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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 o f Animal Physiology, Faculty o f Science, University o f 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. We isolated and sequenced a 543 bp hypothalamic cDN 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 PCR fragment was

obtained and found to have low but significant nucleotide sequence similarity with the 3 'ends of known

MCH-mRNAs. Subsequently, the PCR fragment was used to screen XZAP cDN A libraries constructed from

tilapia hypothalamic poly(A + ) R N A . The cloned tilapia M C H preprohormone cDN 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 75% 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 ADN 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 PCR (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

PCR et l’analyse de sa séquence nucléotidique montre une similitude faible mais significative avec les extré­

mités 3 'des ARNms de M C H connus. Ensuite ce fragment a été utilisé pour cribler des banques de cDN A

118

construites dans XZAP à partir d’A R N poly(A + ) d’hypothalamus de tilapia. L ’ADNc 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 75% 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

Many lower vertebrates are capable of altering the

pigmentation of their skin in response to variations

in background colouration. The physiological

mechanism of background adaptation is of para­

mount importance for these animals and regulation

of this process can occur by both neuronal and en­

docrine mechanisms. In amphibia and reptiles one

pituitary hormone, a-melanocyte-stimulating hor­

mone (af-MSH), is involved in the control of skin

pigmentation. In teleost fish, however, a second

peptide hormone, melanin-concentrating hormone

(M CH), functions in this mechanism. M C H is

produced in the hypothalamus and most of the pep­

tide is axonally transported to the pituitary, where

it is stored in the neural lobe (for reviews see Baker

1991 and Eberle 1988). In addition to the pituitary

projections, some MCH-containing axons project

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

mined following isolation from chum salmon (0 /7-

corhynchus keta) pituitaries (Kawauchi et al. 1983).

It appeared to be a cyclic heptadecapeptide. Subse­

quently, the M C H peptide structure of other

teleosts has been described. Bonito (Katsuwonas pelamis) M C H appeared to be identical to salmon

M C H , whereas only one amino acid substitution at

the amino-terminus was observed in case of the eel

Anguilla japónica (Kawauchi 1989). Recently, the

structures of cDNAs encoding the M C H prepro­

hormone in salmon, rat and man have been eluci­

dated (Ono et al. 1988; Minth et al. 1989; Nahon et al. 1989; Presse et al. 1990) and the M C H peptide

has been purified from rat hypothalamic tissue

(Vaughan et al. 1989). Hence, M C H is not only

present in teleosts but also in other species where its

function is yet unclear. Regulatory roles in water

balance and control of homeostatic functions

(Presse et al. 1990; Zamir et al. 1986) or antagonis­

tic effects on a-MSH-induced behaviour in rats

(Eberle 1988) have been proposed.

At present little is known about the evolutionary

conservation of the M C H preprohormone in fish.

We therefore elucidated the structure of a tilapia

hypothalamic m R N A that encodes the M C H

precursor protein. A comparative structural analy­

sis between tilapia and other known M C H prepro­

hormones is presented. Furthermore, tissue speci­

ficity of tilapia preproMCH m R N A expression has

been investigated by Northern blot analysis.

Materials and methods

Polymerase chain reactions

R N A of tilapia (Oreochromis mossambicus) (ob­

tained from our laboratory stock) was prepared by

the Nonidet P40 method (Sambrook et al. 1989),

followed by purification of m R N A with oligo (dT)

cellulose. For polymerase chain reaction (PCR)

analysis single-stranded cD N A was synthesized

from poly(A + ) R N A by using M L V reverse trans­

119

criptase (BRL) and 50 ng (dT) primer [CCTGCAG-

C G G C C G C A T G C A T T T T T T T T T T T T T T T T T ] in

1 x PCR buffer (Perkin Elmer-Cetus), 1 m M

dNTP, RNAase inhibitor (19 U; Promega), 8.75

m M MgCl2 in a final volume of 20 ¿¿1. The template

was amplified using 50 pmol each of a degenerate

oligonucleotide primer corresponding to a con­

served region of the M C H peptide [T(C/G)GGAT-

C C G T (C /G )T A (C /T )(A /C )G (A /G )C C (A /C /G /

T )TG(C /T)TGG] and the primer CCTGCAGCG-

G C C G C A T G C A for 30 cycles of denaturation

(93°C, 1 min), annealing (50°C, 1.30 min) and ex­

tension (70°C, 1 min) in a Perkin Elmer-Cetus

Thermal Cycler with Ampli-Taq DNA-polymerase

(1 U; Perkin Elmer-Cetus). After agarose gel elec­

trophoresis a 0.2 kb PCR-fragment was extracted

from the gel, digested with BamHl and Notl and

ligated into a pBluescript SK-vector. D N A sequenc­

ing was performed with T7 DNA-polymerase by the

dideoxy chain termination method (Sanger et al. 1977).

Construction o f tilapia hypothalamus cDNA libraries

Two tilapia hypothalamus cD N A libraries were

made. The first one was constructed in the vec­

tor XZAPII according to the manufacturers in­

structions (Stratagene, la Jolla, CA) using 3 ¿tg

poly(A + ) R N A isolated with the Nonidet P40

method. cDN A was synthesized and inserted into

the EcoRI site of the XZAPII vector. The cDN A

library contained 2 x 105 independent clones.

A second tilapia hypothalamus cD N A library

was constructed with a ZAP-cDNA synthesis kit

(Stratagene) using about 4 ^g poly(A + ) R N A , iso­

lated by the acid guanidinium thiocyanate-phenol-

chloroform procedure (Chomczynski and Succhi

1987). cD N A was synthesized using an oligonucleo­

tide that contained a poly dT sequence and a Xhol

restriction site. EcoRI adaptors were ligated and

the cD N A was directionally cloned into the EcoRI-

Xhol sites of the Uni-ZAP X R vector. This cD N A

library contained 4 x 105 independent clones.

Both libraries were amplified.

Screening o f hypothalamus cDNA libraries

Replica nitrocellulose filters of the hypothalamus

cDN A libraries were made. The X-ZAPII library

was screened at 42°C in 6 x SSC (1 x SSC is 0.15

M NaCl, 15 m M sodium citrate, pH 7.0), 1% SDS,

40 m M sodium phosphate buffer, pH 7.0, 2 x Den-

hardt’s solution, 0 .1% sodium pyrophosphate, 1

m M E D T A , 50% formamide and 100 ¿¿g/ml her­

ring sperm D N A . Washing of filters was performed

at room temperature (RT) and 60°C in 2 x SSC,

0.1 % SDS, 0.1 % sodium pyrophosphate and 1 m M

E D T A . As a hybridization probe we used the 0.2 kb

PCR fragment, labeled by nick translation accord­

ing to standard procedures (Sambrook et al. 1989).

The Uni-ZAP X R library was screened at 45°C in

5 x SSPE hybridization solution (5 x SSPE [1 x

SSPE is 0.18 M NaCl, 10 m M sodium phosphate,

pH 7.4, 1 m M EDTA], 5 x Denhardt’s solution,

0 .5% SDS, 50% formamide and 100 /xg/ml herring

sperm DN A ). Washing was performed at RT and

60°C until 0.1 x SSPE, 0.1 % SDS. TMe58, a puta­

tive partial tilapia MCH-cDNA clone isolated from

the first library, was labeled by in vitro cRNA syn­

thesis according to standard procedures (Sambrook

et al. 1989) and used as a hybridization probe.

Hybridization positive phage plaques were purified

and pBluescript D N A was prepared by in vivo excision according to the manufacturers proto­

col (Stratagene). D N A of both strands was se­

quenced.

Northern blot analysis

Tilapia tissues were collected and immediately

frozen on dry ice. Total R N A from tilapia hypo­

thalamus, brain without hypothalamus, ovaria,

intestine, liver, heart, skin, headkidney, was pre­

pared with the acid guanidinium thiocyanate-

phenol-chloroform method. R N A was run on a

horizontal 1% agarose gel in 2.2 M formaldehyde

and M O P S buffer (0.02 M M O P S , 8 m M sodium

acetate, pH 7.0, 1 m M ED TA ). R N A was trans­

ferred to nitrocellulose filter and the filter was

hybridized with a cRNA probe of TMe58 cDN A

in 5 x SSPE hybridization solution with 50%

120

_________________________________ zlS ._________________________________________ z lSer 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 53Patl

1 1 0 . 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 40Thr 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

MetATG

ThrACC

LysAAA

Ser

TCGArgAGA

AlaGCT

ProCCC

ArgAGC

ValGTA

50lieATC

ValCTC

lieATC

AlaGCC

AlaGCT

AspGAT

AlaGCA

AsnAAC

LeuCTC

TrpTGC

60

ArgAGG 232

Sacl

70 T 90AspGAC

LeuCTG

ArgCGC

ValGTG

LeuCTG

HisCAC

AsnAAC

GlyGCC

LeuCTA

ProCCC

LeuCTC

TyrTAC

LysAAG

ArgCGC

ArgAGA

ValCTC

AspGAC

GluGAA

AsnAAC

AsnAAC 292

Sail

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

100AapGAC 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 o f a hypothalamic cD N A clone, TM16f , encoding tilapia p re p ro M C H . The mature horm one M C H is underl ined, the putat ive signal peptide sequence is overlined and the polyadenylat ion signal is underl ined. Putat ive proteolytic cleavage sites are boxed and restriction sites are underl ined. Number ing starts at the first amino acid o f the p ro h o r ­mone. The start o f the partial cDNA clone TMe58 is indicated with an arrow.

formamide at 45°C for 16h. Washing was per­

formed at RT and 60°C until 0.1 x SSPE, 0.1 %

SDS.

Results

PCR analysis

To obtain a tilapia M C H probe, PCR-experi-

ments were conducted on tilapia hypothalamus

m R N A with a degenerate primer corresponding to

preproMCH m R N A and an oligo dT primer. The

resulting 0.2 kb PCR fragment was cloned into

pBluescript and sequenced. The PCR fragment (not

shown) had low but significant similarity with the

3 'end of salmon preproMCH m R N A (Ono et al. 1988; Minth et al. 1989).

Tilapia preproMCH cDNA

Two tilapia hypothalamus cDN A libraries (2 x 105

and 4 x 105 clones respectively) were constructed

and screened under high stringency conditions. For

screening of the first library the 0.2 kb MCH-PCR

fragment was used, which resulted in 12 hybridi­

zation-positive clones with the same 0.6 kb cDN A

insert. The sequence of this cDN A showed similari­

ty in the 0.35 kb 3 'part with salmon preproMCH-

m R N A (Ono et al. 1988; Minth et al. 1989). The 5

end was very A T rich and contained several stop

codons (not shown). A Sail fragment of the puta­

tive MCH-prohormone coding part was subcloned

after removing the poly(A) tail (TMe58). This clone

was used to screen a second hypothalamus Uni-

ZA P X R cDN A library. Two hybridization posi­

tives were purified. The longest cDN A clone (clone

121

T i l a p i a :

S a lm o n :

R a t :

Human:

s i g n a l p e p t i d e _

'F

MRE8MBHV 1130 LfiUST L

MAKMSla

MAKMNlls]

S YMLMLPa SLFSHGH

SYILILTISLFSQG

LSASKSIRWVEDDIVFNTFRMGMA

LSASKSIRNLDDDMVFNTFRLGK

s i g n a l p e p t i d e

T i l a p i a :

S a lm o n :

R a t :

Human :

sllnRe

SLLNQE

TB

vkDnslgJJa ls§^KNPDSV---------

LeGYHM)ESGFMkBi DFT)tKIv

s Jleq y k n d essfm k^: ehnkvskiv

LWRDlflvffHNGL

aGMWKNLr o I ply k

LSLAVKP

LNLAIKPY ALKG

T i l a p i a :

S a lm o n :

R a t :

Human :

Mqrp MCH▼ ▼▼

i r

TT

-----PL

PAVFPAEN

SVAFPAEN

VVEHKD

-----LKAAAAGÜDRALTLDRRE

STC ERP.E

STC ERRE

Tl

S

IPI'iRR— DTMRCMVGRVYHPCWEVIHIBRR —

NSAKFfaj

MSAKrBl

AA A A

DTMRCMVGRVYRPCWEV

RCkBg r v yr pcvJ v

RCMjiGRV YR PCWgV

Fig. 2. Alignment o f the amino acid sequences o f tilapia, salmon (sMCH 1, Minth et al. 1989), rat (Nahon et at. 1989) and hum an (Presse et at. 1990) M C H -prep roho rm ones . The one-letter amino acid notat ion is used. Gaps (-) have been introduced to achieve maximum similarity. Residues identical with the tilapia p rep roh o rm on e are indicated by black boxes; conserved amino acid substi tut ions are indi­cated by hatched boxes. The M C H peptide sequence, the putat ive M C H gene-related peptide (Mgrp) sequence (elongated by an in terrupt­ed line towards the putat ive tilapia cleavage site) are indicated. The putat ive signal peptide sequence o f tilapia is overlined; the in terrupt­ed extension o f the line indicates the salmon signal peptide cleavage site. The mammal ian signal peptide sequence is underlined. A r ro w ­heads indicate potential recognition sites for proteolytic cleavage enzymes.

TM16f, size of 0.58 kb) was selected for further

analysis. Figure 1 shows the nucleotide sequence

and deduced amino acid sequence of TM16f

cDNA . The longest open-reading frame codes for

133 amino acids. The M C H peptide is located at the

carboxy-terminus of the preprohormone, preceded

by two arginine residues, which are proposed to

function as cleavage site for proteolytic processing

to mature hormones (Harris 1989). Three consecu­

tive basic amino acid residues are found at amino

acids 72-74 and cleavage at this site could produce

a 42-amino acid peptide or a 22-amino acid peptide

and mature M C H . At the amino-terminus a se­

quence of characteristics of a signal peptide is

present. The most probable site of signal peptide

cleavage is at alanine - 1 (Heijne 1986; Perlman

and Halvorson 1983), yielding a 116 amino acid

prohormone with a calculated molecular mass of

13,091 D. The 3 'non-coding region of the cDN A

contains 20 to 15 bp upstream of the poly(A)-tail a

polyadenylation consensus sequence (Proudfoot

and Brownlee 1976).

TMe58 cDN A was identical to the corresponding

part of TM16f. Comparison of the amino acid se­

quence deduced from TM16f with salmon and

mammalian MCH-preprohormone structures (Fig.

2) showed a high degree of identity at the carboxy

terminus where the M C H peptide is located (100%

and 75% identity, respectively). The amino acid se­

quence identity between TM16f and the salmon

MCH-preprohormone at the amino-terminus in the

putative signal sequence was also high (65% iden­

tity; Fig. 2). W e therefore conclude that TM16f

cDN A encodes a tilapia MCH-preprohormone.

Northern blot analysis

Northern blot analysis of total R N A isolated from

a variety of tilapia tissues with tilapia proMCH

122

1 2 3 4 5 6 7 8

9.44 —

7.46 —

4.40 —

2.37 —

1.37 —

0.24 —

Fig. 3. Northern blot analysis o f tilapia p re p ro M C H m R N A . Thir ty microgram (unless otherwise mentioned) per sample o f total RNA was subjected to electrophoresis on an 1% agarose gel (20 mM M O P S buffer , 2.2 M formaldehyde), t ransferred to a nitrocellulose filter and hybridized with a tilapia p ro M C H (TMe58) cR N A probe. Lanes 1, ovaria; 2, intestine; 3, liver; 4, heart; 5, skin; 6, headkidney; 7, hypotha lamus (20 ¿ig o f RNA); 8, brain without hypothalamus. Posit ions o f RNA size-markers are indicated.

cDN A clone TMe58 as a probe revealed a single

band at about 850 bases for hypothalamus R N A ,

whereas for brain (minus hypothalamus), ovaria,

intestine, liver, heart, skin, headkidney (Fig. 3A),

fin and muscle R N A (data not shown) no signal

could be detected.

Discussion

Comparison o f tilapia, salmon and mammalian M CH preprohormones

In this report we describe the cloning and expres­

sion of tilapia MCH-preprohormone m R N A . A

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 65% amino acid sequence identity

respectively). In the remaining part the similarity

between the proMCH structures of the two teleost

fishes is remarkably low, with just 26% amino acid

sequence identity and 53% similarity. Thus, except

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.

The similarity between tilapia and mammalian

MCH-preprohormones (Nahon et al. 1989; Presse

et al. 1990) is very low, except in the M C H peptide

part, where 75% 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

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

has substantial physiological importance, although

species-specific functions of the less well conserved

regions cannot be ruled out. Poor conservation of

non-biologically active peptide-coding regions of

prohormones has been reported for other hypotha­

lamic neuropeptide preprohormones like the C R H

precursor (Okawara et al. 1988).

When the sequence comparison of the tilapia and

the salmon M C H preprohormones is studied in

more detail, some marked differences are observed

(Fig. 2). First, based on accepted criteria (Heijne

1986; Perlman and Halvorson 1983), the putative

cleavage site of the signal peptide of the tilapia

MCH-preprohormone is located four amino acids

more amino-terminal than the putative salmon

cleavage site, although in tilapia an alanine residue

corresponding to the salmon cleavage site also lies

in a favourable position for cleavage. It has to be

investigated which of the potential sites in tilapia is

actually used. Second, from the carboxy-terminal

part just preceding the M C H peptide in the tilapia

prohormone structure a 22-residue peptide could be

cleaved off by using a potential proteolytic cleavage

123

site consisting of three consecutive basic amino acid

residues. In salmon, trout (Bird et al. 1990) and

mammals (Nahon et al. 1989; Presse et al. 1990),

however, a smaller 12- to 13-residue or 1.7 kD pep­

tide, called MCH-gene related peptide (Mgrp; Bird

et al. 1990) can be formed. At the cleavage site of

this smaller peptide a single lysine residue is present

in the tilapia precursor. Since monobasic residues

can also serve as cleavage sites (Schwartz 1986) it

cannot be excluded that in tilapia a small 1 1-amino

acid peptide is cleaved off. The functional

relevance of the putative 1 1- or 22-amino acid pep­

tides is still unclear.

Tilapia most probably possesses only one MCH-

precursor gene, since Southern blot analysis of

genomic D N A digested with several restriction en­

zymes that do not cut in the cD N A corresponding

to the proMCH cRNA probe, yields only one band

per lane (data not shown). The presence of one gene

in tilapia is in contrast with the existence of two

genes in salmonids, but this difference can be ex­

plained by the fact that salmonids are tetraploid

(Ohno et al. 1968) and cichlids diploid.

Comparison o f the tilapia M CH preprohormone with the A N P and the Aplysia peptide A prepro­hormone

It has been reported that mammalian preproMCH

shows significant amino acid sequence identity with

the precursor for Aplysia peptide A (Pep A; Nahon

et al. 1989; Presse et al. 1990) and human prepro-

atrial natriuretic peptide (ANP) (Presse et al. 1990).

These observations led to the suggestion that

preproMCH, preproPep A and preproANP are

evolutionarily related, which also might imply some

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;

De Bold 1985) a similar role has been proposed for

mammalian M C H (Presse et al. 1990; Baker 1991).

Computer searches (using the Pearson and Lipman

program) of a NBRF protein data base for homolo­

gies of tilapia preproMCH with other sequences did

not reveal similarities with either preproANP or

preproPep A. When we subsequently compared

tilapia preproMCH with human preproANP, eel

A N P (Takei et al. 1989) and Aplysia preproPep A,

the low degree of resemblance was confirmed. The

identity between the last 13 amino acids of tilapia

M C H and human A N P was 31 % (vs. 38% if the hu­

man peptides are compared; Presse et al. 1990),

whereas identity between tilapia M C H and the cor­

responding part of eel A N P was only 23% . In

the signal peptides the identity between tilapia

preproMCH and human preproANP is 24% (vs.

37% in the human signal peptides; Baker 1991).

The percentages of identity are even lower if tilapia

preproMCH and Aplysia preproPep A are com­

pared (18% identity in the M C H coding region,

21% in 19 amino acids of the,4/?/)>.S7£7 Pep A coding

part and 6% in 17 amino acids of the signal pep­

tides). In addition, it has been observed before that

the similarity between salmon preproMCH and

Aplysia preproPep A is also very low (Baker 1991).

Taken together, these findings suggest that there

has been no extensive selective pressure in teleosts

to maintain structural conservation of these three

peptide precursors.

Tissue distribution o f tilapia preproM CH mRNA

The preliminary findings of a strong preproMCH

m R N A signal in hypothalamic tissue while not in

other brain areas or peripheral tissues are in line

with the tissue distribution reported for salmon

preproMCH m R N A (Ono et al. 1988; Minth et al. 1989). In situ hybridization and dot blot studies

are in progress to determine the exact location of

tilapia preproMCH m R N A and to investigate pre­

proMCH m R N A expression in animals adapted to

a number of environmental challenges such as

changes in background colouration.

Acknowledgements

The authors wish to thank M .C .H .M . van Riel for

technical assistance. This study is financially sup­

ported by the council of Geological and Biological

Sciences of the Netherlands Organization for Scien­

tific Research (N W O ) within the research program

“ Neuropeptides and Behaviour” .

124

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