Proc. Natl. Acad. Sci. USA Vol. 94, pp. 13339–13344, November 1997 Physiology Australian lungfish neurohypophysial hormone genes encode vasotocin and [Phe2]mesotocin precursors homologous to tetrapod-type precursors (molecular cloningyprecursor organizationymolecular evolutionyphylogenetic tree) SUSUMU HYODO*†,SUSUMU ISHII‡, AND JEAN M. P. JOSS§ *Department of Biology, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153, Japan; ‡Department of Biology, School of Education, Waseda University, Nishi-waseda, Shinjuku-ku, Tokyo 169-50, Japan; and §School of Biological Sciences, Macquarie University, New South Wales 2109, Australia Communicated by Howard A. Bern, University of California, Berkeley, CA, September 15, 1997 (received for review August 8, 1997) ABSTRACT In view of the well-established role of neu- which are much larger than the nonapeptides, gives more rohypophysial hormones in osmoregulation of terrestrial ver- precise and reliable information for estimating the molecular tebrates, lungfishes are a key group for study of the molecular evolutionary relationships than does analysis based on substi- and functional evolution of the hypothalamo-neurohypophy- tutions only in the nine amino acid residues. Using statistical sial system. Here we report on the primary structure of the comparison of gene structures and the predicted amino acid precursors encoding vasotocin (VT) and [Phe2]mesotocin sequences of precursors, Urano and colleagues (3–5) have ([Phe2]MT) of the Australian lungfish, Neoceratodus forsteri. proposed that teleost neurohypophysial hormone genes have Genomic sequence analysis and Northern blot analysis con- their own evolutionary history separate from that of the 2 firmed that [Phe ]MT is a native oxytocin family peptide in tetrapod genes. Their hypothesis is further supported by the the Australian lungfish, although it has been reported that the structural characteristics of neurohypophysial hormone pre- lungfish neurohypophysis contains MT. The VT precursor cursors, including composition and the presence or absence of consists of a signal peptide, VT, that is connected to a posttranslational modification sites (2–5). neurophysin by a Gly-Lys-Arg sequence, and a copeptin Lungfishes are the closest surviving freshwater relatives of moiety that includes a Leu-rich core segment and a glycosyl- 2 the Devonian fish group from which early amphibians are ation site. In contrast, the [Phe ]MT precursor does not assumed to have evolved. Acher and his colleagues (6, 7) contain a copeptin moiety. These structural features of the reported that lungfishes have mesotocin (MT), the non- lungfish precursors are consistent with those in tetrapods, but mammalian tetrapod type hormone, as their OT family hor- different from those in teleosts where both VT and isotocin mone, whereas all teleosts examined have isotocin (IT). They precursors contain a copeptin-like moiety without a glycosyl- have proposed an evolutionary lineage of IT (teleosts)-MT ation site at the carboxyl terminals of their neurophysins. (non-mammalian tetrapods)-OT (mammals) and suggest a Comparison of the exonyintron organization also supports close relationship between lungfishes and amphibians. How- homology of the lungfish [Phe2]MT gene with tetrapod oxy- ever, the data on amino acid substitutions in nonapeptides are tocinyMT genes, rather than with teleost isotocin genes. Moreover, molecular phylogenetic analysis shows that neuro- insufficient to estimate the molecular phylogeny of the whole hypophysial hormone genes of the lungfish are closely related neurohypophysial hormone genes. For their VP family pep- to those of the toad. The present results along with previous tide, lungfishes have vasotocin (VT), which is common to all morphological findings indicate that the hypothalamo- non-mammalian vertebrate species. These previous reports led neurohypophysial system of the lungfish has evolved along the us to study lungfish neurohypophysial hormone precursors and tetrapod lineage, whereas the teleosts form a separate lineage, their genes to further clarify the molecular evolution of both within the class Osteichthyes. neurohypophysial hormone genes. As material, we used the Australian lungfish, Neoceratodus forsteri, which is considered to be more closely related to other Neurohypophysial hormones are nonapeptides regulating var- ious physiological events related especially to water and salt freshwater fish than other lungfish species because of the metabolism and reproduction. Twelve distinct nonapeptide well-developed gills on all gill arches. We isolated cDNAs principles have been chemically characterized in a wide variety encoding the neurohypophysial hormone precursors of this of vertebrates, and are classified into two groups: the vaso- species. We report here characteristics of the nucleotide and pressin (VP) and the oxytocin (OT) families. They are believed deduced amino acid sequences of the isolated cDNAs and the to have developed from a common ancestral molecule by gene structural organization of the precursors. These characteris- duplication (1). All vertebrate species, except for the cyclo- tics, together with the molecular phylogenetic analysis, clearly stomes, contain at least one VP family peptide and one OT show that neurohypophysial hormone genes of the Australian family peptide. Several schemes have been proposed as path- lungfish have evolved along the tetrapod lineage, whereas the ways of hormonal molecular evolution based on amino acid teleosts appear to form a separate lineage within the class sequences of nonapeptides and their phyletic distribution (1). Osteichthyes. Furthermore, we present evidence for a peptide, Complementary DNA and genomic analyses have shown [Phe2]MT, as a native neurohypophysial hormone in addition that neurohypophysial nonapeptides are synthesized as large to VT in the Australian lungfish. precursor molecules (2). Analysis of the precursor molecules, Abbreviations: IT, isotocin; MT, mesotocin; OT, oxytocin; VP, vaso- The publication costs of this article were defrayed in part by page charge pressin; VT, vasotocin. Data deposition: The sequences reported in this paper have been payment. This article must therefore be hereby marked ‘‘advertisement’’ in deposited in the GenBank database {accession nos. AB001977 (VT accordance with 18 U.S.C. §1734 solely to indicate this fact. cDNA) and AB001978 ([Phe2]mesotocin cDNA)}. © 1997 by The National Academy of Sciences 0027-8424y97y9413339-6$2.00y0 †To whom reprint requests should be addressed. e-mail: cshyodo@ PNAS is available online at http:yywww.pnas.org. komaba.ecc.u-tokyo.ac.jp. 13339 Downloaded by guest on September 24, 2021 13340 Physiology: Hyodo et al. Proc. Natl. Acad. Sci. USA 94 (1997) MATERIALS AND METHODS which included nucleotide sequences for VT and MT. The cDNAs also included nucleotide sequences for Gly-Lys-Arg Adult lungfish were collected from a tributary of the Mary sequence and the conserved portion of neurophysin, support- River in southeastern Queensland in December 1993. Total ing the notion that the cDNAs encode portions of lungfish RNA was extracted from two hypothalami, weighing 340 and 1 neurohypophysial hormone precursors. By screening the li- 323 mg, with Isogen (Nippon Gene, Tokyo); poly(A) RNA brary with these partial VT and MT cDNAs, 4 VT- and 7 was then prepared by using Oligotex-dT30 (Japan Synthetic MT-positive clones were obtained. One of four VT-positive Rubber, Tokyo). A cDNA library was constructed by using the clones contained a VT-specific sequence. Five of seven MT- cDNA Synthesis System Plus and cDNA Cloning System lgt10 positive clones encoded a MT-like sequence in which the (Amersham). second amino acid residue from the amino terminal was Phe, Complementary DNAs encoding portions of VT and MT- not Tyr. The remaining two MT-positive clones contained no like precursors were amplified by a PCR technique from a sequence encoding neurohypophysial hormone. The MT- mixture of phage DNA prepared from the amplified cDNA positive clones thus encoded a new peptide [Phe2]MT as the library; their sequences were determined and were then used mature OT family hormone instead of MT. Northern blot as screening probes to obtain full-length cDNAs. For PCR analysis revealed one band of 950 bases for the VT probe and amplification, the following three primers were synthesized: one band of 800 bases for the [Phe2]MT probe (data not forward and reverse primers that are complementary to re- shown). gions close to EcoRI ends of the left and right arms of lgt10, VT Precursor. respectively, and a degenerate VTyMT primer that consists of VT precursor is composed of four segments: oligonucleotides predicted from the amino acid sequence of a signal peptide, VT, a neurophysin and a copeptin (Figs. 1–3). VT and MT (residues 1–7). The amplification was performed VT is connected to its neurophysin by the Gly-Lys-Arg se- with the VTyMT primer and either the forward or the reverse quence which serves as a signal for proteolytic processing and primer in the presence of Ampli Taq DNA polymerase (Perkin– carboxyl-terminal amidation (Fig. 1). All Cys residues in the Elmer) using a Program Temp Control System, PC-700 (Astec, neurophysin, which are considered to be important for its Fukuoka). After electrophoresis, major bands were excised conformation, are completely conserved (Fig. 2). Lungfish and ligated into pBluescript II SK1 vector (Stratagene). The copeptin includes a Leu-rich core segment and a glycosylation