GENERAL AND COMPARATIVE ENDOCRINOLOGY 87, 260-265 (1992)
Molecular Cloning of Silver Carp and Bighead Carp Prolactin
Y. S. CHANG,*,~
F. L.
HUANG,*$ ANDT. B. Lo*,?
*Institute of Biological Chemistry, Academia Sinica; )‘Graduate Institute of Biochemical Sciences, and ZDepartment of Zoology, National Taiwan University, P.O. Box 23-106, Taipei, Taiwan
Accepted December 24, 1991
The cDNAs encoding the prolactin of silver carp (scPRL) and bighead carp (bcPRL) have been cloned. Deduced from the nucleotide sequences, both scPRL and bcPRL are com- posed of 187 amino acid residues. Only one residue is different between scPRL and bcPRL. Homology analysis indicates that scPRL and bcPRL are highly homologous to carp PRL (97%), relatively conserved in relation to PRLs of salmon, trout, and tilapia @l-69%), and diversified from avian and mammalian PRL (30-35%). Similar to PRLs of other species of fish, scPRL and bcPRL lack the first 12 N-terminal residues of avian and mammalian PRLs. 0 1992 Academic Press, Inc.
Prolactin
(PRL)
is a polypeptide
hor-
mone secreted from the pituitary gland. To-
gether with growth hormone and placental
somatomammotropin,
it belongs to a family
of hormones
evolved from a common
an-
cestral gene (Miller
and Eberhardt,
1983;
Nicoll
et al., 1986). This family has been
extended
due to the discovery
of mouse
proliferin
(Linzer and Nathans,
1984; Lin-
zer
etal., 1985), bovine PRL-related
cDNA
I (Schuler and Hurley,
1987), rat PRL-like
protein A (Deb
et al.,1989), and somato-
lactins of flounder, Atlantic cod, and chum
salmon (Ono
etal., 1990; Rand-Weaver
etal.,
1991; Takayama
et al.,1991). In fish,
PRL is involved
in many biological
func-
tions such as osmoregulation,
reproduc-
tion, behavior, and metabolism
(Clarke and
Bern, 1980).
The amino acid sequence of PRL had
been determined
chemically
or deduced
from the nucleotide
sequence of cDNA in
many species of vertebrates including
hu-
man (Cooke
et al.,1981), cattle (Miller
etal.,
1981), sheep (Li
et al.,1967; Varma
etal.,
1989), pig (Li, 1976), rat (Cooke
et al.,1980), mouse (Linzer
and Talamantes,
1985), whale (Tsubokawa
et al.,1985),
chicken (Hanks
et al.,1989), carp (Yasuda
et al.,
1987; Chao
et al., 1988),salmon (Ya-
suda
et al.,1986; Song
et al.,1988), rain-
bow trout (Mercier
et al.,1989), and tilapia
(Yamaguchi
et al.,1988; Rentier-Deh-ue
etal.,
1989). In order to obtain more informa-
tion about the evolution
of PRL, we deter-
mined the primary structures of PRL of sil-
ver carp (scPRL) and bighead carp (bcPRL)
by using cDNA cloning technique.
In this
paper, the cDNA nucleotide
sequences and
the deduced
amino
acid sequences
of
scPRL and bcPRL are presented and their
sequences are compared to those of PRLs
from other fish and vertebrates.
MATERIALS AND METHODS
Construction of cDNA library and screening of PRL cDNA. The method used to construct cDNA li- brary was essentially the same as previously described &hang et al., 1990). Briefly, the pituitary polyadenyl- ated RNAs of silver carp (Hypophthalamichthys molitrix) and bighead carp (H. nobilis) were prepared from liquid nitrogen frozen tissues by guanidin- ium/CsCl method (Ullrich et al., 1977) followed by oli- go(dT)-cellulose column chromatography. The double- stranded cDNA, synthesized by the method of Gubler and Hoffman (1983), was ligated with EcoRI linker and subsequently inserted into EcoRI site of XgtlO. To 260
0016~6480/92 $4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
screen the recombinant DNA clones containing the Nucleotide sequencing of cDNAs encoding scPRL PRL cDNA from the cDNA libraries, the cDNA cod- and bcPRL. The cDNAs encoding scPRL and bcPRL ing for carp PRL (cPRL) previously cloned in our lab- were cleaved with AvaII for subcloning. The nucle- oratory was 32P-labeled by nick-translation and used otide sequences were determined by the dideoxy chain as a hybridization probe. Plaque hybridization was termination method @anger et al., 1977) using super- performed according to the method of Benton and coiled plasmid DNA as template (Chen and Seeburg,
Davis (1977). 1985). GGCAAGAGAATAAAGGAACCAGTTAAAATAATAATGGCTGAAGGAT~CAGACTATACTTTGGA MAEGSRLYFA -23 -20 GTGACTGTCCTGATGTGTGCGTTTGTCTCAATCAACGGTGTCGGTCTGAATGATTTACTG V T V LMCAPVS INGVGLNDLL -10 -1 1 GAAAGAGCCTCTCAACTTTCAGACAAACTTCACTCCCTCAGCACCTCTCTCACCAATGAC ERASQLS DKLHSLST S L T N D 10 20 CTGGATTCTCACTTTCCTCCTGTTGGGAGGGTAATGATGCCCCGTCCGTCGATGTGCCAC LDSHFPPVGRVMMPRPSMCH 30 40 ACATCCTCCCTTCAAATTCCCAATGACAAAGACCAAGCCCTGAAAGTGCCGGAGGATGAG TSSLQI PNDKDQALKV P E D E 50 60 TTACTTTCTTTGGCTCGGTCTCTGCTGCTGGCGTGGTCCGCCTCCTCTCC L L S L A R S I, I, L A W S D P I, A L L S 70 80 TCTGAGGCGTCCAGCCTGGCA~AT~~AGAACG~AA~AC~ATTAACAGCAAGAC~AAAGAA
SEASSLAH PERNTI NSKTKE
90 100 C'~GCAAGACAACATCAACAGCCTCGGTGGGTCTGGAGGTCTGGAGCGTGTCGTTCA~AAGATGGGC I Q D N J N S L G A G L E R V V H KM G 110 120 'rCATCC'I'CAGACAACC'I'GT(:CTCTCTCC(:TT?"PTACAGCAACAGCCTTGGCCAGGATAAA S S S D N I, S S LPPYSNS L G Q D K 130 140 ACCTC'I'CGACTTGTCAATTTCCATTTTCTG'PTGTCCTGCTTGCGCAGGGACTCCCACAAA 'I SRLVNFHPL LSCFRRDSHK 150 160 A'I'1'tiACA(:?"rT(:CTCAAAAGTTCTGCGC'rGCCGGGCAGCCAAGAAGAGACCTGAGATGTGC 1 D s F LKVLRCRAAKKRPEMC 170 180 187 TAAACTGAAAATGCTACTCTG~TT~T~T~ATTGTGGATGTTAGGTTAAAATGGCAGAGCA 60 120 180 240 300 360 420 480 540 600 660 720 G'rGAGTGATTTGAAATGTTTCTTTATAATACCGCATGGCAA~ATGCCCTTTATTGTTT 780 CAAAGA'I'GTAGA'rATTTGATTCACTTTCTTATATACCTGGACCAACGG 840 GGATATTATTCTGTCCCCAGAGATAAAATACAACAAAAAGTA~C~AAGTATTTCAATT 900 AAATT'r'l'AAAATTGAAGGTCA'PATAAAAGGTCTGCATGCAGTCATACTGTTTGATGAATA 960 AATGTATATTGAAATGTTTGACCATGCTCAGTTTAATAAGACATTTGTATAGTAAGTATT 1020 TA'rTAAGAAATGATGTTGGATCTTTAACACGTTTATTATTTTAACATTATC~rCTGAAATGCA 1080 A'I'A'l"rTAAAGC'P'PTGCACCAA'I'GTCAATAAATTTCTA'PCATGC-PolyA 1170 FIG. 1. The nucleotide sequence and deduced amino acid sequence of bcPRL cDNA.
262
CHANG, HUANG, AND LORESULTS AND DISCUSSION
When cPRL cDNA was used as a probe,
8 and 9 positive clones were obtained from
5000 and 6000 clones from silver carp and
bighead carp pituitary
cDNA
library,
re-
spectively.
In this investigation,
only the
clone with the largest insert of each library
was chosen for further study.
The cDNA encoding bcPRL investigated
in this study is 1170 bp in length, consisting
of a 5’-untranslated
region of 30 bp, an open
reading frame (ORF) of 630 bp, and a 3’-
untranslated
region of 510 bp. The ORF en-
codes a signal peptide of 23 amino acid res-
idues and a putative
mature PRL of 187
amino
acid residues.
Its nucleotide
se-
quence and deduced amino acid sequence
are presented in Fig. 1.
The cDNA encoding SCPRL investigated
in this study is 1060 bp in length. The nu-
cleotide
sequence
of cDNA
encoding
scPRL
is highly
homologous
to that of
cDNA
encoding
bcPRL
(data not pre-
sented). It contains an ORF of 621 bp and a
3’-untranslated
region
of 436 bp. The
scPRL cDNA thus obtained is not full in
length,
lacking
the translation
initiation
codon, AUG, and the poly(A) tract. Among
the 207 amino acid residues coded by ORF,
a putative mature PRL of 187 residues and
an incomplete
signal peptide of 20 residues
are present.
The scPRL
and bcPRL
are similar
to
each other. Only one of 187 amino acid res-
idues is changed (Fig. 2). When scPRL and
bcPRL were compared to cPRL, from an-
other species of Cyprinidae,
a high extent
of homology
(97%) was found (Yasuda et
al., 1987; Chao et al., 1988). A similar sit-
uation was also found among the PRLs of
three species of Salmonidae:
chum salmon,
chinook
salmon, and rainbow trout where
the homology
of PRL is 98% or more (Ya-
suda et al., 1986; Song et al., 1988; Mercier
et al., 1989). On the contrary, when PRLs
of different
fish families
were compared
(Fig. 2), homology is decreased to the range
of 64 to 74% (Cyprinidae
vs Salmonidae,
69%; Cyprinidae
vs tilapia,
64%; Salmo-
nidae vs tilapia, 74%) (Yasuda et al., 1986,
1987; Song et al., 1988; Yamaguchi
et al.,
1988; Mercier et al., 1989; Rentier-Deh-ue
et al.,
1989).Most fish PRLs thus far investigated
are
composed of 187 to 189 amino acid residues
except 177 residues are found in one form
of two tilapia
PRLs (Yamaguchi
et al.,
1988; Rentier-Deh-ue
et al.,
1989).In com-
parison,
chicken
PRL has 198 residues
while ovine and human PRL have 199 res-
idues. Such difference arises from the ab-
sence in fish PRLs of the first 12 N-terminal
residues of higher vertebrates.
Homology
between fish and higher vertebrate PRL is
low, about 30 to 35% (Li et al., 1967; Li,
1976; Cooke et al., 1980, 1981; Miller et al.,
1981; Linzer and Talamantes,
1985; Tsub-
okawa et al., 1985; Hanks et al., 1989;
Varma et al., 1989). Although the structure
of PRLs of fish and higher vertebrates are
diversified,
two conserved segments of al-
most identical
or chemically
similar
se-
quence, residues 46 to 51 and 160 to 180,
were found in all PRLs thus investigated.
These conserved sequences may be impor-
tant for the biological
activity of PRL. As
shown in Fig. 2, PRLs of fish contain four
cysteine residues while those of higher ver-
tebrates contain six cysteine residues ex-
cept for equine PRL (Li and Chung, 1983).
The first two cysteine residues of higher
FIG. 2. Comparison of the primary structure of scPRL and bcPRL with PRLs of other fish and higher vertebrates. Residues identical to scPRL are indicated by dashes. Gaps, marked by X, are inserted to maximize structural alignment. References used: carp, Yasuda et al. (1987); chum salmon, Yasuda et al. (1986); chinook salmon, Song et al. (1988); rainbow trout, Mercier et al. (1989); 0. mossambicus I and II, Yamaguchi et al. (1988); 0. nilotica I and II, Rentier-Delrue et al. (1989); chicken, Hanks et al. (1989); sheep, Varma et al. (1989); human, Cooke et al. (1981).
Bighead carp (2) ---~----_-_-_---_---~~~~-~-~~-~~~~--~-~~
Carp (3) ---E---
Chum salmon (4) I--S--H---R---K---M--- Chinook salmon (5) I--S--M---R---K---M--- Rainbow trout (6) I--S--M---R---K---M--- 0. mossambicug I (7) -PI-E---H---T--C3g--QE---I- _O. niloticus I (8) -PI-E-F---H---T--QE---.~I~--I-
0. mossambicus II (9) -PI-E-F---H---T---I---I---I- 0. niloticus II (10) -PI---IY----Q---A---M--QE-G-EAF-ID--LA Chicken (11) LFICPIGSVNCQ-S-GE-FD--VK--HYI-Y--SEI'F-EP-ERYAQGRGPIT Sheep (12) TPVCPNGPGDCQ-S-R--FD--VMV-HYI-N--SEMF-EF-KR~AQGKGF~T Human (13) LPICPGGAARCQ-T-R--FD--W--HYI-N--SEMFSEF-YTHGRGFIT 1. 2. 3. 4. 5. 6. I. 8. 9. 10. 11. 12. 13. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 50 60 70 80 90 100 PRPSMCHTSSLQIPNDKDQALKVPEDELLSLARSLLLAWSDPLALLSSEASSL~XPERNT ---X--- ---V---P---X--- ---T-K--EE----s-N--I---N---L---pT-p-X-SNGD ---T-K--E---S-N--I----y---N---L---pT-p-T-SNeD ---T-K--E---S-N--I---N---L---pT-p-X-SNGD ---A---T-I---Q-S-SD-M---Q----W---S--T-P-X-AQSS ---A---T-I---Q-S-SD-M---8---Q---VV---S--T-p-X-AQS- XXXXX---T-T--E---Q-S-SD---Q---EV---STNV-py~SAQs- XXXXX---T-T--E---Q-S-SD---8---Q---E”---STNV-pyXSAQS- KAVNG---TT-E--E--QQIHHED--N-VVGV-RS-N---IH-A--VQRIKEA-DTIL MALNS---PT-E--E--QQTHHEV-M--ILG--RS-N---YH-VT-VRG~KGV-D~IL KAINS---AT-E--E--QQMNQKDF---IV-I-RS-NE--YH-VT-VRGM~EA--AIL 110 120 130 140 150 160 INSKTKELQDNINSLGAGLEHVVHMGSSSDNLSSLPFYSNSLXGQDKTSXRLVNFHFLLSC ---R---X---X--- -D---E---FN--D-T---T---X-E----X--- -S--IR----YSK---D--D,I~-N---p--Qy~--I--KGGD-X-N----X--I---~-- -S--~R----YSK---D--D~~~~---~--Q~~~~~~~~~~~~~~~~~~-~~~~~~~~~~-- -S--IR----YSK---D--DIM-N---p--QyI--I--KGGD-X-N----X--I---M-- -PN-IQ-M-QYSK--KD--DVLSS----PAQAIT---YRGGTNL-H--ITXK-I--N--- -FN-IQ-M-QYSK--KD--DVLSS----PAQAIT---YRGGTNL-H--ITXK-I--N--- LSKTIQKM-EHSKD-KD--DILSS---PAAQTIT----IETNEI---ITXKXXXXXX---- LSKTIQKM-EHSKD-KD--DILSS---PAABTIT---IETN~I----ITX~---- WKAVEI-E-NKRLLE-MEKIVGRVHS-DAGNEIY-HWDGLP--QLA-ED-X--FA-YN--H- SRAIEI-EENKR----MEMIFGEVIP-AKETEPYPVWSGLP--QTK-EDAX-HSA-YN--H- SKAVEI-E-TKRLLE-ME-IVSQVHPETKENEIYPVWSGLP--BMA-EE-X--SAYYN--H- 170 180 187 FRRDSHKIDSFLRVLRCRMKKRPEMC __~----_~-_--~___--_-~---~- ---X--- ---T-"---T- ---TNM---T- ---T-M---A- L---),Q--w L--.---MQ--.s ---A---NHg-BV- ---we---a--NMg-gV- L---NY----K--LI-DSN- L----S---TY--L-N--IIXXYNNN- 263
264
CHANG, HUANG, AND LOvertebrate PRLs are located in the segment
of the first 12 N-terminal
residues which are
lacking in fish PRLs. All the four cysteine
residues of fish PRLs can be aligned at the
same positions
of PRLs of higher verte-
brates. In salmon PRL, two disultide link-
ages are also formed between the corre-
sponding cysteine residues of mammalian
PRLs,
i.e., Cys4c160 and Cy~i~~-is~ (Ya-
suda et al., 1986). Whether
such disultide
linkages are present in cyprinid PRLs re-
mains to be studied.
ACKNOWLEDGMENT
This work was in part supported by the National Science Council, Republic of China.
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