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Department of Anatomy and Cellular Biology, Tufts University Schools of Medicine, Boston, Massachusetts 02111
Address all correspondence and requests for reprints to: Mercedes Jimenez-Liñan, Tufts University Schools of Medicine, Department of Anatomy and Cellular Biology, 136 Harrison Avenue, Boston, Massachusetts 02111.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We therefore isolated the guinea pig LHRH complementary DNA (cDNA) with the aim of examining LHRH gene regulation in this species in future studies. The present study describes the characterization of the cDNA encoding the precursor for LHRH and its associated peptide in guinea pigs. The sequence and identity of the cDNA isolated in this study predicts a unique amino acid sequence of guinea pig LHRH decapeptide and reveals the presence of two LHRH gene transcripts in the brain, differing only in the length of their 3' untranslated regions (3' UTR).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation of total RNA
The brain was dissected generating two tissue blocks, one
containing the preoptic area-anterior hypothalamus (POA-AH) and the
other containing the remainder of the hypothalamus (HYP). Tissue blocks
from each brain were fractionated into nuclear and cytoplasmic phases
using a double detergent lysis buffer and 1-ml tuberculin syringe with
22-gauge needles as homogenizers (3). Briefly, each dissection was
thawed, homogenized in 500 µl lysis buffer (10 mM
Tris-HCl, pH 7.5, 1.5 Mm MgCl2, 0.3 M sucrose,
0.5% Nonidet P-40, 0.25% sodium deoxycholate), and layered on 400
µl cushion buffer (10 mM Tris-HCl, pH 7.5, 1.5
mM MgCl2, 0.4 M sucrose). After
centrifugation at 800 x g for 5 min, the cytoplasmic
fraction was transferred to a new tube and digested with 200 µg/ml
proteinase K for 45–60 min at 45 C. Each sample was extracted once
with phenol-chloroform (1:1) and was precipitated with ethanol. Total
RNA from pituitary, ovary, and testes was extracted using a total RNA
purification system from Qiagen (Santa Clarita, CA). For RNase
protection assay, RNA pellets were resuspended in 20 µl hybridization
solution (0.1 M EDTA, pH 8, and 4 M guanidine
thiocyanate; final pH 7.5).
PCR cloning of guinea pig LHRH
Three oligonucleotide primers were constructed based on the most
conserved regions of the LHRH cDNA sequences of humans, rats, and mice.
Primer 1 was complementary to the first six amino acids of the LHRH
decapeptide, 5'-CAG CAC TGG TCC TAT GG-3'. Primer 2 was complementary
to the sequence immediately upstream to primer 1, 5'-TGG AAG GCT GCT
CCA GC-3', and primer 3 was complementary to the 5' end of exon 4,
5'-TTC CTC TTC AAT CAG AC-3'. One microgram of total RNA from the
POA-AH tissue extracts was reverse transcribed to cDNA and used as
template. The first PCR was performed using an oligo(dT)17 primer and
primer 2. To provide added specificity to the amplification procedure,
a nested second PCR was performed with primers 1 and 3 using the
product of the first PCR as template. For the first PCR, 35 cycles of
amplification were carried out under the following conditions: 94 C, 30
sec; 56 C, 25 sec; and 72 C, 60 sec. For the second PCR, 40 cycles of
amplification were carried out under the following conditions: 94 C, 30
sec; 56 C, 30 sec; and 72 C, 60 sec. Fifteen microliters of each PCR
reaction product (50 µl total volume) were subject to electrophoresis
on a 2% agarose gel for analysis of amplification results. From the
second PCR, a 190-bp band was obtained, cloned in PGEMT-T vector
(Promega, Madison, WI) and sequenced using the Sequenase Kit (U.S.
Biomedicals, Cleveland, OH).
Library construction and screening
The cDNA library was constructed from 5 µg
poly(A)+-messenger RNA (mRNA) isolated from guinea pig
hypothalami using a kit obtained from Stratagene (La Jolla, CA). The
cDNA synthesized with random primers was ligated to EcoRI
adaptors and fractionated to eliminate sequences smaller than 400 bp,
ligated into Uni-ZAP XR vector (Stratagene), and packaged. The primary
library was amplified once, and 106 independent plaques
were screened by standard filter hybridization techniques (4) using the
PCR-generated LHRH cDNA clone as probe. After two successive rounds of
screening, four positive plaques were isolated and purified.
pBluescript SK(±) (Stratagene) containing the insert was excised
in vitro by ExAssist (Stratagene) helper phage.
Double-stranded cDNA from the clones was sequenced using the Sequenase
Kit (U.S. Biomedicals).
Solution hybridization/RNase protection assay
Probe and reference RNA preparation.
The initial template
DNA for in vitro synthesis of probe (antisense) RNA was the
190-bp guinea pig LHRH cDNA fragment extending across exons 2 and 3
that was inserted into the PGEMT-T vector (Promega) described
previously. A second probe containing the last 108 bp of the 3' UTR of
guinea pig LHRH was designed to determine whether more than one LHRH
transcript was expressed in guinea pig brain. This probe was obtained
by PCR using the following primers: 5'-AAA TGA AAT TTG TGA ACC CT-3'
and 5'-CAC TTT ATT TAC AAC ACA GT-3' and one of the positive clones
obtained after screening the cDNA library as template. The template DNA
for reference (sense) RNA was the full-length cDNA coding sequence
inserted into pBluescript SK(±). Probes and reference RNA were
transcribed in vitro from linearized template DNA using
bacteriophage T7 or T3 RNA polymerase (3), and transcription was
terminated by digesting the template DNA with RNase-free DNase I
(Worthington, Freehold, NJ). Probe RNAs were purified by ethanol
precipitation in the presence of 2.5 M ammonium acetate.
Reference RNA was phenol-chloroform extracted and purified through two
sequential ethanol precipitations in the presence of 2.5 M
ammonium acetate. The purified reference RNA was quantified
spectrophotometrically using the A260 reading, aliquoted
and stored at -80 C.
Solution hybridization/RNase protection assay.
Guinea pig
LHRH mRNA was examined in POA-AH and HYP extracts and in other
reproductive tissues (pituitary, ovary, and testes) using a previously
described solution hybridization/RNase protection assay method (3).
Briefly, RNA samples were incubated with 0.2 ng LHRH complementary RNA
probe labeled with [
32P]uridine triphosphate (UTP) to
high specific activity (
1600 cpm/pg). For the standard curves, LHRH
probes were mixed with increasing known amounts of the reference RNA.
The probes were incubated with total RNA samples in a final volume of
25 µl hybridization solution (4 M guanidium thiocyanate,
0.1 M EDTA; pH 7.5) for 16–18 h at 30 C. After
hybridization, the samples were treated with 300 µl RNase solution
(10 mM Tris-HCl, pH 8, 300 mM NaCl, 40 µg/ml
RNase A, 1 µg/ml RNase T1) for 1 h at 30 C, followed by
proteinase-K digestion (140 µg/ml) for 15 min at 45 C. Samples were
extracted with 1 vol of phenol-chloroform (1:1), and the RNA was
precipitated with ethanol, dried, dissolved in 7 µl gel-loading
buffer (25% Ficoll, 0.2 M EDTA, pH 8, 0.25% bromophenol
blue, 0.25% xylene cyanol) and electrophoresed through a 5%
nondenaturing polyacrylamide gel. Gels were mounted on 3 M
(Whatman, Lexington, MA) paper and dried. Protected bands were
visualized by autoradiography.
In situ hybridization
Brains from female guinea pigs housed in the laboratory were
rapidly removed, frozen on crushed dry ice, and 14-µm sections were
cut on a cryostat (Reichert-Jung, Leica Instruments, Heidelberg, West
Germany) at -22 C and mounted onto vectabond coated slides (Vector
Laboratories, Burlingame, CA). Digoxigenin (Dig)-labeled riboprobes
were prepared by in vitro transcription of linearized cDNA
templates for guinea pig LHRH (190 bp) in the presence of Dig-labeled
UTP and unlabeled nucleotides. Following pretreatment, 15 µl
hybridization solution containing Dig-labeled riboprobe at a
concentration of 0.5 µg/ml in hybridization buffer was placed on each
tissue section, and slides were covered with silicon-coated coverslips.
The tissues were hybridized overnight at 55 C in moist chambers. Slides
were washed, treated with RNase, and exposed to a series of washes of
increasing stringencies from 2 x SSC to 0.1 x SSC at 55 C.
Riboprobe/mRNA complexes were visualized by overnight incubation in
alkaline phosphatase conjugated anti-Dig antibody followed by
incubation for 4 h in chromogen solution containing nitroblue
tetrazolium salt and 5-bromo-4-chloro-3-indoyl phosphate toluidine
salt, which imparted a purple color to the hybridization reaction
product.
Southern blot analysis of guinea pig DNA
DNA isolated from guinea pig spleen was digested with
restriction enzymes including BamHI, EcoRI,
HindIII, PstI, SacI, and
XbaI. The hybridization probe used for this analysis was a
genomic fragment of approximately 1.2 kb corresponding to part of exon
2, intron B, and exon 3 of the guinea pig LHRH. The DNA fragment
complementary to this probe contains two PstI sites; one
site for BamHI, EcoRI, and SacI; and
no HindIII or XbaI sites, and was random primed
labeled with 32P.
Initially, the probe was isolated by PCR with two oligonucleotide primers complementary to sequences in exon 2 (5'ATT CCC AAA CTC CTG GCT GG 3') and in exon 3 (5'CCA GAG CTC CTT TCA GGT CC 3') using DNA from the positive clones obtained after screening a lambda FIX II guinea pig kidney genomic library (Stratagene) as template. The library was screened using a cDNA fragment corresponding to exons 1 and 2 of the guinea pig LHRH cDNA. The PCR product was also cloned in a PGEMT-T vector (Promega) for further sequencing.
Digested DNA from guinea pig spleen was loaded onto a 1% agarose gel, 6 µg/lane, electrophoresed, and transferred to a nylon membrane. Hybridization was accomplished using QuikHyb hybridization solution (Stratagene). Low stringency conditions consisted of hybridization at 55 C followed by two washes at room temperature in 2 x SSC + 0.1% SDS and one wash in 0.1 x SSC + 0.1% SDS at 50 C. High stringency conditions included hybridization at 68 C followed by two washes at room temperature in 2 x SSC + 0.1% SDS and one wash in 0.1 x SSC + 0.1% SDS at 60 C. In the high stringency condition, the membrane was exposed to x-ray film with an intensifying screen for 20 h at -80 C, and in the low stringency condition, the membrane was exposed for 48 h at the same temperature.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Deduced sequence of guinea pig LHRH decapeptide
The nucleotide sequencing data obtained predict an amino acid
sequence of guinea pig LHRH decapeptide that differs from mammalian
LHRH by two amino acids (5). The substitutions in the guinea pig
decapeptide are at position 2 (histidine is replaced by tyrosine) and
at position 7 (valine is substituted for leucine). This same nucleotide
sequence was observed in all of the positive clones examined. As shown
in Fig. 1
, the histidine residue at
position 2 is invariant in the primary sequence of all eleven known
forms of LHRH (6, 7, 8). The guinea pig LHRH decapeptide is unique in that
it contains the amino acid valine in position 7 (Fig. 1). The predicted
sequence of guinea pig decapeptide was further confirmed by sequencing
the genomic DNA corresponding to this region of the LHRH precursor. The
genomic sequence corresponding to the decapeptide was identical to the
cDNA sequence initially obtained.
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This PCR product was used to screen the guinea pig cDNA library, and
the sequence analysis of four positive clones revealed the full-length
coding sequence for guinea pig LHRH. The nucleotide sequence of the
guinea pig LHRH and the deduced amino acid sequence are shown in Fig. 2. The cloned cDNA is approximately 500
bp in length and it’s nucleotide sequence contains an open reading
frame of 276 nucleotides encoding proteins of 92 amino acids. The
guinea pig LHRH cDNA revealed sequence homologies at the nucleotide
level with human LHRH and rat LHRH of approximately 80% in the coding
region. In the 3' UTR, homology dropped to approximately 72% compared
with human LHRH and 68% compared with rat LHRH. The 5' untranslated
region of the longest LHRH cDNA isolated was 109 bp.
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) was used as a probe for
RNase protection assay. No protected band was observed with rat
hypothalamic RNA (Fig. 4). When used for
in situ hybridization, the 190-bp probe transcribed from
guinea pig LHRH cDNA clearly labeled LHRH neurons in guinea pig
hypothalamus (Fig. 5).
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). Data from RNase protection analysis
of POA-AH extracts using a probe complementary to the last 108 bp of
the 3' UTR of the longer transcript revealed two protected fragments
that were very similar in size (see Fig. 7). These data were consistent with the
two subspecies of LHRH mRNA obtained by sequencing the cDNA clones. As
shown in Fig. 7, the shorter transcript was more abundant.
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, low
levels of LHRH transcripts were observed in the HYP and in the
pituitary, and levels were undetectable in the ovary and testes. Only
the short form of the LHRH transcript was detected in the HYP and
pituitary. It should be noted that low levels of LHRH mRNA observed in
the HYP were expected because the majority of the LHRH neurons would be
present in the more rostral POA-AH dissections (9). Given that the
pituitaries were obtained from a commercial source, we cannot rule out
the possibility that pituitary stalk was present. Therefore, the LHRH
mRNA present in pituitary extracts may reflect in part contamination
from pituitary stalk LHRH neurons.
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. The predicted guinea pig LHRH
precursor is composed of a signal peptide, the decapeptide, a
Gly-Lys-Arg sequence for proteolytic processing and C-terminal
amidation (10), and an associated peptide (GAP). The predicted guinea
pig precursor protein is identical in length to the human and rat
precursor protein and demonstrates 75% identity (88% similarity) with
human precursor protein and 72% identity (89% similarity) with rat
precursor protein. The signal peptide sequence in the guinea pig has 8
amino acid substitutions relative to human (3 are considered
conservative substitutions) and 10 amino acid substitutions relative to
rat (7 are regarded as conservative). Guinea pig GAP, which is 56 amino
acids in length, has 13 amino acid substitutions compared with humans,
and 14 amino acid substitutions compared with rats (in each case 8 of
the substitutions are considered conservative).
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. Genomic DNA digested with
BamHI, EcoRI, SacI, PstI,
HindIII, and XbaI and hybridized using the probe
previously characterized (see Materials and Methods) yielded
the predicted number of fragments, consistent with a single gene for
LHRH. For a detailed description of the fragments, see Fig. 10.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The sequence and identity of the cDNA isolated in this study has been confirmed at several levels including the comparison of nucleotide and predicted amino acid sequences of the guinea pig LHRH with known sequences of human and rat LHRH. RNase protection assays revealed protected bands of the appropriate size. In situ hybridization clearly revealed LHRH mRNA positive neurons in the spatial distribution expected for neuroendocrine LHRH neurons in the guinea pig preoptic area and hypothalamus. Furthermore, the major DNA fragments detected by genomic Southern blot analysis are consistent with the predominance of a single LHRH gene in guinea pig. However, faint DNA fragments were observed using low stringency Southern analysis. These fragments may reflect the presence of an additional LHRH gene that may be expressed in other regions of the guinea pig neuraxis.
Deduced amino acid sequence of guinea pig LHRH
One striking finding of the studies described is the deduced amino
acid sequence of the guinea pig LHRH decapeptide. Whereas, the
molecular architecture of the predicted LHRH prohormone is identical to
the previously described mammalian forms, the LHRH cDNA isolated
encodes a unique amino acid sequence for the guinea pig LHRH
decapeptide compared with all forms identified to date. Specifically,
guinea pig LHRH decapeptide differs from mammalian LHRH by two amino
acid substitutions. The guinea pig form of LHRH decapeptide as reported
in this study, represents the first peptide of the family that does not
have a histidine in the second position. The histidine residue found at
position 2 in all eleven forms of LHRH identified to date is replaced
by tyrosine in the guinea pig. This represents a nonconservative
substitution. In addition, the amino acid present in position 7,
valine, represents a unique substitution with regard to all forms of
LHRH decapeptide recognized to date. This represents a conservative
substitution. Each of these amino acid changes results from a
single-base nucleotide substitution compared with human and rat LHRH
nucleotide sequences.
Conformation energy analysis of the mammalian form of LHRH suggests that the N- and C-terminals of the molecule are maintained in close proximity (11), and that LHRH interacts with it’s receptor in a folded conformation that is facilitated by the 6–7ß turn (12). All of the vertebrate LHRH receptors require the basic conserved NH2- and COOH-terminal sequences of LHRH for receptor binding and activation. The relationship between biological activity, receptor affinity, and residue substitution has been investigated in detail in the mammalian molecule (13). Initial studies of this peptide suggested that the His2, Tyr5, and Arg8 form a functional unit when bound to the receptor. In particular, the size and the charge of the arginine residue are necessary for full biological activity (14, 15, 16). The Arg8 is conserved in the guinea pig LHRH molecule but not the His2. Although the His2 has been conserved in all LHRH species identified to date, as suggested by Sealfon et al. (17) "residue conservation does not invariably imply functional significance." Substitution of His2 with Trp2 in a synthetic LHRH analog has been shown to retain substantial activity at the LHRH mammalian receptor (18). It is not known whether there is a complementary substitution in the guinea pig LHRH receptor gene that accompanies the His substitution at position 2. Future studies will include the cloning of the guinea pig LHRH receptor to examine this possibility.
Using HPLC and RIA, Sherwood and colleagues (19) demonstrated a dominant form of LHRH in guinea pig tissue extracts that eluted in the same fraction as synthetic mammalian LHRH. This form was immunoreactive with antiserum B-6, which is directed toward the last six amino acids of mammalian LHRH, and therefore should detect forms of LHRH decapeptide with alterations at amino acids 2, 3, or 4. The guinea pig LHRH molecule that we described, with predicted substitutions in positions 2 and 7, could have been the major form of LHRH detected in this earlier study. It is not clear whether binding to the antiserum B-6 would be altered by the conservative amino acid substitution in position 7 (Val for Leu). However, studies in our laboratory have demonstrated that guinea pig LHRH binds to antiserum 1076 (generously supplied by R. M. Millar, University of Capetown Medical School, Capetown, South Africa), which recognizes amino acids 4–8 of mammalian LHRH (20), suggesting that the amino acid substitution at amino acid 7 may not effect binding to some antisera.
Two polyadenylation signals in 3' UTR of guinea pig LHRH
A second striking finding of these studies was the presence of two
canonical polyadenylation signals separated by 13 bp in the 3' UTR of
the guinea pig LHRH gene. The 3' UTR of other mammals, including human,
rat, and mouse (21), contain only a single polyadenylation signal.
Moreover, sequence analysis of the positive clones obtained from the
cDNA library revealed the presence of two subspecies of LHRH mRNA in
guinea pig POA-AH extracts, differing only in the length of their 3'
UTR. Two subspecies of LHRH transcripts would be predicted on the basis
of the two polyadenylation sites observed.
The 3' UTR is thought to influence important characteristics of mRNA, including transport, stability, and translational efficiency (22). If two or more polyadenylation signals exist in a mRNA, they may be alternatively used, leading to different-sized mRNA species that encode the same protein, but they may differ in their stability or translatability. In the case of multiple polyadenylation sites, the mechanisms involved in the selection of one poly(A) site over another remain poorly understood.
The results of RNase protection assays revealed that in the guinea pig POA-AH, the levels of the short transcript of LHRH mRNA were more abundant than the levels of the long transcript. This finding could reflect a preferential utilization of the upstream polyadenylation site or could result from a more complex interaction between transcription, transcript termination, and RNA stability. Low levels of LHRH mRNA transcript were detected in extracts from the HYP and pituitary, and in these extracts only the short transcript was detected. These data suggest that there is no obvious tissue-specific differential expression of the two LHRH transcripts.
The identification of two polyadenylation sites introduces the possibility of differential regulation of LHRH transcript length at different times during development or under certain endocrine conditions that could potentially alter the stability of guinea pig LHRH mRNA. Whereas a second polyadenylation signal has not been described in the LHRH gene of other mammals, it has been observed in some species of fish in which the sequence of the LHRH gene has been elucidated (23, 24). There are, however, no reports regarding the functionality of the two polyadenylation signals in fish. The guinea pig may have maintained a second polyadenylation signal that was subsequently lost by other mammals during evolution.
Guinea pig phylogeny
The fact that the deduced sequence of guinea pig LHRH decapeptide
contains two amino acid substitutions is surprising, but at the same
time this is not the first gene described in guinea pigs with amino
acid substitutions. The guinea pig has been of interest to molecular
evolutionists due to the discovery of unusually high rates of amino
acid replacement in several of its proteins such as insulin (25),
glucagon (26), vasoactive intestinal peptide (27) and factor IX
(28).
The phylogenetic position of the guinea pig as a rodent was recently questioned by Gauer and co-workers (29, 30, 31), who suggested that the guinea pig does not belong to the same order as the myomorphs but represents a separate evolutionary lineage that diverged before the separation of the myomorph rodents from the primates and the artiodactyls. If as suggested by Easteal (32) and Li et al. (33) the myomorphs branched off before the carnivores, artiodactyls, lagomorphs, chiropterans, and primates diverged, then the new order would represent one of the earliest divergence events in evolutionary history of the eutherian mammals. The discovery that guinea pig LHRH decapeptide differs from the form of LHRH observed in all mammals studied to date coupled with the identification of a second polyadenylation sequence in guinea pig LHRH mRNA that is not present in other mammals but is present in some species of fish provides evidence consistent with evolutionary separation of guinea pigs from Old World rodents such as rats. It is unknown whether other species that belong to the same suborder as guinea pigs such as chinchillas, caseraguas, degus, etc., also express the unique LHRH form that we have isolated from guinea pigs.
In summary, we cloned a form of LHRH in guinea pig that is unique among vertebrates. In addition, we observed the existence of two LHRH gene transcripts that differ only in the length of the 3' UTR. These two LHRH transcripts may result from the availability of two polyadenylation sites identified in the guinea pig LHRH gene. Future studies will examine the possibility of differential expression of these two transcripts during development and during the luteal and follicular phases of the estrous cycle of guinea pigs and will characterize LHRH gene expression in general during the reproductive cycle. The long-term goal of these studies is to identify mechanisms that regulate LHRH gene expression in a mammal with a true luteal phase.
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Acknowledgments |
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Footnotes |
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Received April 15, 1997.
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