Download Characterization of Complementary DNA Encoding the Precursor for

Characterization of Complementary
DNA Encoding the Precursor for
Gonadotropin-Releasing Hormone
and Its Associated Peptide from a
Teleost Fish
Chris T. Bond, Richard C. Francis, Russell D. Fernald, and
John P. Adelman
Vollum Institute (C.T.B., J.P.A.)
and the Department of Cell Biology and Anatomy (J.P.A.)
Oregon Health Sciences University
Portland, Oregon 97201
Department of Human Biology
Stanford University (R.C.F., R.D.F.)
Stanford, California 94305
Reproductive maturity among male African cichlids
Haplochromis burtoni is cued by a series of environmental and social interactions and is mediated physiologically by GnRH. A cDNA clone encoding the
precursor for GnRH was isolated from this teleost.
The molecular architecture of the predicted prohormone is analogous to that of the previously characterized mammalian forms; however, the predicted
sequence of the associated peptide is strikingly
different. Attempts to isolate a putative second precursor using low stringency hybridization were not
successful despite evidence that a second related
decapeptide exists in at least some teleost species.
(Molecular Endocrinology 5: 931-937, 1991)
most stable region, residues 1-3, is responsible for
releasing gonadotropins, while the region with the most
changes, residues 5-8, is thought to mediate receptor
binding (2). In some vertebrates, including several teleosts, there is considerable chromatographic and immunological evidence that more than one form of GnRH
exists, and in the chicken two distinct forms have been
isolated, and the amino acid sequences determined (25). These decapeptides differ by three amino acids; one
form is similar to lamprey (chicken II), while the other is
more similar to mammalian GnRH (chicken I), suggesting that chicken II may be the older form. However,
DNA coding sequences have been determined for the
prohormone only in human (6, 7), rat (7), and mouse (8)
species.
In the teleost Haplochromis burtoni, as in many vertebrates, sexual development is regulated by social
interactions (9-11). A normal population of H. burtoni
contains two distinct male types: territorial (machos)
and nonterritorial (wimps). Macho males are brightly
colored, sexually mature, and establish and defend
territories for feeding and breeding. In contrast, wimps
are cryptically colored, sexually immature, and do not
reproduce. In social encounters, machos are dominant
over wimps, and this social dominance is correlated
with the size of forebrain magnocellular neurons in the
preoptic area which are immunoreactive for GnRH (1214). In males that gain territories and become macho,
these GnRH neurons enlarge significantly, while in
males that lose territories and become wimps, GnRH
cells shrink. Thus, an extraordinary neuronal plasticity,
dependent upon social cues and interactions, remains
throughout the adult life of H. burtoni males (Francis,
R. C , and R. D. Fernald, in preparation).
As a first step to understanding the cellular and
molecular basis of this plasticity, we report the isolation
INTRODUCTION
In vertebrates, GnRH plays a central role in development and maintainance of reproductive function. This
decapeptide regulates the release of pituitary gonadotropins, which stimulate the release of steroid hormones
from the gonads. In turn, these steroids feedback to
both the hypothalamus and the pituitary, completing
the hypothalamic-pituitary-gonadal axis, and act to attenuate the release of GnRH and its effects on gonadotroph cells (1). During 500 million yr of vertebrate
evolution, the primary structure of GnRH has been
remarkably conserved. The length of GnRH has remained at 10 amino acids, the amino (pGlu) and carboxy (Gly-amide) residues are unchanged, and five
residues are identical from lamprey to mammals. The
0888-8809/91/0931 -0937$03.00/0
Molecular Endocrinology
Copyright © 1991 by The Endocrine Society
931
MOL ENDO-1991
932
Vol 5 No. 7
shown in Fig. 2. The most 5' AUG initiates an open
reading frame of 90 amino acids. The N-terminal 23
residues comprise a hydrophobic signal sequence that
bears significant homology to cloned mammalian prepro-GnRH and is directly followed by the GnRH decapeptide sequence, identical to that determined from
peptide sequencing of the salmon decapeptide. Analogous to the mammalian forms, a glycine residue in the
11 position, thought to donate the amide group found
at the C-terminus of mature GnRH, is followed by
dibasic residues characteristic of trypsin-like proteolytic
substrates found in many polyprotein hormone precursors (18). The remainder of the open reading frame
predicts a novel associated peptide, cichlid GnRH-associated peptide (cGAP). This peptide is 54 amino acids
in length and bears no homology to the predicted
mammalian GnRH-associated peptides (mGAPs). The
preprohormone is predicted to have a mol wt of 10.1
kilodaltons; there are no consensus sites for N-linked
glycosylation. The structures of the three known mammalian precursors and the teleost precursor are compared in Fig. 3.
and characterization of a cDNA encoding GnRH in this
African cichlid.
RESULTS
Isolation of H. burtoni Pro-GnRH cDNA
The enlarged hypothalamic neurons characteristic of
macho males may reflect an increased level of proGnRH mRNA, and therefore, a cDNA library was constructed from mRNA extracted from the brains of
macho males. Approximately 5 x 105 primary clones
were screened with two pools of oligonucleotides, 17
bases in length, comprised of all possible sequences
encoding the first eight amino acids of salmon GnRH
(Fig. 1) (15). Hybridization specificity was obtained by
washing the filters in 3 M tetramethylammonium chloride
(16, 17), and a single positively hybridizing clone was
isolated. The 435 bases of nucleotide sequence and
the predicted translation product of the cDNA insert are
1a
10
pGlu
Lamprey
pGlu
Chicken II
pGlu
Salmon
pGlu
Chicken I
pGlu
Mammal
pGlu
-
His His
X -
- Tyr -
Ser
- x - x - x - x -
Ser - Leu - Glu - Trp - Lys -
Pro
- GlyNH 2
Pro
- GlyNH 2
His - Trp -
Ser
His - Gly
Trp - Tyr -
Pro
- GlyNH 2
His - Trp -
Ser
Tyr - Gly
Trp - Leu -
Pro
- GlyNH 2
-
His
- Trp -
Ser
Tyr - Gly
Leu - Gin -
Pro
- GlyNH 2
-
His - Trp -
Ser
Tyr - Gly
Leu - Arg -
Pro
- GlyNH 2
-
1b
pGlu - His - Trp - Ser - Tyr - Gly - Trp - Leu - Pro - GlyNH,
CA"
CA;
TGG
AG
GG
C
TCX
TGG
AG,
TA,
GGX
TGG
;TT
TCX
Fig. 1. a, Evolution of the GnRH Decapeptide Family
Primary structure of the five known forms of GnRH. Positions 1, 2, 4, 9, and 10 are invariant, and positions 3 and 7 show only
conservative changes, while positions 5 and 8 tolerate dramatic differences, b, Oligonucleotides used as probes to isolate cichlid
prepro-GnRH cDNA clones. Pools consisted of overlapping 17-mers comprised of all possible coding combinations for eight amino
acids of salmon GnRH.
933
cDNA for a Teleost GnRH Precursor
_23
SIGNAL SEQUENCE
_10
_j
CAGAGTTCTA ATG GAA GCA GGC AGC AGA GTT ATA ATG CAG GTG TTG TTG CTG GCG TTG GTG GTT CAG GTC ACC CTG TCC
MET Glu Ala Gly Ser Arg Val H e Met Gin Val Leu Leu Leu Ala Leu Val Val Gin Val Thr Leu Ser
Processing
+1
GnRH
Site
cGAP
10
20
CAG CAC TGG TCC TAT GGA TGG CTA CCA GGT GGA AAG AGA ACT GTG GGA GAG CTT GAG GCA ACC ATT AGG ATG ATG GGC ACA
| Gin His Trp Ser Tyr Gly Trp Leu Pro Gly] Gly Lys Arg Ser Val Gly Glu Leu Glu Ala Thr H e Arg Met Met Gly Thr
30
40
SO
GGA GGG GTG GTG TCT CTT CCC GAT GAG GCA AAT GCC CAA ATC CAA GAG AGA CTT AGA CCA TAC AAT ATA ATT AAT GAT GAT
Gly Gly Val Val Ser Leu Pro Asp Glu Ala Asn Ala Gin H e
TCC AGT CAC TTT GAC CGA AAA AAA AGG TTC CCT AAT AAT TGA
Gin Glu Arg Leu Arg Pro Tyr Asn lie H e
Asn Asp Asp
AGAGCTACATCATTATCAGCAGCACATGGGAAAGATCGCCCGGCAACAAGA
Ser Ser His Phe Asp Arg Lys Lys Arg Phe Pro Asn Asn
Fig. 2. Nucleotide and Amino Acid Sequence of Cloned H. burtoni cDNA Encoding Prepro-GnRH
Amino acid numbers appear above the sequences, with negative numbers corresponding to the presumed signal sequence. The
functional domains of the precursor are indicated, and the polyadenylation signal is underlined.
It is interesting to note that introduction of a single
base frameshift within the cGAP domain generates a
predicted peptide sequence that contains a significant
block of homology to mGAP, within a region which itself
is highly conserved among the mammalian species. To
assure that the isolated cichlid cDNA did not represent
a cloning artifact in this region, three independent
oligo(dT)-primed reverse transcription reactions were
performed using the same mRNA from which the original cDNA library was constructed. Two independent
aliquots from each of these reactions were subjected
to enzymatic amplification [polymerase chain reaction
(PCR)] (19, 20) in the presence of oligonucleotide
primers specific for untranslated sequences in the proGnRH mRNA, a total of six separate PCR reactions.
The PCR products were cloned and subjected to nucleotide and predicted protein sequence analysis. A
total of four independent clones from each PCR reaction
(24 subclones) were analyzed, and although occasional
disagreements between the sequences were observed,
usually resulting from transitions, incorporation of C in
positions where T was present in the original cDNA
clone and presumed to represent PCR mistakes, in no
case was the reading frame altered.
Northern Blot Analysis of Cichlid Pro-GnRH mRNA
To examine the full-length pro-GnRH mRNA, total and
poly(A)+ RNAs were extracted from whole brains of
macho males and prepared as a Northern blot. Hybridization to a radiolabeled DNA probe derived from the
pro-GnRH cDNA detected a single RNA species approximately 550 nucleotides in length (Fig. 4). Reprobing of this blot at lower stringencies failed to detect
additional RNA species.
Gene Copy Analysis
Chromatographic and immunological data have provided considerable evidence implicating the existence
of a second form of GnRH in several teleost species (2)
(Sherwood, N., personal communication). However, a
second decapeptide form was not found encoded on
the precursor described above. To investigate whether
this second form is encoded by a partially homologous
mRNA transcribed from a distinct gene, 2 x 106 primary
clones from the H. burtoni macho cDNA library were
rescreened at reduced stringency, using a radiolabeled
DNA probe derived from the cichlid pro-GnRH cDNA.
Even employing extremely permissive hybridization
conditions, no related sequences were detected, although several independent pro-GnRH cDNA clones
were isolated. Further, H. burtoni genomic DNA was
digested with a variety of restriction endonucleases,
prepared as a Southern blot, and probed at high and
low stringencies. The results, shown in Fig. 5, indicate
that the gene encoding GnRH in H. burtoni is a single
copy gene; no additional hybridizing bands were detected at any stringency.
DISCUSSION
The cDNA encoding pro-GnRH has been isolated and
characterized for the first time from a nonmammalian
species, the cichlid teleost H. burtoni. The molecular
architecture of the predicted prohormone is identical to
that of the previously described mammalian forms: a
23-amino acid signal sequence in direct linkage with
the decapeptide, which, in turn, is followed by residues
Vol 5 No. 7
MOL ENDO-1991
934
3a
SIGNAL SEQUENCE
n r—i
QHWSYG il PGGKR
SVGE : !ATIR> <G rGGWSLPDEANAQIQERIR ?YNIINDDSSHFDRKKRFPNN
1 1
QHWSYG LR PGGKR
NTEH 1 reSFQE <G (EVDQMAEPQHFECTVHWER 3PLRDLRGALESLIEEEARQKKM
(LMAHVViLITVCLEGCS
3HWSYG LF PGGKR
NTEH 1 rtlSFQE <G (EEDQMAEPQNFECTVHWERSPLRDLRGALERLIEEEAGQKKM
QHWSYG Lf PGGKR
DAEN;CDSFQEIVKEVGQLAETQRFECTTHQE R SPLRDLKGALESLIEEETGQKKI
:J*/IMdVLLl
MIL(LMAGILLITVCLEGCS
< LMAGI L:
IQ < LLAGLI L]
3b
CICHLID
...ERLRPYNIINDD...
. . .HW >RSPI *DIRG. . .
. . .HW >RSPI
. . .HC >RSPI 1DIKG. . .
CICHLID-alt
Fig. 3. a, Comparison of the Mammalian and Cichlid Precursor Proteins for Prepro-GnRH
The single letter code is used to designate amino acids. The three domains of signal sequence, GnRH, and GAP are set apart.
Amino acids that represent the site for enzymatic precursor cleavage and carboxy-terminal amidation (18) are joined to the GnRH
decapeptide. Boxes do not represent homologies between the various mammalian sequences, b, Comparison of GAP domain in
which a frameshift in the cichlid sequence (Cichlid-alt) produces a region of significant homology to the mammalian forms. This
domain is highly conserved among the mammalian species.
involved in proteolytic processing and maturation of
GnRH, and a 54-residue associated peptide. Eight of
23 residues in the signal sequence are shared between
H. burtoni and at least one of the mammalian forms,
not including an analogously placed arginine in the fish
and corresponding lysine in all mamalian sequences.
The known decapeptide sequences vary at two positions, 7 and 8, while the three residues involved in
posttranslational maturation, Gly11, Lys12, and Arg13,
are completely conserved.
Although the signal sequence and decapeptide show
remarkable homology to the mammalian forms, the
associated peptide, cGAP, shows little if any relation to
mGAP; five residues may be appropriately aligned, with
no more than two contiguously. However, a single base
insertion within cGAP generates a potential peptide
sequence with a significant block of homology to a
conserved region of the mammalian forms; a total of
nine residues may be aligned, and allowing substitution
of a lysine for an arginine, this yields a stretch of six of
seven matches. However, analysis of an independent
population of prohormone sequences by PCR demonstrated that the bonifide cGAP does not possess such
homology. It is, therefore, possible that the mammalian
GAP arose by a frameshift event at some point between
the appearance of fish and mammals.
Mammalian GAP has been implicated in the control
of PRL release from pituitary lactotrophs (21). Teleost
fish have two forms of PRL (22,23) which are controlled
by hypothalamic factors (24, 25), and both forms are
thought to function in osmoregulation (26,27). Although
the physiological properties regulated by mammalian
PRL are not germain to teleost physiology (lactation,
parturition), it is possible that cGAP functions to regulate the release of cichlid PRL. Planas et al. (28) recently
reported that human GAP effects PRL release from the
pituitary of the Tilapia, Oreochromis mossambicus.
Since the structure of cGAP, as determined from the
experiments described here, does not resemble that of
mGAP, these results are enigmatic. Alternatively, it is
possible that cGAP does not effect PRL secretion and
that mGAP arose in the tetrapod lineage as PRL came
to serve new functions. Experiments are in progress to
examine the physiological role of this novel peptide,
cGAP.
Chromatographic and immunological methods have
identified two forms of GnRH in several teleost species,
including salmon, trout, goldfish, herring, mullet, siganids, milkfish, and sea bass (2). However, the exact
amino acid sequence of only one form, salmon I, is
known (15). This peptide sequence was used to design
the oligonucleotides that succeeded in detecting the
cDNA encoding pro-GnRH. Reduced stringency cDNA
screening and Northern and Southern analyses failed
to provide evidence for a related gene that may encode
the putative second form of cichlid GnRH. If a second
form is expressed in H. burtoni, it is encoded by DNA
that does not bear strong enough homology to the proGnRH cDNA to be detected by hybridization. Although
two peptides coelute from HPLC columns and demonstrate cross-reactivity with antiserum raised against the
sequenced form of GnRH, data presented here suggest
that the mRNAs encoding these peptides may be very
different in sequence. Without the exact amino acid
935
cDNA for a Teleost GnRH Precursor
H. burtoni Genomic DNA Blot
a
a E2
•
<
,
PQ
H
5
O
o
PQ
u
g
u
w
1400-
780530-
1—
400280-
kb
Fig. 5. Southern Blot Analysis of H. burtoni Genomic DNA
Each lane contains 20 ^g liver DNA cleaved to completion
with the indicated restriction endonucleases. Random primed
cDNA fragment was used as a probe under high or low
stringency conditions, as described in Fig. 4. At lowered
stringency, no additional bands were detected.
Fig. 4. Northern Blot Analysis of Cichlid Prepro-GnRH mRNA
Lane 1 contains 5 ^g poly(A)+, and lane 2 contains 20 /ug
total RNA isolated from the brains of macho males. H. burtoni
pro-GnRH cDNA was random primed and used as probe under
high (50% formamide; 37 C) or low (30% formamide; 25 C)
stringency conditions. At lowered stringency, no additional
bands were detected.
sequence of the second decapeptide form it is difficult
to speculate about possible physiological functions.
However, in the chicken, in which the amino acid sequences of two distinct GnRH-like peptides have been
determined, both forms function to release gonadotropins, although chicken II is 10 times more potent than
chicken I (5). Such a situation is similar to the relationship between the enkephalins and the endorphins; two
peptides with similar biological activities are encoded
by genes that are not able to recognize each other by
cross-hybridization (29, 30). Questions concerning the
comparative biological activities of the GnRH peptide
encoded on the precursor described here and that of
the putative second form, and the evolutionary relatedness of the genes encoding these peptides can only
be addressed, however, when the cDNA encoding the
second form has been isolated and characterized, allowing direct comparison of the peptide moieties, the
nucleic acid milieu harboring each decapeptide-encoding sequence, and experiments to determine the potency of the second form as a GnRH.
MATERIALS AND METHODS
Tissue and RNA Preparation
Social dominance in H. burtoni, a prerequisite for sexual reproduction in the males, is advertised through territorial defense
and bright body colorations (12, 13). Behavioral observations
and these distinctive color patterns were used to select males
from breeding colonies for collection of brain tissue. Sixty
animals were chosen and killed by rapid heart puncture; brains
were quickly removed and stored in liquid nitrogen.
MOL ENDO-1991
936
Total RNA was extracted using guanidine thiocyanate and
purified by centrifugation through a cesium chloride pad, as
previously described (31). The poly(A)+ fraction was purified
by oligo(dT)-cellulose chromatography, as previously described (32).
Complementary DNA Library Construction and Screening
Complementary DNA was synthesized as previously described
(7). After ligation to EcoRI adaptors, synthesized on an Applied
Biosystems PCR Mate DNA synthesizer (Foster City, CA), the
cDNA was size-selected for molecules over 300 basepairs in
length by agarose gel electrophoresis. Eluted material was
ligated to Xgt10 arms (Stratagene, La Jolla, CA) and incorporated into phage particles by in vitro packaging according to
manufacturer's instructions (Stratagene, La Jolla, CA).
Recombinant clones were plaqued on E. Coli C600 hfl at a
density of 40,000/plate, from which filter lifts were prepared
(Genescreen, New England Nuclear-DuPont, Boston, MA.).
Oligonucleotide pools including all possible coding sequences
for the first eight amino acids of the known salmon GnRH
sequence (15) were synthesized on the PCR mate. Chains
were radiolabeled with [7-32P]ATP and polynucleotide kinase
(New England Nuclear-DuPont); filters were prehybridized and
probed at room temperature in 1 M NaCI, 1 % sodium dodecyl
sulfate, 100 ng/m\ yeast RNA (Sigma, St. Louis, MO), and
30% formamide (Bethesda Research Laboratories, Gaithesburg, MD). After a brief rinse at room temperature in 2 x
standard salt citrate solution (SSC), filters were washed with
3 M tetramethylammonium chloride (Aldrich Chemicals, Milwaukie, Wl) at 51 C and exposed to x-ray film for 5 days.
Northern and Southern Blot Analyses
For Northern analysis, 5 ^g poly(A)+ or 20 ^9 total H. burtoni
macho brain RNA were electrophoresed through 1 % agaroseformaldehyde gels and transferred by capillary to Magna NT
nylon membranes (Micro Separations, Inc., Westboro, MA).
For Southern analysis, H. burtoni genomic DNA was extracted from liver tissue, which was quickly frozen and pulverized in liquid nitrogen, followed by treatment with proteinaseK (Boehringer Mannheim, Indianapolis, IN). Nucleic acids were
ethanol precipitated after adjustment to 0.3 M KOAC. The pellet
was resuspended in distilled sterile water, phenol extracted
three times, chloroform extracted twice, and ethanol precipitated in the presence of 0.3 M NaoAc. The pellet was resuspended in 10 mM Tris (pH 7.5), 1 mM EDTA, 25 ng/rr\\ pancreatic RNase (Sigma), and incubated at 37 C. After 1 h, the
reactions were phenol and chloroform extracted, and the DNA
precipitated. After centrifugation, the DNA was dialyzed overnight against excess 10 mM Tris (pH 7.5), 1 mM EDTA, and
10 mM NaCI, reprecipitated, and stored in distilled water.
Twenty micrograms of this material were digested with the
indicated restriction endonucleases (Bethesda Research Laboratories), electrophoresed through a 1 % agarose gel (FMC,
Rockland, ME), and prepared as a Southern blot by capillary
transfer.
Probes for Northern and Southern blots were prepared from
a DNA fragment generated by enzymatic amplification, using
oligonucleotides specific for sequences encoding cichlid GnRH
and Replinase (Bethesda Research Laboratories). After fragment purification over an agarose gel, the eluted DNA was
radiolabeled by random priming with E. coli DNA polymeraseI, Klenow fragment (Bethesda Research Laboratories) to a
specific activity of 5 x 108 cpm/^g. Routinely, 108 cpm were
applied as probe.
cGAP PCR and DNA Sequence Analysis
Reverse transcription and PCRs reactions were performed as
previously described (20), using Replinase. Oligonucleotides
specific for GnRH sequences were initially present at 0.5 ^ M ;
Vol 5 No. 7
these chains contained restriction sites flanking GnRH sequences. After amplification, the reactions were phenol and
chloroform extracted and ethanol precipitated. PCR products
or cDNA clones were cut with appropriate restriction enzymes,
gel purified, and cloned into M13 vectors. DNA sequencing
was performed by the dideoxy chain terminating method (33),
using Sequenase according to manufacturer's instructions
(U.S. Biochemical Corp., Cleveland, OH).
Acknowledgments
We thank Ms. Shiela Vollmer for nurturing the macho male
fish, Drs. Nancy Sherwood and Imogen Coe for fruitful discussions, and the Vollum Illustrations staff for artwork.
Received March 21,1991. Revision received April 22,1991.
Accepted April 22, 1991.
Address requests for reprints to: Dr. John P. Adelman,
Vollum Institute for Advanced Biomedical Research/Oregon
Health Sciences University, L-474, 3181 SW Sam Jackson
Park Road, Portland, Oregon 97201.
This work was supported by NIH Grants HD-23799 (to
R.D.F.) and HD-24562-02 (to J.P.A.).
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