PDF hosted at the Radboud Repository of the Radboud University … · 2017-12-05 · 120 _____zlS._____zl Ser Arg Leu Ser lie lie Phe Ala Ala Ala Leu Phe Phe Lys Cys Tyr Ala 5'--
Post on 19-Jul-2020
0 Views
Preview:
Transcript
PDF hosted at the Radboud Repository of the Radboud University
Nijmegen
The following full text is a publisher's version.
For additional information about this publication click this link.
http://hdl.handle.net/2066/16683
Please be advised that this information was generated on 2017-12-05 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 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
References cited
Baker, B.I. 1991. Melanin-concentrat ing hormone: A general vertebrate neuropeptide. Int. Rev. Cytol. 126: 1 -47 .
Bird, D .J . , Baker, B.I. , Eberle, A. and Swann, R.W. 1990. The biosynthesis o f melanin-concentrat ing horm one in a fish. J. Neuroendocrinology 2: 309-315 .
Chomczynski , P. and Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol- ch loroform extract ion. Anal. Biochem. 162: 156-159.
De Bold, A .J . 1985. Atrial natriuretic factor: a horm one produced by the heart . Science 230: 767-770 .
Eberle, A .N . 1988. Melanin-concentrat ing hormone . In The Melanotropins. Chemistry, Physiology and Mechanisms o f Action, pp. 321-332 . Edited by A .N . Eberle. Karger, Basel.
Harris, R.B. 1989. Processing o f p ro -horm one precursor p ro teins. Arch. Biochem. Biophys. 275: 315-333 .
Heijne, G. von 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14: 4683-4690.
Kawauchi, H. 1989. Structure and biosynthesis o f melanin- concentra t ine hormone . Life Sci. 45: 1133-1140.
Kawauchi, H. , Kawazoe, I., Tsubokawa, M., Kishida, M. and Baker, B.I. 1983. Character izat ion o f melanin-concentrat ing horm one in chum salmon pituitaries. Nature, Lond. 305: 321-323 .
Minth, C .D . , Qui, H. , Akil, H. , Watson , S.J. and Dixon, J .E. 1989. Two precursors o f melanin-concentrat ing hormone: DNA sequence analysis and in situ and immunochemical localization. Proc. Nat. Acad. Sci. U .S .A. 86: 4292-4296.
Nahon , J .L . , Presse, F., Bittencourt, J .C . , Sawchenko, P.E. and Vale, W. 1989. The rat melanin-concentrat ing horm one messenger ribonucleic acid encodes multiple putat ive neuropeptides coexpressed in the dorsolateral hypothalamus. E n docrinology 125: 2056-2065.
Naito, N., Nakai, Y., Kawauchi, H. and Hayashi , Y. 1985. Im- munocytochemical identification o f melanin-concentrat ing horm one in the brain and pituitary gland o f the teleost fishes Oncorhynchus keta and Salrno gairdneri. Cell Tiss. Res. 242: 4 1 -4 8 .
O hno , S., Wolf , U. and Atkin, N.B. 1968. Evolution from fish to mammals by gene duplicat ion. Hereditas 59: 169-187.
Okawara , Y., Morley, S.D. , Burzio, L .O. , Zwiers, H. , Lederis,
K. and Richter, D. 1988. Cloning and sequence analysis of cD N A for corticotropin-releasing factor precursor from the teleost fish Catostomus commersoni. Proc. Nat. Acad. Sci. U.S.A. 85: 8439-8443.
O no , M., W ada , C. , Oikawa, I., Kawazoe, I. and Kawauchi, H. 1988. Structures o f two kinds o f m R N A encoding the chum salmon melanin-concentrat ing horm one . Gene 71: 4 33 -4 3 8 .
Perlman, D. and Halvorson, H .O . 1983. A putat ive signal peptidase recognition site and sequence in eukaryot ic and prokaryotic signal peptides. J. Mol. Biol. 167: 391-409 .
Presse, F., Nahon, J .L . , Fischer, W .H . and Vale, W. 1990. Structure o f the hum an melanin-concentrat ing horm one m R N A . Mol. Endocrinol . 4: 632 -637 .
P roud foo t , N .J . and Brownlee, G .G . 1976. 3 'non-coding region sequences in eukaryot ic messenger RNA. Nature, Lond. 263: 211-214 .
S am brook , J . , Fritsch, E.F. and Maniat is , T. 1989. Molecular Cloning: A Labora to ry Manual (2nd ed.). Cold Spring H a r bor University Press, Cold Spring Harbor .
Sanger, F., Nicklen, S. and Coulson, A .R . 1977. DNA sequencing with chain-terminating inhibitors. Proc. Nat . Acad. Sci. U .S .A. 74: 5463-5467.
Scheller, R .H . , Kaldany, R.R. , Kreiner, T . , M ahon , A .C . , N am bu, J .R . , Schefer, M. and Taussig, R. 1984. N europeptides: mediators o f behaviour in Aplysia. Science 225: 1300-1308.
Schwartz, T .W . 1986. The processing o f peptide precursors. ‘Proline directed arginyl cleavage’ and other monobasic processing mechanisms. FEBS Lett. 200: 1 -10 .
Takei , Y., Takahashi , A. , W atanabe , T .X . , Nakaj ima, K. and Sakakibara , S. 1989. Amino acid sequence and relative biological activity o f eel atrial natr iuretic peptide. Biochem. Biophys. Res. C om m . 164: 537-543 .
Vaughan, J .M . , Fischer, W .H . , Hoeger, C. , Rivier, J. and Vale, W. 1989. Character izat ion o f melanin-concentrat ing h o r mone from rat hypotha lamus. Endocrinology 125: 1660-1665.
Zamir , N., Skofi tsch, G. , Bannon, M .J . and Jacobowitz , D.M. 1986. Melanin-concentra t ing hormone: unique peptide neuronal system in the rat brain and pituitary gland. Proc. Nat . Acad. Sci. U .S .A. 83: 1528-1531.
top related