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Department of Clinical Biochemistry (I.J.R., J.F.R., A.H.J.), Rigshospitalet, and Institute of Medical Anatomy (M.M.), Panum Institute, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark
Address all correspondence and requests for reprints to: A. H. Johnsen, Department of Clinical Biochemistry, Rigshospitalet, KB3011, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. E-mail: johnsen{at}rh.dk
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-amidation, and tyrosyl sulfation (3). The biological
activity of both CCK and gastrin relies on the same C-terminal
tetrapeptide, Trp-Met-Asp-Phe-NH2. As a consequence, it has
been hypothesized that gastrin and CCK share a common evolutionary
origin, perhaps via duplication of an ancestral gene (4, 5). Immunochemical studies suggested gastrin and CCK diverged after the appearance of amphibians (4). However, more recent peptide work indicates that both gastrin and CCK were present as separate peptide systems before this stage in evolution (6, 7). In mammalian species, gastrin is readily distinguished from CCK by two means: 1) gastrin is synthesized in the antral mucosa and regulates gastric acid secretion; and 2) gastrin possesses a tyrosine residue at position six (counting from the C-terminus), whereas in CCK a tyrosine is situated in position seven (8). CCK and gastrin peptides have been purified from avian (9, 10), reptilian, and amphibian (6, 7) species, where the antral gastrin peptide contains a tyrosine in position seven and so is structurally more similar to mammalian CCK than mammalian gastrin. This is most pronounced in the bullfrog where gastrin-8 isolated from the antrum has only one amino acid difference to CCK-8 from the small intestine (6, 7). Hence, the classification of gastrin in nonmammalian vertebrates is based primarily on its antral expression (7).
To gain further insights into the evolution of the CCK/gastrin peptide family, we have identified the gastrin and CCK genes, as well as the complementary DNAs (cDNAs) they encode, from the bullfrog Rana catesbeiana. Analysis of gene structure, gene, and preprohormone sequences and expression patterns uncovered striking parallels when comparing CCK/gastrin in the bullfrog to their mammalian counterparts. Notably, this observation extends to secondary sites of CCK/gastrin production, where gastrin gene expression was found in the bullfrog central nervous system, pituitary gland, colon, duodenum, and testes, while CCK messenger RNA (mRNA) was detected in the lung and testes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA and RNA preparation
Genomic DNA was isolated from R. catesbeiana small
intestine using an SDS/proteinase K/phenol extraction procedure
described by Sambrook et al. (11). Total RNA was purified
from various tissues using a guanidinium thiocyanate method (12). mRNA
was obtained utilizing the QuickPrep Micro system (Pharmacia
Biotech, Uppsala, Sweden). Oligonucleotides were constructed using an
Applied Biosystems (Foster City, CA) 392 DNA/RNA synthesizer.
Genomic library construction and screening
Approximately 500 µg of R. catesbeiana genomic DNA
were partially digested with Sau 3AI (Boehringer Mannheim,
Mannheim Germany) and size fractionated on a sucrose gradient (13). DNA
fragments in the size range of 13–22 kb were selected and ligated into
BamHI prepared arms of 
GEM-11 (Promega). Recombinant
phage DNA was assembled into phage particles using ’Gigapack III
Gold’ packaging extracts (Stratagene, La Jolla, CA). An estimated
1 x 106 individual phage clones were obtained. The
R. catesbeiana genomic library was amplified (11) as 16
independent pools and stored at -80 C for further use.
Recombinant phages were mixed with the E. coli host strain
KW251 and plated at a density of approximately 80,000 pfu on 24 x 24
cm Petri dishes. Phage DNA was transferred in duplicate to
nitrocellulose membrane filters (Schleicher & Schuell, Dassel, Germany)
as described by Sambrook et al. (11). The R.
catesbeiana genomic library was screened for the bullfrog gastrin
gene using degenerate oligonucleotide probes, whereas the CCK gene was
pursued with both oligonucleotide and cDNA probes. Oligonucleotides
were labeled with [
-32P]-ATP (Amersham) using
T4 polynucleotide kinase (Pharmacia) as described by
Sambrook et al. (11). Hybridizations were performed
overnight at 60 C in 2 x Denhardt’s solution (0.04% each of
polyvinylpyrrolidone, BSA, and Ficoll 400), 6 x SSC, 0.1% SDS,
0.1 mg/ml yeast transfer RNA (Boeh-ringer Mannheim) and 0.05% Na
pyrophosphate. cDNA probes were radiolabeled with
[-32P]-dCTP by random primed synthesis using therediprime DNA labeling system (Amersham), and hybridizations were
carried out overnight at 60 C in 5 x Denhardt’s solution, 5
x SSC, 0.1% SDS, 200 µg/ml denatured calf thymus DNA (Sigma) and 1
mM EDTA. For both oligonucleotide and cDNA probes, the
filters were washed twice at room temperature in 2 x SSC/0.1%
SDS followed by two 15 min washes in the same solution at 60 C. Filters
were subjected to autoradiography at -80 C on x-ray film (Fuji, Tokyo,
Japan) for varying lengths of time related to signal strength.
PCR
First-strand cDNA for PCR was synthesized from approximately 1
µg of mRNA or 5 µg of total RNA using the Moloney Murine Leukemia
virus reverse transcriptase (Pharmacia). The reaction was primed
using the XSC-dT17 oligonucleotide (Table 1
). All PCR-based procedures were performed in a final
volume of 50 µl containing; 2.5 U Taq DNA polymerase
(Promega), Taq DNA polymerase buffer (Promega), 1.5
mM MgCl2, 5 mM dNTPs, 50 pmol of
each primer and 100 ng of first-strand or double-stranded cDNA. PCRs
were carried out on a Thermal Reactor (Hybaid) with the following
standard cycling conditions: 95 C for 5 min, 64 C for 5 min (1 cycle);
95 C for 30 sec, 64 C for 45 sec, 72 C for 90 sec (30 cycles); 5 min at
72 C (1 cycle).
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3' RACE (rapid amplification of cDNA ends) PCRs were primed with a
sense gene specific primer (Table 1
) and the XSC oligonucleotide (Table 1) designed from the XSC-dT17 oligonucleotide used to prime
cDNA synthesis. Adapter ligated double-stranded cDNA for 5' RACE was
produced using the Marathon cDNA Amplification Kit (Clontech). 5' RACE
was performed with two PCRs, the first used a gene specific primer
(Table 1) and AP1 (Clontech, Palo Alto, CA), the second contained 1
µl of the initial PCR, a nested gene specific oligonucleotide (Table 1) and AP2 (Clontech). For both 3' and 5'RACE experiments the annealing
temperature of the PCRs was 57 C.
Analysis of isolated genomic DNA and cDNA sequences
Recombinant phage DNA clones were mapped by restriction
endonuclease cleavage (performed using Promega enzymes), Southern
blotting, and DNA hybridization using standard procedures (11).
Fragments containing bullfrog CCK/gastrin sequences were subcloned into
pBluescript SK+ or SK- (Stratagene). Products
from 3' and 5' RACE, as well as inverse PCR, were cloned directly into
the pMOSBlue T-vector (Amersham).
Double-stranded plasmid DNA was routinely sequenced by the dideoxy
chain termination method with the Sequenase 2.0 DNA sequencing
procedure (United States Biochemical, Cleveland, OH). In some
instances, DNA was sequenced using the ABI Prism Dye Terminator Cycle
Sequencing Ready Reaction Kit (Perkin-Elmer, Foster City, CA) and the
reactions resolved on an ABI Prism 377 DNA Sequencer (Perkin-Elmer).
All DNA sequences reported here were confirmed by the sequencing of
both DNA strands.
In situ hybridization
Bullfrog brains were perfused with 4% paraformaldehyde in 0.1
M Na/K-phosphate buffer (pH 7.4) for 15 min and postfixed
for 16 h. After cryoprotection in 20% sucrose for 2 days, the
tissues were frozen on dry ice and cut into 15-µm thick coronal or
sagittal cryostat sections and mounted on gelatinized glass slides.
Four brains were used for in situ hybridization, and two
others were counter stained with cresylviolet for neurohistology.
Five picomoles of antisense oligonucleotides specific for either the
R. catesbeiana CCK or gastrin mRNA (Table 1) were 3' end
radiolabeled via the incorporation of 35S-dATP by calf
thymus terminal transferase (Boehringer Mannheim) during a 60-min
incubation at 37 C. The reaction was stopped by the addition 10 volumes
of TE Buffer (10 mM Tris-HCl (pH 7.4), 1 mM
EDTA), followed by phenol/choloform extraction, ethanol precipitation
(11), and the oligonucleotide pellet resuspended in 100 µl of TE
buffer with 1 mM DTT. The specific activity of the
resulting probes were approximately 1 x 1018
dpm/mol.
Before hybridization, frozen tissues were brought to room temperature, fixed for 5 min in 4% paraformaldehyde in PBS (0.5 M NaCl, 0.1 M phosphate buffer (pH 7.4)), followed by two 1-min washes in PBS. The sections were then acetylated in 0.25% acetic anhydride diluted in 0.1 M trienthanolamine/0.9% NaCl for 10 min, dehydrated in a graded series of ethanols (70% (5 min), 80% (1 min), 95% (2 min), 100% (1 min)), delipidated in 100% choloform (5 min), partially rehydrated in 100% and 95% ethanol (1 min each), and allowed to dry.
For hybridization of the cryostat sections, 10 µl of the labeled probe were diluted in 1 ml of hybridization buffer (50% vol/vol formamide, 2 x SSC, 1.25 x Denhardt’s solution, 10% wt/vol dextran sulfate, 10 mM DTT, 500 µg/ml salmon sperm DNA, and 250 µg/ml yeast transfer RNA). Each section was overlayed with 200 µl of the hybridization buffer containing the labeled probe, covered with parafilm and incubated overnight at 37 C in a humid chamber. The next morning, slides were washed four times at 55 C for 15 min in 1 x SSC, followed by a 30-min wash in 2 x SSC at room temperature, and finally rinsed by two washes in distilled water. The slides were then dried and either exposed to x-ray film (Kodak) for 3 weeks or dipped in Hypercoat LM-1 emulsion (Amersham) at 40 C and exposed for 6 weeks at 4 C. The emulsion-coated slides were developed in Amidol (Merck, Darmstadt, Germany) for 5 min and fixed in 30% thiosulphate. After developing of the emulsion-coated slides, the sections were counterstained with cresyl violet.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), designed against the bullfrog gastrin-47
peptide (6). After three rounds of screening, a single positive clone
(FGas12) containing a 13.1-kb insert was isolated. DNA from this
recombinant phage was subjected to restriction enzyme mapping and
Southern blotting and probed with radiolabeled FGIN1 and FGIN2
oligonucleotides. An approximate 1.3-kb ScaI fragment, which
bound the FGIN1 probe and an 850-bp Sau3AI fragment detected
by the FGIN2 oligonucleotide, were subcloned (Fig. 1A).
Sequence analysis showed that together these two pieces of DNA encoded
the entire bullfrog gastrin-47, including basic proteolytic cleavage
sites and an -amidation substrate (Fig. 1B). Consequently, these
sequence data were used to clone the cDNA encoding bullfrog
gastrin.
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) and the XSC oligonucleotide were used in
3' RACE PCR experiments on first-strand cDNA synthesized from bullfrog
antral mucosa. A single 400-bp product was recovered from the 3' RACE
reaction and found to encompass the 3' end of the bullfrog gastrin cDNA
(Fig. 1B). Similarly, 5' RACE PCR was conducted using two
gastrin-specific oligonucleotides, FGASE33' and FGASE23' (Table 1).
After two nested PCRs, a 210-bp fragment containing the 5' end of the
bullfrog gastrin cDNA was uncovered (Fig. 1B). Together, the RACE
clones encompass a 424-bp bullfrog mRNA encoding preprogastrin that is
102 amino acids in length. R. catesbeiana preprogastrin
putatively contains a hydrophobic N-terminal signal sequence and
all the necessary primary sequence requirements for the
posttranslational production of bullfrog gastrin-47 (Fig. 1B).
The R. catesbeiana gastrin cDNA sequence was used to
determine the structure of the gastrin gene, which included the
subcloning of a 5.1-kb EcoRI/SacI fragment
encoding the 5' untranslated exon (Fig. 1). Consequently, the bullfrog
gastrin gene consists of three exons and two introns (Fig. 1A). The
first exon is 28 bp in length, the second exon possessing the ATG
translation start codon is 210 bp, whereas the third exon is 186 bp in
length and contains the stop codon and AATAAA polyadenylation consensus
sequence (Fig. 1).
Cloning of the R. catesbeiana cholecystokinin gene and cDNA
Two degenerate oligonucleotides designed from R.
catesbeiana CCK-69 (7), FCIN1 and FCIN2 (Table 1), were used in
RT-PCR on bullfrog brain first-strand cDNA to generate a 159-bp CCK
cDNA fragment for genomic library screening. In the first instance,
approximately half of the independent clones from the bullfrog genomic
library were screened for the CCK gene. Following three rounds of
screening, two phage clones were obtained (FCCK6 and FCCK7) from
separate library pools; however, they were determined to contain the
same 12.1 kb insert (Fig. 2A). A single 2-kb
HindIII/SacI fragment was found to hybridize with
the partial CCK cDNA probe. Subcloning and sequencing experiments
revealed the HindIII/SacI fragment encoded the
N-terminus, but not the C-terminus, of bullfrog CCK-69 (Fig. 2B).
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) and the XSC
oligonucleotide. The resulting 400-bp product was subcloned, sequenced,
and found to contain the 3' end of the bullfrog CCK cDNA. In turn, two
nested 5' RACE PCRs using the FCCKE33'I and FCCKE33'II oligonucleotides
(Table 1) were performed to isolate a 690-bp fragment encompassing the
5' region of the R. catesbeiana CCK cDNA. In total, the
bullfrog CCK mRNA was determined to be 693 nucleotides in length,
encoding a 130 amino acid preproCCK molecule (Fig. 2B). This
preprohormone possess an N-terminal hydrophobic signal sequence, as
well as all the necessary primary sequence requirements for the
posttranslational maturation of previously isolated bullfrog CCK-69
(Fig. 2B).
The entire bullfrog genomic library was rescreened with the FCCKE13'
oligonucleotide (Table 1), designed from the 5' end of the R.
catesbeiana CCK cDNA sequence. A single recombinant phage clone
with a 13.2-kb insert was obtained (FCCKB1), which includes 6.5 kb
of the bullfrog genome present in clones FCCK6 and FCCK7 (Fig. 2A). A 701-bp BglII fragment was subcloned, sequenced, and
found to code for the 5' end of the CCK gene (Fig. 2). However, despite
numerous efforts using both oligonucleotide and cDNA probes, we were
unable to obtain a genomic clone encoding the 3' end of the bullfrog
CCK mRNA. To circumvent this problem, inverse PCR was performed using
two oligonucleotides, FCCKE35'op and FCCKE33'op (Table 1), designed
from the 3' region of the CCK cDNA sequence. The resulting 1.4-kb
product was sequenced and found to encode the remainder of the bullfrog
CCK cDNA sequence (Fig. 2B). Consequently, like the gastrin gene, the
bullfrog CCK gene consists of three exons, 45, 251, and 394 bp in
length, respectively. Long-range PCR using the Expand Long Template PCR
System (Boehringer Mannheim) was performed in an attempt to
characterize the length of intron 2 of the R. catesbeiana
CCK gene. Unfortunately, these experiments were unsuccessful (data not
shown), but this intron is clearly very large, exceeding 8.1 kb (Fig. 2A).
Comparison of R. catesbeiana CCK/gastrin preprohormone sequences to
those from other species
Alignment of the bullfrog and human preprogastrin shows the most
conserved region of this preprohormone surrounds the C-terminal
bioactive region of the gastrin peptide (Fig. 3).
Analysis of bullfrog and human CCK preprohormones shows a similar
region of conservation; however, this homology extends throughout the
C-terminal flanking peptide (Fig. 3). Comparison of bullfrog gastrin
and CCK to those characterized from other species indicates that
preproCCK sequences have been conserved to a greater degree than
preprogastrin (Table 2). This is partly due to a shift
in the locality of a tyrosine in gastrins from position 7 (from the
C-terminus) in frog and chicken gastrin to position 6, typical of
mammalian gastrins (Fig. 3).
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). In addition, degenerate
oligonucleotides designed from a Xenopus laevis cytoplasmic
actin cDNA, which give rise to a 500-bp R. catesbeiana
product, were used to ensure equal quantities of starting material
(data not shown). Following agarose gel electrophoresis, the PCR
products were transferred to a Nytran membrane and hybridized with
radiolabeled cDNA probes as described in Materials and
Methods. Gastrin cDNA was detected from bullfrog antrum, duodenum,
colon, pancreas, brain, and testes, but not from kidney, liver, lung,
skin, or the distal two-thirds of the small intestine (Fig. 4A). In comparison, CCK expression was observed in the
brain, lung, testes, and throughout the length of the small intestine
(Fig. 4B).
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), with
moderate expression in the neural lobe. In addition, gastrin gene
expression was detected in the brain stem and certain cells of the
tuberal part of the hypothalamus. In contrast, CCK expression was
predominantly found in the optic tectum and certain areas of the brain
stem (Fig. 5B). In each instance, CCK and gastrin expression patterns
in the brain and pituitary gland were confirmed by using two
independent gene specific antisense oligonucleotides.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The proximal promoter region of the bullfrog gastrin and CCK genes were analysed for cis-acting regulatory sequences known to influence the expression of these genes in other species. Even though the transcriptional start sites of the bullfrog CCK and gastrin genes have not been determined experimentally, the 5' untranslated regions of both the RACE PCR products have lengths similar to other members of the CCK/gastrin family (22, 23, 25, 26, 27). Thus is appears that we have identified very close to the entire transcription product. The bullfrog gastrin promoter does not contain an obvious TATA element, which is also lacking in the chicken gastrin promoter (19), but present in mammalian gastrin genes (30). Putative purine-pyrimidine stretches and G/C rich regions, which possibly play a role in the regulation of chicken gastrin gene transcription (19), do not seem to be present in the bullfrog gastrin promoter region. Transgenic mice studies have already shown that significant differences exist in the specifities of the human and rat gastrin promoters (31), indicating considerable species variations in factors regulating gastrin gene expression. In contrast, the R. catesbeiana CCK gene possess at least four highly conserved regulatory motifs present in mammalian CCK genes, namely a TATA box, Sp1 site, AP-1 binding site (also known as a CRE/TRE binding element), and an E-box (32, 33). In fact, the order and spacing of these elements relative to the transcription start site in the bullfrog CCK gene is similar to the human CCK promoter region. Furthermore, the bullfrog CCK promoter contains a second E-box, in between the AP-1 and Sp1 sites, that is also present in the rat CCK promoter (32). Because no CCK-specific elements have been identified in the promoter region of the human CCK gene, it has been hypothesized that complex interactions may occur between the many transcription factors that bind the above motifs, resulting in the developmental- and cell-specific regulation of CCK gene transcription in humans (33). The obvious conserved nature of the Sp1 site, AP-1 site, and E-box DNA binding motifs between mammals and the bullfrog strongly suggests these elements play a crucial role in dictating CCK gene expression patterns in vertebrates.
The deduced preprohormones of both R. catesbeiana gastrin and CCK appear to contain all the necessary primary sequence requirements for the posttranslational maturation of gastrin-47 and CCK-69 previously isolated from this species (6, 7). These features include a hydrophobic N-terminal sequence for directing the prohormone through the secretory pathway, di-basic endoproteolytic cleavage sites, and immediately C-terminal to the bioactive peptide sequence a glycine residue, which acts as an amide donor. Comparison of the preprohormone sequences to some of their mammalian counterparts shows that preproCCK has been more conserved during evolution than preprogastrin. In both cases, the greatest degree of homology is around the C-terminus of the bioactive peptide sequence, although one other notable conserved region is the C-terminal flanking peptide of proCCK. This acidic region, which contains two tyrosine residues in bullfrog proCCK, is almost identical to that seen in human proCCK and is also similar to the C-terminal flanking peptide of preprocionin. Current evidence indicates that sulfation of the two tyrosine residues of proCCK is important for the correct sorting and processing of human CCK (34).
By several criteria, CCK has been more conserved during evolution than gastrin: 1) the position of a tyrosine residue in relation to the C-terminal end of the bioactive gastrin peptide sequence has changed from +7 to +6 in mammals; 2) the number of noncoding nucleotides in exon 2 differs between nonmammalian and mammalian gastrin genes, but not between the CCK genes; 3) proximal regulatory motifs of gastrin genes are less conserved than those seen in CCK genes; and 4) between bullfrog and mammalian sequences the preproCCKs show a higher degree of similarity than do the preprogastrins. Taken together, these features indicate that CCK is phylogentically older than gastrin. Parallels can be drawn to the neuropeptide Y (NPY)/peptide Y (PYY)/pancreatic polypeptide (PP) family. At the amino acid level, NPY is the most conserved and is believed to be the oldest member of this peptide family, whereas pancreatic polypeptide, which has only been found in tetrapods, shows much greater sequence variations than either NPY or PYY (35). However, sequences from several more species will be needed before it can be determined if the same relationship is valid for the CCK/gastrin family.
The tissue distribution of gastrin and CCK gene expression in the
bullfrog was analyzed by RT-PCR. Typical to that seen in other species,
CCK was expressed in the small intestine and brain, whereas gastrin
production was found in the antral mucosa. However, bullfrog gastrin
and CCK gene expression was detected in other tissues, which in mammals
can be considered as secondary sites of gastrin/CCK production.
Firstly, CCK expression was detected in the R. catesbeiana
lung. Cholecystokinin mRNA has been observed in the lung of X.
laevis (18) and small quantities of CCK peptide have been noted in
mammalian lung tissue (36), although its biological significance is
unknown. Both CCK and gastrin gene expression was found in the testes.
Previously, CCK production has been shown in the peripheral regions of
the seminiferous tubules of five mammalian species (37), as well as
gastrin mRNA in the same tissue of humans (38). Consistent with peptide
purification studies (7) gastrin mRNA was detected in the bullfrog
duodenum, as found in mammalian species (39, 40). Furthermore, gastrin
gene expression was observed in the colon of R. catesbeiana.
The rat and sheep colon have been found to express both gastrin and CCK
throughout development, with the highest concentrations of -amidated
gastrin being found in the fetus where it may act as a growth factor
(39, 41). Lastly, bullfrog gastrin gene transcription was also detected
in the pancreas. Again this parallels mammalian gastrin expression
patterns (42, 43, 44), with the majority of gastrinomas being found in the
pancreas (45).
Along with CCK, RT-PCR also detected gastrin expression in the R.
catesbeiana brain. To further investigate the distribution of CCK
and gastrin production in the bullfrog central nervous system and
pituitary gland, in situ hybridization was performed with
mRNA specific radiolabeled antisense oligonucleotides. Completely
distinct areas of the bullfrog brain were found to express the two
genes. In mammals, CCK is the most abundant neuropeptide in the brain
with regards to both quantity and distribution (46, 47) and is involved
in numerous functions including sleep, memory, and anxiety (for review,
see Ref.2). Therefore, it was not surprising to find CCK gene
expression in many areas of the bullfrog brain, present at the highest
levels in the optic tectum and brain stem, a similar pattern to that
observed in X. laevis (18). In contrast, the majority of
bullfrog gastrin expression was detected in the intermediate lobe of
the pituitary gland and the infundibular area of the hypothalamus.
Gastrin peptides have been found in the pituitary gland of all
mammalian species examined, with the intermediate and neural lobes
containing the highest concentrations of -amidated gastrin (48, 49).
Furthermore, SI nuclease mapping of total RNA from porcine tissues
detected gastrin mRNA in the pig pituitary (50). A small quantity of
gastrin peptide has also been found in the porcine hypothalamus (48).
Because the gastrointestinal G- and I-cells produce significantly
greater quantities of gastrin than the CNS and pituitary, it is
believed that the latter tissues would have little influence on
circulating gastrin levels and thereby probably not affect gut function
(51). Consequently, gastrin synthesized in the pituitary gland, and
possibly the hypothalamic region, is more likely to be involved in
local neurotransmitter activity (48). The occurrence of gastrin
production in the pituitary gland and central nervous system of both
amphibian and mammalian species suggests an important function of
gastrin in these tissues that is probably separate from the role of
CCK.
As noted above, the previous identification of two different peptides from distinct organs of R. catesbeiana strongly indicated the presence of two members of the CCK/gastrin family in amphibian species (6, 7). However, this information was overlooked in a recent review, where it was argued that amphibian species possess a single gene encoding a CCK-like peptide (52). Now the present study unequivocally shows that distinct gastrin and CCK genes exist in the bullfrog, R. catesbeiana. Consequently if the CCK/gastrin peptide family arose through a gene duplication, it appears that this event occurred before, or perhaps during, amphibian evolution. In fact, preliminary work in our laboratory has uncovered several CCK-like peptides in both the rainbow trout (Oncorhynkus mykiss) and the spiny dogfish (Squalus acanthias) (53 and Johnsen et al., unpublished observations). Most interestingly, the expression patterns of gastrin and CCK genes appears highly conserved between the bullfrog and mammalian species, strongly suggesting widespread important roles for gastrin outside the antrum and for CCK beyond the small intestine and central nervous system.
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Acknowledgments |
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Footnotes |
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2 Supported by grants from The Danish MRC, The Danish Biotechnology
Program for Signal Peptide Research, The Velux Foundation, The John and
Birthe Meyer Foundation, and The Novo Nordisk Foundation.
Received October 7, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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