Fmrfamide) and Novel Related Peptides Are Encoded in Multiple Copies by a Single Gene in the Snail Lymnaea Stagnalis

The Journal of Neuroscience, February 1990, IO(2): 412-419

Cardioactive Neuropeptide Phe-Met-Arg-Phe-NH, (FMRFamide) and Novel Related Peptides Are Encoded in Multiple Copies by a Single Gene in the Snail Lymnaea stagnalis

A. Linacre,2 E. Kellett,’ S. Saunders,’ K. Bright,* P.R. Benjamin,* and J.F. Burke’ Departments of ‘Biochemistry and *Sussex Invertebrate Neuroscience Group, School of Biological Sciences, Falmer, Brighton, East Sussex, BNl 9QG, United Kingdom

The neuropeptide Phe-Met-Arg-Phe-NH, (FMRFamide) is a FMRFamide-like immunoreactivity has been found in a potent cardioactive neuropeptide in Lyfnnaea sfagnalis. Iso- number of neurons in the CNS of Lymnaea (Schot and Boer, lation and sequencing of 2 cDNAs and a genomic clone shows 1982), and recent work using immunocytochemistry and ra- that a single gene encodes a precursor protein which con- dioimmunoassay has identified a pair of heart motoneurons tains 9 copies of the FMRFamide peptide, 2 copies of the whose excitatory effects on the heart appear to be due to the related peptide Phe-Leu-Arg-Phe-NH, (FLRFamide), and sin- releaseof FMRFamide or a related peptide (Buckett, 1987; Ben- gle copies of the putative pentapeptides Gln-Phe-Tyr-Arg- jamin et al., 1988). The electrophysiologicalevidence combined Ile-NH, (posttranslationally modified to pQFYRlamide) and with pharmacology strongly indicated that FMRFamide is the Glu-Phe-Leu-Arg-lie-NH, (EFLRlamide). The gene is tran- principal excitatory neurotransmitter peptide usedby Lymnaea scribed in the CNS and gives rise to a single RNA of 1.7 kb heart motoneurons. In addition, a uniquely identifiable inter- in size. The organization of the Lymnaea gene is significant neuron (VWI) has been found in the Lymnaea CNS which has with respect to the evolution of FMRFamide and related pep- widespread synaptic connections with other central neurons tides in other organisms. (Benjamin, 1984) and also contains FMRFamide-like peptides (Benjamin et al., 1988). It hasbeen proposedthat FMRFamide- The discovery that neuropeptidescan act as neurotransmitters, like peptides might mediate thesesynaptic connections. While neuromodulators, or hormones has had a great impact on the a physiological role for FMRFamide in cardioexcitation in Lym- field of neurobiology. We are interested in understanding the naea is now well established, FMRFamide has a variety of role of identified neuropeptides in neuronal circuits of known actions in other systems. It has been shown to modulate the function usingthe pulmonate mollusc, Lymnaea stagnalis.This activity of specific central neurons (Stone and Mayeri, 1981), snail has previously been usedas a model system for analyzing as well as the L2 neuron (Thompson and Ruben, 1988) and the way in which peptides mediate simple behaviors (Vreug- R14 neuron (Ichinose and McAdoo, 1988) in Aplysia. denhil et al., 1988) and for studies on the structure and orga- FMRFamide causesseveral kinds of membrane conductance nization of invertebrate neuropeptide genes(Smit et al., 1988). changesin the CNS (Cottrell et al., 1984; Boyd and Walker, In this study we describe the structure of the molluscan gene 1985) and shortensthe presynaptic action potential in sensory encodingthe neuropeptide Phe-Met-Arg-Phe-NH, (FMRFam- neurons (Abrahams et al.; 1984). FMRFamide has also been ide). shown to act in peripheral systemsby inhibiting spontaneous FMRFamide was first discovered as a cardioexcitatory agent activity of the gut (Austin et al., 1982) and salivary gland (Bul- in the Venusclam Macrocallista nimbosa(Price and Greenberg, loch et al., 1988). It also stimulates contractions of the buccal 1977), and although the distribution of FMRFamide itself ap- mass (Richmond et al., 1984) and gill (Weiss et al., 1984) in pearsto be limited to molluscs(Price, 1986), immunoreactivity Aplysia. to FMRFamide has been reported in all the major animal phyla FMRFamide is not the only member of this family of and a number of FMRFamide-like peptides have been isolated neuropeptides to have been shown to be present in Lymnaea. from coelenterates(Grimmelikhuijzen and Graff, 1985), arthro- Ebberink et al. (1987) have isolated 2 heptapeptidesfrom the pods (Boer et al., 1980), and chordates (Dockray et al., 1983; central ganglia, SDPFLRFamide (Ser-Asp-Pro-Phe-Leu-Arg- Yang et al., 1985),indicating that FMRFamide is one of a family Phe-NH,) and GDPFLRFamide (Gly- . . . etc.). In previous of related neuropeptides.However, the antibody usedin many investigations of the genetic organization of families of struc- of these surveys doescross-react with other Arg-Phe-NH, pep- turally related neuropeptides, various forms have been found tides. to be encoded by the same gene and are present on the same precursor.The questiontherefore arisesin Lymnaea asto whether the 3 known forms of FMRFamide-related peptides are present on the samegene and/or different transcripts. Received June 9, 1989; revised July 19, 1989; accepted July 28, 1989. We report here on the isolation of 2 cDNA clones and a This work was supported by MRC project grant G8622759N and SERC stu- genomic clone that encode9 copies of FMRFamide in addition dentships to A.L. and K.B. We thank Dr. Mike Powell for help with the RNA analysis and Nicola Ford for preparation of the manuscript. to novel forms of FMRFamide-like peptides. The Lymnaea Correspondence should be addressed to Dr. J. F. Burke at the above address. genomecontains a single copy of the gene that gives rise to a Copyright 0 1990 Society for Neuroscience 0270-6474/90/l 004 12-08$02.00/O unique transcript within the CNS. Our data from in situ hy- The Journal of Neuroscience, February 1990, IO(2) 413 bridization suggest that the gene is expressed in specific neurons throughout the Lymnaea CNS. Results Isolation of FMRFamide cDNA clones Materials and Methods A mixture of 256 different degenerate oligonucleotides, complementary to the coding sequence for Phe-Met-Arg-Phe-Gly Molecularprocedures. General procedures such as growth of phage, Southern transfers, and plasmid preparations were carried out as de- plus the first 2 complementary bases for lysine, was used to scribed by Maniatis et al. (1982). screen a Lymnaea CNS cDNA library (described in Vreugdenhil Constructionof oligonucleotide.Mixed oligonucleotides were synthe- et al., 1988, and kindly donated by E. Vreugdenhil). The codon sized on an Applied Biosystems 38 1A DNA synthesizer according to for Gly was included as this amino acid is thought to be required the manufacturer’s instructions. The sequence of the oligonucleotide was (5’ to 3’) (T/C)TTNCC(A/G)AANC(G/T)CAT(A/G)AA (where N for amidation (Loh and Gainer, 1983) and Lys is an amino acid represents A, G, C, or T). commonly found at a site of proteolytic cleavage within hor- Extraction of genomicDNA. The ring of ganglia that forms the CNS mone precursor molecules (Douglass et al., 1984). Approxi- (the “brain”) was dissected from animals and non-neuronal tissue and mately 20,000 recombinant clones were screened with the ra- mucus strands carefully removed from the ganglia using forceps. The diolabeled oligonucleotide from which 2 independent clones brains were then washed in Lymnaeasaline (Benjamin and Winlow, 198 1) (3 changes). The tissues (10 brains/ 100 ~1) were gently homoge- were isolated. Subsequent restriction analysis indicated that the nized in 1 x PK buffer (Burke and Ish-Horowitz, 1982) containing 2% 2 cDNAs contained inserts of 1.2 and 0.9 kb. The 2 clones were SDS. Two drops ofAntifoam A (Sigma) were added per 100 ~1 of buffer. designated Dl and D3, respectively. Nucleotide sequence data This was followed by phenol extraction and ethanol precipitation of the revealed that both D 1 and D3 contained sequences that encode DNA, which was found to be of high molecular weight and free from FMRFamide. As neither cDNA included an initiator methio- contaminating nucleases. Isolationof FMRFamide-encodingcDNA and genomicclones. Ap- nine that was in frame with the FMRF sequences, it was con- proximately 20,000 plaques from an amplified XgtlO LymnaeaCNS cluded that both Dl and D3 were partial cDNA clones. An cDNA library (Vreugdenhil et al., 1988) were bound to Hybond-N additional 20,000 clones were screened with hexamer labeled (Amersham) and screened with the mixed oligonucleotides labeled with Dl but no further positives were identified. +*P-ATP using T4 polvnucleotide kinase. Hvbridizations were carried out at 37°C in a solution containing 6 x SSC, 5 x Denhardt’s solution, and 0.5% SDS. Filters were washed at 42°C in 6 x SSC, 0.5% SDS, dried, Isolation of a FMRFamide genomic clone and autoradiographed. Two positive clones, designated Dl and D3, Southern blot analysis (Fig. 3) had indicated the presence of a were obtained. When the entire EcoRI insert of Dl was subcloned into single EcoRI genomic fragment of approximately 4.1 kb in size either M 13mp 18 or M 13mp 19, it was found to be unstable and readily deleted. The EcoRI inserts ofD1 and D3 were subcloned into Bluescribe containing FMRFamide sequences. A Lymnaea genomic library (Stratagene) and subsequently digested independently with the restric- was constructed from EcoRI digestedDNA in Xgt10 from which tion enzymes TaqI and Sau3A. The smaller fragments that this gen- approximately 20,000 cloneswere screened.This resultedin the erated were ligated into M 13mpl8 and M 13mpl9 for single-strand isolation of one positive phage which hybridized with Dl and sequencing. D3 and was found to contain an insert of the expected size (4.1 To isolate a genomic clone the D3 insert was hexamer labeled (Fein- berg & Vogelstein, 1983) and used to screen 20,000 clones of an EcoRI kb). A 0.9 kb EcoRIIBamHI restriction fragment from this genomic library in Xgt 10. A positive clone containing a single insert of genomic clone was found by hybridization and sequenceanal- 4.1 kb was identified and isolated. The DNA from this clone was di- ysis to encode the 5’ end of the cDNAs including an in frame gested by EcoRI and BarnHI, and the resulting fragments were ligated AUG start codon. into M13mp18 and M13mp19. Nucleotidesequenceanalysis. Phage inserts, or fragments derived from Deduced structure of the FMRFamide mRNA preprohormone them, were subcloned into either Bluescribe or M13mp18/19 vectors for sequence analysis in both orientations and were also double-strand The Dl and D3 cDNAs were completely sequencedby the sequenced. Sequencing using &S-dATP was performed as described strategy outlined in Figure 1. This showedthat the D3 sequence by Sanger et al. (1977) or by using a commercially available kit (Se- was contained within D 1 and was identical except that D3 con- quenase, U.S. Biochemicals). The samples were separated on a 7 M urea, 6% acrylamide gel. Ambiguities were resolved bv resequencing using tained a run of 17As (position 986, Fig. 2), whereasD 1 contained synthetic oligonucleotides- derived from known -sequences, 5; CCAI 14. The overlap between the cDNA clones Dl, D3, and the AGCCGGCCAGACTCGTTGGCC 3’ and 5’ GGAAGAGATAT- genomic clone is also illustrated in Figure 1. The longest open GAGTGACGTGGAC 3’. reading frame (ORF) in the 2 cDNA clones contained the DNA blot hybridization.Genomic DNA, 5 pg, was fully digested with a restriction enzyme and separated on a 0.5% agarose gel. The DNA FMRFamide sequences(Fig. 2). There was no N-terminal me- was then transferred to Hybond-N (Amersham). The D3 cDNA insert thionine in either cDNA, indicating that both were partial at was hexamer labeled in the presence of &2P-dCTP and hybridized at the 5’ end. The cDNA clone Dl contains 11 nucleotides at the 65°C overnight. The filter was washed in 0.3x SSC (Maniatis et al., extreme 5’ end which do not correspond to the genomic se- 1982) 0.5% wt/vol SDS, at 65°C and autoradiographed. quence. This could be a polymorphism or evidence of splicing RNA blot hybridizations.Total RNA was isolated from 10 CNS by the standard method of homogenizing in 2 x PK buffer followed by or a cloning artifact. The first stop codon (TAA) in the phenol extraction. Some 10 pg of total RNA was run on a formaldehyde FMRFamide reading frame is at position 942-944. This is fol- gel essentially as described in Maniatis et al. (1982) and transferred to lowed by multiple stop codons in all 3 reading frames and a Hybond-N. The D3 cDNA insert clone was hexamer labeled in the stretch of 14As starting at nucleotide 986. This is unlikely to presence of olJ2P-dCTP and hvbridized to the filter at 42°C ovemiaht in 4 x SSC, 10 &ml denatured salmon sperm DNA, 0.1 x Denhardt’s be the posttranslational polyA tail asextra nucleotidesare pres- solution, 12.5% foramide, 10% wt/vol Dextran sulfate. The filter was ent 3’ to this. Furthermore, from Southern analysis (data not washed in 2 x SSC at 60°C and then autoradiographed. shown) these 3’ sequencesare contiguous in the genomewith In situ hybridizations.CNS from Lvmnaeawere dissected, fixed, and sequencesin the FMRFamide ORF. The FMRFamide ORF in sectioned essentially as described by.Dirks et al. (1989). Sections were the Xgt 10 genomic clone extends 5’ to a stop codon at nucleotide hvbridized with c@S-dATP nick-translated D3 cDNA insert overnight af 37°C. Slides were exposed for autoradiography for 3 weeks. ” 5 l-53, suggestingthat the methionine at 82-84 is the initiating Proteinsequence comparisons. Comparisons and alignments were car- methionine. As is typical of many membrane or secretedpro- ried out using the Lipman and Pearson (1985) FASTP program. teins, a signal sequencecontaining, in this case, 6 hydrophobic 414 Linacre et al. * Lymnaea FMRFamide Gene

A I------4 Genomlc 01 03 UQnt B Figure 1. Organization of cloned sequences. A, Alignment of XgtlO geno- w mic clone and cDNAs showing regions R ST T S 5 BS T 5 TS TS TSS R I I I II I I II II I II sequenced (-). B, Part of the strategy I rI I II II II II II III I for sequencing the insert of cDNA Dl. c * Arrows indicate direction and extent of - sequencing runs for TuqI (T) and Suu3A (S) fragments cloned into M13mp18/ 19 (B, BumHI). c

amino acids follows the methionine codon. This N-terminal result of this Southern blot is shown in Figure 3. Single digests sequence is significant (Kyte and Doolittle, 1982) as it is the with DraI, BamHI, and EcoRI when hydridized with D3 give only hydrophobic region in the whole precursor protein. Inter- single fragments of 1.1, 3.4, and 4.1 kb. A double digest with estingly, 6 hydrophobic amino acids makesthe Lymnaea signal DraIIBamHI gives a single fragment of 1.0 kb and a double sequencethe least hydrophobic of over 300 eukaryotic signal digest with BamHIIEcoRI gives 3.2 kb fragment. The obser- sequencesrecently surveyed (von Heijne, 1985, 1986). The hy- vation that the BamHI site is asymmetric in the 4.1 kb EcoRI drophobic leader also conforms to the -1, -3 rule for signal fragment which is present only once in the genomeand that peptidecleavage. Residue - 1 must be small and residue-3 should BamHI, which also cuts in the cDNA, produced a single frag- not be aromatic, charged, or large and polar. In the precursor, ment that hybridizes with D3, suggeststhat the sequenceen- Thr (- 1) and Ala (-3) at positions 18 and 16, respectively, fulfill coding D3 is present only once in the genome. Furthermore, thesecriteria, predicting cleavagebetween Thr and Lys at amino the size of the bandswas consistent with the size of the genomic acids 18-19. It cannot be discounted that the first potential fragmentsprepared from the EcoRI Xgt10 genomic library, sug- cleavage site of Lys-Arg at positions 19-20 may act as the site gestingthat there are no large introns in the genomic sequence. for removal of the signal sequence. Nine copies of Phe-Met-Arg-Phe (FMRF) are contained on Transcription of the FMRFamide precursor the precursor (Fig. 2). Eight of the FMRF sequencesare flanked Northern blot analysis(Fig. 4) was usedto indicate the number on their amino termini by Lys-Arg, the other by Lys-Ser. The and size of transcripts produced by the FMRFamide genein the carboxy termini of the 9 FMRF sequencescontain the residues CNS. Total RNA was isolated from 10 complete CNS, frac- Gly-Arg (4 out of 9), Gly-Lys-Ser (4 out of 9) and Gly-Lys-Arg tionated according to size, transferred to Hybond-N, and hy- (1 out of 9). The Gly is required for amidation of the peptides bridized to D3 cDNA insert. The result shown in Figure 4 and the basic residues,which serve as proteolytic cleavagesig- indicates that this gene gives rise to a unique 1.7 kb mRNA nals, are similar to those found on the Aplysia FMRFamide specieswithin the CNS. precursor (Taussigand Scheller, 1986). Spacer peptides sepa- rating the 9 FMRF sequencesvary in size and composition of The FMRFamide gene is expressedthroughout the Lymnaea amino acids and probably do not have any biological function CNS other than to separatethe FMRFamide sequences. In order to determine more precisely the location of the The precursorprotein alsocontains 2 Phe-Leu-Arg-Phe(FLRF) FMRFamide transcripts in the CNS, in situ hybridization was sequencesat positions 21-24 and 57-60. These sequencesare performed (Fig. 5). Preliminary analysisshows that at least 100 flanked by a probable cleavage signal Lys-Arg on the amino neurons expressthe FMRFamide gene and these neurons are termini and Gly-Arg on the carboxy termini and would therefore distributed throughout the CNS. be amidated if cleaved from the precursor. Other putative peptides may be contained on the precursor Discussion protein. Theseincluded the pentapeptidesGln-Phe-Tyr-Arg-Ile We have characterized 2 cDNA clonesand a genomic clone that (QFYRI) at position 4 l-44 which was bounded by Arg-Arg and encodes9 copies of FMRFamide, 2 copies of FLRFamide, and Gly-Arg at its amino and carboxy termini, respectively, and 2 novel pentapeptides(summarized in Fig. 6). We have yet to Glu-Phe-Leu-Arg-Ile (EFLRI) at position 262-266 and bound- determine how many of these individual peptides are released ed by the cleavage signalsArg-Arg on the amino side and Gly- from the precursor protein. However, the basicamino acidsthat Arg on the carboxy side. These 2 pentapeptideswould be ami- serve as recognition sitesfor cleavageare similar to those of the dated if processedfrom the precursor and glutamine if present Aplysia FMRFamide precursor with the exception of the Lys- at the amino terminal of a peptide can circularize to form pyro- Ser seenon the amino side of one FMRFamide. Zn vivo labeling glutamine. This would produce pQFYRIamide and EFLRIam- experiments using 3H-phenylalanine have indicated that most ide. of the 28 FMRFamides encoded by the Aplysia FMRFamide geneare cleaved from the precursor(Taussig and Scheller, 1986). A single gene encodesthe FMRFamide precursor Therefore, it is a reasonableassumption that most of the pep- Genomic DNA was digestedwith various restriction enzymes, tides on the Lymnaea precursor will also be cleaved. blotted, and probed with hexamer labeledD3 cDNA insert. The The presenceof the highly related peptide FLRFamide has The Journal of Neuroscience, February 1990, IO(2) 415

1 60 ATTTGTAACTTTACTTTAGGGTTGTAATATGTATGTATGCAAGCTGTTTTTAATTTAAAA 120 GTCTTTCCAGTTTTTTTGTTTATGTATTCCCCAACACTGATAG~GT~GTCTTTCTTT MetTyrSerProThrLeuIleValCysLeuSerPhePhe 1 180 CACTCTGCAGTGACCAAACGCTTTTTGAGGTTTGGCCGAGCTCTGGACACTACGGATCCT HisSerAlaValThrLysArgPheLeuArgPheGlyArgAlaLeuAspThrThrAspPrO 20 240 TTCATACGGTTGAGAAGGCAGTTCTATCGAATTGGCCGAGGGGGCTATCAGCCTTACCAG PheIleArgLeuArgArgGlnPhe~rArgIleGlyArgGlyGlyTyrGlnProTyrGln 40 300 GACAAGAGATTCTTGCGGTTCGGCCGGTCTGAGCAACCGGATGTCGATGACTACCCTAGA AspLysArgPheLeuArgPheGlyArgSerGluGlnProAspValAspAspTyrProArg 60 360 GATGTTGTCCTCCAGTCAGAGGAGCCGCTGTACAGAAAACGGAGATCCACGGAGGCGGGT AspValValLeuGlnSerGluGluProLeuTyrArgLysArgArgSer~rGluAlaGly 80 420 GGCCAATCAGAAGAGATGACACACAGAACAGCGAGATCAGCTCCCGAGCCAGCAGCTGAG GlyGlnSerGluGluMetThrHisArgThrAlaArgSerAlaProGluProAlaAlaGlu 100 480 AACAGAGAGATTATGAAAAGAGAAACCGGTGCGGAAGATCTAGATGAGGA~AGAGG~C AsnArgGluIleMetLysArgGluThrGlyAlaGluAspLeuAspGluGluLysArgPhe 120 540 ATGAGGTTCGGAAGAGGAGACGAAGAGGCTGAAAAGAGG~TATGAGGTTCGG~~AGT netArgPheGlyArgGlyAspGluGluAlaGluLysArgPhenetArgPheGlyLysSer 140 600 TTCATGAGGTTCGGAAGAGATATGAGTGACGTGGACAAAAGGTTCATGAGGTTCGGTAAA PheUetArgPheGlyArgAspMetSerAspValAspLysArgPheNetArgPheGlyLys 160 660 CGATTCATGAGGTTCGGACGAGAGCCTGGAACAGACAAGAGGTTCATGAGGT~GGAAGA ArgPheUetArgPheGly:~~GluProGlyThrAspLysArgPheUetArgPheGlyArg 720 GAACCTGGAGCCGACAAGAGGTTCATGAGGTTCGGGAAAAGCTTCGATGGAGAAGAAGAG GluProGlyAlaAspLysArgPhe~etArgPheGlyLysSerPheAspGlyGluGluGlu 200 780 AACGACGACGATCTCTACTACAACGAGAGTGACGCGGACTCAAACGATGACGTGGACAAG AsnAspAspAspLeuTyrTyrAsnGluSerAspAlaAspSerAsnAspAspValAspLys 220 840 CGGTTTATGCGCTTTGGGAAAAGCGCAGAGGAAAAGCGATTTATGAGATTCGGGAAAAGC ArgPhenetArgPheGlyLysSerAlaGluGluLysArgPheUetArgPheGlyLysSer 240 900 CAGGACGCTAGCAGAGATAAGAAAGAGTTCTTGCGCATTGGGAAGCGGGAGTCAAGGTCG GlnAspAlaSerArgAspLysLysGluPheLeuArgIleGlyLysArgGluSerArgSer 260 960 GCGGAAGTGGAAAACAATATCCAAATTGCTGCCAAACAGTCGTAATCAAAATTAGAGCTA AlaGluValGluAsnAsnIleGlnIleAlaAlaLysGlnSer*** Figure 2. Compilation ofcDNA and 280 genomic sequences encoding the 1120 FMRFamide precursor. Nucleotide 1 GGGGGGCTTGAAGAGACAGTTGAGTAAAAAAAAAAAAAAGCCGAAAATATCACCGTCTTA corresponds to a Mae111 site in the ge- 1180 nomic XgtlO clone which was se- TAGTAATATCATATTTGGCAATCAGTATCGAAGATCGTTGTCAGTTCTAAGTCACAGCGG quenced out to nucleotide 178. cDNA 1240 Dlextendsfromnucleotide 135to 1382 GAAACTCGATCTTAAAATACTTTTTTCCCCCTTTAAATATAGCTTTTCATGTCTTAACCA and cDNA D3 extends from 380 to 1300 ACAAATCGAGCGAAAACGAAAAATATGTTGTATCCGCAACTTTATTAAATAAAAAGAGTG 1352 (a short sequence, S'GGCCA- 1360 GTATGA3' at the start of the cDNA ATGACTTTTAGCTGTATTATTGCCCGTTTATTTGTAAAATAATATATGTGATCCCAATTC DIdoesnotcorrespondtothegenomic 1382 sequence and is not included in the GGCACACAGAATAGCGGAGATC compilation). not been detected by HPLC analysisof the Lymnaea CNS (Eb- The unique position (Fig. 6) of the 2 FLRFamide peptides in berink et al., 1986). A similar situation arose in Aplysia where the Lymnaea precursor suggeststhat they have some biological FLRFamide remained undetected by HPLC analysis (Lehman function and are not just simple methionine-leucine substitu- et al., 1984)but waslater found to be presenton the FMRFamide tions. The substitution of a leucine for a methionine may confer precursor by sequencingofa cDNA (Taussigand Scheller, 1986). greater stability on the peptide as leucine is lesssusceptible to 416 Linacre et al. l Lymnaea FMRFamide Gene ABCDEF KB KB

23.l-

9.L 6.6- 4.3-

2.3- 2.0-

0.9-

Figure 3. Southerntransfer of genomicLymnaea DNA digestedwith variousrestriction enzymes and hybridized with D3 insert.A, Undigest- ed DNA, B, DraI; C, DraIIBamk; D, BamHI; E, EcoRIIBamHi; F, EcoRI. Size markers are from a Hi&III digest of XDNA and the D3 cDNA insert (0.9 kb).

Figure 4. Northern transfer of total Lymnaea CNS RNA hybridized with D3 cDNA insert. The size is estimated from the migration in adjacent tracks of yeast mRNAs Ura4 and hsp70. oxidation than methionine. This greater stability may be important in the potential hormonal actions of FLRFamide (Price, 1986). Lymnaeu has also been shown to possess2 amino terminally Comparison of the Aplysia CRF-like peptide with the extended forms of FLRFamide, SDP- and GDPFLRFamide. corresponding Lymnaea sequences Extensions at the amino terminal may give the peptide even The Aplysia FMRFamide precursor was reported to contain greater stability by protecting against enzymatic degradation. sequencesthat are homologous to mammalian brain peptide The fact that the N-terminal-extended FLRFamides are not sequences, including corticotropin-releasing factor (CRF); presentin the FMRFamide precursor protein supportsthe prop- oc-melanocyte-stimulatinghormone (orMSH), and corticotropin- osition of Price (1986) that after a duplication of the ancestral like intermediate-lobe peptide (CLIP) (Taussig and Scheller, FMRFamide gene, one gene became specialized for the pro- 1986). duction of transmitters (FMRFamide) and the other for the The Aplysia CRF-like peptide consists of 42 amino acids production of extended FMRFamide hormones. situated between the FLRFamide and the FMRFamides on the The 2 pentapeptides,pQFYRIamide and EFLRIamide, are precursor. Twelve of these amino acids show homology to the previously unreported. They are bounded by basic amino acid ovine CRF. At a similar position (63-104) on the Lymnaeu cleavage signalsand therefore likely to be processed. FMRFamide precursor, there is also a sequenceof 42 amino The Northern blot analysis indicated that there is only one acids that is bounded on the amino and caboxy sidesby Arg- mRNA produced. Therefore, any neurons that express the Ser (62-63 and 105-106). The comparison with the Aplysia FMRFamide gene are likely to synthesizethe precursor, so any CRF-like peptide, ovine CRF, frog sauvagine,and carp uroten- differential usageof the peptides probably occurs as a result of sin-l is shown in Figure 7. The Lymnaea and Aplysia genes posttranslational processing. share 6 1% homology at the amino acid level with a total of 25 The Journal of Neuroscience, February 1990, 1~32) 417

* * * * * * *

Lymnaea SEQPDVDDYPRDVVLQ-SEEPLYRKBRSTEAGGQSEEMTERTA Aplysia SQEPDIEDYARAIALIESEEPLYRKRRSADADGQSEKVLMRA Ovine SQEPPISLDLTFHLLREVLEM-TKADQLAQQANSNRKLLDIA Frog -QGPPISIDLSLELLRKMIEI-EKQEKEKQQAANNRLLLDTI Carp NDDPPISIDLTFHLLRNMIEM-ARNENQREQAGLNRKYLDEV Dros. QAEQLPPEGSYAGSDELEGMAKRAAMDRYGR

Figure 7. Comparison of Lymnueu CRF-like peptide with the anal- ogous sequence in the Aplysiu Fh4RFamide precursor, ovine CRF, frog sauvagine, carp urotensin and the CRF-like sequence in the Drosophila precursor. Bold letters show amino acids conserved between Lymnaeu and Aplysiu, and asterisks show amino acids in Lymnueu CRF which are in common with CRF from at least one other organism other than Aplysiu. Sequences from Taussig and Scheller (1986) and Schneider and Taghert (1988). Figure 5. Illustration of the localized expression of FMRFamide encoding mRNA to specific neurons in the CNS of Lymnaeu stagnaZis as shown by in situhybridization. %-labeled probe was prepared by nick translation of the gel-purified insert from the cDNA D3. gested (Taussigand Scheller, 1986). Comparison of the same region in Lymnaea (amino acids 15-37) showsthat only amino acids serine (15), valine (17) threonine (18), and arginine (36) positional matches out of 42. However, the degree of homology are conserved between Lymnaea and Aplysia, and only valine between the Lymnaea sequence and any of the vertebrate pep- (17) is common to all 3 sequences.The 15 amino acid otMSH- tides is even less than that of the ApZysia CRF-like peptide and like sequencein Aplysia (45-59) correspondsto Lymnaea 40- the vertebrate peptides. 48, a 9-amino acid region containing a run of 6 gapswhich are Taussiget al. (1988) concluded that if the Aplysia CRF-like required to maximize alignment in the first 100 amino acids of peptide was indeed related to the vertebrate CRFs, then there the sequence.There is therefore no sequenceof any significant would have beenan amino acid substitution every 17.5 million homology of (wMSH. years on the average. On this basis, the degreeof divergence between Lymnaea and Aplysia sequencesover the 350 million Does the Lymnaea FMRFamide represent the ancestral years sincetheir divergence (Moore and Pitrat, 1960)should be precursor? 22 out of 42. The observed difference is 17, indicating that this Previously, the only other molluscanFMRFamide precursor to is a good approximation to what is expected. The degree of be examined at the genetic level is that of Aplysia (Schaeferet homology throughout the 42 amino acids however is not uni- al., 1985;Taussig and Scheller, 1986). The most striking feature form. There is a sequence(79-89) of Ser-Glu-Glu-Pro-Leu-Tyr- of that precursor protein is its highly repeated nature. This is Arg-Lys-Arg-Arg that is 100%homologous between the 2 mol- especiallytrue betweenthe 7th FMRFamide and the 26th, where luscan speciesand is 79% homologousat the nucleotide level. the FMRFamide and its spaceris composedof 15 or 16 amino This exceptionally high level of conservation indicatesthat this acids. Many of these sequencesare identical at the amino acid sequencemay have somefunctional importance. The tetra basic level, and several show high homology at the nucleotide level. sequenceArg-Lys-Arg-Arg is reminiscent of the Arg-Lys-Arg- The repeatednature of the Aplysia FMRFamide precursor led Arg sequencein the Aplysia ELH prohormone (Scheller et al., Price et al. (1987) to speculateon the origin of the precursor. 1983). The processingof this prohormone has been studied by They proposeda model to showhow the modem opisthobranch Fischer et al. (1988) who suggestthat this sequenceis the first gene could have arisen from a hypothetical pulmonate gene. to be recognizedand cleaved. The tetra basiccleavage site may They concludedthat thesehighly repeatedsequences could have not only serve to generatethis novel peptide but could also be arisen from an intragenic duplication event; presumably by the important in the differential processingof the precursor by sep- sequencesencoding the 7th FMRFamide and its spacerbeing arating the FLRFamide and FMRFamides. amplified 19 times. With the removal of this iterative portion The Lymnaea precursor, unlike that of Aplysia, has no se- of the Aplysia precursor, a model of the ancestralprecursor may quenceswith any significanthomology to either vertebrate arMSH be generated. In ApZysia this would contain 9 FMRFamides, 1 or CLIP. Apparent homology between the mammalian CLIP FLRFamide, and the “vertebrate”-like peptides.In this respect, peptide and the Aplysia sequenceresidues 20-42 has been sug- it shouldbe noted that the Lymnaea genecontains 9 FMRFam-

Figure 6. Summary of the organization of putative peptides in the Lym- naeu FMRFamide precursor. Symbols:- H, hydrophobic signal sequence; MI, FLRFamide; K&I,pQFYRIamide; CRF-like peptide; Rl, FMRFamide; and q D,EFLRIamide. The positions of lysine or arginine residues in the sequence are indicated by asterisks. 418 Linacre et al. - Lymnaea FMRFamide Gene

J. Salanki and KS. Rozsa, eds., pp. 415431, Akademiai Kaido, Budapest. (a) 30 40 50 Boer, H. H., L. P. C. Schot, J. A. Vennstra, and D. Reichelt (1980) TTDPFIRLRRQP ------YRIGRGGY Immunocytochemical identification of neural elements in the central . . . . . nervous systems of a snail, some insects, a fish, and a mammal with C~~G;~~~~~S~SVEEPH~~~~~R~~ antiserum to the molluscan cardioexcitory tetrapeptide FMRFamide. 35 45 55 60 Cell. Tissue Res. 213: 21-27. Boyd, P. J., and R. J. Walker (1985) Actions of the molluscan neuropeptide FMRFamide on neurones in the suboesophageal ganglia of (b) the snail Helix aspersa. Comp. Biochem. Physiol. 81: 379-386. 261 272 Buckett, K. J. (1987) Neuronal and Peptidergic Control of the Heart ------KEFLRIGKRESR------in the Snail Lymnaea stagnalis. D. Phil. Dissertation, University of :.:.::: Sussex, England. GTEDVD;RFMRFGKRFM:FGRSVGDS Bulloch, A. G. M., D. A. Price, A. D. Murphy, T. D. Lee, and H. N. Bowes (1988) FMRFamide peptides in Helisoma: Identification and 550 560 570 physiological actions at a peripheral synapse. J. Neurosci. 8: 3459- Figure 8. Comparison of Lymnaea and Aplysia sequences surrounding 3469. the novel peptides in Lymnaea. Alignment is by means of FASTP Burke, J. F., and D. Ish-Horowitz (1982) Expression of Drosophila (Lipman and Pearson, 1985). (:), identical amino acid, (.), conservative heat shock genes is regulated in Rat-l cells. Nucleic Acids Res. 10: substitution. 3821-3830. Cottrell, G. A., N. W. Davies, and K. A. Green (1984) Multiple actions of a molluscan cardioexcitory neuropeptide and related peptides on identified Helix neurones. J. Physiol. (Lond.) 356: 3 15-333. Dirks, R. W., D. K. Raap, J. van Minnen, E. Vreugdenhil, A. B. Smit, ides. It may therefore be the case that the Lymnaea FMRFamide and M. der Ploeg (1989) Detection of mRNA molecules for neu- gene bears a close relationship to the original molluscan ropeptide hormones of the pond snail Lymnaea stagnalis by radio- FMRFamide gene. The Lymnaea FMRFamide gene, like that active and non-radioactive in situ hybridization: A model study for of Aplysia, also shows similarities in organization to the Dro- mRNA detection. J. Histochem. Cytochem. 37: 7-14. Dockray, G. J., K. R. Reeve, J. Shively, R. J. Gayton, and C. S. Barnard sophila DPKQDFMRFamide precursor protein (Schneider and (1983) A novel active pentapeptide from chicken brain identified by Taghert, 1988). antibodies to FMRFamide. Nature 305: 328-330. A comparison of the Lymnaea and Aplysia amino acid se- Douglass, J., 0. Civelli, & E. Hervert (1984) Polyprotein gene expres- quence around 2 of the novel Lymnaea predicted peptides sug- sion: Generation of diversity of neuroendocrine peptides. Annu. Rev. Biochem. 53: 665-7 15. gests how these novel peptides may have arisen (Fig. 8). The Ebberink, R. H. M., D. A. Price, H. van Looenhout, K. E. Doble, J. P. predicted peptide Gln-Phe-Tyr-Arg-Ile-NH, corresponds to the Reihm, W. P. M. Geraerts, and M. J. Greenberg (1987) The brain region of Aplysia encoding the cYMSH-like peptide. It is possible of Lymnaea contains a family of FMRFamide-like peptides. Peptides that this sequence was present in Lymnaea at one time and a 8: 5 15-522. deletion and some substitutions gave rise to the new sequence. Feinberg, A. P., and B. Vogelstein (1983) A technique for radiola- belling DNA restriction endonuclease fragments to high specific ac- The other novel predicted peptide Glu-Phe-Leu-Arg-Ile-NH, tivity. Anal. Biochem. 132: 6-13. could have arisen from a FMRFamide sequence as a result of Fischer, J. M., W. Sossin, R. Newcombe, and R. H. Scheller (1988) substitutions. 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