The second exon of an additional CfAFP gene, 2.7a, encoding a new + 9-kDa isoform, was found 3.7 kb upstream of Afp-Lul and demonstrates that some AFP family members are linked in tandem
Trang 1Eur J Biochem 269, 38-46 (2002) © FEBS 2002
A family of expressed antifreeze protein genes from the moth,
Choristoneura fumiferana
Daniel Doucet, Michael G Tyshenko, Peter L Davies and Virginia K Walker
Department of Biology, Queen’s University, Kingston, Ontario, Canada
The freeze-intolerant insect, Choristoneura fumiferana
(spruce budworm), produces multiple antifreeze protein
(AFP) isoforms for protection during the overwintering
stage We now report the cloning of AFP genes from insects;
Afp-Lul encodes a ~9-kDa AFP isoform, and 4ƒ?-ïu1
encodes a ¥ 12-kDa AFP isoform Both CfAFP genes have
similar structures with a single 3- to 3.6-kb intron inter-
rupting the coding region The second exon of an additional
CfAFP gene, 2.7a, encoding a new + 9-kDa isoform, was
found 3.7 kb upstream of Afp-Lul and demonstrates that
some AFP family members are linked in tandem This gene
appears to encode an AFP with 68-76% identity to previ-
ously isolated CfAFPs With its eight Cys residues necessary
for disulfide bonding and five perfectly conserved “Thr
button’ (Thr-Xaa-Thr) ice-binding motifs, it can be modeled
as a functional AFP Southern blot analysis shows that there are ~ 17 genes in this AFP family, with each of the isoforms represented by two to five gene copies Transcript accumu- lation from Afp-Lul and Afp-Iul (or closely related genes) was maximal during the overwintering stage, while 2.7a transcripts were only detected in first instars, larvae that are normally found only in the summer Contrary to expecta- tions, this differential expression demonstrates that Cf/AFP gene family transcripts are primarily regulated during development, rather than by seasonally low temperatures Keywords: antifreeze protein; gene family; cold stress; moth development
Insects have developed multiple adaptations to changes in
their environment Environmental stress of a predictable
nature such as seasonal drought, heat or cold may be
endured by entering diapause, a physiological state charac-
terized by low metabolic activity [1] Many species of insects
prepare for overwintering by triggering the diapause
response, but freeze-intolerant species such as the spruce
budworm, Choristoneura fumiferana, must also have some
protection from freezing at subzero temperatures Their
diapause in the second larval instar is accompanied by the
synthesis of antifreeze proteins [2] that depress the freezing
point of extracellular fluids and allow the larvae to
supercool [3] Over the course of the winter, larvae also
increase the synthesis of the low molecular mass cryopro-
tectant, glycerol, from glycogen stores [4]
Antifreeze proteins (AFPs) adsorb to microscopic ice
crystals and prevent their growth, thus lowering the freezing
point of solutions [5] While this adsorption depresses the
freezing point, it leaves the melting point unaffected,
generating a thermal hysteresis AFPs are found in different
organisms including fish, insects, plants, bacteria and fungi,
but are best known in fish [6] Four different types of
antifreeze proteins (AFPs I to IV) and one antifreeze
glycoprotein have been characterized in bony fish from the
icy seas of both the northern and southern hemispheres At
high concentrations they offer protection to the freezing
point of seawater (—1.9 °C) AFPs isolated from beetles and
Correspondence to V K Walker, Department of Biology, Queen’s
University, Kingston, Ontario, Canada K7L 3 N6
Fax: + 1 613 533 6617, Tel + 1 613 533 6123,
E-mail: walkervk@biology.queensu.ca
Abbreviations: AFP, antifreeze protein
(Received 30 August 2001, accepted 19 October 2001)
spruce budworm are 10-100 times more active on a molar basis than the fish AFPs or antifreeze glycoproteins [2,7] This presumably reflects the need for high TH in freeze- susceptible insects that have to cope with the unpredictable, lower temperatures of the terrestrial environment The structures of the insect AFPs are unique and unlike the fish AFPs [8,9]; they are B helices with two rows of Thr residues down one side of the protein that make a good match to the ice surface on both prism and basal planes
Thermal hysteresis activity increases with AFP concen- tration and both fish and insects are dependent on high circulating AFP concentrations for full protection against freezing In some fishes, the demand for AFP has been largely met by increasing the number of AFP genes Ocean pout and wolffish from cold coastal waters have = 150 and
80 AFP gene copies, respectively [10,11] Other fishes such as the sea raven [12] and winter flounder [13] also have multiple AFP gene copies Insects may also use gene families for AFP production Numerous AFP isoforms differing slightly in primary sequence or mass have been found in the beetles, Tenebrio molitor and Dendroides canadensis as well as in
C fumiferana [14-16] In Tenebrio, Southern hybridizations with AFP gene probes showed numerous bands, reflecting the presence of a multiple copy AFP gene family [14]
In C fumiferana, at least seven different AFP (CfAFP; previously designated sbwAFP) isoforms are known at the cDNA level These different cDNAs encode proteins of two distinct sizes, + 9 and = 12 kDa The larger variants contain
a 30- or 3l-amino-acid insertion creating two additional 15- or 16-amino-acid loops in the B helix [16] The 9-kDa proteins are encoded by cDNAs possessing either a long (+ 1000 nucleotides) or a short (= 200 nucleotides) 3’ UTR, while the 12-kDa CfAFPs are encoded by cDNAs with 3’ UTRs of intermediate size (~ 450 nucleotides) The existence of CfAFP variants, differing in both protein size
Trang 2and message length strongly suggests that a gene family
encodes these AFPs This paper reports the cloning of AFP
genes from an insect As well as revealing additional isoform
variation, the isoforms show an unexpected differential
transcript accumulation pattern, especially during diapause
MATERIALS AND METHODS
Nucleic acid probes
DNA probes for the detection and isolation of specific
members of the CfAFP gene family were generated from
cDNA clones /0 and 337 [16] or genomic sequences
A cDNA 10-specific probe encompassed the entire 414-bp
coding region of the isoform A probe specific for the 337
cDNA isoform was generated by PCR with SPF and SPR
primers as previously described [16] A sequence 2.7-specific
probe was generated with the SPF primer and primer 2.7R
(5’-TCAGGACACTACTTTCAC-3’), located 58 nucleo-
tides downstream of the putative termination codon
Genomic DNA isolation and Southern blots
C fumiferana were obtained from the Canadian Forest
Service (Sault Ste-Marie, ON, Canada) Genomic DNA was
purified from a single sixth instar larva by proteinase K
digestion and phenol/chloroform extraction using the
method of Blin & Stafford [17] DNA samples (20 pg) were
digested with the restriction enzymes HindIII, EcoRI, S1,
and BamHI (Gibco BRL, Burlington ON, USA), electro-
phoresed in a 0.9% agarose gel, and subsequently trans-
ferred to Hybond N nylon membrane (Pharmacia, Baie
d’Urfe, Quebec, Canada), following the manufacturer’s
protocol Hybridization was carried in 0.25 mM Na;HPO/,/
7% SDS [18] or 5 x NaCl/Cit/0.5% SDS/5 x Denhardt’s
solution for a minimum of 12 h [19] To examine the
complexity of the gene family, the blot was hybridized with
the 337 isoform probe and washed under low stringency (0.1
M NazHPO,/5% SDS, 65 °C, 2x 15 min) To detect
specific hybridization signals for the 337, 10 and 2.7a
probes, the blots were hybridized successively with each
probe and washed under high stringency conditions
(0.2 x NaCl/Cit/0.1% SDS, 65 °C 15 min) After washing,
blots were exposed to a Phosphorimager screen (Pharmacia)
at room temperature or to Biomax MS film (Eastman
Kodak, Rochester, NY, USA) at —-80 °C
Genomic DNA library screening
A C fumiferana genomic library constructed in ADASH II
(Stratagene, LaJolla, CA, USA) was plated, and 6.5 x 10°
plaques were screened using the 337 cDNA fragment probe
Filters were washed using the low stringency conditions
described above Positive clones were randomly picked, and
their DNA isolated and digested with HindIII, SstIl and
XhoI restriction enzymes to generate restriction enzyme
maps DNA fragments, obtained by restriction digests or
long PCR (Expand™ Long Template PCR, Boehringer
Mannheim, Laval, QC, USA) using vector primers, were
subcloned into pBluescript (Stratagene) or pCR2.1 (Invi-
trogen, Carlsbad, CA, USA), respectively DNA encoding
AFPs was sequenced on both strands Noncoding DNA
was sequenced in one direction only
RNA isolation and Northern blots
C fumiferana used for developmental studies were reared
on an artificial diet [20] All individuals were maintained at
23 °C under a 16-h light/8-h dark photoperiod regime, except for diapausing second instar larvae (L2) that were switched to 2 °C and constant darkness after 2 weeks Eggs were collected 3—5 days after oviposition and first instar (L1) samples were sacrificed 48-51 h after egg hatching L2s were collected from cheesecloth at various times after hibernaculum (cocoon) spinning and molting: after 1 and
2 weeks at 23 °C and 1, 5, 10, 15 and 30 weeks after storage
at 2 °C Post-diapausing instars (L3—L6) were sacrificed 24-48 h after molting Animals were frozen in liquid nitrogen and stored at —80 °C Total RNA was isolated with Trizol (Gibco BRL) using a modification of the manufacturer’s protocol [21] RNA (10 ug) was loaded onto a 1.2% agarose/formaldehyde gel and transferred to Hybond N according to established methods [19] Blots were hybridized successively with the appropriate AFP probes in 50% formamide/5 x NaCl/Cit/5 x Denhardt’s solution/0.5% SDS and 100-150 pg of sheared salmon sperm DNA (Gibco BRL) for a minimum of 12 h The blots were also probed with an o-tubulin fragment from Drosophila mela- nogaster as a RNA loading control High stringency washes were carried out in 0.1 x NaCl/Cit/0.5% SDS at 65 °C fora minimum of 20 min Detection of signal on X-ray film was carried out as described for Southern hybridizations
RESULTS
Isolation and characterization of C fumiferana AFP genes
When C fumiferana genomic libraries were hybridized with the 337 isoform CfAFP probe, a total of 165 positive plaques were identified Of these, two were chosen and mapped using restriction enzymes (Fig 1) One of the two, 2.7, had a 13.9-kb insert that contained the complete coding, upstream and downstream nucleotide sequence correspond- ing to a member of the long 3’ UTR class of isoform This gene, designated Afp-Lul (Long UTR 1) has six nucleotide substitutions in the coding region compared to 337 cDNA (three are nonsilent: Glu36 — Asp, Thr67 > Ser and Ser76 — Leu) and two substitutions compared to another isoform of the long UTR class, 333 [16] (both substitutions are nonsilent, with Glu36— Asp and Ser84 > Thr changes) It is possible that Afp-Lu/ is an allelic variant of either of these +9-kDa isoform proteins, or represents a closely related isoform A single 3.6-kb intron, identified by comparison with the cDNA sequence, is positioned after the first nucleotide of the Vall6 codon in the signal sequence, and possesses conserved splice junction sequences found in Drosophila and in the C fumiferana trypsinogen gene [22,23] (GenBank accession no AF325859)
Afp-LuIl contains several kb of 5’ flanking DNA that was examined for potential regulatory regions A putative TATA box was found at position —84 relative to the translation start site, and a putative CAAT box at position
—116 The clone contained 6 kb of 3’ flanking DNA, of which 1.4 kb contiguous to Afp-Lul, were sequenced This section was very similar to the 3’ UTR of isoform 337, except for an insertion of an additional 517 nucleotides No
Trang 340 D Doucet et al (Eur J Biochem 269)
A)
À2.7
S H 8 XH HH HHHHAHH HS HX SH
L( "é ¬" 72 et a
2.74 Afp-Lul
H H XS S§ S XH 5 H
| | 1 \f Xứ 1 i i
*
Ỷ
—
Afp-Tul
Fig 1 Restriction maps of C fumiferana genomic clones encoding the
AFP genes Afp-Lul and 2.7a (A) and Afp-lul (B) Horizontal black
boxes below the long horizontal lines indicate the coding regions of the
AFP genes, and horizontal thin lines, regions sharing homology with
cDNA untranslated regions The dashed horizontal line for the gene
2.7a indicates the putative 3’ UTR of the gene, based on the transcript
size estimation The white box in the 3’ end of Afp-Lul is a 517-
nucleotide nonintronic sequence that is not present in the 3’ UTR of
cDNA 337 Vertical arrows point to putative polyadenylation sites and
the stars indicate those that are used based again on transcript size
estimation V-shaped thin lines joining exons represent spliced regions
of the AFP genes, and the inward pointing triangles in clone 2.7 rep-
resent a + 500 bp inverted repeat The restriction enzymes used were:
H, Hind ll; S, Sstl; X, Xhol
splice junctions were found within this 3’ UTR insertion
indicating that it 1s not an intron Sequencing also allowed
the identification of putative polyadenylation signals at
positions 4548, 4650 and 4785 Upstream of the Afp-Lul
gene at —641 there was a large inverted repeat spanning
about 500 bp (Fig 1) Approximately 200 nonoverlapping
nucleotides of this palindrome were sequenced and it
appeared to be unrelated to any repetitive or transposable
element in the GenBank database
Clone 2.7 also contained a sequence which, when
conceptually translated, showed 87% identity with the
second exon of Afp-Lul This sequence, tentatively desig-
nated 2.7a (as it should not be designated a formal gene
symbol without the corresponding cDNA), was located
~ 3.7 kb upstream from the translation start of Afp-LuI
The sequence does not have the obvious features of a
pseudogene such as stop codons or frameshifts It has
conserved Cys residues and putative “Thr button’ 1ce-
binding motifs (Thr-Xaa-Thr; where Xaa 1s any amino acid)
every 15-17 residues (Fig 2A) and therefore probably
encodes a functional CfAFP With a predicted mass of
9320 Da, it would be placed either in the long or short
3’ UTR isoform class (~ 1000 and 200 nucleotides,
respectively) and indeed it shows similar amino-acid identity
(68-73%) with most members of both classes As four
potential polyadenylation sites were found within 1.4 kb
downstream of the stop codon at 845, 1102, 1304 and 1760,
© FEBS 2002
it has been provisionally placed in the long UTR 1soform class based on 1ts nucleic acid sequence and the potential size
of its mRNA Molecular modeling of this new, putative isoform, based on the NMR-derived structure of the 337 isoform, 1s consistent with the 2.7a sequence encoding a functional AFP (Fig 2B) Indeed, this gene would encode
an isoform containing all ‘perfect’ Thr-Xaa-Thr repeats Of
a dozen previously identified AFP isoforms, all contain imperfect repeats of the ice-binding motifs, except for 2.7a (Fig 2A; M G Tyshen, unpublished results)
The second phage plaque characterized from the genomic library, clone 2.26, had a 13.9-kb insert which included the complete sequence for a gene of the intermediate size 3’ UTR class of AFP (Fig 1) With the exception of the longer coding region, the overall structure of the AFP-Iu/ (inter- mediate UTR 1) gene ts very similar to Afp-Lul The open reading frame is interrupted by a single phase | intron at the conserved Val codon (Vall5 in Afp/u-/) At 3 kb, this intron
is only 600 bp smaller than that of Afp-Lul The interme- diate UTR isoform class encodes AFPs that have two additional structural repeats (loops of the B helix), and thus have a 30-amino-acid insertion in the coding region The coding sequence of the Afp-JuJ (GenBank accession
no AF325857) matched the larger 12-kDa isoforms with
10 nucleotide differences when compared to cDNA 10 (four resulted 1n amino-acid changes: Asp1l3 —> Asn, Gln88 — Arg, Tyr89 — Phe and Asn101 — Ser) A puta- tive TATA box was found at —79, but no evidence of a CAAT box could be found AFP-IuJ had the same polyadenylation signal, AATATA, found in the cDNA 70 isoform
Southern analysis
In order to estimate the copy number of the cloned genes, probes representing cDNA 337 and J0 clones, as well as the newly discovered 2.7a sequence, were hybridized to blots of digested C fumiferana genomic DNA (Fig 3A,B) South- ern hybridization with the 337 probe, washed at low stringency, showed that the AFP gene sequence is present In multiple copies Washing conditions were chosen to detect hybridization equivalent to a T,, ~20 °C below that of routine washes with the 337 probe Assuming a 1% mismatch per degree of difference [24], 1t was estimated that even the more divergent intermediate UTR class [16] would be detected Examination of Fig 3A demonstrates that bands corresponding to those seen with the interme- diate UTR class probe, /0, could also be seen 1n the blot washed at low stringency, verifying this procedure After washing at low stringency, approximately 17 bands (consistent with the number of hybridizing plaques/genome
in the library screens) of various intensities could be resolved
in the Sstl, HindIII and EcoRI digests of the DNA from individual animals Reprobing the same blots with sequenc-
es specific for each of the three genes and washing under conditions of high stringency yielded different overall banding patterns As would be expected, however, the bands were a subset of those seen after low stringency washes of the heterologous probe (Fig 3A) Southern blots using DNA isolated independently from five animals (Fig 3B) showed that there are at least two copies of the gene hybridizing with the 337 probe (one of which would correspond to Afp-Lul) and two copies hybridizing to the
Trang 4© FEBS 2002
A
Afp-Iul
cDNA 10
cDNA 104
cDNA 501
Afp-Lul
cDNA 4
cDNA 337
cDNA 333
cDNA 339
2.7a
Afp-Iul
cDNA 10
cDNA 104
cDNA 501
Afp-Lul
cDNA 4
cDNA 337
cDNA 333
cDNA 339
2.7a
Antifreeze protein genes in Choristoneura (Eur J Biochem 269) 41
STRISGPACSISR GVAAPSAACRISGCTLRAN 137 eeeseeee eee ee oMoMe ee ee eee eee eee eeeeee 137
-8- -VT- Neecteeee I 138
‹T- ‘*T*A++-K-+++S-S'M 138
*T- P eee wa K “+8 FS * 108 -.8- P K eee FS 108
-T‹ Pp s0 ge6`:0: 8 K: FS 108 - 8- Pp K ove ó 6346 FS 108
-s- P -:P ® Ằ@:0:©-©:08:0:0-6:0.6 107 :H- Ss “AP: *K ee eee Ss 92
Fig 2 Compendium of CfAFP sequences (A) Amino-acid alignment of sequences retrieved from clones 2.7 (Afp-Lul and 2.7a) and clone 2.26 (Afp-IuJ) as well as representatives of the intermediate (cDNAs 10, 105 and 507), long (cDNAs 4, 337 and 333), and short (CDNA 339) UTR classes
of CfAFP cDNAs Amino acids identical to Afp-Zul are represented by dots while dashes indicate gaps in the alignment Afp-Iu/ and cDNAs 10,
104 and 50] each encode a longer (= 12-kDa) isoform than the other cDNA or gene sequences, which encode 9-kDa AFPs The shaded boxes in the alignments indicate the conserved putative ice-binding ‘threonine buttons’ of the Thr-Xaa-Thr motifs Residues in italics make up the signal peptide (B) Molecular model of the putative mature AFP encoded by the 2.7a gene with Thr residues pointing upward, compared to the 337 isoform
2.7a gene probe Hybridization to the cDNA 10 probe
(corresponding to Afp-Iu/) shows that there are at least five
copies (Fig 3A,B)
Northern analysis
The probes used for Southern hybridizations were also used
to study the pattern of expression of each isoform
throughout C fumiferana development The sizes of tran- scripts corresponding to Afp-Lul and Afp-Iu/ are consistent with the size class of the previously cloned cDNA homo- logues, 337 and 10, respectively (Fig 4) However, North- ern blots probed with the 337 probe showed an additional, smaller mRNA of | kb as well as the expected transcript at 1.4 kb There was a single 1-kb transcript detected with the cDNA /0 probe, although a faint band representing
Trang 542 D Doucet et al (Eur J Biochem 269)
A
Low stringency 337
S E H B S E H B
-
-
94— ~~ = = ° -
6.6 — PA = -
= -
44 — s
ý
«- -
20—
B
Low stringency 337
© FEBS 2002
S E H B S E H B
.ẳẲỒ Ñ
acs
ø ~-
S`
o- = ~
- ’ «
-
ˆ
~
- ein |:: |:
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-— § Re ã |
~ 822} ưu
20—=
Fig 3 Southern hybridization of three Cf/AFP sequences to restriction-digested C fumiferana DNA (A) DNA from a single sixth instar larva was digested with four restriction enzymes The first panel shows low stringency washes of the Cf/AFP 337 isoform probe after hybridization Hybridization and high stringency washes for probes of isoforms 337 (corresponding to Afp-Lu1), 10 (to identify Afp-IuJ) and 2.7a are shown in the second, third and fourth panels, respectively DNA was digested with: S, SsI; E, EcoRI; H, HindIII and B, BamH1 Molecular mass markers (in kb) for the restriction fragments are indicated on the left (B) Southern hybridization of five different individual sixth instar DNA under the same conditions as in (A) above Genomic DNAs were digested with the restriction enzyme Ss/I
differentially processed transcripts or a related, uncloned
isoform could be seen late in second instars (see Fig 4,
arrow) The strongest hybridization signals for both probes
were detected in the larval diapausing stage, from 1 week to
30 weeks after transfer to 2 °C Unexpectedly, however, the
transcripts that correspond to the Afp-Lul and Afp-lul
genes also accumulated to relatively high levels in first
instars and in diapausing second instars maintained at
23 °C A faint Afp-Lul signal could even be detected in egg
mRNA No 4fp-Iul signals were seen in eggs but low transcript levels were seen in the fifth and sixth larval instars kept at 23 °C
In order to examine the expression of 2.7a, for which no cDNA has been previously described, a probe was synthe- sized encompassing the majority of the putative coding sequence, and a small portion of the 3’ UTR Northern analysis showed the accumulation of two transcripts, at 1.4kb and 1 kb (Fig 4) In contrast to the expression
Trang 6
L2 (Diapause)
Probe
(Kb)
— 1.1
— 14 2.7a
w — 1.0
Se == == eeeee~ -
Fig 4 Expression of C/4FP isoforms 33”, 70 and 2.7ø and a control gene (z-tubulin) during development Egøss, all six larval instars (indicated with the ‘L’ prefix and a number), pupal (Pu) and adult (Ad) stages of the insects were tested for expression by northern hybridization Sizes of the AFP mRNAs (in kb) are indicated at the right Second instars in diapause, 1 and 2 weeks (w) after hibernaculum spinning (L2, 23 °C) as well as 1, 5, 10,
15 and 30 weeks after transfer to cold storage (L2, 2 °C) were sampled The arrow on the isoform 10 panel indicates a 1.4-kb transcript seen later during diapause The 337 probe hybridizes with transcripts corresponding to the Afp-LuJ gene and the /0 probe hybridizes with transcripts corresponding to the Afp-Iul gene
pattern of the previous described genes, however, the two
2.7a mRNAs accumulated to higher levels in the first instar
than in the second instar where the abundance appeared to
be independent of diapause status No transcripts were seen
at any other stage As previously mentioned there were four
potential polyadenylation sites identified in 2.7a Assuming
that the gene has a 5’ exon and 5’ UTR of roughly the same
length as the other AFP genes (100-115 bp depending on
the isoform), transcript sizes of 0.9, 1.1, 1.3 and 1.8 kb
would be expected, suggesting that the first and third sites
are recognized
DISCUSSION
AFP gene families
Based on the discovery of several CfAFP isoforms differing
in sequence, it was postulated in an earlier study [16] that
members of a multigene family would encode them Here we
have addressed this hypothesis by cloning genomic frag-
ments containing insect AFP genes There are + 17 unique
loci, making this a low abundance gene family, and one that
appears to be developmentally regulated Gene families can
often be found where high production levels of certain proteins must be achieved at a particular developmental stage [25-27] Multiple gene copies also appear to be selected
in response to environmental stress [28] Even in Lepi- dopteran (moths) and Dipteran (flies) orders that have a compact genome [29], gene amplification can occur if the selection pressure is strong, such as in the amplification of esterase genes in the mosquito organophosphate insecticide resistance phenotype [30], the metallothionein gene dupli- cation in metal resistant Drosophila [31] and the magnifica- tion of ribosomal DNA in bb mutants [32]
In fish genomes there may be even less constraint to increasing gene copy number by selection Indeed, AFPs in fish are often encoded by moderately sized multigene families Ocean pout caught in the cold waters off the Newfoundland coast have ~ 150 AFP genes, and those caught in a more southern latitude have ~ 40 AFP genes [10] This high gene dosage is presumably required to maintain temperature-appropriate serum AFP concentra- tions (20-25 mg-mL“') during the winter [33] Analogously, multiple gene copies may have been selected in this
C fumiferana population, collected from the northern boreal forest, to satisfy a similar demand for elevated levels
Trang 744 D Doucet ef al (Eur J Biochem 269)
of AFP in the hemolymph of overwintering larvae Insect
AFP is hyperactive compared to fish AFP, but nevertheless,
selection for increasing gene copy number would be strong
presumably because of the more extreme subzero terrestrial
temperatures
The beetle, Tenebrio molitor, also has a hyperactive AFP
and has 30-50 gene copies as detected by Southern
hybridization [14] Curiously then, 7 molitor with at least
twice the number of AFP genes is a domestic species that
overwinters in granaries at more moderate temperatures
than C fumiferana, which undergoes diapause at the tips of
coniferous tree branches in the boreal forest Diapausing
C fumiferana, however, as well as synthesizing AFPs, spin a
silk hibernaculum, which may prevent inoculative freezing
During the winter they also increase the concentration of
the cryoprotectant, glycerol, 10-fold and desiccate to 40%
of the prediapause water content [4] Taken together,
these adaptations may explain the impressive ability of
C fumiferana to survive temperatures of —30 °C or lower
The existence of a C fumiferana AFP sequence upstream
of the gene Afp-Lul on clone 2.7 provides the first direct
evidence that some AFP genes are tightly linked in this
insect However, very large arrays of tandem genes as found
in fish type I and IIT AFPs [11,13], and hypothesized for
Tenebrio AFP [14], are unlikely Southern blots indicate a
somewhat smaller AFP gene family (Fig 3A,B), and
because the hybridization signals were distributed between
several, nonidentical large fragments of DNA, it is likely
that at least some of the ~ 17 genes of the family are spaced
several kb apart (as for Afp-Lu/ and 2.7a), even though they
may be linked
In both Afp-Lul and Afp-Iul, the majority of the
sequence is taken up by a single, intervening sequence of
at least 3 kb This is a relatively large intron for species with
a compact genome like C fumiferana, and often such large
introns are characteristic of developmentally regulated genes
[34,35] In addition, a large palindrome of ~ 500 bp (of
which ~200 bp at each extremity was sequenced) was
found 641 bp upstream of Afp-Lul in clone 2.7 (Figs 1A
and 2A) There are many examples of palindromic
sequences or inverted repeats associated with tandem
amplicons in other systems [36-38] and it is possible that
the CfAFP-associated sequence could promote the rear-
rangement of the DNA as well as mediate gene conversion
events in the AFP gene cluster
AFP gene expression
Transcripts corresponding to Afp-Lul and Afp-lul accu-
mulate in the second larval instar This is consistent with
the detection of C/AFP isoforms in larval extracts of the
same stage, 12 weeks after storage at 2 °C [2,17];
Although AFP would be obviously required during the
obligate diapause where subzero temperatures are nor-
mally experienced, AFP messages are not diapause-specific
because approximately equivalent levels were detected in
first instars, which in the wild, are exposed to the high
temperatures of mid and late summer Temperature, and
constant darkness, did not affect transcript accumulation
of Afp-Lul or Afp-Iul either, as no difference could be
detected when diapausing larvae were transferred to the
2 °C incubator This was also apparent after the transfer
of L2s, kept at 2 °C for 10 weeks, to 15 °C for 1 week;
© FEBS 2002
this treatment had no effect on transcript accumulation (not shown) Expression in other stages was low and isoform-specific, as exemplified by the transcript corre- sponding to Afp-Lul found in eggs and the Afp-Iul transcripts found in third, fifth and sixth instars The cloned AFP genes are thus developmentally regulated Although synthesis of cryoprotectants in some insect species appears to be controlled by temperature or photoperiod [4], a number of ‘stress genes’ such as the AFP genes from 7 molitor [39], the heat shock protein gene, hsp70, from the flesh fly, Sarcophaga crassipalpis [40], and the AFP genes studied here, appear to be developmentally regulated but sometimes with enhanced transcript accumulation during cold or desiccation stress [39]
Northern analysis of the 2.7a sequence, upstream of Afp- Lul confirmed that it was transcribed and therefore not a pseudogene but transcript accumulation was different than for Afp-Lul and Afp-Iul1 As transcript levels for 2.7a were low in L2s, it is not surprising that an isoform correspond- ing to this gene was not recovered in plaque lifts of acDNA library made from second instars [16] The reason for this differential pattern of expression in these AFP genes, however, is unclear as the insects would not normally encounter subzero temperatures for extended periods after hatching from the egg in late summer [41] It is possible then, that this gene (tentatively grouped with the long 3’ UTR class) encodes a protein that accumulates in early second instars to protect against late summer frosts
Two differently sized transcripts were seen for each of Afp-Lul and 2.7a As Southern blots showed that there were two different gene copies, these different transcript lengths could be encoded by distinct loci It must be noted, however, that transcript diversity can also be generated by alternative polyadenylation [42] and several sites containing the canonical sequence AATAAA, or a single nucleotide variant of it, were found downstream of both Afp-Lul and 2.7a genes, followed within 30 bp by the “CA” dinucleo- tides necessary for primary transcript cleavage and poly(A) attachment [43] AFP gene regulation by the hormonal regime preceding and during diapause would be attractive,
as Afp-Lul and Afp-Iul appear to be similarly regulated, but these AFP gene family members have no obvious regulatory elements in upstream or intron regions
It is thus apparent that C fumiferana evolved multiple AFP gene copies as part of a strategy to survive extreme winter temperatures Naively, one might assume that the copy number was increased during evolution simply to provide for an increased titre of hemolymph AFP It now appears that not only was copy number increased, but differences in protein structure, most obviously represented
by larger proteins encoded by the intermediate 3’ UTR class, as well as subtle differences in gene regulation seen during development, may all be part of a complex solution
to selective forces exerted by seasonal environmental stress
ACKNOWLEDGEMENTS
Dr Michael Kuiper is thanked for generously modeling the 2.7a protein One of two genomic libraries used was a gift from Dr Donal Hickey (University of Ottawa) The research was supported by scholarships from the Fonds pour la Formation des Chercheurs et I’Aide a la Recherche and the Ontario Government to D D and a grant from the
Trang 8National Science and Engineering Research Council of Canada to
V.K.W P.L.D is funded by Canadian Institutes of Health
Research
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