Proc. Natd. Acad. Sci. USA Vol. 86, pp. 5242-5246, July 1989 Biochemistry Vasotocin and isotocin precursors from the white sucker, Catostomus commersoni: Cloning and sequence analysis of the cDNAs (neuropeptide/hypohlamic hormone precursor/teleos neurophysin/glycopeptide/copeptin) JORG HEIERHORST*, STEVEN D. MORLEY*, JAIME FIGUEROA*, CHRISTIANE KRENTLER*, KARL LEDERISt, AND DIETMAR RICHTER*t *Institut fur Zellbiochemie und klinische Neurobiologie, Universitits-Krankenhaus Eppendorf, Martinistrasse 52, 2000 Hamburg 20, Federal Republic of Germany; and tDepartment of Pharmacology and Therapeutics, Health Science Centre, The University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1 Communicated by Berta Scharrer, March 27, 1989

ABSTRACT The nucleotide sequences of cloned cDNAs In teleost fish, vasotocin and isotocin are, respectively, the encoding the precursors for vasotocin and isotocin have been counterparts of mammalian and (1, 6). elucidated by analyzing a Agtll library constructed from However, neither their precursor structures nor their neuro- poly(A)' RNA from the hypothalamic region of the teleost fish physin sequences have been analyzed to date. Here we report Catostomus commersom'. Screening of the library was carried the identification of cDNA clones encoding the teleost fish out with synthetic oligonucleotide probes deduced from the vasotocin and isotocin precursors, the structural organiza- amino acid sequences of the nonapeptides vasotocin and iso- tion of the predicted proteins, and their relationship to their tocin. The cDNA nucleotide sequences predict isotocin and mammalian counterparts. § vasotocin prohormone precursors each consisting of a signal peptide, a hormone moiety, and a neurophysin-like molecule. However, in comparison to their mammalian counterparts, MATERIALS AND METHODS both fish are extended at their C termini by an =30 amino acid sequence with a -rich core segment. Materials. Tetramethylammonium chloride was purchased These extensions show striking similarities with the glycopep- from Merck-Schuchardt (Hohenbrunn, F.R.G.). Endonu- tide moiety (the so-called copeptin) present in mammalian clease-free Escherichia coli DNA polymerase I (Klenow vasopressin precursors, except that they lack the consensus enzyme) and restriction enzymes were obtained from Boeh- sequence for N-glycosylation. These data suggest that mam- ringer Mannheim; reverse transcriptase, RNase H, phage T4 malian copeptin is derived from the C terminus of an ancestral DNA ligase, and T4 DNA polymerase, from P. H. Stehelin neurophysin. (Basel); E. coli DNA ligase and T4 polynucleotide kinase, from BRL (Neu Isenburg, F.R.G.); and (dT)12_18, oligo(dT)- cellulose, and EcoRI adaptors (9-mer, CTCGTGCCG; 13- The vasopressin/oxytocin hormone superfamily is widely mer, AATTCGGCACGAG), from Pharmacia. Thermus distributed throughout the animal kingdom. It comprises aquaticus (Taq) DNA polymerase was from Cetus. EcoRI- more than a dozen variants, all containing a 9-amino acid digested, dephosphorylated Agtll arms were from Promega peptide backbone with several highly conserved residues Biotec. Gigapack Gold in vitro packaging extracts were including the two at positions 1 and 6, which form bought from Stratagene. 2'-Deoxyadenosine 5'-[a-35S]tri- a disulfide bridge: Cys-Xaa-Xaa-Xaa-Asn-Cys-Pro-Xaa- phosphate (500 Ci/mmol; 1 Ci = 37 GBq) was from Du Pont, Gly-NH2 (1). Their endocrine activities in mammals- and adenosine 5'-[-32P]triphosphate (6000 Ci/mmol) came namely, control of water retention (vasopressin) and smooth from Amersham-Buchler (Braunschweig, F.R.G.). Oligonu- muscle contraction (oxytocin)-are well documented, cleotides were synthesized in an automated DNA synthesizer whereas their functions in invertebrates such as the coelen- using phosphoramidites. terate Hydra (2) or the fish-hunting cone snail Conus (3) Construction of a Agtll Library. Total RNA was isolated remain to be elucidated. from hypothalamic fragments, containing the nucleus preop- Sequence analysis of cDNAs encoding the vasopressin or ticus regions, of 225 suckers (Catostomus commersoni) (7), oxytocin precursors have been carried out for several mam- and poly(A)+ RNA was purified by oligo(dT)-cellulose chro- malian species (4). They predict that the vasopressin precur- matography (8). cDNA was prepared from 5 ,ug of poly(A)+ sor consists of the hormone, a -rich protein called RNA according to the method of Gubler and Hoffmann (9) neurophysin, and a glycopeptide. While the neurophysin and inserted via EcoRI adaptors into the EcoRI site of Agtll probably acts as carrier protein in the transport of the (10). The resulting library consisted of 107 independent hormone from the to the neurohypophysis, the recombinants and was screened without resort to amplifica- glycopeptide, or "copeptin," is thought to function as a tion. prolactin-releasing factor (5). The oxytocin precursor has a Identification of cDNAs Encoding the Sucker Vasotocin and similar structure except that the copeptin moiety is absent Isotocin Precursors. For each screen 2.5 x 105 recombinants and the neurophysin sequence is slightly shorter than its were analyzed by in situ hybridization on nitrocellulose vasopressin-associated counterpart. The central regions of replica filters (=25,000 phage per 150-mm plate) with fully the two neurophysins are extremely well conserved between degenerate synthetic 20-mer oligonucleotide pools corre- different mammals, with variations occurring predominantly sponding to the first 7 amino acids ofthe respective hormone at the N and C termini. Abbreviation: PCR, polymerase chain reaction. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. M25144 and M25145). 5242 Downloaded by guest on September 23, 2021 Biochemistry:Biochemistry:HeierhorstHeierhorstetetal.al.Proc. Nati. Acad. Sci. USA 86 (1989) 5243 sequences. For isotocin two pools were generated: pool I, Polymerase Chain Reaction (PCR) and Genomic DNA Se- 5'-GG-RCA-RTT-NGA-DAT-RTA-RCA-3'; pool II: 5'- quencing. One microgram of sucker liver genomic DNA (15) GG-RCA-RTT-RCT-DAT-RTA-RCA-3'. For vasotocin a was subjected to 30 cycles of PCR amplification (16) using single pool was synthesized: 5'-GG-RCA-RTT-YTG-DAT- oligonucleotide primers deduced from the vasotocin- or iso- RTA-RCA-3'. (R = A or G; N = A, C, G, or T; D = A, G, tocin-encoding cDNA sequences. The reaction mixture (100 or T; Y = C or T.) The oligonucleotides were 5'-end-labeled 1ud) contained 2.5 units of Taq polymerase, 100 pmol each of with T4 polynucleotide kinase to a specific activity of >108 an appropriate "forward" and "reverse" oligonucleotide cpm4&g and filters were screened using conditions under primer (Fig. 1, arrows). Annealing was at 550C for 120 sec, which only exact matches with the target sequence should be extension was at 720C for 75 sec (except for the last cycle, detected (11). In brief, after nonstringent hybridization, fil- where 5 mmn was used), and denaturation was at 920C for 75 ters were washed in a solution containing tetramethylammo- sec. Agarose gel (2%) electrophoresis and ethidium bromide nium ions, the binding of which eliminates the preferential staining revealed in each case a single amplified DNA band melting of A-T versus G-C base pairs, allowing the stringency corresponding to the expected size of 160 base pairs (bp) for of washing to be controlled solely as a function of probe vasotocin and 195 bp for isotocin. length and independently of base composition. The hybrid- For direct sequencing of amplified genomic DNA, single- ization and washing conditions used reproduced those de- stranded DNA template was generated (17) and the PCR scribed previously (11), except that 50 pmol of end-labeled protocol was modified by using 0.5 pmol and 50 pmol of oligonucleotide pool was used per hybridization; the final appropriate forward and reverse primers and 35 amplification stringent wash steps were carried out at 580C, a temperature cycles. After an ethanol/ammonium acetate precipitation 2-40C below the dissociation temperature calculated for step, the amplified DNA was sequenced by using the forward 20-mer oligonucleotides in 3 M tetramethylammonium chlo- oligonucleotide as primer (13). Sequencing of the opposite ride. strand was performed by reversing the oligonucleotide con- Nucleotide Sequence Analysis. Inserts from positive clones centrations in the PCR and using the reverse oligonucleotide were subcloned into Bluescript plasmids (Stratagene) or into as pnimer. M13 phage vectors (12) and their identities were confirmed by Northern Blot Analysis. Poly(A)' RNA (10 ,&g) was dena- sequence analysis on both strands (13). The precursor struc- tured with glyoxal (18), electrophoresed in a 1.2% agarose tures of sucker vasotocin and isotocin were compared with gel, and transferred to Hybond-N membranes (Amersham) those for their mammalian counterparts, vasopressin and according to the supplier's instructions. Hybridization was oxytocin, by alignment of nucleotide and predicted protein carried out with labeled (19) cDNA probes (106 cpm/mi) sequence, using the ALIGN program ofthe DNASTAR (Mad- specific for either vasotocin (nucleotides 440-535; Xba I- ison, WI) sequence analysis software package, which is EcoRI fragment) or isotocin (nucleotides 420-692; Pvu II- based on the algorithm of Wilbur and Lipman (14). EcoRI fragment) mRNA. isotocin 5' CTGAATCGTTTCACTACCTGTCAATTTCAGCCTTTGCAAAAT vasotocin C

i s ot oc in N P m s g s m f s v f 5 11 y 1 1 s v c s a cy i s n cp i g g k r ai 34 43 AGCGAGAGTTATTTCCGTTTTCGCGCGTACTTAACCACGCCTGTGAAGGC 2 GGTTTATTTTGCAGGACTTTTCCCCCCTTCTCAACAACGCGGGAGAGGTT msd s f1 p tc ii1 cli a i s s a cy i qn cp r gg k r s q 33 v as ot o ci n N P q d sp srq cm s cg pg dr gr cf gps i cc g eg 1 g cl 67 143 TCGATACTTGCGGAGCTCGCGGGTGGCGTTTGTCATTTCGGTAGTTGCGC 10? AGCCAGACACC- --AGCAGAGAGTGTGTGTCGTGTGGTCCTGGGAATGCAGGCCGTTGTTACGGTCCCAGTATCTGCTGTGGAGCGGCTCTGGGTTGTCT p dt s re cv s cgp g nag r cy g psi c cga al1gcI 65 1 g s p e t q r c 1 e e d f 1 p s p c e a g g k v c g y e g r c a 100 243 GTGCCCGAATACAGCGAGGATTTCCCCGGGGCGTGAATTCGTTAGCGTTC 199 CTGTCCAAAGTACGAGAGGACCTCGGCCGGGCGAGAACTTGGTAGAGTTC v g s p e t . s c m e e n h 1 p s p c e t g g r p c g d e g r c a 98 a p g v c c d s e g c s v q9s vd g d g d-at- a v s q p a s s q 134 343 GCCAGGCGTTATAAGCGA 7TACGCT-- ATGGCGGCCAACGCGCACGCGA C 299 GCCCGAGCTTGTACCGTATTCGTTGACCGATT__GAACTCGTCGACGTTAAATCAGAGCCAG a p g v c c d s v s c v m d s e c 1 e d v r s d q s e d p s r 1 k t 132 d Ill k 11I hI s np a h py r h q 154

399 CTTTCGGGAACCTCGGTTTTAATGGGTTGAGCGCGGCTCTAACTTATCTGGTTGCCACCGC v sg ei 1 In 1 nlIa s rgr r df 152

499 GTGTtTCCAGTGTGTAAATATGTAATAAATGGTCAAATGTGAAAAAAAAAAAAAA 643 TTTACTGTGCAGCTATTAAAGTCAGAGAGAACGTAAAAAAAAAAAAAAA 3' FIG. 1. Comparison of nucleotide sequences and deduced amino acid sequences of the sucker isotocin and vasotocin precursor cDNAs. Polyadenylylation signals are underlined. Nucleotide sequences used for synthetic PCR primers (Fig. 2) are marked by arrows. Amino acids are represented by lowercase one-letter symbols. Hormone amino acid sequence [residues 21-29 (isotocin) or 20-28 (vasotocin)] is followed by a Gly-Lys-Arg sequence and then the neurophysin (NP) sequence, which extends to the C terminus. Broken lines show amino acid sequences where a G -.+ A point mutation would generate a potential glycosylation site (additional sites are located further upstream, but these are not marked because they are not the right distance from the leucine-rich core segment). Numbers on the left indicate nucleotide positions; those on the right are for amino acid sequences. Three dashes in the vasotocin cDNA sequence indicate a gap introduced to optimize the alignment. Downloaded by guest on September 23, 2021 5244 Biochemistry: Heierhorst et al. Proc. Natl. Acad. Sci. USA 86 (1989) RESULTS quences for both precursors predict larger neurophysin-like proteins that are extended at their C termini by a stretch of Cloning and Nucleotide Sequence Analysis. A cDNA library =30 amino acid residues including a leucine-rich core seg- representing mRNA from the sucker brain hypothalamic region was constructed in Agtll and was screened with two 1 2 3 4 5 6 7 20-mer oligonucleotide pools deduced from the first 7 amino A acids of the isotocin and vasotocin sequences, respectively. In each case, analysis of 2.5 x 105 recombinants gave rise to 4 positively hybridizing clones. Subsequent sequence anal- ysis confirmed the validity of our oligonucleotide screening strategy by revealing two families ofclones, one encoding the isotocin precursor and the other the vasotocin precursor. The largest cDNA clone encoding the isotocin precursor consists of 691 bp, while that for the vasotocin precursor is 553 bp long (Fig. 1). The isotocin-encoding insert exhibits a single open reading frame of 462 nucleotide residues predict- ing a protein of 154 amino acid residues with Mr 16,055. The vasotocin counterpart encodes 152 amino acid residues giv- ing rise to a calculated Mr of 16,011. The isotocin cDNA includes a putative 5' untranslated _,195 bp region with the first possible initiation codon at position 43. ' 160 bp - However, the 5' region ofthe vasotocin cDNA is incomplete, with the first ATG triplet encountered at position 5. Neither of the potential initiation codons are preceded by the con- sensus sequences for initiation found in most eukaryotes (20), but they are succeeded by putative signal sequences (see B 3' 3' below). The 3' untranslated region of isotocin cDNA is significantly longer than that for vasotocin, suggesting a T L e u T\ somewhat larger isotocin mRNA. This was confirmed by C C Northern blot analysis of sucker poly(A)+ RNA, which revealed RNA bands of 775 bases for vasotocin mRNA and A A ,,G A r g G \ 920 bases for isotocin mRNA (Fig. 2). Provided that the A A isotocin cDNA represents the full-length mRNA, it should contain a poly(A) tail of -220 residues. Consensus sequences T -1' for polyadenylylation are present at positions 573-578 and G S e r G 657-662 in isotocin cDNA and at positions 522-527 in vaso- A A tocin cDNA (Fig. 1). Structural Organization of the Isotocin and Vasotocin Pre- C cursors. As shown in Fig. 1, the two precursors possess C P1 r o C similar structures, each consisting ofa putative signal peptide C C followed directly by the hormone moiety and then by a cysteine-rich protein with many characteristics of the neu- A A s p A rophysin family. G The predicted signal sequences of the two precursors contain a high proportion of hydrophobic amino acids, a A A feature characteristic for signal peptides. Each nonapeptide A G I u A hormone is connected to the rest of the precursor by the G G residues Gly-Lys-Arg, known to serve as potential signals for .T hormone amidation (Gly) and precursor processing (Lys- \C S e r C/ Arg), respectively (21). I Comparison of the sucker vasotocin- and isotocin- 5 associated neurophysins reveals a highly conserved central 5 region at amino acid residues 10-76 (isotocin neurophysin) or 9-75 (vasotocin neurophysin), which also shows consider- FIG. 3. (A) Ethidium bromide staining (Left) and Southern blot able homology to their mammalian (1), avian (22), and analysis (Right) of PCR-amplified sucker liver genomic DNA encod- the cDNA se- ing the vasotocin precursor. Lane 1; Hae 111-digested 4X174 DNA amphibian (23) counterparts. Unexpectedly, markers; the following lengths in base pairs are indicated (top to bottom) by horizontal lines: 310, 281/271, 234, 194, 118, and 72; lanes _1 -2 2 and 5, cDNA encoding the vasotocin precursor; lanes 3 and 6, I -3 cDNA encoding the isotocin precursor; lanes 4 and 7, sucker liver V _ -4 genomic DNA. The "forward" and "reverse" primers used for amplification are indicated by arrows in Fig. 1. Amplification of the isotocin cDNA (lane 3) gave rise to the expected band of 195 bp. The amplified DNA was electrophoresed in a 2% agarose gel and South- ern blotted (15). The blot was hybridized to the vasotocin-specific probe (see Fig. 2). No cross-hybridization was observed with iso- FIG. 2. Northern blots of sucker poly(A)' RNA. Blots were tocin cDNA (lane 6). (B) Comparison of cDNA nucleotide residues hybridized with either a vasotocin (V)- or an isotocin (I)-specific 374-394 (Fig. 1) and amplified genomic DNA sequence encoding the probe. Hae 111-digested, 32P-labeled OX174 DNA provided molec- sucker vasotocin precursor. (Right) Genomic DNA was amplified ular size markers: 1, 1353 bp; 2, 1078 bp; 3, 872 bp; 4, 603 bp. The and sequenced as outlined in Materials and Methods. (Left) Corre- differences in the intensity ofthe signals for the two mRNAs are due sponding cDNA sequence. Lanes (from left to right) show products to different specific activities of labeled probes used. of T, C, G, and A sequencing reactions. Downloaded by guest on September 23, 2021 Biochemistry: Heierhorst et A Proc. Natl. Acad. Sci. USA 86 (1989) 5245

rat AVP AREQ$:NA~~~~AtQlOCP.A::RElLLRLVQLAGTQESVDSAKPRVY bovine AVP NORE-S: NA- LL : At LL R L V QtLAC A P E P A E PAQ P G V Y human AVP ^|St $:sR" A ~TQt -0 6 A- :0 eiL L R L V; Q: L A 6 A P E fP F f P A Q P D A Y toad vasotocin A VTP E Q : " MKA....:X:IQ M :0 A S A. :S : D -t t t Rt L M V M- A N 07RQ Q Q S K :H Q F ::Y sucker vasotocin S Q$E PS R [K T V : E LR LINLA S R G R R D F sucker isotocin ::D G: : A. T A V SQ P A S 5 Q0 tt .I I At: : S: N::: P H P Y R L H Q FIG. 4. Comparison of glycopeptide (copeptin) sequences of various species with the C-terminal region of sucker vasotocin and isotocin precursors. Aligned amino acid residues that are homologous in the sucker and the glycopeptide sequences are shown in bold type. Colons indicate gaps introduced to optimize homology. The sequences are from refs. 4 and 23. AVP, vasopressin.

ment. This part of the neurophysin sequence shows a re- ation site in the sucker vasotocin precursor, there is an markable resemblance to that of the glycopeptide (copeptin) arginine residue that may have become a processing signal in of the vasopressin precursor family except that the teleost the corresponding precursors of higher eukaryotes. fish sequence lacks a potential glycosylation site. Further, our data refine the hypothesis that the two hor- Analysis of the C Terminus of Sucker Neurophysin. Inspec- mone precursors are derived from an ancestral molecule, tion of the amino acid and nucleotide sequences of the presumably by duplication ofthe vasotocin gene encoding the vasotocin and isotocin precursors reveals sites within the nonapeptide and a larger neurophysin protein. In the course C-terminal regions that, by a single mutation, could be altered of evolution the C-terminal part of the neurophysin eventu- to a glycosylation site (Fig. 1, broken lines). In the case ofthe ally became a separate glycopeptide with distinct biological isotocin precursor, the respective codons (nucleotides 409- function(s) (vasopressin precursor) or was simply discarded 417) read Asp-Ala-Thr, and those of the vasotocin precursor because of lack of need (oxytocin precursor). Nevertheless, (nucleotides 380-388) read Asp-Pro-Ser. In either case, a it should be kept in mind that teleost have evolved for single G -- A mutation would change the aspartic codon a long time (>400 million years; ref. 24) in a phylogenetic (GAC or GAT) into an asparagine-encoding triplet (AAC or branch quite remote from the line ofdescent ofthe tetrapods. AAT), thus establishing the N-glycosylation consensus se- quence Asn-Xaa-Thr/Ser. We thank Mrs. Helen Wilson of Nanton (Alberta) for help in fish To show that the sequence Asp-Pro-Ser determined from collection; Henk Zwiers, Evelyn and David Ko, Ilona Berbeckar, the vasotocin cDNA did not derive from a cDNA synthesis and Estrella Gonzalez for assistance with the collection of fish artifact but was also present in the genomic DNA encoding hypothalami and preparation oftotal RNA; Werner Rust and Marion the respective precursor, sucker liver DNA was amplified by Daumigen-Kullmann (Hamburg) for technical assistance; and Dr. the PCR technique (16). Two synthetic 24-mer oligonucleo- Willi Kullmann (Hamburg) for synthesizing the oligonucleotides. tide primers were synthesized from the previously deter- This work was supported by the Deutsche Forschungsgemeinschaft mined vasotocin cDNA sequence up- and downstream ofthe (D.R.) and the Medical Research Council of Canada (K.L.). site encoding Asp-Pro-Ser (Fig. 1, arrows). The amplified 1. Acher, R. & Chauvet, J. (1988) Biochimie 70, 1197-1207. DNA, consisting of 160 bp, was shown to have a sequence 2. Grimmelikhuijzen, C. J. P., Dierickx, K. & Boer, G. J. (1982) identical to that obtained for the respective cDNA region Neuroscience 7, 3191-3199. (Fig. 3). This experiment independently confirmed at the 3. Cruz, L. J., de Santos, V., Zafaralla, G. C., Ramilo, C. A., genomic level that the sequence reads Asp-Pro-Ser. In anal- Zeikus, R., Gray, W. R. & Olivera, B. M. (1987) J. Biol. Chem. ogy the sequence Asp-Ala-Thr of the isotocin precursor was 262, 15821-15824. found to be encoded in the amplified genomic DNA fragment 4. Rehbein, M., Hillers, M., Mohr, E., Ivell, R., Morley, S., (195 bp) ofthe isotocin gene (data not shown). Hence both the Schmale, H. & Richter, D. (1986) Biol. Chem. Hoppe-Seyler vasotocin and isotocin precursor truly lack an N-linked 367, 695-704. glycosylation site. 5. Nagy, G., Mulchahey, J. J., Smyth, D. G. & Neill, J. D. (1988) Biochem. Biophys. Res. Commun. 151, 524-529. DISCUSSION 6. Gill, V. E., Burford, G. D. & Lederis, K. (1977) Gen. Comp. Endocrinol. 32, 505-511. The data show that vasotocin and isotocin are expressed as 7. Okawara, Y., Morley, S. D., Burzio, L. O., Zwiers, H., Led- composite precursors, together with their respective neuro- eris, K. & Richter, D. (1988) Proc. Natl. Acad. Sci. USA 85, physins, in the hypothalamic neurosecretory system of the 8439-8443. teleost fish C. commersoni. Although the primary structure 8. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, of the two identified neurophysins indicates an organization 1408-1412. similar to their counterparts from mammals (1), birds (22), 9. Gubler, U. & Hoffman, B. J. (1983) Gene 25, 263-269. and amphibians (23), they exhibit features not previously 10. Huynh, T. V., Young, R. A. & Davis, R. W. (1985) in DNA found in other neurophysin molecules. Both sucker neuro- Cloning, ed. Glover, D. M. (IRL, Oxford, U.K.), Vol. 1, pp. physins are extended by -30 amino acids at their C termini, 49-78. and these the 11. Wood, W. I., Gitschier, J., Lasky, L. A. & Lawn, R. M. (1985) extensions show striking similarities with Proc. Natl. Acad. Sci. USA 82, 1585-1588. glycopeptide ofthe vasopressin precursor family, except that 12. Yanish-Perron, C., Vieira, J. & Messing, J. (1985) Gene 33, the sucker sequences lack a glycosylation site (Fig. 4). In rats 103-119. this glycopeptide, also known as copeptin, has been shown to 13. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. be involved in prolactin release (5), a function that has not Acad. Sci. USA 74, 5463-5467. been determined in the sucker endocrine system. 14. Wilbur, D. J. & Lipman, W. J. (1983) Proc. Natl. Acad. Sci. The cDNA sequences encoding the two sucker precursors USA 80, 726-730. predict that a single nucleotide mutation could change an 15. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular aspartic residue into an asparagine residue to generate a Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold site Spring Harbor, NY). potential glycosylation (Asn-Xaa-Thr/Ser) within the 16. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, C-terminal region of the neurophysin molecules (Fig. 1). The R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science potential glycosylation site and the leucine-rich core se- 239, 487-491. quence would be separated by roughly the same number of 17. Gyllensten, U. B. & Erlich, H. A. (1988) Proc. Natl. Acad. Sci. amino acid residues as they are in the mammalian (4) and toad USA 85, 7652-7656. (23) glycopeptides. Also, upstream of the potential glycosyl- 18. Thomas, P. S. (1983) Methods Enzymol. 100, 255-266. Downloaded by guest on September 23, 2021 5246 Biochemistry: Heierhorst et al. Proc. Nadl. Acad. Sci. USA 86 (1989)

19. Feinberg, A. P. & Vogelstein, B. (1984) Anal. Biochem. 137, 22. Lazure, C., Saayman, H. S., Naud6, R. J., Oelofsen, W. & 266-267. Chrdtien, M. (1987) let. J. Peptide Protein Res. 30, 634-645. 20. Kozak, M. (1984) Nucleic Acids Res. 12, 857-872. 23. Nojiri, H., Ishida, I., Miyashita, E., Sato, M., Urano, A. & 21. Richter, D. (1986) in Molecular Cloning ofHormone Genes, ed. Deguchi, T. (1987) Proc. Natl. Acad. Sci. USA 84, 3043-3046. Habener, J. F. (Humana, Clifton, NJ), pp. 173-206. 24. Acher, R. (1980) Proc. R. Soc. London Ser. B 210, 21-43. Downloaded by guest on September 23, 2021