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Department of Biology, University of Victoria, Victoria, British Columbia, Canada, V8W 2Y2
Address all correspondence and requests for reprints to: Dr. Nancy Sherwood, University of Victoria, Department of Biology, P.O. Box 1700, Victoria, British Columbia, Canada V8W 2Y2. E-mail: Nsherwoo{at}uvic.ca
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Introduction |
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To understand the evolution of the nervous system, one approach is to
compare the structure and location of neuropeptides in protochordates
and mammals. However, limited structural data exists in protochordates
for genes, complementary DNAs (cDNAs), and proteins with homology to
their vertebrate counterparts. Studies have shown that protochordates
produce neuropeptides that cross-react with antisera raised against
mammalian peptides (4), including LHRH, SS (5), and neurotensin-like
peptides (5). Also, glucagon (6) and 
MSH (7) immunoreactivity have
been detected within the protochordate Ciona intestinalis.
In the same tunicate, cross-reactivity is detected in the nervous
system and alimentary tract with antisera raised against mammalian
insulin (8, 9, 10), neuropeptide Y (5, 11), peptide histidine isoleucine
(12), vasoactive intestinal peptide (VIP), secretin, and pancreatic
polypeptide (5). Evidence relating to immunocytochemical localization,
tissue expression, and nucleotide and amino acid sequences of
protochordate genes and peptides should clarify aspects of the
evolution of the vertebrate nervous system. To date, the sequences for
three protochordate neuropeptides with identity to their mammalian
counterparts have been determined. The first tunicate neuropeptide to
be identified was cionin (13), which is a unique hybrid of
cholecystokinin and gastrin. The other two tunicate neuropeptides were
GnRH-like peptides associated with tunicate neural structures (14, 15).
Mackie (16) showed that immunoreactive GnRH cells form a neural plexus
surrounding the dorsal strand. Determination of the peptide sequence of
two forms of tunicate GnRH made it possible to prepare synthetic forms
of GnRH (17). Injections of these two tunicate GnRH forms resulted in
an increase in the content of estradiol in the gonads (Sherwood,
Rivier, and Mackie; unpublished observation). Hence, a separation of
approximately 600 million yr (18) did not obscure the origin of GnRH,
in that the tunicate GnRH peptides each has a sequence identity (60%)
and a conserved function to that found for mammalian GnRH.
The primary structure of members of the glucagon superfamily, however, has not been identified in protochordates. In humans, the glucagon superfamily is composed of GRF (19, 20), glucagon (21), glucagon-like peptides (GLP-1 and GLP-2) (22), secretin (23), VIP (24), glucose-dependent insulin-releasing polypeptide (GIP) (25), and pituitary adenylate cyclase-activating polypeptide (PACAP) (26, 27). PACAP, the newest family member, is of particular interest because: 1) the nucleotide and amino acid sequence is highly conserved among mammals (26, 27), birds (28), and fish (29); and 2) in addition to its role as a releaser of pituitary hormones, it seems to have a role as a growth factor in the developing nervous system (30, 31) and primordial germ cells (32). It is assumed that the glucagon superfamily members share a common ancestor, based on similar amino acid sequences and intron/exon structure (33, 34). However, the hypothesis that extant superfamily members originated from a common ancestor is speculative, because structural evidence is not available. To investigate the origin of the glucagon superfamily, we used molecular techniques to determine the nucleotide sequence and tissue expression of PACAP within the protochordate Chelyosoma productum. This paper reports the structure of two protochordate cDNAs and two partial genes in which the first gene, pacap1, encodes both a GRF1–27-like peptide and PACAP1–27, whereas the second gene, pacap2, encodes two structurally-related peptides of the glucagon superfamily.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Amplification of tunicate pacap1 cDNA by PCR
DNA was amplified using a cDNA library lysate and the degenerate
primers Tun 1 (5'-cattcggatgggatcttcacggatag) and Tun 2
(5'-catgtttggacagaacacaacgtgagcg). First round amplification was done
in a 50-µl vol reaction that contained 0.2 µg cDNA, 5U Taq, 1
x Taq buffer (Pharmacia, Baie d’urfé, Québec, Canada),
200 mM dNTPs (2'-deoxynucleoside-5'-triphosphates), 2
mM MgCl2, 20 pmol of primers Tun 1 and Tun-2
with 40 cycles at 94 C for 1.5 min, 45 C for 2 min, and 72 C for 2.5
min. PCR reactions were electrophoresed on a 2% agarose gel. The cDNA
in the bands from the gel was cloned into pBluescript KS+ (Stratagene),
electroporated into XL-1 Blue (MRF’) cells and prepared for sequencing
with an alkaline hydrolysis method (36). Both strands of the plasmid
were sequenced with [-35S] dATP
(2'-deoxyadenosine-5'-triphosphate) using the chain termination method
(37) with Sequenase 2.0 (US Biochemical Corp., Cleveland, OH) and Vent
(exo-) (New England Biolabs, Beverly, MA). All sequencing gels were 6%
polyacrylamide/7 M urea wedge gels, dried under vacuum at
80 C and exposed to Kodak XAR-5 film for 12–24 h.
Tissue assay by RT/PCR
mRNA was isolated from the following tissues: neural gland,
dorsal strand/neural ganglion, gonad, gonad/digestive gland, intestine,
heart, tunic, and branchial basket. Single-stranded cDNA was
synthesized from 0.5 µg mRNA for each tissue, using 200 U avian RT
(H- Superscript RT, Gibco-BRL, Burlington, Ontario,
Canada), 10 mM dithiothreitol, 1 mM each dNTP,
160 U RNA guard, 2 mM oligo dT20, and 1 x
H- RT buffer, to a total reaction vol of 20 µl. The
reaction proceeded for 90 min at 41 C, followed by 5 min at 90 C. DNA
amplifications were done in a 50-µl vol that contained 0.5 µg cDNA,
1 x Taq buffer (Pharmacia), 200 µM dNTPs, 1.5
mM MgCl2, and 20 pmol of each primer. The
pacap-specific primers were Tun 3,
(5'-tacactggattgtcttggccgcc) and Tun 4 (5'-cgctcagcatgagttctgtc). The
vip-specific primers were Tun 5 (5'-gacggtaacgattcttatgc)
and Tun 6 (5'-gcctaacagatagcctagtc). All reagents, except the Taq
enzyme, were mixed, layered with mineral oil, and heated to 95 C for 5
min. The mixture was frozen rapidly in a dry ice/ethanol bath and Taq
DNA polymerase (5U, Promega) was added. The tubes were replaced in the
preheated 95 C thermal cycler where 40 cycles at 95 C for 1 min, 55 C
for 2 min, and 74 C for 1.5 min were completed. Tubulin PCR
amplifications were in a 50-µl reaction that contained 0.5 µl cDNA
from each tissue, 5U Taq DNA polymerase, 1 x Taq buffer
(Pharmacia), 200 µM each dNTP, 3 mM
MgCl2, and 20 pmol of each tubulin primer. The reactions
were carried out for 35 cycles of 94 C for 1 min, 55 C for 1.5 min, and
72 C for 1.5 min.
Screening of cDNA library
A total of 5 x 104 pfu from the tunicate
library was screened. Duplicate nylon membrane (Bio-Rad, Mississauga,
Ontario, Canada) lifts were prehybridized at 50 C in 6 x SSC,
5 x Denhardt’s solution, and 0.5% SDS for 4 h. The
hybridization solution consisted of 6 x SSC and 0.5% SDS, to
which the probe was added. The probe was a 163-bp product amplified by
PCR with primers Tun-1 and Tun-2. The probe was labeled with
[-32P]dCTP (7.8 x 106 cpm/ml), and
the membranes and the probe were incubated at 50 C overnight. The
membranes were washed under high stringency (0.1 x SSC/0.1% SDS)
for 50 min at 65 C, then exposed to Kodak XAR-5 film for 5 days at -80
C. Isolated single positive clones were cored and inserts rescued with
in vitro excision.
Genomic DNA amplification
Tunicate genomic DNA of high molecular weight was digested with
proteinase K (Sigma), followed by repeated phenol:chloroform:isoamyl
alcohol (24:24:1) washes. The extract was dialyzed against 0.01
M Tris-HCl/EDTA. Primers (Tun 3 and 4) to the 5'
untranslated region (5'UTR) and 3'UTR of the tunicate pacap
cDNA or primers Tun 5 and 6 to the vip cDNA were used in the
amplification of the tunicate genes. Amplifications were done in a
50-µl vol (1.0 µg DNA, 5U Taq, 1 x Taq buffer, 200
µM dNTPs, 2 mM MgCl2, 20 pmol of
each primer) with 40 cycles at 94 C for 1.5 min, 45 C for 2 min, and 72
C for 2.5 min. The reaction was electrophoresed through a 1.5% agarose
gel. A band was cloned into pBluescript KS+ (Stratagene); the plasmid
and insert were electroporated into XL-1 (MRF’) competent cells; and
the extracted DNA was prepared for sequencing with an alkaline
hydrolysis method (35).
Zoo blot and Southern analysis
For the zoo blot, DNA (10 µg) was prepared from rat
(Rattus norviegus), starling (Sturnus vulgarus),
chicken (Gallus domesticus), alligator (Alligator
mississippiensis), Pacific salmon (Oncorhynchus nerka),
catfish (Clarias macrocephalus), reedfish
(Calamoichthys calabaricus), tunicate (Chelyosoma
productum), urchin (Strongylocentrotus purpuratus),
Drosophila (Drosophila melanogaster), yeast
(Saccharomyces cerevisiae), and bacteria [Escherichia
coli (E. coli)]. DNA for the zoo blot and Southern
blot (tunicate DNA only) were digested to completion with
EcoRI and electrophoresed in a 0.8% agarose gel. The DNA
was transferred, as suggested by the manufacturer (Bio-Rad), for the
alkaline Zeta-Probe GT membrane. Prehybridization was in 7% SDS, 0.5
M NaH2PO4, and 1 M EDTA
at 65 C for 15 min. Hybridization was overnight (14 h) at 65 C in fresh
prehybridization solution plus the 163-bp probe labeled with
[-32P]dCTP. The hybridized membranes were rinsed with
5% SDS, 40 mM NaHPO4, and 1 mM
EDTA and then washed for 45 min at 45 C with fresh solution. The wash
solution was then changed to 1% SDS, 40 mM
NaHPO4, and 1 mM EDTA and washed twice for 45
min at 65 C with fresh solution. After washing, the membrane was sealed
in plastic and exposed at -80 C for 8 days to Kodak BIOMAX (Rochester,
NY) film.
In situ hybridization of tunicate pacap1 and pacap2 mRNA
Localization of the tunicate pacap1 or
pacap2 mRNA in sections from the neural ganglion of
Cheylosoma productum was done by in situ
hybridization using a digoxigenin (DIG)-labeled tunicate
pacap1 or pacap2 cRNA probe. All RNA probes were
synthesized, purified, and tested in accordance with the
manufacturer’s (Boehringer Mannheim, Laval, Québec, Canada)
instructions. The changes in protocol for fixation, prehybridization,
and hybridization are listed below. The tunicate neural gland and
ganglia were dissected and pinned on Sylgard-coated dishes and fixed
for 3 h in 4% paraformaldehyde in PBS (pH 7.4). The fixed tissues
were washed in PBS and soaked overnight in 30% sucrose. Tissue was
embedded in Tissue-Tek O.C.T. compound (Miles Inc., Elkhart, IN), then
sectioned (10 µm), and allowed to dry on
poly-L-lysine-coated slides. Sections were fixed again in
4% paraformaldehyde for 5 min, washed three times in PBS (5 min each),
and placed for 10 min in 2 x SSC. Prehybridization was in 2
x SSC for 2 h at room temperature. This solution was exchanged
for the hybridization solution that consisted of 2 x SSC with a
DIG-labeled RNA probe diluted 1:200. The hybridization solution was
incubated overnight at 42 C. The sections were washed with SSC
(0.5 x SSC) followed with 2% normal goat serum in TBS buffer for
30 min at room temperature. The remaining steps, involving the anti-DIG
antibody and the substrate detection, were performed in accordance with
the manufacturer’s (Boehringer Mannheim) instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Encoded within
the pacap cDNA is another peptide that could be a GRF-like
peptide (59% identity to human GRF) or tunicate glucagon (67%
identity to human glucagon). However, with GRF as the 5' adjacent
peptide in preproPACAP in birds and fish, the peptide in the same
position in tunicates is most likely a GRF-like peptide and not
glucagon. Another clone, distinct from the pacap clone, was
isolated from the tunicate cDNA library (Fig. 1B). This clone,
pacap2, also encodes a PACAP-like peptide but is not the
same as the pacap1 cDNA clone, because of nucleotide changes
within the exons and differences in the exon/intron borders of the two
isolated genes.
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).
The remaining 5 phage DNA clones that hybridized to the probe were
purified, and 1 encoded a different cDNA. The latter clone is referred
to as tunicate pacap2 cDNA, because it has 1 exon with 85%
sequence identity to human PACAP1–27; another exon has
59% identity with human GRF1–27 and 63% identity with
human glucagon (Fig. 1B). Therefore, tunicates contain 2 different
mRNAs; both encode tunicate PACAP1–27, named because of
the high identity to the mammalian PACAP1–27 protein. In
addition, the 2 exons in each clone have identity with other family
members: the PACAP exons have 67% identity with VIP, and the adjacent
exons have 63–67% identity with glucagon.
The tunicate pacap1 cDNA clone was 507 bp long and encodes a
signal peptide, a GRF-like peptide1–27 and
PACAP1–27; no other proteins are encoded (Fig. 1A). The
tunicate cDNA does not encode the longer version of
PACAP1–38 or the GRF-like peptides of 43–46 amino acids
that are found in vertebrates.
The tunicate pacap2 mRNA was 883 bp and encoded a signal
peptide, possibly a cryptic peptide, and 2 peptides with identity to
members of the glucagon superfamily (Fig. 1B). Encoded within the
tunicate pacap2 cDNA sequence, but not found in the tunicate
pacap1 cDNA, are 159 nucleotides that encode a 53-amino-acid
cryptic peptide, if the first ATG codon is assumed to be the correct
start site. However, if the 3rd ATG start site is correct, then a
cryptic peptide does not exist, but the 5'UTR is longer. The tunicate
peptide encoded immediately after the cryptic peptide has amino acid
identity (33–63%) to all superfamily members in humans.
The two cDNAs are similar in sequence. The nucleotides in the coding regions for the tunicate PACAP1–27 and the corresponding region of the pacap2 gene are 93% identical, that is, only six bases are different. Identity of the tunicate PACAP amino acids to other members of the glucagon superfamily ranges from 96% with human PACAP to only 19% with human glucose-dependent insulin-releasing polypeptide (GIP).
Isolation of the tunicate pacap1 and pacap2 genes
The partial tunicate pacap1 gene isolated was
1590 bp long and encoded three exons, as deduced from the cDNA (Fig. 2). Located on the 1st exon is the 5'UTR and the signal
peptide. The 2nd exon encodes GRF-like peptide, and the 3rd exon
encodes PACAP1–27. No exon encoding a cryptic peptide was
found between the signal and the bioactive peptides in the 14 cDNA
clones isolated. In addition, a partial tunicate pacap2 gene
of 1435 bp was isolated and, as deduced from its cDNA clone, contains 4
exons (Fig. 3A). Within the 1st intron of the
pacap1 gene, nucleotides exist (position 313–442) that have
a high identity (93%) with the encoded pacap2 cryptic
segment (Fig. 3B). The 4 exons contain nucleotides encoding a signal
peptide, possibly a cryptic peptide, 2 peptides that are similar to
tunicate GRF1–27 and PACAP1–27, and a 3'UTR.
Whether the tunicate pacap2 gene encodes a cryptic peptide,
depends on the translation start site. The nucleotides encoding the
cryptic peptide have high sequence identity to nucleotides within
intron 1 of the pacap1 gene. However, corresponding
exon/intron splice sites (exon/gt–intron–ag/exon) are not found in
the pacap1 gene.
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). If the first
ATG is the correct start site, the prepropeptide is as described above,
and a cryptic peptide is encoded. The second ATG site would mean that
the 5'UTR is longer and the cryptic peptide is shorter than in the
first prepropeptide. The third ATG site would result in the longest
5'UTR of the three cDNAs and loss of a cryptic peptide, compared with
the other possible preprohormones. In contrast, there is only one
possible ATG codon as a start site for the pacap1
prepropeptide (Fig. 2).
Tissue expression of tunicate pacap1 and pacap2 mRNA by a PCR
method
A sensitive PCR detection method for the presence of
tunicate pacap1 and pacap2 in various tissues was
developed. For each cDNA clone, primers Tun 3 and 4 and Tun 5 and 6
(Fig. 1, A and B) were designed for the 5' and 3' untranslated regions.
These regions were distinct and allowed the specific detection of
either pacap1 or pacap2 mRNA/cDNA. Reverse
transcribed cDNA of various tunicate tissues was amplified with
clone-specific primers, and the results are shown in Fig. 4, A and B. Tunicate pacap1 mRNA was detected
specifically in the neural ganglion but not in the reaction containing
the tunicate neural gland, gonad, gonad/digestive gland, intestine,
heart, branchial basket, negative control, or the reaction containing
the tunicate pacap2 clone. Tunicate pacap2 mRNA
was detected (Fig. 4B) in the neural ganglion, dorsal strand,
intestine, and the reaction containing the tunicate pacap2
cDNA (positive control). Bands were not detected in the lanes
containing the mRNA/cDNA from the neural gland, gonad, gonad/digestive
gland, heart, branchial basket, negative control, or the lane
containing the tunicate pacap1 clone.
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, A and B). Both mRNAs were localized in cortical
cells of the neural ganglion (Fig. 5, C and E). Hybridization was not
seen, using the sense RNA probe (Fig. 5D and F). The in situ
hybridization evidence confirms the presence of both tunicate mRNAs in
the cells of the neural ganglion.
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).
This 163-bp probe corresponds to the nucleotides encoding for tunicate
PACAP. This probe not only has high sequence identity to all known
pacap cDNAs, but may hybridize to other family members such
as VIP. The tunicate 163-bp probe detected two bands in all lanes
except the Drosophila, yeast, and E. coli, where
bands were not detected. The two bands produced in the zoo blot may be
caused by the conservation of the pacap and vip
nucleotides; both pacap and vip are single-copy
genes, at least in mammals, or the probe may have hybridized to the
pacap1 and pacap2 genes in tunicates. In Fig. 7, tunicate DNA was digested to completion with
EcoRI, and each lane was probed with one of four different
cDNA probes. Lane A was probed with the 163-bp pacap PCR
cDNA fragment and, because of a high identity between the two clones,
is thought to have hybridized to both the pacap2 and
pacap1 genomic sequences. Lane B was probed with a tunicate
pacap1-specific probe (bases 387–531) that hybridized only
to pacap1 genomic sequences. Specific hybridization to the
tunicate pacap2 gene was obtained with a
pacap2-specific probe (bases 713–907). In lane D, a
clone-encoding tunicate pacap2 cDNA was used, and as
expected, hybridization to both tunicate pacap1 and
pacap2 occurred. Therefore, the two bands can be identified
as pacap1 (5-kb band) and pacap2 (7.5-kb band)
when digested with EcoRI.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and partial gene (Fig. 2) that encodes
PACAP1–27. This finding is striking, in that the deduced
amino acids from the tunicate pacap1 have 96% (26/27)
identity, in comparison with the human and five other
PACAP1–27 peptide sequences. The amino acids and
nucleotides that encode PACAP have been maintained for over 600 million
years (Fig. 8), a degree of identity unknown for any
hormone of comparable size. PACAP was initially isolated and purified
by Miyata (38) as a 38-amino-acid peptide. In the following year,
another shorter form was found to be identical, in comparison with the
first 27 amino acids of PACAP1–38 (39). A cDNA-encoding
PACAP has been isolated from human (26, 27), sheep (26), rat (40),
chicken (28), salmon (41), and catfish (29) brains, and interestingly,
all PACAP peptides isolated have an amino acid sequence that is highly
conserved. The isolation of tunicate pacap1 cDNA and partial
gene is the first report of any superfamily member to be isolated from
an invertebrate, and the deduced amino acids encode a peptide that is
virtually identical to the vertebrate PACAP peptides.
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) and RT/PCR (Fig. 4) methods, show
that tunicate pacap1 cDNA is expressed specifically in the
neural ganglion. Therefore, the role of PACAP in protochordates is more
likely to be that of a factor for cellular proliferation or growth than
a releaser of pituitary hormones.
Tunicate pacap1 and pacap2 genes are related to other family
members
A feature of the tunicate pacap1 and pacap2
genes is the high sequence identity found between the encoded peptides.
The tunicate pacap2 mRNA encodes amino acids that have high
sequence identity to the tunicate PACAP peptide (89%) and, in
addition, to the human PACAP peptide (85%) (Fig. 9, A
and B). This similarity is indicative of a gene duplication of the
parent pacap gene, resulting in two tunicate genes. We argue
that a duplication allowed one of the two genes, presumably
pacap2, to evolve into a distinct gene, encoding related,
but different peptides.
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Exon and gene duplication produced two tunicate genes
The identity shared between exons on the same gene suggests exon
duplication occurred before the gene duplication (Fig. 9B). Within the
pacap2 gene, the similarity of amino acids between exons 3
and 4 suggest an exon duplication: the nucleotides encoding exons 3 and
4 are similar in length, the nucleotides are 57% identical, and the
encoded amino acids have 48% identity. This data from the
pacap2 gene is the best proof to date to confirm the
speculation that the two exons originated by an exon duplication. Based
on amino acid identity, we argue that exon duplication preceded the
gene duplication (Fig. 9B).
pacap1 gene organization provides insight in glucagon superfamily
evolution
Examination of the pacap2 gene shows how the
next stage of gene organization may have evolved. The second exon of
pacap2 seems to have resulted from the addition of splice
sites within intron 1 of pacap1 gene (
Figs. 1–3). This
would explain the origin of the cryptic peptide in the tunicate gene,
in that the nucleotide coding is present in the intron and requires
only a few base changes to create an intron/exon splice site on each
side, resulting in a new exon (Fig. 3B). With the exception of the
human grf gene, where a recent report shows that the cryptic
peptide has a role in stimulating sertoli cell activity (45), the true
function of the cryptic peptides is not known for any member of the
superfamily. An additional exon seems to have occurred after the
divergence of the pacap2 and pacap1 genes.
Alternatively, the exon encoding the cryptic peptide may have been
present before the protochordates evolved, but changed from an exon to
an intron in the tunicate pacap1 gene because the
splice-site nucleotides were altered. Within both tunicate genes
reported here, consensus intron/exon splice sites are present at all
intron/exon boundaries for proper intron removal.
This lack of an exon within the pacap1 gene is interesting because it demonstrates the evolution of an exon in relation to an intronic sequence. The ancestral gene is assumed to have contained a nucleotide sequence that is either being incorporated or lost as an exon. Depending on the translation start site, the exon encodes a cryptic peptide or 5'UTR. It was speculated by Campbell and Scanes (33) that the ancestral gene giving rise to the present day family contained only three exons. One possibility is that the tunicate pacap1 gene is derived from such an ancestral gene, because it contains only three exons.
Conservation of PACAP may extend to taxons predating tunicates
A feature of the many pacap genes is the
conservation not only of the amino acids, but also the nucleotides
encoding PACAP. The nucleotides that encode tunicate PACAP are highly
conserved, 90% (73/81), in comparison with human
PACAP1–27 (Fig. 8). Further evidence of
pacap’s nucleotide conservation is shown by hybridization
of the tunicate pacap probe within the zoo-blot. Using the
tunicate pacap1 PCR fragment as a probe, we were able to
detect a pacap gene in rat, starling, chicken, alligator,
salmon, catfish, tunicate, reedfish, and sea urchin. Also, conservation
of the chordate pacap gene sequence is suggested by the
conservation of the restriction enzyme sites (EcoRI) used
for the zoo-blot (Fig. 6) and the migration of the PACAP fragments at
approximately the same position. Among the species represented in the
zoo blot, conservation for the pacap cDNA sequences among
the untranslated regions, both 3'UTR (41) and 5'UTR (46), has been
reported. However, the extent of PACAP’s sequence conservation between
species that are separated by 700 million years of evolution was
unexpected. No other known hormone of comparable size has such high
sequence conservation, which is even more intriguing because the basic
function of PACAP is still speculative.
The other band that hybridized to the probe in the lanes containing the
vertebrate DNA may be the vip gene, because the tunicate
pacap probe has a high degree of identity to vertebrate VIP.
However, because the lane containing the sea urchin DNA (lane I) also
had two bands, it would be expected that sea urchins would have
pacap1 and pacap2 genes similar to the tunicate
genes. Echinoderms branched from a stem line that led to vertebrate
evolution about 100 million years before tunicates. It is possible that
sea urchin pacap1 and pacap2 genes have a higher
degree of sequence identity than found in tunicates, provided there has
not been a high number of substitutions in these genes between the
present-day and ancestral sea urchins. One can speculate that some
organisms, evolving before the sea urchins, contain a single parent
pacap gene (Fig. 9).
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Acknowledgments |
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Footnotes |
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Received October 30, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 J C R Cardoso, M S Clark, F A Viera, P D Bridge, A Gilles, and D M Power The secretin G-protein-coupled receptor family: teleost receptors J. Mol. Endocrinol., June 1, 2005; 34(3): 753 - 765. [Abstract] [Full Text] [PDF]
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J C R Cardoso, D M Power, G Elgar, and M S Clark Duplicated receptors for VIP and PACAP (VPAC1R and PAC1R) in a teleost fish, Fugu rubripes J. Mol. Endocrinol., October 1, 2004; 33(2): 411 - 428. [Abstract] [Full Text] [PDF]
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 I. Harfi, S. D'Hondt, F. Corazza, and E. Sariban Regulation of Human Polymorphonuclear Leukocytes Functions by the Neuropeptide Pituitary Adenylate Cyclase-Activating Polypeptide after Activation of MAPKs J. Immunol., September 15, 2004; 173(6): 4154 - 4163. [Abstract] [Full Text] [PDF]
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 S. L. Gray, K. J. Cummings, F. R. Jirik, and N. M. Sherwood Targeted Disruption of the Pituitary Adenylate Cyclase-Activating Polypeptide Gene Results in Early Postnatal Death Associated with Dysfunction of Lipid and Carbohydrate Metabolism Mol. Endocrinol., October 1, 2001; 15(10): 1739 - 1747. [Abstract] [Full Text] [PDF]
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 M. Lee, V. Lelievre, P. Zhao, M. Torres, W. Rodriguez, J.-Y. Byun, S. Doshi, Y. Ioffe, G. Gupta, A. E. de los Monteros, et al. Pituitary Adenylyl Cyclase-Activating Polypeptide Stimulates DNA Synthesis But Delays Maturation of Oligodendrocyte Progenitors J. Neurosci., June 1, 2001; 21(11): 3849 - 3859. [Abstract] [Full Text] [PDF]
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 N. M. Sherwood, S. L. Krueckl, and J. E. McRory The Origin and Function of the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)/Glucagon Superfamily Endocr. Rev., December 1, 2000; 21(6): 619 - 670. [Abstract] [Full Text]
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D. Vaudry, B. J. Gonzalez, M. Basille, L. Yon, A. Fournier, and H. Vaudry Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: From Structure to Functions Pharmacol. Rev., June 1, 2000; 52(2): 269 - 324. [Abstract] [Full Text] [PDF]
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D. Alexandre, Y. Anouar, S. Jegou, A. Fournier, and H. Vaudry A Cloned Frog Vasoactive Intestinal Polypeptide/ Pituitary Adenylate Cyclase-Activating Polypeptide Receptor Exhibits Pharmacological and Tissue Distribution Characteristics of Both VPAC1 and VPAC2 Receptors in Mammals Endocrinology, March 1, 1999; 140(3): 1285 - 1293. [Abstract] [Full Text]
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M. Montero, L. Yon, K. Rousseau, A. Arimura, A. Fournier, S. Dufour, and H. Vaudry Distribution, Characterization, and Growth Hormone-Releasing Activity of Pituitary Adenylate Cyclase-Activating Polypeptide in the European Eel, Anguilla anguilla Endocrinology, October 1, 1998; 139(10): 4300 - 4310. [Abstract] [Full Text] [PDF]
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A. O. L. Wong, M. Y. Leung, W. L. C. Shea, L. Y. Tse, J. P. Chang, and B. K. C. Chow Hypophysiotropic Action of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in the Goldfish: Immunohistochemical Demonstration of PACAP in the Pituitary, PACAP Stimulation of Growth Hormone Release from Pituitary Cells, and Molecular Cloning of Pituitary Type I PACAP Receptor Endocrinology, August 1, 1998; 139(8): 3465 - 3479. [Abstract] [Full Text] [PDF]
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