The primary structures of the precursors of neurohypophysial hormones vasotocin (VT) and mesotocin (MT) in the hypothalamus of the toad Bufo japonicus were determined by analyzing the nucleotide sequences of the cloned cDNAs encoding them. The MT precursor consists of 125 amino acid residues containing a signal peptide followed directly by MT, which in turn is connected to the MT neurophysin by Gly-Lys-Arg, a processing and carboxylterminal amidation signal. In contrast, the VT precursor includes a glycoprotein of 36 amino acids following the VT neurophysin. Except for glycoprotein, the structures of MT and VT precursors are quite similar. RNA transfer blotting analysis showed that both MT and VT mRNAs are present in the brain but not in the liver, ovaries, and testes ofthe toad. The sequences and the structural organizations of the MT and VT precursors are highly homologous to those of their mammalian counterparts, oxytocin and arginine vasopressin precursors, respectively. This fact suggests that, in the evolutionary pathway of neurohypophysial hormones, VT is the ancestor molecule of vasopressin, while MT is that of oxytocin.Vasotocin (VT) and mesotocin (MT) are neurohypophysial peptides produced by magnocellular preoptic neurons in the hypothalamus in amphibians, reptiles, and birds. They have important physiological roles in the regulation of water balance in anuran amphibians (1). VT is probably an antidiuretic hormone, while MT may be a diuretic one. Recent neuroethological studies have suggested a further physiological significance of VT in anuran reproductive behavior (2).Ten distinct nonapeptide principles have been characterized in a wide variety of vertebrates (3). The molecular evolution of neurohypophysial hormones has attracted many investigators, and many schemes have been proposed for an evolutionary pathway based on their amino acid structures and phyletic distributions (4 MT are the most widely distributed neurohypophysial principles in lower vertebrates, elucidation of the structures of their precursors would provide valuable information as to the evolutionary relationship of these peptides.We report here the isolation and characterization of the cDNAs encoding the mRNAs for VT and MT precursors of the toad, Bufo japonicus. The elucidated nucleotide sequences show considerable homology in both organizations and sequences among the four precursors of neurohypophysial hormones (VT, MT, [Arg8]VP, and OT).
MATERIALS AND METHODSProcedures for constructing a cDNA library from the toad diencephalons, screening of the library, analyzing the nucleotide sequences, and RNA transfer-blot analysis followed those of Ishida et al. (10). In brief, the diencephalons (15 g) were collected from 300 toads captured in the breeding season in March 1985, and a total RNA (5.1 mg) was extracted from them in 4 M guanidinium thiocyanate buffer (11). Poly(A)+ RNA (115 ug) was separated by oligo(dT)-cellulose column chromatography (12). A cDNA library was constructed by the method of Okayama and Berg (13) with 10