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Please be advised that this information was generated on 2021-09-27 and may be subject to change. Fish Physiology and Biochemistry vol. 11 no. 1-6 pp 117-124 (1993) Kugler Publications, Amsterdam/New York Cloning and sequence analysis of hypothalamus cDNA encoding tilapia melanin-concentrating hormone

Diet Gröneveld, Mark J. Hut, Paul H .M . Balm, Gerard J.M . Martens and Sjoerd E. Wendelaar Bonga Department of Animal Physiology, Faculty of Science, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands

Keywords: melanin-concentrating hormone, M C H , teleost, tilapia, Oreochromis mossambicus, messenger R N A , complementary D N A , hypothalamus, background adaptation

Abstract

Melanin-concentrating hormone (M C H ) is a neuroendocrine peptide involved in the regulation of skin pig­ mentation in teleosts. W e isolated and sequenced a 543 bp hypothalamic c D N A encoding the MCH- preprohormone of tilapia (Oreochromis mossambicus). Initially, polymerase chain reaction (PCR) experi­ ments were performed on hypothalamic R N A with a synthetic oligonucleotide primer corresponding to a con­ served region of salmon and mammalian M C H peptide and an oligo dT primer. A 0.2 kb P C R fragment was obtained and found to have low but significant nucleotide sequence similarity with the 3 'ends of known M CH- m RNAs. Subsequently, the P C R fragment was used to screen X ZA P c D N A libraries constructed from tilapia hypothalamic poly(A + ) R N A . The cloned tilapia M C H preprohormone c D N A encodes a 133-amino acid protein of which 17 amino acids belong to the signal peptide. The M C H peptide sequence is located at the carboxy terminus of the preprohormone structure and is preceded by a pair of arginine residues which can serve as a proteolytic cleavage site. 23 to 25 amino acids further upstream in the prohormone structure three consecutive basic residues are present. Cleavage at this site would yield a 22-amino acid M C H gene- related peptide (Mgrp), which is much larger than (12- to 13-amino acid) salmon and mammalian Mgrp. A comparative structural analysis between tilapia preproMCH and its salmon and mammalian counterparts revealed that the M C H peptide sequence is very well conserved (100% identity with salmon and 7 5 % identity with both rat and human M C H ). In contrast, the remaining parts of the preproMCH structures have diverged considerably. Northern blot analysis revealed the presence of tilapia preproMCH m R N A in the hypothala­ mus and not in other brain regions nor in several peripheral tissues.

Résumé

La M C H (melanin-concentrating hormone) est un peptide neuroendocrinien impliqué dans la régulation de la pigmentation de la peau chez les Téléostéen. Nous avons isolé et séquencé un A D N c hypothalamique de 543 pairs de bases codant pour la préprohormone de la M C H de tilapia (Oreochromis mossambicus). Ce travail a débuté par des expériences de P C R (Polymerase Chain Reaction) qui ont été réalisées sur des A R N m hypothalamiques avec comme amorces un oligonucléotide synthétique correspondant à une région conservée du peptide M C H de saumon et de mammifère et un oligo dT. Un fragment de 0.2 kb a ainsi été obtenu par P C R et l’analyse de sa séquence nucléotidique montre une similitude faible mais significative avec les extré­ mités 3 'des AR N m s de M C H connus. Ensuite ce fragment a été utilisé pour cribler des banques de c D N A 118

construites dans XZA P à partir d’A R N poly(A + ) d’hypothalamus de tilapia. L ’A D N c de la préprohormone de M C H de tilapia code pour une proteine de 133 acides aminés dont 17 constituent le peptide signal. Le pep- tide de la M C H est localisé dans l’extrémité carboxy de la préprohormone et se trouve précédé par une pair de résidus arginine qui servent de site de coupure protéolitique. De plus, 3 résidus basiques consécutifs sont présents à 23-25 acides aminés en amont de la structure de la prohormone. La coupure à ce site permettrait d’obtenir un peptide associé au gène de la M C H (Mgrp) de 22 acides aminés, ce qui est beaucoup plus grand (de 12 à 13 acides aminés) que les Mgrp de saumon ou de mammifères. Une analyse structurale comparée entre la preproMCH de tilapia et la preproMCH de saumon et de mammifères montre que la séquence du peptide M C H est très conservée (100% d’identité avec le saumon et 7 5 % d’identité avec la M C H humaine ou de rat). Par contre, les autres parties de la structure de la preproMCH ont divergé considérablement. L ’analyse par “ Northern Blot” met en évidence la présence d’A R N m de preproMCH dans l’hypothalamus de tilapia mais pas dans les autres régions du cerveau ni dans plusieurs tissus périphériques.

Introduction dated (Ono et al. 1988; Minth et al. 1989; Nahon et al. 1989; Presse et al. 1990) and the M C H peptide Many lower vertebrates are capable of altering the has been purified from rat hypothalamic tissue pigmentation of their skin in response to variations (Vaughan et al. 1989). Hence, M C H is not only in background colouration. The physiological present in teleosts but also in other species where its mechanism of background adaptation is of para­ function is yet unclear. Regulatory roles in water mount importance for these animals and regulation balance and control of homeostatic functions of this process can occur by both neuronal and en­ (Presse et al. 1990; Zamir et al. 1986) or antagonis­ docrine mechanisms. In amphibia and reptiles one tic effects on a-MSH-induced behaviour in rats pituitary hormone, a-melanocyte-stimulating hor­ (Eberle 1988) have been proposed. mone (af-MSH), is involved in the control of skin At present little is known about the evolutionary pigmentation. In teleost fish, however, a second conservation of the M C H preprohormone in fish. peptide hormone, melanin-concentrating hormone W e therefore elucidated the structure of a tilapia (M C H ), functions in this mechanism. M C H is hypothalamic m R N A that encodes the M C H produced in the hypothalamus and most of the pep­ precursor protein. A comparative structural analy­ tide is axonally transported to the pituitary, where sis between tilapia and other known M C H prepro­ it is stored in the neural lobe (for reviews see Baker hormones is presented. Furthermore, tissue speci­ 1991 and Eberle 1988). In addition to the pituitary ficity of tilapia preproMCH m R N A expression has projections, some MCH-containing axons project been investigated by Northern blot analysis. into the brain, where the role of M C H is still unknown (Naito et al. 1985). The primary structure of M C H was first deter­ Materials and methods mined following isolation from chum salmon (0 /7- corhynchus keta) pituitaries (Kawauchi et al. 1983). It appeared to be a cyclic heptadecapeptide. Subse­ Polymerase chain reactions quently, the M C H peptide structure of other teleosts has been described. Bonito (Katsuwonas R N A of tilapia (Oreochromis mossambicus) (ob­ pelamis) M C H appeared to be identical to salmon tained from our laboratory stock) was prepared by M C H , whereas only one amino acid substitution at the Nonidet P40 method (Sambrook et al. 1989), the amino-terminus was observed in case of the eel followed by purification of m R N A with oligo (dT) Anguilla japónica (Kawauchi 1989). Recently, the cellulose. For polymerase chain reaction (PCR) structures of cDNAs encoding the M C H prepro­ analysis single-stranded c D N A was synthesized hormone in salmon, rat and man have been eluci­ from poly(A + ) R N A by using M L V reverse trans­ 119 criptase (BRL) and 50 ng (dT) primer [CCTGCAG- Screening o f hypothalamus cDNA libraries CGGCCGCATGCATTTTTTTTTTTTTTTTT] in 1 x P C R buffer (Perkin Elmer-Cetus), 1 m M Replica nitrocellulose filters of the hypothalamus dN TP, RNAase inhibitor (19 U; Promega), 8.75 c D N A libraries were made. The X-ZAPII library m M MgCl2 in a final volume of 20 ¿¿1. The template was screened at 42°C in 6 x SSC (1 x SSC is 0.15 was amplified using 50 pmol each of a degenerate M NaCl, 15 m M sodium citrate, pH 7.0), 1% SDS, oligonucleotide primer corresponding to a con­ 40 m M sodium phosphate buffer, pH 7.0, 2 x Den- served region of the M C H peptide [T(C/G)GGAT- hardt’s solution, 0.1% sodium pyrophosphate, 1 CCGT(C/G)TA(C/T)(A/C)G(A/G)CC(A/C/G/ m M E D T A , 5 0 % formamide and 100 ¿¿g/ml her­ T)TG(C/T)TGG] and the primer CCTGCAGCG- ring sperm D N A . Washing of filters was performed G C C G C A T G C A for 30 cycles of denaturation at room temperature (RT) and 60°C in 2 x SSC, (93°C, 1 min), annealing (50°C, 1.30 min) and ex­ 0.1 % SDS, 0.1 % sodium pyrophosphate and 1 m M tension (70°C, 1 min) in a Perkin Elmer-Cetus E D T A . As a hybridization probe we used the 0.2 kb Thermal Cycler with Ampli-Taq DNA-polymerase P C R fragment, labeled by nick translation accord­ (1 U; Perkin Elmer-Cetus). After agarose gel elec­ ing to standard procedures (Sambrook et al. 1989). trophoresis a 0.2 kb PCR-fragment was extracted The Uni-ZAP X R library was screened at 45°C in from the gel, digested with BamHl and Notl and 5 x SSPE hybridization solution (5 x SSPE [1 x ligated into a pBluescript SK-vector. D N A sequenc­ SSPE is 0.18 M NaCl, 10 m M sodium phosphate, ing was performed with T7 DNA-polymerase by the pH 7.4, 1 mM EDTA], 5 x Denhardt’s solution, dideoxy chain termination method (Sanger et al. 0 .5 % SDS, 5 0 % formamide and 100 /xg/ml herring 1977). sperm D N A ). Washing was performed at RT and 60°C until 0.1 x SSPE, 0.1 % SDS. TMe58, a puta­ tive partial tilapia M C H - cDN A clone isolated from Construction of tilapia hypothalamus cDNA the first library, was labeled by in vitro c R N A syn­ libraries thesis according to standard procedures (Sambrook et al. 1989) and used as a hybridization probe. Two tilapia hypothalamus c D N A libraries were Hybridization positive phage plaques were purified made. The first one was constructed in the vec­ and pBluescript D N A was prepared by in vivo tor XZAPII according to the manufacturers in­ excision according to the manufacturers proto­ structions (Stratagene, la Jolla, C A ) using 3 ¿tg col (Stratagene). D N A of both strands was se­ poly(A + ) R N A isolated with the Nonidet P40 quenced. method. c D N A was synthesized and inserted into the EcoRI site of the XZAPII vector. The c D N A library contained 2 x 105 independent clones. Northern blot analysis A second tilapia hypothalamus c D N A library was constructed with a ZAP- cDNA synthesis kit Tilapia tissues were collected and immediately (Stratagene) using about 4 ^g poly(A + ) R N A , iso­ frozen on dry ice. Total R N A from tilapia hypo­ lated by the acid guanidinium thiocyanate-phenol- thalamus, brain without hypothalamus, ovaria, chloroform procedure (Chomczynski and Succhi intestine, liver, heart, skin, headkidney, was pre­ 1987). c D N A was synthesized using an oligonucleo­ pared with the acid guanidinium thiocyanate- tide that contained a poly dT sequence and a Xhol phenol-chloroform method. R N A was run on a restriction site. EcoRI adaptors were ligated and horizontal 1 % agarose gel in 2.2 M formaldehyde the c D N A was directionally cloned into the EcoRI- and M O P S buffer (0.02 M M O P S , 8 m M sodium Xhol sites of the Uni-ZAP X R vector. This c D N A acetate, pH 7.0, 1 m M E D T A ). R N A was trans­ library contained 4 x 105 independent clones. ferred to nitrocellulose filter and the filter was Both libraries were amplified. hybridized with a c R N A probe of TMe58 c D N A in 5 x SSPE hybridization solution with 5 0 % 120

______z lS .______z l Ser Arg Leu Ser lie lie Phe Ala Ala Ala Leu Phe Phe Lys Cys Tyr Ala 5'-- G TCG CGC CTG TCC ATC ATC TTT CCT GCA GCG CTC TTT TTC AAG TGC TAC GCT 53 Patl

1 10 . 2 0 Leu Thr Val Ala Leu Pro Met Ala Lys Ala Glu Asp Gly Ser Leu Glu Lys Asp Ala Phe CTG ACA GTG GCA TTA CCC ATG GCC AAG GCT GAA GAT GGC TCC TTG GAG AAG GAT GCT TTT 112

30 40 Thr Ser Leu Leu Asn Asp Glu Ala Thr Glu Asn Ser Leu Gly Asp Ala Glu Leu Ser Ser ACC TCC CTG CTG AAC GAT GAG GCC ACG GAA AAC AGC CTA GGC GAT GCA GAG CTG TCC TCC 172

50 60 Met Thr Lys Ser Arg Ala Pro Arg Val lie Val lie Ala Ala Asp Ala Asn Leu Trp Arg ATG ACC AAA TCG AGA GCT CCC AGC GTA ATC CTC ATC GCC GCT GAT GCA AAC CTC TGC AGG 232 Sacl

70 T 90 Asp Leu Arg Val Leu His Asn Gly Leu Pro Leu Tyr Lys Arg Arg Val Asp Glu Asn Asn GAC CTG CGC GTG CTG CAC AAC GCC CTA CCC CTC TAC AAG CGC AGA CTC GAC GAA AAC AAC 292 Sail

90 100 Gin Val Val Glu His Lys Asp Val Gly Gin Asp Leu Thr lie Pro lie Leu Arg Arg Aap CAG CTC GTC GAG C AC AAA GAT GTC GCA CAG GAC CTC ACC ATC CCC ATC CTC AGC AGC GAC 352

110 ■hr Mac Arg Cy« Mat Val Gly Arg Val Tyr Arg Pro Cy« Trp Glu Val ••• lCC ATG AGG TGC ATG GTG GGA CGA GTG TAT CGG CCA TGC TOO GAG GTG TAG GACAGTTTGCT 414 MCH

rTTCTCCTCAAGGAGTCCAAACAGAGGATAGCTGTCAAGTCACCTTACACTGAGTTGCAAACTAATCCAAAAATGTGTG 4 93

Fig. 1. Nucleotide sequence and deduced amino acid sequence of a hypothalamic cDNA clone, TM16f, encoding tilapia preproMCH. The mature hormone MCH is underlined, the putative signal peptide sequence is overlined and the polyadenylation signal is underlined. Putative proteolytic cleavage sites are boxed and restriction sites are underlined. Numbering starts at the first amino acid of the prohor­ mone. The start of the partial cDNA clone TMe58 is indicated with an arrow. formamide at 45°C for 16h. Washing was per­ Tilapia preproMCH cDNA formed at RT and 60°C until 0.1 x SSPE, 0.1 % SDS. Two tilapia hypothalamus c D N A libraries (2 x 105 and 4 x 105 clones respectively) were constructed and screened under high stringency conditions. For Results screening of the first library the 0.2 kb M CH- PCR fragment was used, which resulted in 12 hybridi­ PCR analysis zation-positive clones with the same 0.6 kb c D N A insert. The sequence of this c D N A showed similari­ To obtain a tilapia M C H probe, PCR-experi- ty in the 0.35 kb 3 'part with salmon preproMCH- ments were conducted on tilapia hypothalamus m R N A (Ono et al. 1988; Minth et al. 1989). The 5 m R N A with a degenerate primer corresponding to end was very A T rich and contained several stop preproMCH m R N A and an oligo dT primer. The codons (not shown). A Sail fragment of the puta­ resulting 0.2 kb P C R fragment was cloned into tive MCH-prohormone coding part was subcloned pBluescript and sequenced. The PC R fragment (not after removing the poly(A) tail (TMe58). This clone shown) had low but significant similarity with the was used to screen a second hypothalamus Uni- 3'end of salmon preproMCH mRNA (Ono et al. Z A P X R c D N A library. Two hybridization posi­ 1988; Minth et al. 1989). tives were purified. The longest c D N A clone (clone 121

signal peptide _ T i l a p i a : 'F Salm on: MRE8MBHV 1130 LfiUST L R at : MAKMSla S YMLMLPa SLFSHGH LSASKSIRWVEDDIVFNTFRMGMA Human: MAKMNlls]SYILILTISLFSQG LSASKSIRNLDDDMVFNTFRLGK

signal peptide

T i l a p i a : sllnRe TBnslgJJa^ls§^ ^LWRDlflvffHNGL Salm on: SLLNQEvkDKNPDSV------aGMWKNLr ^ o I plyk R at : LeGYHM)ESGFMkBi^DFT)tKIv LSLAVKP

Human : s Jleqykndessfmk^: ehnkvskiv LNLAIKPY ALKG

Mqrp MCH i r ▼ ▼▼ TT T i l a p i a : -----PL VVEHKD TlIPI'iRR— DTMRCMVGRVYHPCWEV Salm on: -----LKAAAAGÜDRALTLDRRE SIHIBRR — DTMRCMVGRVYRPCWEV R at : PAVFPAEN STC ERP.E NSAKFfaj RCkBg r v yr pcvJ v Human : SVAFPAEN STC ERRE MSAKrBl RCMjiGRV YR PCWgV

AA A A

Fig. 2. Alignment of the amino acid sequences of tilapia, salmon (sMCH 1, Minth et al. 1989), rat (Nahon et at. 1989) and human (Presse et at. 1990) MCH-preprohormones. The one-letter amino acid notation is used. Gaps (-) have been introduced to achieve maximum similarity. Residues identical with the tilapia preprohormone are indicated by black boxes; conserved amino acid substitutions are indi­ cated by hatched boxes. The MCH peptide sequence, the putative MCH gene-related peptide (Mgrp) sequence (elongated by an interrupt­ ed line towards the putative tilapia cleavage site) are indicated. The putative signal peptide sequence of tilapia is overlined; the interrupt­ ed extension of the line indicates the salmon signal peptide cleavage site. The mammalian signal peptide sequence is underlined. Arrow­ heads indicate potential recognition sites for proteolytic cleavage enzymes.

TM16f, size of 0.58 kb) was selected for further polyadenylation consensus sequence (Proudfoot analysis. Figure 1 shows the nucleotide sequence and Brownlee 1976). and deduced amino acid sequence of TM 16f TMe58 c D N A was identical to the corresponding c D N A . The longest open-reading frame codes for part of TM16f. Comparison of the amino acid se­ 133 amino acids. The M C H peptide is located at the quence deduced from TM 16f with salmon and carboxy-terminus of the preprohormone, preceded mammalian MCH-preprohormone structures (Fig. by two arginine residues, which are proposed to 2) showed a high degree of identity at the carboxy function as cleavage site for proteolytic processing terminus where the M C H peptide is located (100% to mature hormones (Harris 1989). Three consecu­ and 7 5 % identity, respectively). The amino acid se­ tive basic amino acid residues are found at amino quence identity between TM 16f and the salmon acids 72-74 and cleavage at this site could produce MCH-preprohormone at the amino-terminus in the a 42-amino acid peptide or a 22-amino acid peptide putative signal sequence was also high (65% iden­ and mature M C H . At the amino-terminus a se­ tity; Fig. 2). W e therefore conclude that TM 16f quence of characteristics of a signal peptide is c D N A encodes a tilapia MCH-preprohormone. present. The most probable site of signal peptide cleavage is at alanine - 1 (Heijne 1986; Perlman and Halvorson 1983), yielding a 116 amino acid Northern blot analysis prohormone with a calculated molecular mass of 13,091 D. The 3 'non-coding region of the c D N A Northern blot analysis of total R N A isolated from contains 20 to 15 bp upstream of the poly(A)-tail a a variety of tilapia tissues with tilapia proM CH 122

1 2 3 4 5 6 7 8 structural comparison of the tilapia M C H prepro­ hormone with its salmon counterpart (Ono et al. 1988; Minth et al. 1989) shows a high identity in the M C H peptide and the signal peptide sequence (100% and 6 5 % amino acid sequence identity respectively). In the remaining part the similarity

9.44 — between the proM CH structures of the two teleost 7.46 — fishes is remarkably low, with just 2 6 % amino acid sequence identity and 5 3 % similarity. Thus, except

4.40 — for the MCH- and signal-peptide regions the M C H preprohormones of cichlids (tilapia) and the more primitive salmonids have diverged considerably during evolution. 2.37 — The similarity between tilapia and mammalian MCH-preprohormones (Nahon et al. 1989; Presse 1.37 — et al. 1990) is very low, except in the M C H peptide part, where 7 5 % amino acid sequence identity oc­ curs. It can therefore be concluded that in general the M C H preprohormone is a very poorly con­ served peptide precursor, with only high identity in the M C H peptide coding part. Since functionally 0.24 — significant protein regions as a rule are strongly conserved during evolution, it is obvious that only the MCH-coding part of the M C H preprohormone Fig. 3. Northern blot analysis of tilapia preproMCH mRNA. has substantial physiological importance, although Thirty microgram (unless otherwise mentioned) per sample of species-specific functions of the less well conserved total RNA was subjected to electrophoresis on an 1% agarose regions cannot be ruled out. Poor conservation of gel (20 mM MOPS buffer, 2.2 M formaldehyde), transferred to non-biologically active peptide-coding regions of a nitrocellulose filter and hybridized with a tilapia proMCH (TMe58) cRNA probe. Lanes 1, ovaria; 2, intestine; 3, liver; 4, prohormones has been reported for other hypotha­ heart; 5, skin; 6, headkidney; 7, hypothalamus (20 ¿ig of RNA); lamic neuropeptide preprohormones like the C R H 8, brain without hypothalamus. Positions of RNA size-markers precursor (Okawara et al. 1988). are indicated. When the sequence comparison of the tilapia and the salmon M C H preprohormones is studied in c D N A clone TMe58 as a probe revealed a single more detail, some marked differences are observed band at about 850 bases for hypothalamus R N A , (Fig. 2). First, based on accepted criteria (Heijne whereas for brain (minus hypothalamus), ovaria, 1986; Perlman and Halvorson 1983), the putative intestine, liver, heart, skin, headkidney (Fig. 3A), cleavage site of the signal peptide of the tilapia fin and muscle R N A (data not shown) no signal MCH-preprohormone is located four amino acids could be detected. more amino-terminal than the putative salmon cleavage site, although in tilapia an alanine residue corresponding to the salmon cleavage site also lies Discussion in a favourable position for cleavage. It has to be Comparison o f tilapia, salmon and mammalian investigated which of the potential sites in tilapia is MCH preprohormones actually used. Second, from the carboxy-terminal part just preceding the M C H peptide in the tilapia In this report we describe the cloning and expres­ prohormone structure a 22-residue peptide could be sion of tilapia MCH-preprohormone m RNA. A cleaved off by using a potential proteolytic cleavage 123 site consisting of three consecutive basic amino acid tilapia preproMCH with human preproANP, eel residues. In salmon, trout (Bird et al. 1990) and A N P (Takei et al. 1989) and Aplysia preproPep A , mammals (Nahon et al. 1989; Presse et al. 1990), the low degree of resemblance was confirmed. The however, a smaller 12- to 13-residue or 1.7 kD pep­ identity between the last 13 amino acids of tilapia tide, called MCH-gene related peptide (Mgrp; Bird M C H and human A N P was 31 % (vs. 3 8 % if the hu­ et al. 1990) can be formed. At the cleavage site of man peptides are compared; Presse et al. 1990), this smaller peptide a single lysine residue is present whereas identity between tilapia M C H and the cor­ in the tilapia precursor. Since monobasic residues responding part of eel A N P was only 2 3 % . In can also serve as cleavage sites (Schwartz 1986) it the signal peptides the identity between tilapia cannot be excluded that in tilapia a small 1 1-amino preproMCH and human preproANP is 24% (vs. acid peptide is cleaved off. The functional 3 7 % in the human signal peptides; Baker 1991). relevance of the putative 11- or 22-amino acid pep­ The percentages of identity are even lower if tilapia tides is still unclear. preproMCH and Aplysia preproPep A are com­ Tilapia most probably possesses only one MCH- pared (18% identity in the M C H coding region, precursor gene, since Southern blot analysis of 2 1 % in 19 amino acids of the,4/?/)>.S7£7 Pep A coding genomic D N A digested with several restriction en­ part and 6% in 17 amino acids of the signal pep­ zymes that do not cut in the c D N A corresponding tides). In addition, it has been observed before that to the proM CH c R N A probe, yields only one band the similarity between salmon preproMCH and per lane (data not shown). The presence of one gene Aplysia preproPep A is also very low (Baker 1991). in tilapia is in contrast with the existence of two Taken together, these findings suggest that there genes in salmonids, but this difference can be ex­ has been no extensive selective pressure in teleosts plained by the fact that salmonids are tetraploid to maintain structural conservation of these three (Ohno et al. 1968) and cichlids diploid. peptide precursors.

Comparison of the tilapia MCH preprohormone Tissue distribution of tilapia preproMCH mRNA with the ANP and the Aplysia peptide A prepro­ The preliminary findings of a strong preproMCH hormone m R N A signal in hypothalamic tissue while not in other brain areas or peripheral tissues are in line It has been reported that mammalian preproMCH with the tissue distribution reported for salmon shows significant amino acid sequence identity with preproMCH mRNA (Ono et al. 1988; Minth et al. the precursor for Aplysia peptide A (Pep A; Nahon 1989). In situ hybridization and dot blot studies et al. 1989; Presse et al. 1990) and human prepro- are in progress to determine the exact location of atrial natriuretic peptide (A N P ) (Presse et al. 1990). tilapia preproMCH m R N A and to investigate pre­ These observations led to the suggestion that proM CH m R N A expression in animals adapted to preproMCH, preproPep A and preproANP are a number of environmental challenges such as evolutionarily related, which also might imply some changes in background colouration. functional relationship. Since both A N P and Aplysia Pep A are involved in regulation of water balance and vascular functions (Scheller et al. 1984; Acknowledgements De Bold 1985) a similar role has been proposed for mammalian M C H (Presse et al. 1990; Baker 1991). The authors wish to thank M .C .H .M . van Riel for Computer searches (using the Pearson and Lipman technical assistance. This study is financially sup­ program) of a N B R F protein data base for homolo­ ported by the council of Geological and Biological gies of tilapia preproMCH with other sequences did Sciences of the Netherlands Organization for Scien­ not reveal similarities with either preproANP or tific Research (N W O ) within the research program preproPep A. When we subsequently compared “ Neuropeptides and Behaviour” . 124

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