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in the Small Spotted Catshark (Scyliorhinus canicula)1
*
Department of Zoology, University of Aberdeen, Aberdeen, United Kingdom; and
Sars International Center for Marine Molecular Biology, Bergen, Norway
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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gene is presented that contains a short 5' untranslated region
(54 bp), a 903-bp open reading frame, a 379-bp 3' untranslated region,
a polyadenylation signal, and eight mRNA instability motifs. The
predicted translation (301 amino acids) has highest identity to trout
IL-1 (31.7%), with greatest homology within the putative 12
-sheets. The IL-1 family signature is also present, but there is no
apparent signal peptide. As with other nonmammalian IL-1 sequences,
the IL-1-converting enzyme cut site is absent. Expression of the
IL-1 transcript is detectable by RT-PCR in the spleen and testes,
induced in vivo with LPS. Furthermore, a 7-fold increase of transcript
levels in splenocytes incubated for 5 h with LPS was seen. The
genomic organization comprises six exons and five introns with highest
homology seen in exons encoding the largest amount of secondary
structure per amino acid. Southern blot analysis suggests at least two
copies of the IL-1 gene or genes related to the 3' end of the
IL-1 sequence are present in the catshark. The cloning of IL-1 in
S. canicula, the first cytokine sequenced within
cartilaginous fish, verifies previous bioactivity evidence for the
presence of inflammatory cytokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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45 families of sharks and their
relatives were known to have lived during the late Palaeozoic and
Mesozoic eras. Members of the class Chondrichthyes still in existence
have been shown to be derived from Palaeozoic ancestors and can be
divided into two subclasses: the elasmobranchs (sharks and rays) and
the holocephalans (chimaeras). Modern elasmobranchs (the Neoselachii)
radiated particularly during the Jurassic and Cretaceous periods
to give 760 species present today. It has been suggested that this
radiation was in response to the appearance of abundant sources of
food, which was brought about by the radiation of the actinopterygian
bony fish (1). Present-day elasmobranchs possess a complex immune system typical of all jawed vertebrates (2), and the continued study of this group will give a valuable insight into the origins of specific immunity. Elasmobranchs also possess a variety of nonspecific mechanisms, which act as a first line of defense. The first antimicrobial agent to be identified was squalamine (3), a steroid with broad-spectrum antibiotic activity. It may serve as a systemic antimicrobial agent in elasmobranchs and has been shown to act against bacteria such as Escherichia coli, strains of Staphylococcus and Streptococcus, and fungi (4). Lysozyme activity has also been detected in shark leukocyte lysates (5).
The initiation and regulation of these elasmobranch immune
responses is likely to require intercellular signaling molecules,
cytokines, as seen in mammals. Recently a cytokine was sequenced in
Agnathans and identified as a member of the chemokine family, having
significant homology with IL-8 (6). Many cytokine genes
have also been isolated from different species of bony fish over the
last few years. For example, in rainbow trout, two IL-1 genes
(7, 8), three isoforms of TGF- (9, 10, 11),
two TNF-
genes (12), and several chemokines and their
receptors (13, 14) have been cloned and sequenced. In
cartilaginous fish no cytokine or cytokine receptor genes have been
cloned or sequenced to date. The discovery of cytokine homologs
within both jawless fish and bony fish suggests the existence of these
molecules in cartilaginous fish.
IL-1 is a primary regulator of inflammatory and immune responses in
mammals (15). Until recently this important cytokine had
only been sequenced in mammals. Over the last few years bony fish and
amphibian IL-1 has been extensively studied, and a number of regions
conserved throughout vertebrates were identified (16, 17).
Previous work has provided evidence for the presence of inflammatory
cytokines in elasmobranchs (18, 19). In this study,
primers designed to these conserved regions of IL-1 were used for
the homology cloning of the small spotted catshark (Scyliorhinus
canicula) IL-1 gene, the first cytokine gene sequenced in a
cartilaginous fish.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sexually mature male S. canicula weighing 900-1000 g were obtained from the North Sea around Aberdeen, U.K. Adults were maintained in recirculating artificial saltwater (Peacock, Glasgow, U.K.) at 9–10°C and were fed squid three times weekly.
cDNA production
Splenocytes were isolated from individual animals and
suspended in L-15 medium (Life Technologies, Paisley, U.K.)
adjusted to cartilaginous fish osmolarity (20, 21),
containing 0.2 M NaCl (Sigma-Aldrich, St. Louis, MO), 0.35 M urea
(Sigma-Aldrich), 10% FCS (Life Technologies), 100 µg/ml streptomycin
(Life Technologies) and 100 U/ml penicillin (Life Technologies). The
cells were incubated at 25°C and used unstimulated or after
stimulation for 4 h with LPS from E. coli, serotype 0127:B8
(Sigma-Aldrich) at 10 µg/ml, a concentration known to be
stimulatory for IL-1 expression in trout leukocytes
(7). Total RNA was then isolated using RNAzol (Biogenesis,
Poole, U.K.) following the manufacturer’s instructions and poly(A)
mRNA was purified using an Oligotex mRNA kit (Qiagen, Valencia, CA).
cDNA was synthesized from 2 µg of mRNA by Moloney murine leukemia
virus reverse transcriptase (Life Technologies) at 42°C for 50
min with oligo(dT) primer (Life Technologies) and used as a template
for PCR.
Cloning and sequencing
Initially, PCR was performed using the cDNA prepared above, with
primers universal-F17 and -R (Table I
;
Fig. 1) that were designed to conserved
regions of all known IL-1 sequences, to allow isolation of the
S. canicula IL-1 gene. Obtained PCR products were ligated
into the pGEM-T Easy vector (Promega, Madison, WI). Following
transfection into competent E. coli cells, recombinants were
identified through red-white color selection when grown on MacConkey
agar (Sigma-Aldrich). Plasmid DNA from at least three independent
clones was recovered using an alkaline lysis-based method
(22) and sequenced using an ABI 377 Automated Sequencer
(Applied Biosystems, Foster City, CA). Sequences generated were
analyzed for similarity with other known sequences using the
FASTA (23) and basic local alignment search
tool (24) suite of programs. Direct comparison
between cDNA sequences was performed using the GAP program
(25) within the Wisconsin Genetics Computer Group Sequence
Analysis Software Package (version 10.0) and multiple sequence
alignments generated using CLUSTAL W (version 1.74) (26).
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sequence the
5' and 3' ends were obtained by RACE-PCR, using gene-specific primers
(Table I
). In 3' RACE-PCR, cDNA was transcribed from poly(A) mRNA using
an oligo(dT) adapter primer. PCR was performed with a S.
canicula IL-1-specific forward primer (dogfish-F1) and the
adapter primer and further seminested with a second S.
canicula IL-1-specific forward primer (dogfish-F2) and the
adapter primer (Fig. 1). In 5' RACE-PCR, cDNA was transcribed from
poly(A) mRNA using an oligo-dT primer (Life Technologies), treated with
E. coli RNase H (Promega), purified using a PCR Purification
kit (Qiagen), and tailed with poly(C) at the 5' end with terminal
deoxynucleotidyl transferase (TdT; Promega). PCR was performed
initially with a S. canicula IL-1-specific reverse primer
(dogfish-R1) and the oligo(dG) primer and further seminested with a
second S. canicula IL-1-specific reverse primer
(dogfish-R2) and the oligo(dG) (Fig. 1). S. canicula
IL-1-specific reverse primers (dogfish-R3 and -R4) were used to
fully sequence the amplified product.
Finally, phylogenetic analysis was performed on the full-length amino
acid sequences of the known IL-1 molecules using the
neighbor-joining method (27) and was drawn using the
TreeView program v1.6.1 (28), and confidence limits were
added (29). Predicted amino acid sequence was analyzed by
SignalP v1.1 (30) and the hydrophobicity profile was
determined using Kyte and Doolittle plots (31).
Expression studies
RT-PCR with primers IL-1SC-F and IL-1SC-R
(Table I) was performed with cDNA from a range of tissues (peripheral
blood leukocytes, brain, gill, heart, kidney, Leydig organ, liver,
muscle, spleen, testes) isolated 24 h following peritoneal
injection of S. canicula with 200 µg of LPS. cDNA was also
produced from tissues taken from noninjected S. canicula and
used as a control. After isolation of total RNA (as described above), 5
µg was reverse-transcribed to cDNA. Primers for S.
canicula -actin (GenBank accession no. AJ312004) were
used as a positive control for RT-PCR (Table I), because the gene is
expressed constitutively in the tissues examined. Tissue expression was
studied in three noninjected and injected S. canicula.
Northern blot analysis was also performed on total RNA from splenocytes
stimulated in vitro at 25°C for 0, 1, 2, 3, 4, and 5 h with 10
µg/ml LPS (as described above). Unstimulated cells were also cultured
for a variety of the above time periods. A probe was prepared from the
PCR product using primers IL-1SC-F and IL-1SC-R. The probe was
then labeled with 32P and used for hybridization
to RNA (20 µg per lane) at 65°C for 4 h. A
32P-labeled S. canicula -actin
probe was used as a control to ensure that any increases seen were not
a result of a general increase in mRNA sample loading. Following
stringent washing, the membrane was put into an x-ray cassette and film
(Kodak, Rochester, NY) was exposed for 4 h. The relative levels of
mRNA were quantified by densitometric scanning of exposed film, using a
UVP imaging system (Ultraviolet Products, Cambridge, U.K.) and UVP
Gelworks ID advanced software v3.01 (Ultraviolet Products), and
expressed relative to the transcript in unstimulated cells. Northern
blotting was performed from three S. canicula.
Genomic DNA extraction
Liver tissue was removed from an individual catshark and left to dry for 10 min in a vacuum concentrator. After drying, an extraction mix was added to each tube, consisting of 500 µl 10x TNE buffer (0.01 M Trizan base-HCl (pH 8; Sigma Aldrich), 0.1 M NaCl, 0.5 M EDTA (pH 8; Sigma Aldrich)), 50 µl of 1 M Trizan base-HCl (pH 8; Sigma Aldrich) and 20 µl 25% SDS (Sigma Aldrich), followed by 20 µl of proteinase K (20 mg/ml; Promega). The tubes were then incubated at 55°C for 3 h, with vortexing every 30 min. A layer of oil formed at the surface (due to the presence of squalene in the liver), which was removed by careful pipetting. Next, 5 µl of RNase A (10 mg/ml; Promega) was added to each tube and was incubated at 37°C for 1 h to digest the RNA. To separate the DNA, 300 µl phenol (Sigma Aldrich) and 300 µl chloroform (Sigma Aldrich) were added. The tubes were rotated for 10 min to mix well and centrifuged at 10,000 x g for 15 min, and the DNA in the aqueous layer was carefully aspirated. To this aqueous phase 600 µl chloroform/isoamyl alcohol (Sigma Aldrich) was added and the tubes were again rotated and centrifuged. The aqueous layer was collected, the DNA was precipitated with 50 µl 3 M sodium acetate (pH 8; Sigma-Aldrich) and 1 ml (2 volumes) of cold 100% ethanol (BDH Chemicals, Poole, U.K.), and the precipitate was pelleted by centrifugation. The pellet was washed in cold 70% ethanol and centrifuged at 10,000 x g for 5 min, and the ethanol was removed. Finally, the pellet was resuspended in 30–100 µl of autoclaved ddH20 and incubated at 4°C overnight before determining the 260:280 ratio. The quality of the genomic DNA was checked by running 1 µl of DNA (0.25 µg/µl) on an ethidium bromide (0.1 µg/ml)-stained agarose gel (0.5%). The DNA was placed at -20°C for long-term storage. Genomic DNA was also digested with several restriction enzymes (EcoRV, ScaI, DraI; Promega) and ligated into the GenomeWalker adapter (Clontech Laboratories, Palo Alto, CA) following the manufacturer’s instructions.
Genomic PCR
Based on the S. canicula IL-1 cDNA sequence and
genomic organization of IL-1 from mammals, several primers were made
to characterize the genomic structure of S. canicula IL-1
(see Fig. 7 and Table I). Where possible, gene-specific primers were
designed in the same exon to avoid intron polymorphism. Gene walking in
the 5' direction was performed using a nested approach with primers
DB-R1, DB-R2, AP-1, and AP-2. The first-round PCR used the outer
adapter primer (AP-1) and the outer gene-specific primer (DB-R2), and
the second round used the nested adapter primer (AP-2) and the inner
gene-specific primer (DB-R1). Gene walking in the 3' direction was
performed using a nested approach with primers dogfish-F1, dogfish-F2,
AP-1, and AP-2. The first-round PCR used the outer adapter primer
(AP-2) and the outer gene-specific primer (dogfish-F1), and the second
round used the nested adapter primer (AP-1) and the inner gene-specific
primer (dogfish-F2). Wax-mediated hot start and touchdown PCR were
performed using an enzyme mixture of Taq DNA polymerase
(Bioline, London, U.K.) and PFU DNA polymerase (Promega) in a 50:1 U
ratio. First- and second-round PCR amplification was performed using
one cycle of 4 min (2 min for second round) at 94°C, 10 cycles of 1
min at 94°C, 1 min at 68°C (62°C for second round), and 3 min at
68°C, 10–20 cycles of 30 s at 94°C, 30 s at 62°C, and
3 min and 4 s per cycle at 68°C, with a final extension step of
5 min at 68°C. Specific primers DB-F7, -F8, -F9, -R5, -R6, and -R7
were used to fully sequence the amplified 5' product. A product that
overlapped the two gene walking products was also amplified with
gene-specific PCR using DB-F1, DB-F2, dogfish-R1, and dogfish-R2.
Specific primers DB-F3, -F4, -F5, -F6, -R3, -R4, -R8, and -R9 were used
to fully sequence the amplified product.
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S. canicula genomic DNA extracted from spleen (as above) was digested completely with BamHI (Promega) and EcoRI (Promega). The digested DNA samples were electrophoresed (15 µg per lane) on a 1% agarose gel, transferred to a nylon membrane, and hybridized at 65°C for 4 h with the same probe used for the Northern blot analysis. After stringency washing the membrane was treated as for the Northern blot, except that it was exposed to film for 2 days. Southern blotting was performed from two S. canicula.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three overlapping products were obtained using
PCR and universal and specific primers (Fig. 1) that contained the
full-length S. canicula IL-1 cDNA (Fig. 2). The transcript consisted of 1351 nt
that translated in a single reading frame to give a predicted 301-aa
IL-1 molecule. The gene had a 54-bp 5' untranslated region (UTR) and
a 379-bp 3' UTR. The 3' UTR contains eight RNA instability motifs
(attta) and a polyadenylation signal 14 bp upstream from the poly(A)
tail. The translated molecule contained two potential
glycosylation sites and a readily identifiable IL-1 family
signature(L e S A m y R g w Y V S T s r r n r q p I).
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amino acidsequences
(Fig. 3) revealed many areas of strong
amino acid conservation throughout the vertebrates. Good conservation
is seen especially in the regions that would be expected to represent
the 12 -sheets found in the -trefoil family members
(32). As with other nonmammalian IL-1 genes sequenced
to date, the S. canicula IL-1 gene lacks an aspartic acid
found at the cut site region of mammalian IL-1s. This site is
required for cleavage by IL-1-converting enzyme (ICE), a member of the
caspase family.
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sequence had highest
nucleotide identity (45.3%) and amino acid similarity (43%) and
identity (31.7%) with trout IL-1 (Table II), with chicken amino acid identities
next highest, followed by Xenopus. All amino acid identities
were <32%, indicating low homology with other known IL-1 genes.
The closer relationship between the trout and chicken IL-1 with
S. canicula IL-1 was also apparent in the phylogenetic
tree obtained using amino acid data (Fig. 4). In this study the S.
canicula sequence branches with the fish and chicken sequences
away from the mammalian IL-1s, although the genetic distance of
S. canicula from the trout and chicken sequence is
high.
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amino acids were hydrophobic there was
no suggestion that a signal peptide was present, in common with other
known IL-1 sequences. This was also confirmed using SignalP
v1.1. Expression studies
Following injection of 200 µg LPS 24 h earlier,
up-regulation of IL-1 transcripts could be detected by RT-PCR in
only the spleen and testes (Fig. 5). No
expression was seen in control S. canicula gill, heart,
kidney, and muscle tissue. Slight background expression of IL-1 was
found in the blood, brain, Leydig organ, and liver, but no increase in
expression was detectable in response to LPS. Northern blot analysis
(Fig. 6) of samples from splenocytes
stimulated in vitro with LPS confirmed that IL-1 expression could be
up-regulated in these cells over a 5-h period. A transcript of the
correct size (1.35 kb) was detectable, with optimum expression seen at
5 h, where a 7-fold increase of expression was detected relative
to RNA from control 0-h splenocytes. A second, smaller transcript of
0.9 kb was also detected with the probe, which followed a similar
expression pattern as the 1.35-kb transcript, but it remains to be
determined whether this molecule is related to IL-1.
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gene organization
Three overlapping products were obtained using PCR and a gene
walking approach with gene-specific primers (Fig. 7). PCR with primers DB-F2 and dogfish-R1
gave a 3.3-kb product. Gene walking in the 5' direction with primers
DB-R2 and DB-R1 using nested PCR gave a 3.1-kb product. Gene walking in
the 3' direction with primers dogfish-F1 and dogfish-F2 using nested
PCR gave a 0.7-kb product. The full gene sequence obtained by PCR is
shown in Fig. 8, with the genomic
organization of S. canicula IL-1 consisting of five
introns and six exons (Table III).
S. canicula has the same genomic organization as chicken
when compared with other known IL-1 genomic organizations (Fig. 9), which, relative to the human gene, is
missing an intron within the 5' UTR. The IL-1 introns in S.
canicula were similar to mammalian introns in size relative to
other known IL-1 introns. Multiple alignment of known IL-1 amino
acid sequences by exons (Fig. 10)
showed that the junction amino acids between exons are quite conserved
within exon 5 (exon 4 in S. canicula) only. Table IV shows the amino acid and nucleotide
homologies of the coding regions of known IL-1 exons using the GAP
program in theGenetics Computer Group package. The highest amino
acid homology was between chicken and S. canicula exon 3
(exon 4 in mammals), with the next highest between Xenopus
exon 6 and S. canicula exon 5 (exon 6 in mammals). The
highest nucleotide homology was between mouse exon 6 and S.
canicula exon 5 (exon 6 in mammals). Generally, on comparison of
each individual exon the highest homologies for S. canicula
amino acid and nucleotides was with nonmammalian vertebrates.
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To determine the number of IL-1 gene copies in the S.
canicula genome, genomic DNA was digested with BamHI
and EcoRI and hybridized with the IL-1 cDNA probes (Fig. 11). Using these enzymes two bands were
expected, one of 1.8 kb and one >2.7 kb, because the cDNA probe would
potentially bind to exon 4 before the EcoRI cut site and to
exons 5 and 6 after the EcoRI cut site. Hybridization
revealed two clear bands; one was of the expected size (3.6 kb) for
hybridization to exons 5 and 6 the other was and smaller (0.9 kb)
than the expected (1.8 kb) for the region containing exon 4. The 1.8-kb
band was probably too weak to be detected, due to the exon region that
the probe binds to being very short (40 bp).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene in S. canicula, the first cytokine gene to be cloned
within cartilaginous fish. S. canicula is found within the
group Galeomorphs and belongs to the order Carcharhiniformes, family
Scyliorhinidae. They are not true dogfish and it is more accurate to
call them catsharks due to a number of different morphological
characteristics (33). They were used in this investigation
as they were an abundant representative from the cartilaginous group of
fishes, available locally and easily kept in an aquarium.
The S. canicula IL-1 gene cDNA had highest nucleotide
(45.3%) and amino acid (31.7%) identity to trout IL-1 (7, 34). Chicken (35) and Xenopus
(17) IL-1 had the next highest amino acid identities. A
closer relationship with the trout and chicken IL-1 was also
apparent in the phylogenetic tree, where S. canicula
branched with them away from the mammals, although in each case the
genetic distance between the sequences was still quite high. Amino acid
homology was highest in regions containing the secondary structure of
the molecule, the 12 -sheets characteristic of the -trefoil
cytokines (32). The predicted mature peptide also contains
the IL-1 family signature, which spans -sheets 9–11. The IL-1
family signature pattern or motif has been taken from a selected
conserved region in the C-terminal section, which has the following
consensus pattern:
[FC]-x-S-[ASLV]-x(2)-P-x(2)-[FYLIV]-[LI]-[SCA]-T-x(7)-[LIVM]
and can be found in the PROSITE database (36). All
mammalian, chicken, and carp IL-1 sequences contain this exact
motif, although to allow the inclusion of the trout and S.
canicula IL-1 sequences slight changes are required in the
consensus sequence. The consensus sequence would need to read
[FCL]-x-S-[ASLV]-x(2)-[PR]-x(2)-[FYLIV]-[LIV]-[SCA]-T-x(7)-[LIVMK].
The observation of homology within the predicted -sheets, along with
the presence of the IL-1 family signature in the predicted mature
protein, suggests that the S. canicula protein is also a
-trefoil cytokine.
When analyzed using SignalP v1.1 it was shown that no apparent signal
peptide was present in S. canicula IL-1. This is in
common with other IL-1 sequences (37), indicating that
the molecule is secreted through a nonclassical pathway that does not
involve the Golgi/endoplasmic reticulum route (38). Also
found in the 3' UTR of S. canicula IL-1 are numerous
copies of an mRNA instability motif (attta). The degradation of mRNA in
eukaryotic cells is a regulated process and can determine the level of
expression of the gene. The presence of the consensus sequence, attta,
in the 3' UTR of mRNA can play a role in the degradation of mRNA
(39). The presence of this motif is typical of genes
coding for inflammatory mediators (40) and suggests this
gene will be biologically functional.
Until the cloning of trout IL-1 (7), the processing of
the precursor IL-1 molecule was not in question. In all mammalian
IL-1 homologs ICE or caspase-1 cuts the precursor at an aspartic
acid, Asp116 (41). Mammal
pro-IL-1 is biologically inactive until ICE cleaves it
(42). Like trout and carp IL-1, S. canicula
IL-1 has no identifiable sequence that corresponds to an ICE cut
site in the region where mammalian IL-1 is cleaved to release the
functional, mature peptide. Ala145 aligned with
the first residue of the mammalian peptides, but it remains to be
determined whether the S. canicula precursor molecule is
cleaved naturally at this residue and whether this predicted "mature
peptide" is active biologically.
Localization of IL-1 expression in S. canicula was also
investigated and indicates that the IL-1 molecule is biologically
relevant to cartilaginous fish immune responses to Gram-negative
bacteria. Expression of the S. canicula IL-1 transcript
was clearly detectable by RT-PCR in the spleen and testes, tissues
known to contain a variety of leukocytes (43), and was
shown to be inducible in vivo following injection with LPS 24 h
earlier. Stimulation of splenocytes in vitro with LPS for 0–5 h also
resulted in a significant increase in transcript expression, with
maximal expression seen at 5 h (the last time point examined).
Northern blot analysis showed that there was a 7-fold increase in the
transcript level after a 5-h incubation with LPS. Similar patterns of
expression have also been seen in tissues of bony fish (34, 44). In carp, administration of PMA and LPS to phagocytes
isolated from the head kidney induced expression of IL-1 in these
cells after 2 h of stimulation (44). In trout, head
kidney leukocytes and isolated macrophages express IL-1 after
stimulation with LPS (34). A total of 5 µg/ml LPS was
shown to produce maximal expression, which was first detectable 1–2 h
poststimulation, with maximal induction at 4 h. While the kidney
is a major source of phagocytes in bony fish, in cartilaginous fish it
has been shown to contain lymphohemopoietic tissue only during
embryonic development (45). In elasmobranchs, tissues such
as the Leydig organ and testis (epigonal tissue) are found to contain
lymphohemopoietic tissue. The lack of expression of IL-1 in the
Leydig organ in this study may reflect a redundancy between this tissue
and the testis, as noted for B cell development in the clearnose skate
(46). Temperature has also been shown to have a marked
effect on trout IL-1 expression (47) because fish are
poikilothermic. While this is likely to be true also for cartilaginous
fish, the temperatures used reflect those encountered naturally by this
species.
Similar to the human IL-1 gene (48), the carp IL-1
gene has seven exons and six introns (44), whereas
S. canicula, chicken, trout IL-11 (34), and
trout IL-12 (8) have six exons and five introns. In
S. canicula and chicken it is clear that the first intron is
missing in the 5' UTR, whereas in trout the equivalent of an exon and
an intron appear to be missing at the 5' end (34). In
S. canicula and chicken the first exon is translated, unlike
all other known IL-1 genes where it is part of the 5' UTR. The sizes
of the introns in S. canicula are more similar to those in
humans, although introns 2, 3, and 4 (3, 4, and 5 in mammals) are
smaller and 1 and 5 (2 and 6 in mammals) are larger than their human
counterparts. All the S. canicula intron and exon boundaries
follow the GT-AG consensus (49). The sizes of the coding
exons 4, 5, and 6 (5, 6, and 7 in mammals) are similar to the exon
sizes of mammalian IL-1, whereas exons 2 and 3 (3 and 4 in mammals)
are larger. Exon 1 (2 in mammals) is also larger, but this is due to
the missing intron. Generally, the highest homology seen between the
different exons using both nucleotide and amino acid data was with
S. canicula exon 5 (6 in mammals), chicken and trout exon 5,
and mammalian exon 6. This exon is also known to have highest homology
within mammals, and it contains the largest amount of secondary
structure (four -sheets) per amino acid coded, even relative to the
coding region of exon 7, which contains the IL-1 motif region. This
adds further evidence to the statement that the most conserved region
of the IL-1 molecule is found within the 12 -sheets that form the
secondary structure. S. canicula amino acid homology for
exons 1 and 2 (exons 2 and 3 in mammals) showed very little homology to
other species.
Southern blot analysis suggests there is a second S.
canicula IL-1 gene, or at least a related gene with homology to
the 3' end of S. canicula IL-1. The presence of two
IL-1 genes has beenknown in mammals for some time
(50), and in bony fish it is now clear that more than one
gene can exist for IL-1 (Ref. 8 ; GenBank accession no.
AJ401030). Until it is sequenced this gene could also be a second
member of the IL-1 gene family present in S. canicula, such
as the IL-1R antagonist or IL-1, which would potentially show
homology with the 3' end of the gene. Whatever the outcome, it is clear
that at least one member of the IL-1 gene family exists in
cartilaginous fish.
In conclusion, previous bioactivity evidence for the presence of
inflammatory cytokines in elasmobranchs has now be verified with the
cloning of IL-1 in S. canicula. The discovery of the
S. canicula IL-1 gene will greatly increase knowledge on
the role of cytokines in the cartilaginous fish immune system. Future
work will involve the production of the recombinant protein and the
study of its immunological role directly. In addition it should be
feasible to use the S. canicula IL-1 gene to probe for
other IL-1 family members to determine whether they had evolved before
the divergence of bony and cartilaginous fish.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chris J. Secombes, Department of Zoology, University of Aberdeen, Tillydron Avenue, Aberdeen AB24 2TZ, U.K. E-mail address: c.secombes{at}abdn.ac.uk
3 Abbreviations used in this paper: Myr, million year; UTR, untranslated region; ICE, IL-1-converting enzyme.
Received for publication November 7, 2001. Accepted for publication January 18, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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from rainbow trout Oncorhynchus mykiss reveals no evidence of an ICE cut site. Cytokine 11:552.[Medline]
gene in rainbow trout (Oncorhynchus mykiss). Immunogenetics 51:1002.[Medline]
gene: analysis reveals tissue specific expression and a potential regulatory sequence in rainbow trout (Oncorhynchus mykiss). Cytokine 10:555.[Medline]
3 exists in bony fish. Vet. Immunol. Immunopathol. 72:45.[Medline]
are present in plaice (Pleuronectes platessa) DNA. Fish Shellfish Immunol. 10:261.[Medline]
. Eur. J. Biochem. 268:1315.[Medline]
from Xenopus laevis and analysis of expression in vivo and in vitro. Immunogenetics 49:332.
gene has a different organisation to mammals and undergoes incomplete splicing. Eur. J. Biochem. 259:901.[Medline]
: cDNA cloning and purification of active recombinant protein. Eur. J. Biochem. 258:994.[Medline]
, a protein lacking a signal sequence. EMBO J. 9:1503.[Medline]
precursor at two distinct sites and does not cleave 31-kDa IL-1. J. Immunol. 147:2964.[Abstract]
: two cytokine substrates for ICE (caspase-1). J. Clin. Immunol. 19:1.[Medline]
RNA expression in the common carp Cyprinus carpio L. Dev. Comp. Immunol. 25:195.[Medline]
in cultured rainbow trout (Oncorhynchus mykiss) leucocytes. Dev. Comp. Immunol. 24:575.[Medline]
gene. Gene 52:95.[Medline]
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