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Fundulus heteroclitus gonadotropins. 3. Cloning and sequencing of gonadotropic hormone (GTH) I and II β-subunits using the polymerase chain reaction
Molecular and Cellular Endocrinology, 85 (1992) 127-139 0 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/92/$05.00
127
MOLCEL 02757
Fundulus heteroclitus gonadotropins. 3. Cloning and sequencing of gonadotropic hormone (GTH) I and II P-subunits using the polymerase chain reaction Y.-W.P. Lin a, B.A. Rupnow a, D.A. Price a, R.M. Greenberg
a and R.A. Wallace a,b
a Whitney Laboratory, Uniuersiry of Florida, St. Augustine, FL 32086, USA, and b Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesuille, FL 32610, USA
(Received 19 December 1991; accepted 31 January 1992)
Key words: Gonadotropin; Nucleotide (Fundulus heteroclitus)
sequence;
Amino acid sequence;
Polymerase
chain reaction;
Pituitary cDNA library;
Summary
mRNA was isolated from Fundulus heteroclitus pituitaries and used to construct a cDNA library in hgt22A. A series of synthetic oligonucleotides, based on conserved regions of teleost gonadotropic hormone (GTH) P-subunits, were constructed and used as primers in the polymerase chain reaction (PCR) to amplify GTH cDNAs. Appropriate length PCR products were subcloned and sequenced. Eight clones were eventually identified as cDNAs encoding two distinct P-subunits of F. heteroclitus, GTH I and GTH II. By comparison with known GTH sequences, putative signal sequences of 19 end 21 amino acids and mature p-subunits of 95 and 115 amino acids were found for GTH I and GTH II, respectively. Both p-subunits had well conserved cysteine positions when aligned with other members of the glycoprotein family. The elucidation of the complete nucleotide sequences of two types of F. heteroclitus GTH provides definitive proof that in this species there are at least two distinct forms of pituitary GTH analogous to the classical luteinizing hormone-follicle stimulating hormone family.
Introduction
The pituitary gonadotropins, luteinizing hormone (LH or lutropin) and follicle-stimulating hormone (FSH or follitropin), together with
Correspondence to: Dr. Y.-W. Peter Lin, Whitney Laboratory, 9505 Ocean Shore Blvd., St. Augustine, FL 32806, USA. Tel. (904) 461-4000, Fax (904) 461-4008. The nucleotide sequences reported in this paper have been deposited in the GenBank database (accession Nos. M87014 and M87015 for GTH I p and GTH II p, respectively).
chorionic gonadotropin (CG) of placental origin and pituitary thyroid-stimulating hormone (TSH) constitute a family of glycoprotein hormones with diverse physiological functions. All the members of this glycoprotein hormone family share important structural similarities and are heterodimers that consist of two chemically distinct, noncovalently bound polypeptide chains (designated as aand P-subunits). Both subunits contain multiple intramolecular cross-linked disulfide bonds and are glycosylated at specific sites. Within a given species, the a-subunit is essentially identical among the glycoprotein hormones and is highly conserved even between distantly related species,
128
whereas the @-subunit is unique to each hormone and apparently bestows the biological specificity (Pierce and Parsons, 1981). In most tetrapod species (birds, amphibians and reptiles), two distinct pituitary gonadotropic hormones (GTHsI have been identified and are generally agreed to be related to the ‘classical’ mammalian LH and FSH glycoprotein hormone family (Licht et al., 1977; Pierce and Parsons, 1981; Sairam, 1983). In teleostean species, evidence for the duality of pituitary GTHs has not been as forthcoming, and strong sentiment historically favored the notion that only a single GTH was present in fish (Fontaine and BurzawaGerard, 1978; Burzawa-Gerard, 1982; Fontaine and Dufour, 1987). More recently, a dual GTH system (based on concanavalin A-Sepharose separation) consisting of maturational or ‘carbohydrate-rich’ GTH (CR-GTH) and vitellogenic or ‘carbohydrate-poor’ GTH (CP-GTH) has been described for a variety of fish (Idler, 1982; Idler and Ng, 1983; Idler and So, 1987). Presumably, the CR-GTH corresponds to a tetrapod GTH such as LH or FSH. However, the CP-GTH has no comparable counterpart, since the GTHs isolated thus far have all been heavily glycosylated. In addition, a second dual GTH system that is more analogous to the classical tetrapod glycoprotein hormone family has also been identified in teleosts. Distinct GTHs with different P-subunit primary structures, designated GTH I and GTH II (structurally more FSH-like and LH-like, respectively), have been isolated and characterized from chum and coho salmon (Itoh et al., 1988; Suzuki et al., 1988a, b; Sekine et al., 1989; Swanson, 1991; Swanson et al., 1991). Recently, cDNAs encoding the (Y- and P-subunits of GTH I and GTH II of chum salmon have been cloned and sequenced (Kawauchi et al., 1989; Sekine et al., 1989), thus providing definitive proof that there are two distinct pituitary GTHs in fish. However, the significance of two GTHs in regulating teleost reproductive processes remains unclear. In order to better understand the fundamental mechanisms involved in teleost reproduction and to provide a broader basis for comparative study of the teleost GTHs, we have initiated a series of studies focusing on Fundufus heteroclitus GTHs and their role in con-
trolling the cyclic reproductive activity characteristic of this species. These have included the development of a homologous bioassay for GTHs using oocyte maturation and steroid production by isolated ovarian follicles (Lin et al., 1987), a characterization of the steroidogenic responses to homologous GTH by ovarian follicles (Petrino et al., 1989a, b, 1990), and a demonstration that different steroidogenic activities were distinguishable from F. heterocfitus pituitary extracts by a variety of chromatographic (FPLC) procedures (Lin and Wallace, 1988). In this paper, we report the construction of a F. heteroclitus pituitary cDNA library and the isolation and identification of sequences encoding the p-subunits of two distinct GTHs (GTH I and GTH II) by polymerase chain reaction (PCR) and sequence analysis. Materials
and methods
Animals F. heteroclitus (killifish)
were collected in minnow traps placed around salt marshes in the Matanzas River near the Whitney Laboratory (St. Augustine, FL, USA). Captured animals were kept in indoor fiberglass tanks with running seawater under controlled laboratory conditions. Our routine husbandry regimen was able to maintain the fish in a reproductively healthy state throughout the year (Lin et al., 1989). Pituitary glands were collected on dry ice from freshly decapitated, reproductively active fish (gonadosomatic index = N 10 for females and N 5 for males) and stored at -80°C until used (usually within 2-4 months).
F. heteroclitus pituitary cDNA library construction Total RNA was prepared from several batches of frozen pituitary glands (a total of about 3000 glands, collected during 1990 and 1991) by the guanidine/CsCl method (Chirgwin et al., 1979). From each total RNA preparation, the polyadenylated mRNA was isolated by oligo (dT)-cellulose affinity chromatography (Poly (A) Quik mRNA purification kit (Stratagene, La Jolla, CA, USA)). mRNAs were then pooled and used for the construction of a directional cDNA library. cDNA synthesis and A-cloning were accomplished using the Superscript lambda system
129 TABLE PRIMERS
1 (OLIGONUCLEOTIDES)
USED
IN THE POLYMERASE
CHAIN
REACTION
(PCR)
(*) Refers to primers which correspond to the inverse and compliment of the target sequence. PEL5 and PEL6 are the exact primers, respectively, to the P-subunits of F. heteroclitus GTH II and GTH I. A(F) and h(R) denote external primers corresponding to the vector sequences upstream and downstream, respectively, of the GTH inserts. PO(T) is the external primer corresponding to the sequence of NotI/poly dT primer-adaptor. Numeral above the target sequence refers to the position of the amino acid (see Fig. 6, target sequences are underlined). Nucleotide residue: I = inosine.
Target Sequence (Amino Acid)
Oligonucleotide
No. of Bases
Direction of Primer
PE1;3
23
Reverse
3’ TAC CT6
P&
24
Forward
Pz6
25
/;i;,
Primer
#92 *MDTSDCTI
$99
3’ 5’ -IT GTC ACC TAC CCC GTT GCC CTG A
#76 VVTYPVAL
~83
Reverse
5’ 3’ G GAA ATA CGT GTG TAT GGG TCG ACA
#88 *LYAHIPSC
25
Forward
3’ 5’ TTGACACCAGACCAACTGGTAATGG
AF)
24
Reverse
5’ 3’ GCCCGAGGTCCTCAGCAGCGGTGG
*Vector
P:(T)
28
Reverse
3’ (-f),,CGCCGGCGCTTk?
* Poly-A Tail
TGI
+;I
(BRL Life Technologies, Gaithersburg, MD, USA). The first strand was synthesized using a NotI/oligo dT primer-adaptor and a cloned Moloney murine leukemia virus reverse transcriptase (Superscript RT). Second strand synthesis was by nick translational replacement of the mRNA (Gubler and Hoffmann, 1983). The termini of the double-stranded cDNA were blunted by adding T4 DNA polymerase and Sal1 adaptors were then ligated to the blunt-ends of the cDNA. The asymmetric termini were then exposed by digestion with Not1 restriction enzyme. The resulting cDNA with Not I-Sal1 termini were sizefractionated on a Sephacryl S-500 HR column (Pharmacia LKB Biotechnology, Piscataway, NJ, USA), ligated with N&I/Sal1 arms of Agt22A and packaged in vitro (Gigapack II gold packag-
CT;
5’ A cTGI TA ACG
Vector
lf95
(dgt22A)
(kJ22A)
ing extract; Stratagene). The library was amplified in Escherichia coli strain Y1090 (r -). Synthetic oligonucleotide primers Conserved regions based on known P-subunit sequences (amino acid and/or nucleotide) of mammalian and teleost GTHs were used to design a series of synthetic oligonucleotide primers (Table 1, Fig. 6) for use in the polymerase chain reaction (PCR). Primers of various lengths, degeneracies and directions (forward and reverse) were obtained from the DNA Synthesis Core facility at the University of Florida. Inosines (I) were inserted at some of the most variable positions to reduce the degeneracy of the oligonucleotide primers. Lambda vector (Agt22A) sequences upstream (h(F)) and downstream (h(R))
130
A lgt22A 4c--r
GTH II p Insert
lgt22A t- ---t
I<-_--_-----_---_-----+_I Clones: PTA XXV-2, PTA XXV-3 -I t Clone: pTA XXIV-3 .I PEL5
PEI&3
PO(T)
-
Clone: PTA XVII-2 I
f3 ltgt22A -u!--t
;lgt22A t_--*
G-m I j5 Insert
I~_-__-__-_---__---_--_“I Clone: PTA XXIV-1 1
--I
PEM -
I-:
PO(T) -
-
Clones: PTA XXVI-2, PTA XXVI-3, PTA XXVI-4
Fig. 1. Schematic representation of GTH If (A) and GTH I (3) p-subunit DNA inserts (with asymmetric termini, SalI and Nor1 arms) in lambda vector, Agt22A (not drawn to scale). Striped regions depict the relative locations of the target sequences for the oligonucleotide primers used in the PCR reaction. Directions of the primers are indicated by arrows above. Clones of the PCR products are indicated by solid lines above the diagram. A(F) and h(R) represent primers corresponding to hgt22A vector sequences located, respectively, upstream and downstream of the GTH cDNA inserts. PO(T): primer corresponding to poly dT/NotI primer-adaptor. WI : Sal1arm.
of the cDNA inserts and a sequence corresponding to the NotJ/oligo dT primer-adaptor (PO(T)) utilized in the library construction were also synthesized for use as external primers.
Polymerase chain reaction The overall PCR strategy empioyed to isolate cDNAs encoding the CTH p-subunits is depicted in Fig. 1. We had designed a series of appropriate
131
primers in both forward and reverse directions. These primers were comprised of either the putative GTH sequences (such as PEL3 or PEW) or the vector sequences (such as h(F) or h(R)). According to this scheme, a PCR product will be formed if the internal primers have enough homology to the F. heterociitus GTH sequences, and eventually both the 5’- and 3’-end of the GTH inserts can be specifically amplified. In order to facilitate the above scheme, an aliquot (5 ~1) of the amplified F. heteroclitus pituitary cDNA library was incubated at 70 o C to disrupt the lambda phage and the released DNA was employed as template for PCR using an automatic thermocycler (Tempcycler Model 50, Coy Laboratory Products, Grass Lake, MI, USA). Reaction mixture for PCR contained 50 mM KCI, 10 mM Tris-Cl (pH 9.01, 1.5 mM MgCl,, 0.01% gelatin, 0.1% Triton X-100, together with 200 JLM of each dNTP, 25 pmol each of forward and reverse primers, and 2.5 U of Tuq DNA polymerase (Promega, Madison, WI, USA) in a total volume of 100 ~1. After overlaying the PCR mixture with mineral oil (100 & 30-35 cycles of amplification were carried out with the following conditions: denaturation at 94 o C for 2 min, annealing at 50°C (a lower or higher temperature was used depending on the primers) for 2 min and extension at 72 o C for 3 min. At the end of the last cycle, an additional 15 min incubation at 72 ’ C was implemented to ensure the completion of final extension (Sambrook et al., 1989). Subcloning and DNA sequence determination To visualize the PCR products, an aliquot (10 ~1) of the reaction mixture was run on a 1.5% agarose gel with EcoRI X ~~ndlII-cut lambda as standard. PCR products of the expected size were subcloned into pCR 1000 vector and transformed into INV(ar)F’-competent E. coli (TA Cloning system, Invitrogen, San Diego, CA, USA). Transformed cells were plated on LB plates supplemented with kanamycin (50 pgg/ml) and X-Gal (40 mg/ml). Resulting white colonies were picked and screened for inserts by PCR using primers to the plasmids (Innis et al., 1990). The isolated DNA was alkaline-denatured (0.2 M NaOH, 0.2 mM EDTA, 30 min at 37 “C> and both strands were sequenced by the dideoxy chain te~ination
method (Sanger et al., 1977) using the Sequenase sequencing system (United States BiochemicaI, Cleveland, OH, USA). DNA sequence analysis was performed using the PC/GENE program (IntelliGenetics, CA, USA) and sequence comparisons were done with the program FASTA (Pearson and Lipman, 1988). Results
F. heteroclitus pituitary cDNA library Total RNA (853 fig) was obtained from about 3000 pituitaries (0.2 mg wet weight per gland) and of this, 4.8 hg of poly A+ RNA was isolated and used to generate a directional cDNA library containing 1.05 X lo6 recombinants. This cDNA library in hgt22A was amplified in E. coli strain Y 1090(r _ ). PCR amplification and sequencing of the clones that encode the P-subunit of F. heteroclitus GTH II Initial attempts to isolate the P-subunits of F. heteroclitus GTH from the cDNA library using oligonucleotides based on conserved teleost GTH nucleotide sequences failed to yield any detectable products. We changed our strategy in designing the primers and deliberately constructed an oligonucfeotide (PEL3, Table I> which was more degenerate to cover all possible variations. Inosines were introduced in some of the most variable positions to help reduce the degeneracy. PCR products of expected size (300-400 bp) were generated using PEL3 with A(F) (Fig. 1A). Amplified DNAs of appropriate size were cloned and sequenced. Using the FASTA program to search the GenBank data base (Pearson and Lipman, 19881, we found that the DNA sequence of clone PTA XVII-2 has substantial homology with the &subunits of the GTH II of other teleosts (see Fig. 6). Tentatively, clone PTA XVII-2 was designated as the cDNA encoding the 5’ end of the P-subunit of F. heteroclitus GTH II (Fig. 1A). Its nucleotide sequence (1 to 388) is shown in Fig. 2. Based on the nucleotide sequence found in PTA XVII-2, an exact oligonucleotide primer (PELS) to the F. heteroclit~ GTH II /3 sequence was synthesized (Table 1). This exact primer, PEL5, which is upstream from PEL3 (Fig. l&I,
132
was used to specifically amplify the 3’ end of the GTH II p insert and to confirm the sequence covered by the original priming site of PEL3 (by examining the resulting overlapping clones with PTA XVII-21. From this series of PCRs, four relevant clones (about 200-300 bp) were obtained. Clone PTA XXIV-l and PTA XXIV-3 were generated by primer PELS and h(R),
whereas PTA XXV-2 and pTA XXV-3 were produced using primer PEL.5 and PO(T) (Fig. 1 A). Sequence analysis of these clones indicated that pTA XXIV-3, PTA XXV-2 and pTA XXV-3 all coded (based on sequence homology) for the 3’ end of the F. heterocfitus GTH II p-subunit. The nucleotide sequences of these three clones (represented by PTA XXV-3, nucleotide 319 to 482,
Fundulus heteroclitus GTH II P-subunit -20 CTCAGGATAGAGGACTGAAGTTTTCACTG
ATG
GTG
M
GGA
GCC
G
A
TTT
CAG
TCC S
CTC
TCA
TTC
S
F
-10 ATT I
TGG
TCC
CTG
w
s
L
V
GCA
TGT C
TTG
TTT
CTG
L
F
L
47
CCC
GCG
GCA
GCA
-1 GCC
ACA
ATA
TCT
CTA
137
92
CCT
CGC
TGT
GAG
CTC
CTC
3’“““” 10 AAC CAG
TCT S
GGC G
TGT C
CAC H
AGA R
GTG V
GAA E
ACC T
ACC T
30 ATC I
182
GAT
40 CCC
AAC
TAT
AAG
ACA
TCA
227
P
N
iQLPRCQLLNQT1.L
GAG
AAG
AGG
GGC
E
K
R
G
20 TGT C
TGC
AGC
GGG
TAC
TGC
GCG
ACC
AAG
c
S
G
Y
C
A
T
K
TAT
AAC
AAA
GCA
CAG
CAT
GTG
A
50 ATC I
TTC
GAG
TTT
Y
TAC Y
N
K
AAG
ACA
K
T
F
Q
E
F
H
V
CCT
GAA
P
E
D
TGC C
TGT C
ACT
TAT
T
Y
Y
GGG G
70 GTG
CCC
GGA
V
P
G
K
GAT D
GTT V
T
TTG
S 60 TAC
L
Y
GAC
CCA
D
272
317
P
362
GCC
#$&
: ;j~~$$?::&@&
PZ3 ._,. .(.,,., ::~~.~i.~~~~~~~~~~~~~~~~:~:~~~C
A
N’
A
T
S
TTT
TGC
ATG
AAT
110 GAC
F
C
M
N
D
ATC
D
AAAAAAAAAAAAAAAAAAAAAAAAAAA
I
100 GAG
AG C
CT T
fJ A G
CCT
GA C
S
L
Q
P
D
C
T
F
E
a,b CC@
TTT
TAC
CAO
P
407
a F
Y
H
TAG
CCATGTGTAAAAAAA
455
* 482
Fig. 2. The nucleotide and deduced amino acid sequence of cDNA encoding F. heteditus GTH II p-subunit. Numbering on the right-hand side refers to the nucleotide sequence and numbers on top of the sequence refer to the amino acid sequence. The first amino acid of the putative mature @,ubunit (circled phenylalanine, Fl is numbered as $1. Minus numbers are used to indicate the amino acids that comprise the signal sequence. Two overlapping cDNA clones, TA XVII-2 (nucleotide 1 to 388) and TA XXV-3 (319 to 4821, were used to construct the nucleotide sequence shown above. Shaded regions are the target sequences for the primers PEW and PEL5. Sites of nucleotide substitutions in clone TA XXIV-3 and TA XXV-2 are marked with boxes (al and (bl respectively. Details of the nucleotide substitutions of the various clones are shown in Fig. 4A. (*l Indicates the stop codon. Arrowhead indicates site of putative N-gIy~osylation.
133
The complete nucleotide sequence (482 residues), which includes the 5’-untransIated region and the 3’-polyadenylated region (comprised of 34 A), for the cDNA encoding the F. heteroclitus GTH II P-subunit was then compiled from the data derived from the various clones and is shown in Fig. 2. An N-linked glycosylation site was predicted to be located at the number 10 asparagine residue (under the arrowhead in Fig. 2 and Fig. 6). The presumed cleavage site for the
in Fig. 2) were exactly the same except at two locations (nucleotide 428 and 437, and are marked with box (a) and (b) in Fig. 2 and Fig. 4A) near the carboxy-terminal. These nucleotide substitutions ((a) 428 (A to G) and 437 (C to Tf in PTA XXIV-3; and (b) 428 (A to C) in PTA XXV-2 (as compared with PTA XXV-3 in Fig. 4A)) were silent mutations that did not affect the amino acid residues (aa 112 (P> and aa 115 (HI, Fig. 4A).
-10 ATG
CAG
M
TTG
Q
GGA
GTT
L
TGC
TTC
CTC
V
L
-1 GGT
1 TGC
ATG
GCG
GCG
A
A
CTG
AAG
TGC
M a C&
CTG
GCG
L
A
r AAT
GTC
AGC
ATC
CCC
10 ATG
GAG
ATC
20 CAC
ACC
ACG
ATC
TGC
GAA
GGG
H
T
T
I
E
G
GAA
AGC
CCT
GAC
40 GAG
GCG
P
5
E
A
TAC
GAG
GTG
AAA
C
F
AGA
TGC
GGC
CAG
AGG
R
C
G
Q
R
V
C
I
CTG
TGC
TTC
30 TCT
GAG
GAC
GCC
GTC
L
C
F
S
GAG
CAT
AGG
E
V
CTG L
GCA A
GAA E
GTG
45
V
90
GOHLKNVSIPME
G
CCT I?
GTC
H
GTG
E
R
A
V
F
AAC
GGG
50 GAC D
D
GTC
TGC
V
TTT
C
N
G
E
TGG
S
TCC
W
S
Y
C
E
V
135
180
225
K
270
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c
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G’3i.“:‘:’
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N
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C
C T
A C T T A CA T
TGT C
ACC
315
T
GC A C T T A A
364
*
AATCCTCGCGTCTCTTTGTCTGACGCTGAAATAAAAGAAGATACTGCTCGTAAAAAAAA
4,2 3
AAAAAAAAAA
433
Fig. 3. The nucleotide sequence and the deduced amino acid sequence of cDNA encoding the P-subunit of F. heteroclitus GTH 1. The nucleotide sequence shown is composed of two overlapping cDNA clones, TA XXVI-3 (nucleotide 1 to 342), and TA XXIV-1 (248 to 433). Numbers on the right-hand side refer to the nucleotide sequence and numbering on top of the sequence refers to the amino acid sequence. The putative mature GTH I p-subunit starts with a cysteine (circled) and is numbered as + 1. Amino acids that comprise the signal sequence are indicated with minus numbers. Target sequences for primers PEL5 and PEL6 are shaded. Boxes (a) and (b) represent sites of nucleotide substitutions in clone TA XXVI-4 and clone TA XXVI-Z, respectively. Details of the nucleotide substitutions of the various clones are shown in Fig. 4B. Stop codon is marked with a (* 1 and consensus sequence (AATAAA) for ~lyadenylation signal is underlined. Putative N-glycosylation site is marked with an arrowhead.
134
signal sequence was determined by comparison with other known GTH sequences. A signal sequence of 21 amino acids was located and the proposed mature P-subunit of F. heteroclitus GTH II starts with a phenylalanine (circled F in Fig. 2) and consists of 115 amino acids. PCR amplification and sequencing of the clones that encode the P-subunit of F. heteroclitus GTH I Of the four clones that were generated by using the primer PELS, one of them, PTA XXIV-l (Fig. lB), was radically different from the other three clones that were described in the previous section. The DNA sequence of PTA XXIV-l was then used to search the GenBank data base with the FASTA program (Pearson and Lipman, 1988) and significant homology was found to the p-subunit of Oncorhynchus keta GTH I (see Fig. 6). Tentatively, clone PTA XXIV-1 was designated as the cDNA encoding the 3’ end of the putative F. heteroclitus GTH I p-subunit. Its nucleotide sequence (248 to 433) is shown in Fig. 3. This clone included the 3’-untranslated region, and in it the consensus sequence (AATAAA, underlined in Fig. 3) for the polyadenylation signal was found to be 48 residues downstream from the stop codon (TAA). Based on this new sequence information from clone PTA XXIV-l, an exact oligonucleotide primer to the putative F. heteroclitus GTH I P-sequence (downstream of PELS) was created (PEL6, Table 1 and Fig. 1B). Eventually, three pertinent clones (PTA XXVI-2, PTA XXVI-3, PTA XXVI-4), encoding the 5’ end of F. heterocfitus GTH I p-subunit, were obtained using PEL6 and A(F) (Fig. 1B). Their nucleotide sequences (as represented by PTA XXVI-3 (nucleotide 1 to 342) in Fig. 3) were all similar to each other except at three locations (nucleotide 62, 229, 306 and are marked with box (a) and (b) in Fig. 3 and Fig. 4B). Two nucleotide substitutions ((a) 62 and 229, both of them were A to G changes, Fig. 48) in clone PTA XXVI-4 resulted in the replacement of two amino acids (aa 2 (H to R), and aa 58 (I to V>, (compared with PTA XXVI-31 Fig. 4B), whereas the one nucleotide substitution in PTA XXVI-2 ((b) 306 (A to G)) did not alter the amino acid residue (aa 83 CT)) (Fig. 4B).
A
q m
pTA XXIV-3
GTH II B-subunit v ATC
pTAXXV-2
ATC
C&I
PTA XXV-3
h4C
c
B
CA&
TAG
440
TTT
TAC
CAC
TAG
440
P
TTT F
Th0
CAC
TAG
440
H
*
Y
Ii
c
L
K
PTA XXVI-3
@Qt.
TilC
c
Cft:
X&G
69
PTA XXVI-4
ggt
TGC C
CMC
CTG
AAG
69
(R)
L
K
“I
Q
G
G
Y56 K
PTA XXVI-3 PTA XXVI-4
PTA XXVI-3 •j
TAC
GTH IB-subunit G
q
TTT fl
I
q
CC
PTA XXVI-2
H
160
Mu
ChO
c
cxa
dwJ
237
AAA K
CAC H
G C Ir (V)
CAG
GGG
237
AAh
G&C
A
Q
c
TAi?
T@T
312
TAC
TGT
312
v ¶ AAA
GAC
K
D
(81
ACM
T
Y
C Y85
Fig. 4. Nucleotide substitutions in various clones encoding the P-subunits of (A) GTH II and (B) GTH I. Shaded sequences are part of the clones shown in Fig. 2 and Fig. 3 and are used for comparison with other clones in this figure. Arrowheads indicate the location of the nucleotide replacements and consequent amino acid changes are shown in parentheses. (#) Numbers refer to amino acid sequence (see Fig. 2 and Fig. 3). Numerals on the right-hand side denote the nucleotide sequence. Boxed a and boxed b: Clones that have nucleotide substitutions in the corresponding boxed locations in Fig. 2 and Fig. 3. ( *) Denotes stop codon.
In addition to the three nucleotide changes described above, we found that the long 5’-untranslated region of F. heteroclitus GTH I P-subunits were extremely variable (data not shown). By comparing our sequence with other known glycoprotein hormones (see Fig. 61, a signal sequence of 19 amino acids was predicted for F. heterocfitus GTH I p-subunit and the N-glycosylation site was assumed to be at the fifth asparagine residue (marked by arrowhead in Fig. 3 and Fig. 6). We concluded that there are 95 amino acids in the putative mature p-subunit of F. heteroclitus GTH I.
135
Discussion
199
(92
A
s
D
c
T
lPEL3 Target ATG
G ; ACI
;?I
GA:
TG;
$1
ATG M
Glti C ACA A ‘p
TCC s
GAC D
TGC C
ACC T
"
Y
P
v
A
TAC
CCC
GT
GCC
TAC Y
CCC P
GT 1 v
A GCC D A 7' 171
M
In this study, we have cloned and sequenced of the cDNAs encoding the putative p-subunits (GTH I two distinct F. heteroclitus gonadotropins and GTH II) analogous to the classical LH-FSH glycoprotein hormone family. Although GTH duality has been described in a number of species (for review see Scott et al., 1991), two distinct GTHs have only been isolated in two closely related fish (chum and coho salmon) (Itoh et al., 1988; Suzuki et al., 1988a, b; Sekine et al., 1989; Swanson et al., 1991). Recently, Kawauchi and his colleagues successfully cloned and sequenced the (Y-and p-subunits of two distinct chum salmon GTHs (Kawauchi et al., 1989; Sekine et al., 1989). Our ability to achieve a similar goal in F. heteroclitus - to clone and sequence cDNAs encoding two distinct GTH P-subunits - represents the second molecular demonstration of GTH duality for any teleost. Preliminary PCR experiments using oligonucleotide primers based on conserved nucleotide sequences of teleost GTHs were unavailing. Apparently, the F. heteroclitus GTH nucleotide sequence is very different from other known teleost sequences. In order to overcome this difficulty, we designed degenerate GTH oligonucleotides (such as PEL3, with inosines to help reduce degeneracy) for use as internal primers. Together with the vector or oligo dT external primers, we were able to specifically amplify the cDNAs that encoded the P-subunits of F. heteroclitus GTH II and, fortuitously, we also captured the GTH I P-sequence (by PELS) using PCR. In retrospect, the oligonucleotide PELS (Table 1) was more useful than we had imagined. Originally, PEL.5 was designed as an exact primer based on the nucleotide sequence deduced from clone pTA XVII-Z (Fig. 11, which coded for the P-subunit of Fundulus GTH II. Comparison of the target sequences for primer PEL3 (Fig. 5A) and PEL.5 (Fig. 5B) with the actual F. heteroclitus GTH sequences resulting from the PCR procedures revealed mismatches of two and six (out of 23 and 24) residues, respectively. Both F. heterocfitus GTH II and hCG P-subunits have particularly short 3’-untranslated regions (8 and 16 nucleotides respectively). In the
F. he GTH II
B
D
#16 "
PEL5
'l'r 6
Target
GE n:
F. he GTH I
s
T C
A
T
ACC
C RCC I T
164 Fig. 5. Comparison
of the target
(Al
with the corresponding
and PEL5
(Bl,
sequences found in F. heteroclirus pTA
XXV-31
respectively.
and GTH
(* )
Indicates
primer. acids.
nucleotides Numbers Numeral
GTH
I p-subunit
denote
right-hand
sequence.
P
1183 L A
of primer
II p-subunit
342
271
PEL.7
(in clone XXVI-31,
sequence shown is
of the actual mismatches
388
II
actual nucleotide
primer
sequence.
with oligonucleotide
on top of the sequences to the
388
(in clone pTA
that nucleotide
the inverse and complement Shaded
sequence
I
refer
side denotes
to amino nucleotide
I: inosine.
case of hCG, there is an extension of 24 amino acids (when compared with hLH1 at the carboxyterminal, which appears to have arisen by a readthrough event caused by a single base deletion (Talmadge et al., 1984). This readthrough event incorporated the 3’-untranslated region of the ancestral LH /?-gene into the coding region of hCG, hence shortening considerably the 3’-untranslated region. However, no such similar readthrough event can be detected in F. heteroclitus. We cannot disregard the possibility that the short 3’-untranslated region in the F. heteroclitus GTH II p-subunit (as deduced from clones pTA XXIV-3, PTA XXV-2 and PTA XXV-31 is an artifact due in part to mispriming or a copying error in the reverse transcriptase reaction during the cDNA library construction. Likewise, the six nucleotide substitutions observed in the various clones (Fig. 4) could be due to a reading error of the reverse transcriptase or, more likely, to heterogeneity in the mRNA populations (since they were obtained from more than 3000 fish). Comparison of the F. heterocfitus GTH I and GTH II P-sequences with other members of the glycoprotein hormone family is shown in Fig. 6. Their homology became immediately apparent when the cysteine residues were aligned. In F. heteroclitm GTH II P-sequence, a gap of two
1
v
2
3
h LHj3 h CG,B b LH_8 0. ke 0. ts
i
30 30
30 21 27 27 27 27 27 27
C. ca H. mo M. ci A. an Ehe
GTH II-,9
1
h FSH#? b FSH# h TSH-3 b TSH$ GTH I-,4
_[
24 22 23 23 27 20
$ 2 . 4
5
6
h LH$ h CG_B b LH$ 0. ke 0. fs c. ca fimo M ci
GTHW
i
-I
7 PRGVDPVVSFPVALS
01
87 87 84 84 84 84 84 84 84
A. an F. he
h FSH# b FSH+ h TSH$ b TSHB GTHI$3
V
81 79 82 82 83 72
$2
8
3
10 121 145 121 119 119 118 117 113 116 115
GmI,${
0l;e &
d
CDCIK KTDNT CDRISMATPS IVNPLEM ... .. .. . ....i...~ i\/,.i....,.,....... $$%,S ',, :@z@T-z :(.) ;$&.%&K.zf:&j
~.~..~
118 109 112 113 113 95
137
Dendrogram
of Pituitary
and Charionic
Glycoprotein
Human
__~.......~..l*...‘~.***~.*.......~...*
i.l._._.......;
LH-/3
:................. .................... .. Human
---_-: 1
i .
... .... . . .... ... _......... ,.... . . . . ... ...
P-Subunits
CG-/j
Bovine
LH-/3
I
Oncot-hynchus keta 0. is~~a~ts~ha
jL
Cyprinus ca rpio Hypophthahnichthys
molitrix GTH
II-/3
Muraenesox cinereus Anguilla anguilla
..,.,..,..............
1
’ 1
---------<
FSH-fj
Human
~““...“‘.‘“““~““‘......“’
L
Human FSH-/3
_................_.................... Bovine
1--------I
1
f... .. . .. ... .
._...__.....t.._.........~._-Bovine
TSH-P TSH_/j
I
~~~orhy~~~~s keta
GTH
~~~d~~~~ h~~~r~c~it~sF Fig. 7. Dendrogram
of mammalian
(phylogenetic
is formed
tree)
(.
. ‘1 and
by clustering
teleost (-----
) pituitary
the corresponding
amino
and chorionic glycoprotein
acid sequences
1-p
P-subunits.
based on overall
This dendrogram
similarity
using ‘average
linkage cluster analysis’ fSncath and Sokal, 1973).
Fig. 6. Comparison
of the amino acid sequences of the P-subunits
teteost and mammalian
glycoprotein
p-subunits
of F. ~~~~~~~jf~.~ GTHs
using the ‘Clustal’
program
in PC/GENE
(GTH
II and GTH
(Higgins
I, shaded) with other
and Sharp,
1988). The teleost
CF. he) are chum salmon, Oncorhynchas keta (0. ke) (Sekine et al., 1989), chinook salmon, 0. fschawyrscha (0. ts) (Trinh et al., 19X6), common carp, Cyprinus carpio (C, ca) (Changet al., 198X), silver carp, H~~c~&thnlmichthys molitrir (N. mo) (Chang et al., 1990), pike eel, Mrtruenesox cinereus CM. ci) (Liu et al., 1989) and European eel, Anguilhzz ~~g~j~~u (A. an) (Q&rat et al., 1989). The mammalian P-subunit sequences used are human lut~inizin~ hormone fhLH-6) GTH
sequences used for comparison
(Talmadge (Maurer,
with F. heteroclitus
et al., 1984), human chorionic gonadotropin 1983,
human follicle-stimulating
hormone
(hCG+) (hFSHj3)
CbFSH-j3) (Esch et al., 1986), human thyroid-stimulating ing hormone
(bTSH/3)
of the mature mature GTH
proteins,
glycoproteins 1-p. Amino
for the primers
(Maurer
et al., 1984). Numbers
Lowercase
19811), bovine luteinizing lY76),
and Rathnam,
hormone (hTSH-/3) at the right-hand
(Hayashizaki
accordingly
et al., 198.5), and bovine thyroid-stimulat-
The aspargine
The
on the top of the boxes. ( 1:) Denotes
12 cysteinr
residues in the
atypical cysteine location
1-p are shown in parentheses.
residues (N) under the arrowheads
glycosylation sites. Dash line f-) denotes gap introduced
(hLH+‘) hormone
side refer to the amino acids (single letter code in capital)
in the various clones encoding the K he GTH
used in the PCR are underlined.
hormone
bovine follicle-stimulating
single letter code denotes amino acid of the signal sequences.
are boxed and are numbered
acid substitutions
(Fiddes and Goodman, (Saxena
Target
may be the putative
during alignment to maximize
homology.
in
sequences N-linked
residues needed to be included only when compared with TSH, otherwise perfect alignment of the 12 cysteines was achieved when compared with other GTHs (LH, FSH, hCG and teleost GTHs). However, alignment for E heteroclitus GTH 1-B was not as flawless. In GTH 1-p of both 0. keta and F. heteroclitus, several gaps (such as between cysteine 7 and 8, 3 and 4) were required (Fig. 6). As evident from the dendrogram (Fig. 71, the teleost GTH 1-p seems to be more distantly related to the other members of the glycoprotein hormone family. Since the identity of the teleost GTH I has become apparent only recently, much more sequence data are needed before the evolution of structure and function relationships within the glycoprotein family can be appraised. In view of the significant role played by the cysteine residues in the assembly of the (rp dimer and their presumed highly conserved positions in the /?-sequence (Beebe et al., 19901, it is intriguing to find that in the ~-subunits of the GTH I from both 0. kern and F. ~zeteroelit~s, the cysteinc residues near the amino-terminal did not quite align with the other members of the glycoprotein hormones family (Fig. 6). In the P-subunit of 0. keta GTH I, cysteine 3 (C3> is missing from the customary position and is relocated four amino acids upstream of C’ (new location is designated as 1* in Fig. 61, whereas in F. heteroclitus GTH I, cysteine 2 (C2> is displaced two residues upstream from the conventional C’ position (becomes 2* in Fig. 61. How these cysteine residue displacements and the consequent threedimensional folding of the P-chain affect the assembly of the biologically functional ap heterodimer remains to be investigated.
Acknowledgements
This study was supported by NSF grants No. DCB-8819005 awarded to R.A.W. and DIR8914602 awarded to members of the Whitney Laboratory. We wish to thank Dr. Marion Byrne for the design of some oligonucleotides, Dr. Sandy Yosha for providing veterinary care, Mr. Scott Van Arnam for collection of F. hereroclitus, Ms. Louise
MacDonald for secretarial assistance, and Ms. Lynn Milstead for graphic assistance. Oligonucleotide primers were provided by the DNA Synthesis Core, Interdisciplinary Center for Biotechnology Research, University of Florida. References Beebe, J.S., Mountjoy, K., Krzesicki, R.F., Perini, F. and Ruddon, R.W. t 1990) J. Biol. Chem. 265, 3 12-3 17. Burzawa-Gerard, E. (1982) Can. J. Fish. Aquat. Sci. 39,80-91. Chang, Y.S., Huang, C.J.. Huang, F.L. and Lo, T.B. (1988) Int. .I. Pept. Protein Res. 32, SSh-564. Chang, V.S.. Huang, C.J.. Huang, F.L., Liu, C.S. and Lo, T.B. (1990) Gen. Camp. Endocrinol. 78. 23-33. Chirgwin, J.M., Przybyla, A. E., MacDonald, R.J. and Rutter. W.J. (1979) Biochemistry 18. 529445299. Esch F.S., Mason, A.J.. Cooksey, K.. Mercado, M. and Shimasaki, S. (1986) Proc. Natl. Acad. Sci. USA 83, 661% 6621. Fiddes, J.C. and Goodman, H.M. (19X0) Nature 286, 684-687. Fontaine, Y.A. and Burznws-Gerard, E. (1978) in Structure and Function of the Gonadotropins (McKerns, M.W., ed.f. pp. 36 l-380, Plenum, New York. Fontaine, Y.A. and Dufour. S. (19871 in Proceedings of the Third Snt~rn~lti(~nal Symposium on the Reproductive Physiology of Fish (Idler, D.R.. Grim, L.W. and Walsh, J.M., eds.), pp. 45-56, Memorial University of Newfoundland, St. John’s, Newfoundland. Gubler, U. and Hoffman, B.J. (1983) Gene 25. 263-269. Hayashizaki, V.. Miyai, K.. Kato. K. and Matsuhara. K. (19X5) FEBS Lett. 188, 394-400. Higgins, D.G. and Sharp, P.M. (1988) Gene 73. 237-244. Idler, D.R. (1982) in Proceedings of the Third International Symposium on the Reproductive Physiology of Fish (Richter, C.J.J. and Goos, H.J.T., eds.), pp. 4-13, Pudoc. Wageningen. Idler. D.R. and Ng, T.B. (198?) in Fish Physiology (Hair, W.S., Randall, D.J. and Donaldson, EM., edsf, Vol. 9A, pp. 187-221, Academic Press, New York. Idler, D.R. and So, Y.P. (1987) in Proceedings of the Third International Symposium on the Reproductive Physiology of Fish (Idler, D.R.. Grim, L.W. and Walsh, J.M., eds.). pp. 5740, Memorial University of Newfoundland. St. John’s, Newfoundland. Innis, M.A., Gelfand. D.H.. Sninsky, J.J. and White. T.J. (1990) PCR Protocols: A Guide to Methods and Applications, Acndamic Press, San Diego. Itoh. H., Suzuki, K. and Kawauchi, H. (198X) Gen. Comp. Endocrinol. ?1, 438-451. Kawauchi, Ii.. Suzuki. K.. Itoh, H.. Swanson, P.. Naito. N.. Nagahama, Y., Nozaki, M., Nakai, Y. and Itoh. S. (19891 Fish Physiol. Biochem. 7, 29-X. Licht, P., Papkoff, H., Farmer, S.W.. Muller. C.H., Tsui, H.W. and Crews, D. (1977) Recent Prog. Horm. Res. 33. 16% 248. Lin, Y.-W.P. and Wallace. R.A. (1988) Am. 2001. 28, A641.
139 Lin, Y.-W.P., LaMarca, M.J. and Wallace, R.A. (1987) Gen. Comp. Endocrinol. 67, 126-141. Lin, Y.-W.P., Greeley, Jr., M.S. and Wallace, R.A. (1989) Fish Physiol. Biochem. 6. 139-148. I.iu, C.S., Huang, F.L., Chang, Y.S. and Lo, T.B. (1989) Eur. J. Biochem. 186, 105-114. Maurer, R.A. (1985) J. Biol. Chem. 260, 4684-4687. Maurer, R.A., Croyle, M.L. and Donelson, J.E. (1984) J. Biol. Chem. 259, 5024-5027. Pearson, W.R. and Lipman, D.J. (1988) Proc. Natl. Acad. Sci. USA 85, 2444-2448. Petrino, T.R., Lin, Y.-W.P. and Wallace, R.A. (1989a) Gen. Comp. Endocrinol. 73, 147-156. Petrino, T.R., Greeley, Jr., MS., Selman, K., Lin, Y.-W.P. and Wallace, R.A. (1989b3 Gen. Comp. Endocrinol. 76, 230-240. Petrino, T.R., Hoch, K.L., Lin, Y.-W.P. and Wallace, R.A. (1990) J. Exp. Zool. 253, 177-185. Pierce, J.G. and Parsons, T.F. (1981) Annu. Rev. Biochem. 50, 465-495. Q&rat, B., Moumni, M., Jutisz, M., Fontaine, Y.A. and Counis, R. (1990) J. Mol. Endocrinol. 4, 257-264. Sairam, M.R. (1983) in Hormonal Proteins and Peptides (Li, C.H., ed.), Vol. 11, pp. l-79, Academic Press, New York. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Nat]. Acad. Sci. USA 74, 5463-5467. Saxena, B.B. and Rathnam, P. (1976) J. Biol. Chem. 251, 993-1005. Scott, A.P., Sumpter, J.P., Kime, D.E. and Rolfe, M.S. (eds.) (1991) Proceedings of the Fourth lnternational Symposium on the Reproductive Physiology of Fish, pp. l-28, FishSymp91, Sheffield. Sekine, S., Saito, A., ltoh, H., Kawauchi, H. and ltoh, S. (1989) Proc. Nat]. Acad. Sci. USA 86, 8645-8649. Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy, Freeman, New York. Suzuki, K., Kawauchi, H. and Nagahama, Y. (1988a) Gen. Comp. Endocrinol. 71, 292-301. Suzuki, K., Kawauchi, H. and Nagahama, Y. (1988b3 Gen. Comp. Endocrinol. 71, 302-306. Swanson, P. (1991) in Proceedings of the Fourth International Symposium on the Reproductive Physiology of Fish (Scott, A.P., Sumpter, J.P., Kime, D.E. and Rolfe, M.S., eds.), pp. 2-7. FishSymp91, Sheffield. Swanson, P., Suzuki, K., Kawauchi, H. and Dickhoff, W.W. (1991) Biol. Reprod. 44, 29-38. Talmadge, K., Vamvakopoulos, N.C. and Fiddes, J.C. (1984) Nature 307, 37-40. Trinh, K.Y., Wang, N.C., Hew, C.L. and Crim, L.W. (1986) Eur. J. Biochem. 159, 619-624.
127
MOLCEL 02757
Fundulus heteroclitus gonadotropins. 3. Cloning and sequencing of gonadotropic hormone (GTH) I and II P-subunits using the polymerase chain reaction Y.-W.P. Lin a, B.A. Rupnow a, D.A. Price a, R.M. Greenberg
a and R.A. Wallace a,b
a Whitney Laboratory, Uniuersiry of Florida, St. Augustine, FL 32086, USA, and b Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesuille, FL 32610, USA
(Received 19 December 1991; accepted 31 January 1992)
Key words: Gonadotropin; Nucleotide (Fundulus heteroclitus)
sequence;
Amino acid sequence;
Polymerase
chain reaction;
Pituitary cDNA library;
Summary
mRNA was isolated from Fundulus heteroclitus pituitaries and used to construct a cDNA library in hgt22A. A series of synthetic oligonucleotides, based on conserved regions of teleost gonadotropic hormone (GTH) P-subunits, were constructed and used as primers in the polymerase chain reaction (PCR) to amplify GTH cDNAs. Appropriate length PCR products were subcloned and sequenced. Eight clones were eventually identified as cDNAs encoding two distinct P-subunits of F. heteroclitus, GTH I and GTH II. By comparison with known GTH sequences, putative signal sequences of 19 end 21 amino acids and mature p-subunits of 95 and 115 amino acids were found for GTH I and GTH II, respectively. Both p-subunits had well conserved cysteine positions when aligned with other members of the glycoprotein family. The elucidation of the complete nucleotide sequences of two types of F. heteroclitus GTH provides definitive proof that in this species there are at least two distinct forms of pituitary GTH analogous to the classical luteinizing hormone-follicle stimulating hormone family.
Introduction
The pituitary gonadotropins, luteinizing hormone (LH or lutropin) and follicle-stimulating hormone (FSH or follitropin), together with
Correspondence to: Dr. Y.-W. Peter Lin, Whitney Laboratory, 9505 Ocean Shore Blvd., St. Augustine, FL 32806, USA. Tel. (904) 461-4000, Fax (904) 461-4008. The nucleotide sequences reported in this paper have been deposited in the GenBank database (accession Nos. M87014 and M87015 for GTH I p and GTH II p, respectively).
chorionic gonadotropin (CG) of placental origin and pituitary thyroid-stimulating hormone (TSH) constitute a family of glycoprotein hormones with diverse physiological functions. All the members of this glycoprotein hormone family share important structural similarities and are heterodimers that consist of two chemically distinct, noncovalently bound polypeptide chains (designated as aand P-subunits). Both subunits contain multiple intramolecular cross-linked disulfide bonds and are glycosylated at specific sites. Within a given species, the a-subunit is essentially identical among the glycoprotein hormones and is highly conserved even between distantly related species,
128
whereas the @-subunit is unique to each hormone and apparently bestows the biological specificity (Pierce and Parsons, 1981). In most tetrapod species (birds, amphibians and reptiles), two distinct pituitary gonadotropic hormones (GTHsI have been identified and are generally agreed to be related to the ‘classical’ mammalian LH and FSH glycoprotein hormone family (Licht et al., 1977; Pierce and Parsons, 1981; Sairam, 1983). In teleostean species, evidence for the duality of pituitary GTHs has not been as forthcoming, and strong sentiment historically favored the notion that only a single GTH was present in fish (Fontaine and BurzawaGerard, 1978; Burzawa-Gerard, 1982; Fontaine and Dufour, 1987). More recently, a dual GTH system (based on concanavalin A-Sepharose separation) consisting of maturational or ‘carbohydrate-rich’ GTH (CR-GTH) and vitellogenic or ‘carbohydrate-poor’ GTH (CP-GTH) has been described for a variety of fish (Idler, 1982; Idler and Ng, 1983; Idler and So, 1987). Presumably, the CR-GTH corresponds to a tetrapod GTH such as LH or FSH. However, the CP-GTH has no comparable counterpart, since the GTHs isolated thus far have all been heavily glycosylated. In addition, a second dual GTH system that is more analogous to the classical tetrapod glycoprotein hormone family has also been identified in teleosts. Distinct GTHs with different P-subunit primary structures, designated GTH I and GTH II (structurally more FSH-like and LH-like, respectively), have been isolated and characterized from chum and coho salmon (Itoh et al., 1988; Suzuki et al., 1988a, b; Sekine et al., 1989; Swanson, 1991; Swanson et al., 1991). Recently, cDNAs encoding the (Y- and P-subunits of GTH I and GTH II of chum salmon have been cloned and sequenced (Kawauchi et al., 1989; Sekine et al., 1989), thus providing definitive proof that there are two distinct pituitary GTHs in fish. However, the significance of two GTHs in regulating teleost reproductive processes remains unclear. In order to better understand the fundamental mechanisms involved in teleost reproduction and to provide a broader basis for comparative study of the teleost GTHs, we have initiated a series of studies focusing on Fundufus heteroclitus GTHs and their role in con-
trolling the cyclic reproductive activity characteristic of this species. These have included the development of a homologous bioassay for GTHs using oocyte maturation and steroid production by isolated ovarian follicles (Lin et al., 1987), a characterization of the steroidogenic responses to homologous GTH by ovarian follicles (Petrino et al., 1989a, b, 1990), and a demonstration that different steroidogenic activities were distinguishable from F. heterocfitus pituitary extracts by a variety of chromatographic (FPLC) procedures (Lin and Wallace, 1988). In this paper, we report the construction of a F. heteroclitus pituitary cDNA library and the isolation and identification of sequences encoding the p-subunits of two distinct GTHs (GTH I and GTH II) by polymerase chain reaction (PCR) and sequence analysis. Materials
and methods
Animals F. heteroclitus (killifish)
were collected in minnow traps placed around salt marshes in the Matanzas River near the Whitney Laboratory (St. Augustine, FL, USA). Captured animals were kept in indoor fiberglass tanks with running seawater under controlled laboratory conditions. Our routine husbandry regimen was able to maintain the fish in a reproductively healthy state throughout the year (Lin et al., 1989). Pituitary glands were collected on dry ice from freshly decapitated, reproductively active fish (gonadosomatic index = N 10 for females and N 5 for males) and stored at -80°C until used (usually within 2-4 months).
F. heteroclitus pituitary cDNA library construction Total RNA was prepared from several batches of frozen pituitary glands (a total of about 3000 glands, collected during 1990 and 1991) by the guanidine/CsCl method (Chirgwin et al., 1979). From each total RNA preparation, the polyadenylated mRNA was isolated by oligo (dT)-cellulose affinity chromatography (Poly (A) Quik mRNA purification kit (Stratagene, La Jolla, CA, USA)). mRNAs were then pooled and used for the construction of a directional cDNA library. cDNA synthesis and A-cloning were accomplished using the Superscript lambda system
129 TABLE PRIMERS
1 (OLIGONUCLEOTIDES)
USED
IN THE POLYMERASE
CHAIN
REACTION
(PCR)
(*) Refers to primers which correspond to the inverse and compliment of the target sequence. PEL5 and PEL6 are the exact primers, respectively, to the P-subunits of F. heteroclitus GTH II and GTH I. A(F) and h(R) denote external primers corresponding to the vector sequences upstream and downstream, respectively, of the GTH inserts. PO(T) is the external primer corresponding to the sequence of NotI/poly dT primer-adaptor. Numeral above the target sequence refers to the position of the amino acid (see Fig. 6, target sequences are underlined). Nucleotide residue: I = inosine.
Target Sequence (Amino Acid)
Oligonucleotide
No. of Bases
Direction of Primer
PE1;3
23
Reverse
3’ TAC CT6
P&
24
Forward
Pz6
25
/;i;,
Primer
#92 *MDTSDCTI
$99
3’ 5’ -IT GTC ACC TAC CCC GTT GCC CTG A
#76 VVTYPVAL
~83
Reverse
5’ 3’ G GAA ATA CGT GTG TAT GGG TCG ACA
#88 *LYAHIPSC
25
Forward
3’ 5’ TTGACACCAGACCAACTGGTAATGG
AF)
24
Reverse
5’ 3’ GCCCGAGGTCCTCAGCAGCGGTGG
*Vector
P:(T)
28
Reverse
3’ (-f),,CGCCGGCGCTTk?
* Poly-A Tail
TGI
+;I
(BRL Life Technologies, Gaithersburg, MD, USA). The first strand was synthesized using a NotI/oligo dT primer-adaptor and a cloned Moloney murine leukemia virus reverse transcriptase (Superscript RT). Second strand synthesis was by nick translational replacement of the mRNA (Gubler and Hoffmann, 1983). The termini of the double-stranded cDNA were blunted by adding T4 DNA polymerase and Sal1 adaptors were then ligated to the blunt-ends of the cDNA. The asymmetric termini were then exposed by digestion with Not1 restriction enzyme. The resulting cDNA with Not I-Sal1 termini were sizefractionated on a Sephacryl S-500 HR column (Pharmacia LKB Biotechnology, Piscataway, NJ, USA), ligated with N&I/Sal1 arms of Agt22A and packaged in vitro (Gigapack II gold packag-
CT;
5’ A cTGI TA ACG
Vector
lf95
(dgt22A)
(kJ22A)
ing extract; Stratagene). The library was amplified in Escherichia coli strain Y1090 (r -). Synthetic oligonucleotide primers Conserved regions based on known P-subunit sequences (amino acid and/or nucleotide) of mammalian and teleost GTHs were used to design a series of synthetic oligonucleotide primers (Table 1, Fig. 6) for use in the polymerase chain reaction (PCR). Primers of various lengths, degeneracies and directions (forward and reverse) were obtained from the DNA Synthesis Core facility at the University of Florida. Inosines (I) were inserted at some of the most variable positions to reduce the degeneracy of the oligonucleotide primers. Lambda vector (Agt22A) sequences upstream (h(F)) and downstream (h(R))
130
A lgt22A 4c--r
GTH II p Insert
lgt22A t- ---t
I<-_--_-----_---_-----+_I Clones: PTA XXV-2, PTA XXV-3 -I t Clone: pTA XXIV-3 .I PEL5
PEI&3
PO(T)
-
Clone: PTA XVII-2 I
f3 ltgt22A -u!--t
;lgt22A t_--*
G-m I j5 Insert
I~_-__-__-_---__---_--_“I Clone: PTA XXIV-1 1
--I
PEM -
I-:
PO(T) -
-
Clones: PTA XXVI-2, PTA XXVI-3, PTA XXVI-4
Fig. 1. Schematic representation of GTH If (A) and GTH I (3) p-subunit DNA inserts (with asymmetric termini, SalI and Nor1 arms) in lambda vector, Agt22A (not drawn to scale). Striped regions depict the relative locations of the target sequences for the oligonucleotide primers used in the PCR reaction. Directions of the primers are indicated by arrows above. Clones of the PCR products are indicated by solid lines above the diagram. A(F) and h(R) represent primers corresponding to hgt22A vector sequences located, respectively, upstream and downstream of the GTH cDNA inserts. PO(T): primer corresponding to poly dT/NotI primer-adaptor. WI : Sal1arm.
of the cDNA inserts and a sequence corresponding to the NotJ/oligo dT primer-adaptor (PO(T)) utilized in the library construction were also synthesized for use as external primers.
Polymerase chain reaction The overall PCR strategy empioyed to isolate cDNAs encoding the CTH p-subunits is depicted in Fig. 1. We had designed a series of appropriate
131
primers in both forward and reverse directions. These primers were comprised of either the putative GTH sequences (such as PEL3 or PEW) or the vector sequences (such as h(F) or h(R)). According to this scheme, a PCR product will be formed if the internal primers have enough homology to the F. heterociitus GTH sequences, and eventually both the 5’- and 3’-end of the GTH inserts can be specifically amplified. In order to facilitate the above scheme, an aliquot (5 ~1) of the amplified F. heteroclitus pituitary cDNA library was incubated at 70 o C to disrupt the lambda phage and the released DNA was employed as template for PCR using an automatic thermocycler (Tempcycler Model 50, Coy Laboratory Products, Grass Lake, MI, USA). Reaction mixture for PCR contained 50 mM KCI, 10 mM Tris-Cl (pH 9.01, 1.5 mM MgCl,, 0.01% gelatin, 0.1% Triton X-100, together with 200 JLM of each dNTP, 25 pmol each of forward and reverse primers, and 2.5 U of Tuq DNA polymerase (Promega, Madison, WI, USA) in a total volume of 100 ~1. After overlaying the PCR mixture with mineral oil (100 & 30-35 cycles of amplification were carried out with the following conditions: denaturation at 94 o C for 2 min, annealing at 50°C (a lower or higher temperature was used depending on the primers) for 2 min and extension at 72 o C for 3 min. At the end of the last cycle, an additional 15 min incubation at 72 ’ C was implemented to ensure the completion of final extension (Sambrook et al., 1989). Subcloning and DNA sequence determination To visualize the PCR products, an aliquot (10 ~1) of the reaction mixture was run on a 1.5% agarose gel with EcoRI X ~~ndlII-cut lambda as standard. PCR products of the expected size were subcloned into pCR 1000 vector and transformed into INV(ar)F’-competent E. coli (TA Cloning system, Invitrogen, San Diego, CA, USA). Transformed cells were plated on LB plates supplemented with kanamycin (50 pgg/ml) and X-Gal (40 mg/ml). Resulting white colonies were picked and screened for inserts by PCR using primers to the plasmids (Innis et al., 1990). The isolated DNA was alkaline-denatured (0.2 M NaOH, 0.2 mM EDTA, 30 min at 37 “C> and both strands were sequenced by the dideoxy chain te~ination
method (Sanger et al., 1977) using the Sequenase sequencing system (United States BiochemicaI, Cleveland, OH, USA). DNA sequence analysis was performed using the PC/GENE program (IntelliGenetics, CA, USA) and sequence comparisons were done with the program FASTA (Pearson and Lipman, 1988). Results
F. heteroclitus pituitary cDNA library Total RNA (853 fig) was obtained from about 3000 pituitaries (0.2 mg wet weight per gland) and of this, 4.8 hg of poly A+ RNA was isolated and used to generate a directional cDNA library containing 1.05 X lo6 recombinants. This cDNA library in hgt22A was amplified in E. coli strain Y 1090(r _ ). PCR amplification and sequencing of the clones that encode the P-subunit of F. heteroclitus GTH II Initial attempts to isolate the P-subunits of F. heteroclitus GTH from the cDNA library using oligonucleotides based on conserved teleost GTH nucleotide sequences failed to yield any detectable products. We changed our strategy in designing the primers and deliberately constructed an oligonucfeotide (PEL3, Table I> which was more degenerate to cover all possible variations. Inosines were introduced in some of the most variable positions to help reduce the degeneracy. PCR products of expected size (300-400 bp) were generated using PEL3 with A(F) (Fig. 1A). Amplified DNAs of appropriate size were cloned and sequenced. Using the FASTA program to search the GenBank data base (Pearson and Lipman, 19881, we found that the DNA sequence of clone PTA XVII-2 has substantial homology with the &subunits of the GTH II of other teleosts (see Fig. 6). Tentatively, clone PTA XVII-2 was designated as the cDNA encoding the 5’ end of the P-subunit of F. heteroclitus GTH II (Fig. 1A). Its nucleotide sequence (1 to 388) is shown in Fig. 2. Based on the nucleotide sequence found in PTA XVII-2, an exact oligonucleotide primer (PELS) to the F. heteroclit~ GTH II /3 sequence was synthesized (Table 1). This exact primer, PEL5, which is upstream from PEL3 (Fig. l&I,
132
was used to specifically amplify the 3’ end of the GTH II p insert and to confirm the sequence covered by the original priming site of PEL3 (by examining the resulting overlapping clones with PTA XVII-21. From this series of PCRs, four relevant clones (about 200-300 bp) were obtained. Clone PTA XXIV-l and PTA XXIV-3 were generated by primer PELS and h(R),
whereas PTA XXV-2 and pTA XXV-3 were produced using primer PEL.5 and PO(T) (Fig. 1 A). Sequence analysis of these clones indicated that pTA XXIV-3, PTA XXV-2 and pTA XXV-3 all coded (based on sequence homology) for the 3’ end of the F. heterocfitus GTH II p-subunit. The nucleotide sequences of these three clones (represented by PTA XXV-3, nucleotide 319 to 482,
Fundulus heteroclitus GTH II P-subunit -20 CTCAGGATAGAGGACTGAAGTTTTCACTG
ATG
GTG
M
GGA
GCC
G
A
TTT
CAG
TCC S
CTC
TCA
TTC
S
F
-10 ATT I
TGG
TCC
CTG
w
s
L
V
GCA
TGT C
TTG
TTT
CTG
L
F
L
47
CCC
GCG
GCA
GCA
-1 GCC
ACA
ATA
TCT
CTA
137
92
CCT
CGC
TGT
GAG
CTC
CTC
3’“““” 10 AAC CAG
TCT S
GGC G
TGT C
CAC H
AGA R
GTG V
GAA E
ACC T
ACC T
30 ATC I
182
GAT
40 CCC
AAC
TAT
AAG
ACA
TCA
227
P
N
iQLPRCQLLNQT1.L
GAG
AAG
AGG
GGC
E
K
R
G
20 TGT C
TGC
AGC
GGG
TAC
TGC
GCG
ACC
AAG
c
S
G
Y
C
A
T
K
TAT
AAC
AAA
GCA
CAG
CAT
GTG
A
50 ATC I
TTC
GAG
TTT
Y
TAC Y
N
K
AAG
ACA
K
T
F
Q
E
F
H
V
CCT
GAA
P
E
D
TGC C
TGT C
ACT
TAT
T
Y
Y
GGG G
70 GTG
CCC
GGA
V
P
G
K
GAT D
GTT V
T
TTG
S 60 TAC
L
Y
GAC
CCA
D
272
317
P
362
GCC
#$&
: ;j~~$$?::&@&
PZ3 ._,. .(.,,., ::~~.~i.~~~~~~~~~~~~~~~~:~:~~~C
A
N’
A
T
S
TTT
TGC
ATG
AAT
110 GAC
F
C
M
N
D
ATC
D
AAAAAAAAAAAAAAAAAAAAAAAAAAA
I
100 GAG
AG C
CT T
fJ A G
CCT
GA C
S
L
Q
P
D
C
T
F
E
a,b CC@
TTT
TAC
CAO
P
407
a F
Y
H
TAG
CCATGTGTAAAAAAA
455
* 482
Fig. 2. The nucleotide and deduced amino acid sequence of cDNA encoding F. heteditus GTH II p-subunit. Numbering on the right-hand side refers to the nucleotide sequence and numbers on top of the sequence refer to the amino acid sequence. The first amino acid of the putative mature @,ubunit (circled phenylalanine, Fl is numbered as $1. Minus numbers are used to indicate the amino acids that comprise the signal sequence. Two overlapping cDNA clones, TA XVII-2 (nucleotide 1 to 388) and TA XXV-3 (319 to 4821, were used to construct the nucleotide sequence shown above. Shaded regions are the target sequences for the primers PEW and PEL5. Sites of nucleotide substitutions in clone TA XXIV-3 and TA XXV-2 are marked with boxes (al and (bl respectively. Details of the nucleotide substitutions of the various clones are shown in Fig. 4A. (*l Indicates the stop codon. Arrowhead indicates site of putative N-gIy~osylation.
133
The complete nucleotide sequence (482 residues), which includes the 5’-untransIated region and the 3’-polyadenylated region (comprised of 34 A), for the cDNA encoding the F. heteroclitus GTH II P-subunit was then compiled from the data derived from the various clones and is shown in Fig. 2. An N-linked glycosylation site was predicted to be located at the number 10 asparagine residue (under the arrowhead in Fig. 2 and Fig. 6). The presumed cleavage site for the
in Fig. 2) were exactly the same except at two locations (nucleotide 428 and 437, and are marked with box (a) and (b) in Fig. 2 and Fig. 4A) near the carboxy-terminal. These nucleotide substitutions ((a) 428 (A to G) and 437 (C to Tf in PTA XXIV-3; and (b) 428 (A to C) in PTA XXV-2 (as compared with PTA XXV-3 in Fig. 4A)) were silent mutations that did not affect the amino acid residues (aa 112 (P> and aa 115 (HI, Fig. 4A).
-10 ATG
CAG
M
TTG
Q
GGA
GTT
L
TGC
TTC
CTC
V
L
-1 GGT
1 TGC
ATG
GCG
GCG
A
A
CTG
AAG
TGC
M a C&
CTG
GCG
L
A
r AAT
GTC
AGC
ATC
CCC
10 ATG
GAG
ATC
20 CAC
ACC
ACG
ATC
TGC
GAA
GGG
H
T
T
I
E
G
GAA
AGC
CCT
GAC
40 GAG
GCG
P
5
E
A
TAC
GAG
GTG
AAA
C
F
AGA
TGC
GGC
CAG
AGG
R
C
G
Q
R
V
C
I
CTG
TGC
TTC
30 TCT
GAG
GAC
GCC
GTC
L
C
F
S
GAG
CAT
AGG
E
V
CTG L
GCA A
GAA E
GTG
45
V
90
GOHLKNVSIPME
G
CCT I?
GTC
H
GTG
E
R
A
V
F
AAC
GGG
50 GAC D
D
GTC
TGC
V
TTT
C
N
G
E
TGG
S
TCC
W
S
Y
C
E
V
135
180
225
K
270
,‘.... #AC N
c
TGC C
G’3i.“:‘:’
.
.
.
.
.
TGC
Y
_.: : .
:
R
TAC
.
.
.
.
_.
,._.........
TCT
C
GCG
TGC
AAC
80 ACC
AAA
A
C
N
T
K
S
.?..! ..:.. .......... :::... 0..
..,.:,.
... .... .. .
..::....
.,......L..
‘..
./.....
.. .
GAC
..I
..:_
.,
.:
.:.
L
:.
.
:..,
.
.
.
.:_:_:.,.:
Y
.
.
.::...
..
:.
:.
.:::
A
..__,_.
.,..
,.
H
::
.:.
. .. .
I
. ..i
.:
-.._.
.
P
. . .
.i.
:.
.
.:.-:
S
T
TAC Y
. .
a~:~:,i.~~~~~~~~~~~~~~~:~~~~~:~~:~~~~.~~~,~~~:::~~~~~~:~ :;q$JA! T A A A
‘2.:.
D
b ACM
.-...A..
: ::_.;
C
C T
A C T T A CA T
TGT C
ACC
315
T
GC A C T T A A
364
*
AATCCTCGCGTCTCTTTGTCTGACGCTGAAATAAAAGAAGATACTGCTCGTAAAAAAAA
4,2 3
AAAAAAAAAA
433
Fig. 3. The nucleotide sequence and the deduced amino acid sequence of cDNA encoding the P-subunit of F. heteroclitus GTH 1. The nucleotide sequence shown is composed of two overlapping cDNA clones, TA XXVI-3 (nucleotide 1 to 342), and TA XXIV-1 (248 to 433). Numbers on the right-hand side refer to the nucleotide sequence and numbering on top of the sequence refers to the amino acid sequence. The putative mature GTH I p-subunit starts with a cysteine (circled) and is numbered as + 1. Amino acids that comprise the signal sequence are indicated with minus numbers. Target sequences for primers PEL5 and PEL6 are shaded. Boxes (a) and (b) represent sites of nucleotide substitutions in clone TA XXVI-4 and clone TA XXVI-Z, respectively. Details of the nucleotide substitutions of the various clones are shown in Fig. 4B. Stop codon is marked with a (* 1 and consensus sequence (AATAAA) for ~lyadenylation signal is underlined. Putative N-glycosylation site is marked with an arrowhead.
134
signal sequence was determined by comparison with other known GTH sequences. A signal sequence of 21 amino acids was located and the proposed mature P-subunit of F. heteroclitus GTH II starts with a phenylalanine (circled F in Fig. 2) and consists of 115 amino acids. PCR amplification and sequencing of the clones that encode the P-subunit of F. heteroclitus GTH I Of the four clones that were generated by using the primer PELS, one of them, PTA XXIV-l (Fig. lB), was radically different from the other three clones that were described in the previous section. The DNA sequence of PTA XXIV-l was then used to search the GenBank data base with the FASTA program (Pearson and Lipman, 1988) and significant homology was found to the p-subunit of Oncorhynchus keta GTH I (see Fig. 6). Tentatively, clone PTA XXIV-1 was designated as the cDNA encoding the 3’ end of the putative F. heteroclitus GTH I p-subunit. Its nucleotide sequence (248 to 433) is shown in Fig. 3. This clone included the 3’-untranslated region, and in it the consensus sequence (AATAAA, underlined in Fig. 3) for the polyadenylation signal was found to be 48 residues downstream from the stop codon (TAA). Based on this new sequence information from clone PTA XXIV-l, an exact oligonucleotide primer to the putative F. heteroclitus GTH I P-sequence (downstream of PELS) was created (PEL6, Table 1 and Fig. 1B). Eventually, three pertinent clones (PTA XXVI-2, PTA XXVI-3, PTA XXVI-4), encoding the 5’ end of F. heterocfitus GTH I p-subunit, were obtained using PEL6 and A(F) (Fig. 1B). Their nucleotide sequences (as represented by PTA XXVI-3 (nucleotide 1 to 342) in Fig. 3) were all similar to each other except at three locations (nucleotide 62, 229, 306 and are marked with box (a) and (b) in Fig. 3 and Fig. 4B). Two nucleotide substitutions ((a) 62 and 229, both of them were A to G changes, Fig. 48) in clone PTA XXVI-4 resulted in the replacement of two amino acids (aa 2 (H to R), and aa 58 (I to V>, (compared with PTA XXVI-31 Fig. 4B), whereas the one nucleotide substitution in PTA XXVI-2 ((b) 306 (A to G)) did not alter the amino acid residue (aa 83 CT)) (Fig. 4B).
A
q m
pTA XXIV-3
GTH II B-subunit v ATC
pTAXXV-2
ATC
C&I
PTA XXV-3
h4C
c
B
CA&
TAG
440
TTT
TAC
CAC
TAG
440
P
TTT F
Th0
CAC
TAG
440
H
*
Y
Ii
c
L
K
PTA XXVI-3
@Qt.
TilC
c
Cft:
X&G
69
PTA XXVI-4
ggt
TGC C
CMC
CTG
AAG
69
(R)
L
K
“I
Q
G
G
Y56 K
PTA XXVI-3 PTA XXVI-4
PTA XXVI-3 •j
TAC
GTH IB-subunit G
q
TTT fl
I
q
CC
PTA XXVI-2
H
160
Mu
ChO
c
cxa
dwJ
237
AAA K
CAC H
G C Ir (V)
CAG
GGG
237
AAh
G&C
A
Q
c
TAi?
T@T
312
TAC
TGT
312
v ¶ AAA
GAC
K
D
(81
ACM
T
Y
C Y85
Fig. 4. Nucleotide substitutions in various clones encoding the P-subunits of (A) GTH II and (B) GTH I. Shaded sequences are part of the clones shown in Fig. 2 and Fig. 3 and are used for comparison with other clones in this figure. Arrowheads indicate the location of the nucleotide replacements and consequent amino acid changes are shown in parentheses. (#) Numbers refer to amino acid sequence (see Fig. 2 and Fig. 3). Numerals on the right-hand side denote the nucleotide sequence. Boxed a and boxed b: Clones that have nucleotide substitutions in the corresponding boxed locations in Fig. 2 and Fig. 3. ( *) Denotes stop codon.
In addition to the three nucleotide changes described above, we found that the long 5’-untranslated region of F. heteroclitus GTH I P-subunits were extremely variable (data not shown). By comparing our sequence with other known glycoprotein hormones (see Fig. 61, a signal sequence of 19 amino acids was predicted for F. heterocfitus GTH I p-subunit and the N-glycosylation site was assumed to be at the fifth asparagine residue (marked by arrowhead in Fig. 3 and Fig. 6). We concluded that there are 95 amino acids in the putative mature p-subunit of F. heteroclitus GTH I.
135
Discussion
199
(92
A
s
D
c
T
lPEL3 Target ATG
G ; ACI
;?I
GA:
TG;
$1
ATG M
Glti C ACA A ‘p
TCC s
GAC D
TGC C
ACC T
"
Y
P
v
A
TAC
CCC
GT
GCC
TAC Y
CCC P
GT 1 v
A GCC D A 7' 171
M
In this study, we have cloned and sequenced of the cDNAs encoding the putative p-subunits (GTH I two distinct F. heteroclitus gonadotropins and GTH II) analogous to the classical LH-FSH glycoprotein hormone family. Although GTH duality has been described in a number of species (for review see Scott et al., 1991), two distinct GTHs have only been isolated in two closely related fish (chum and coho salmon) (Itoh et al., 1988; Suzuki et al., 1988a, b; Sekine et al., 1989; Swanson et al., 1991). Recently, Kawauchi and his colleagues successfully cloned and sequenced the (Y-and p-subunits of two distinct chum salmon GTHs (Kawauchi et al., 1989; Sekine et al., 1989). Our ability to achieve a similar goal in F. heteroclitus - to clone and sequence cDNAs encoding two distinct GTH P-subunits - represents the second molecular demonstration of GTH duality for any teleost. Preliminary PCR experiments using oligonucleotide primers based on conserved nucleotide sequences of teleost GTHs were unavailing. Apparently, the F. heteroclitus GTH nucleotide sequence is very different from other known teleost sequences. In order to overcome this difficulty, we designed degenerate GTH oligonucleotides (such as PEL3, with inosines to help reduce degeneracy) for use as internal primers. Together with the vector or oligo dT external primers, we were able to specifically amplify the cDNAs that encoded the P-subunits of F. heteroclitus GTH II and, fortuitously, we also captured the GTH I P-sequence (by PELS) using PCR. In retrospect, the oligonucleotide PELS (Table 1) was more useful than we had imagined. Originally, PEL.5 was designed as an exact primer based on the nucleotide sequence deduced from clone pTA XVII-Z (Fig. 11, which coded for the P-subunit of Fundulus GTH II. Comparison of the target sequences for primer PEL3 (Fig. 5A) and PEL.5 (Fig. 5B) with the actual F. heteroclitus GTH sequences resulting from the PCR procedures revealed mismatches of two and six (out of 23 and 24) residues, respectively. Both F. heterocfitus GTH II and hCG P-subunits have particularly short 3’-untranslated regions (8 and 16 nucleotides respectively). In the
F. he GTH II
B
D
#16 "
PEL5
'l'r 6
Target
GE n:
F. he GTH I
s
T C
A
T
ACC
C RCC I T
164 Fig. 5. Comparison
of the target
(Al
with the corresponding
and PEL5
(Bl,
sequences found in F. heteroclirus pTA
XXV-31
respectively.
and GTH
(* )
Indicates
primer. acids.
nucleotides Numbers Numeral
GTH
I p-subunit
denote
right-hand
sequence.
P
1183 L A
of primer
II p-subunit
342
271
PEL.7
(in clone XXVI-31,
sequence shown is
of the actual mismatches
388
II
actual nucleotide
primer
sequence.
with oligonucleotide
on top of the sequences to the
388
(in clone pTA
that nucleotide
the inverse and complement Shaded
sequence
I
refer
side denotes
to amino nucleotide
I: inosine.
case of hCG, there is an extension of 24 amino acids (when compared with hLH1 at the carboxyterminal, which appears to have arisen by a readthrough event caused by a single base deletion (Talmadge et al., 1984). This readthrough event incorporated the 3’-untranslated region of the ancestral LH /?-gene into the coding region of hCG, hence shortening considerably the 3’-untranslated region. However, no such similar readthrough event can be detected in F. heteroclitus. We cannot disregard the possibility that the short 3’-untranslated region in the F. heteroclitus GTH II p-subunit (as deduced from clones pTA XXIV-3, PTA XXV-2 and PTA XXV-31 is an artifact due in part to mispriming or a copying error in the reverse transcriptase reaction during the cDNA library construction. Likewise, the six nucleotide substitutions observed in the various clones (Fig. 4) could be due to a reading error of the reverse transcriptase or, more likely, to heterogeneity in the mRNA populations (since they were obtained from more than 3000 fish). Comparison of the F. heterocfitus GTH I and GTH II P-sequences with other members of the glycoprotein hormone family is shown in Fig. 6. Their homology became immediately apparent when the cysteine residues were aligned. In F. heteroclitm GTH II P-sequence, a gap of two
1
v
2
3
h LHj3 h CG,B b LH_8 0. ke 0. ts
i
30 30
30 21 27 27 27 27 27 27
C. ca H. mo M. ci A. an Ehe
GTH II-,9
1
h FSH#? b FSH# h TSH-3 b TSH$ GTH I-,4
_[
24 22 23 23 27 20
$ 2 . 4
5
6
h LH$ h CG_B b LH$ 0. ke 0. fs c. ca fimo M ci
GTHW
i
-I
7 PRGVDPVVSFPVALS
01
87 87 84 84 84 84 84 84 84
A. an F. he
h FSH# b FSH+ h TSH$ b TSHB GTHI$3
V
81 79 82 82 83 72
$2
8
3
10 121 145 121 119 119 118 117 113 116 115
GmI,${
0l;e &
d
CDCIK KTDNT CDRISMATPS IVNPLEM ... .. .. . ....i...~ i\/,.i....,.,....... $$%,S ',, :@z@T-z :(.) ;$&.%&K.zf:&j
~.~..~
118 109 112 113 113 95
137
Dendrogram
of Pituitary
and Charionic
Glycoprotein
Human
__~.......~..l*...‘~.***~.*.......~...*
i.l._._.......;
LH-/3
:................. .................... .. Human
---_-: 1
i .
... .... . . .... ... _......... ,.... . . . . ... ...
P-Subunits
CG-/j
Bovine
LH-/3
I
Oncot-hynchus keta 0. is~~a~ts~ha
jL
Cyprinus ca rpio Hypophthahnichthys
molitrix GTH
II-/3
Muraenesox cinereus Anguilla anguilla
..,.,..,..............
1
’ 1
---------<
FSH-fj
Human
~““...“‘.‘“““~““‘......“’
L
Human FSH-/3
_................_.................... Bovine
1--------I
1
f... .. . .. ... .
._...__.....t.._.........~._-Bovine
TSH-P TSH_/j
I
~~~orhy~~~~s keta
GTH
~~~d~~~~ h~~~r~c~it~sF Fig. 7. Dendrogram
of mammalian
(phylogenetic
is formed
tree)
(.
. ‘1 and
by clustering
teleost (-----
) pituitary
the corresponding
amino
and chorionic glycoprotein
acid sequences
1-p
P-subunits.
based on overall
This dendrogram
similarity
using ‘average
linkage cluster analysis’ fSncath and Sokal, 1973).
Fig. 6. Comparison
of the amino acid sequences of the P-subunits
teteost and mammalian
glycoprotein
p-subunits
of F. ~~~~~~~jf~.~ GTHs
using the ‘Clustal’
program
in PC/GENE
(GTH
II and GTH
(Higgins
I, shaded) with other
and Sharp,
1988). The teleost
CF. he) are chum salmon, Oncorhynchas keta (0. ke) (Sekine et al., 1989), chinook salmon, 0. fschawyrscha (0. ts) (Trinh et al., 19X6), common carp, Cyprinus carpio (C, ca) (Changet al., 198X), silver carp, H~~c~&thnlmichthys molitrir (N. mo) (Chang et al., 1990), pike eel, Mrtruenesox cinereus CM. ci) (Liu et al., 1989) and European eel, Anguilhzz ~~g~j~~u (A. an) (Q&rat et al., 1989). The mammalian P-subunit sequences used are human lut~inizin~ hormone fhLH-6) GTH
sequences used for comparison
(Talmadge (Maurer,
with F. heteroclitus
et al., 1984), human chorionic gonadotropin 1983,
human follicle-stimulating
hormone
(hCG+) (hFSHj3)
CbFSH-j3) (Esch et al., 1986), human thyroid-stimulating ing hormone
(bTSH/3)
of the mature mature GTH
proteins,
glycoproteins 1-p. Amino
for the primers
(Maurer
et al., 1984). Numbers
Lowercase
19811), bovine luteinizing lY76),
and Rathnam,
hormone (hTSH-/3) at the right-hand
(Hayashizaki
accordingly
et al., 198.5), and bovine thyroid-stimulat-
The aspargine
The
on the top of the boxes. ( 1:) Denotes
12 cysteinr
residues in the
atypical cysteine location
1-p are shown in parentheses.
residues (N) under the arrowheads
glycosylation sites. Dash line f-) denotes gap introduced
(hLH+‘) hormone
side refer to the amino acids (single letter code in capital)
in the various clones encoding the K he GTH
used in the PCR are underlined.
hormone
bovine follicle-stimulating
single letter code denotes amino acid of the signal sequences.
are boxed and are numbered
acid substitutions
(Fiddes and Goodman, (Saxena
Target
may be the putative
during alignment to maximize
homology.
in
sequences N-linked
residues needed to be included only when compared with TSH, otherwise perfect alignment of the 12 cysteines was achieved when compared with other GTHs (LH, FSH, hCG and teleost GTHs). However, alignment for E heteroclitus GTH 1-B was not as flawless. In GTH 1-p of both 0. keta and F. heteroclitus, several gaps (such as between cysteine 7 and 8, 3 and 4) were required (Fig. 6). As evident from the dendrogram (Fig. 71, the teleost GTH 1-p seems to be more distantly related to the other members of the glycoprotein hormone family. Since the identity of the teleost GTH I has become apparent only recently, much more sequence data are needed before the evolution of structure and function relationships within the glycoprotein family can be appraised. In view of the significant role played by the cysteine residues in the assembly of the (rp dimer and their presumed highly conserved positions in the /?-sequence (Beebe et al., 19901, it is intriguing to find that in the ~-subunits of the GTH I from both 0. kern and F. ~zeteroelit~s, the cysteinc residues near the amino-terminal did not quite align with the other members of the glycoprotein hormones family (Fig. 6). In the P-subunit of 0. keta GTH I, cysteine 3 (C3> is missing from the customary position and is relocated four amino acids upstream of C’ (new location is designated as 1* in Fig. 61, whereas in F. heteroclitus GTH I, cysteine 2 (C2> is displaced two residues upstream from the conventional C’ position (becomes 2* in Fig. 61. How these cysteine residue displacements and the consequent threedimensional folding of the P-chain affect the assembly of the biologically functional ap heterodimer remains to be investigated.
Acknowledgements
This study was supported by NSF grants No. DCB-8819005 awarded to R.A.W. and DIR8914602 awarded to members of the Whitney Laboratory. We wish to thank Dr. Marion Byrne for the design of some oligonucleotides, Dr. Sandy Yosha for providing veterinary care, Mr. Scott Van Arnam for collection of F. hereroclitus, Ms. Louise
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