THE BRAIN

Three peptides derived from diazepam-binding inhibitor (DBI) were isol ated in pure form from the brain of the frog Rana ridibunda. The prima ry structures of these peptides showed that they correspond to mammali an DBI-(1-39), DBI-(58-87), and DBI-(70-87). A set of degenerate prime rs, whose design was based on the amino acid sequence data, was used t o screen a frog brain cDNA library. The cloned cDNA encodes an 87-amin o acid polypeptide, which exhibits 68% similarity with porcine and bov ine DBI. Frog DBI contains two paired basic amino acids (Lys-Lys) at p ositions 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 absen t in peripheral tissues. The distribution of DBI mRNA and DBI-like imm unoreactivity in the frog brain was studied by in situ hybridization a nd immunocytochemistry. Both approaches revealed that the DBI gene is expressed in ependymal cells and circumventricular organs lining the v entricular cavity. Since amphibia diverged from mammals at least 250 m illion years ago, the data show that evolutionary pressure has acted t o conserve the structure of DBI in the vertebrate phylum. The distribu tion of both DBI mRNA and DBI-like immunoreactivity indicates that DBI is selectively expressed in glial cells.