Tài liệu Báo cáo Y học: A family of expressed antifreeze protein genes from the moth, Choristoneura fumiferana ppt

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 t he overwintering
stage. We now report the cloning of AFP genes from insects;
Afp-Lu1 encodes a  9-kDa AFP isoform, and Afp-Iu1
encodes a  12-kDa AFP isoform. Both CfAFP genes have
similar structures w ith 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 k b upstream of Afp-Lu1 and demonstrates that
some AFP family members are linked in tandem. This gene
appears t o encode an AFP w ith 68±76% identity to p revi-
ously isolated CfAFPs. With i ts eight Cys residues necessary
for disul®de bonding and ®ve perfectly conserved ÔThr
buttonÕ (Thr-Xaa-Thr) ice-binding motifs, it c an be modeled
as a functional AFP. Southern blot analysis shows that there
are  17 genes in this AFP family, w ith e ach of t he isofo rms
represented by two to ®ve gene copies. Transcript accumu-
lation from Afp-Lu1 and Afp-Iu1 (or closely related genes)
was maximal during the overwintering stage, while 2.7a
transcripts were only detected in ®rst instars, larvae that are
normally found only in the summer. Contrary to expecta-
tions, this dierential expression demonstrates that CfAFP
gene family transcripts are primarily regulated during
development, rather than by seasonally low temperatures.
Keywords: antifreeze protein; gene family; cold stress; mo th
development.

Insects have developed m ultiple adaptations to changes in
their environment. Environmental stress of a predictable
nature such as seasonal d rought, h eat or cold may be
endured by entering diapause, a physiological state charac-
terized by low m etabolic activity [1]. Many species of insects
prepare for overwintering by triggering t he diapause
response, but freeze-intolerant species such as the spruce
budworm, Choristoneura fumiferana, must a lso 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 f reezing
point of extracellular ¯uids and a llow 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) a dsorb to microscopic ice
crystals and p revent their g rowth, 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 a re found in differen t
organisms including ®sh, insects, plants, bacteria and fungi,
but are best known in ®sh [6]. Four different types of
antifreeze proteins (AFPs I to IV) and one antifreeze
glycoprotein have been characterized in bony ®sh from t he
icy seas of both the northern a nd southern hemispheres. At
high concentrations they offer protection to the freezing
point of seawater ()1.9 °C). AFPs isolated f rom beetles and
spruce budworm are 10±100 times more active on a molar
basis than the ®sh AFPs or antifreeze glycoproteins [2,7].
This p resumably re¯ects t he 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 ®sh
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 t he
ice surface on both prism and basal planes.
Thermal hysteresis activity increases with AFP concen-
tration and bo th ®sh and insects are dependen t on high
circulating AFP concentrations for full protection against
freezing. In some ®shes, the demand for AFP has been
largely met by increasing the number of AFP genes. Ocean
pout and wolf®sh from cold coastal waters have  150 and
80 AFP gene copies, respectively [10,11]. O ther ®shes such as
the s ea raven [12] and winter ¯ounder [13] a lso h ave multiple
AFP g ene copies. I nsects may a lso use ge ne families f or AFP
production. Numerous AFP isoforms differing slightly in
primary sequence or m ass have b een found in the beetles,
Tenebrio molitor and Dendroides c anadensis as well as in
C. fumiferana [14±16]. In Tenebrio, Southern hybridizations
with AFP gene probes showed numerous bands, re¯ecting
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 d ifferent cDNAs e ncode p roteins o f two
distinct size s,  9and 12 kDa. The larger variants c ontain
a 30- or 31-amino-acid insertion creating two additional
15- or 16-amino-acid loops in the b helix [16]. The 9-kDa
proteins are e ncoded by cDNAs possessing either a long
( 1000 nucleotides) or a short (  200 nucleotides) 3¢ UTR,
while the 12-kDa CfAFPs are e ncoded by c DNAs w ith

3¢ UTRs of intermediate size ( 450 nucleotides). The
existence of Cf AFP v ariants, differing in both protein s ize
Correspondence to V. K. Walker, D ep artment of B iology, Queen's
University, Kingston, Ontario, Canada K7L 3 N6.
Fax: + 1 613 533 6617, Tel. + 1 613 533 6123,
E-mail:
Abbreviations: AFP, antifreeze protein.
(Received 30 August 2001, accepted 19 October 2001)
Eur. J. Biochem. 269, 38±46 (2002) Ó FEBS 2002
and message length strongly suggests that a gene family
encodes these AFPs. This paper reports the cloning of AFP
genes f rom a n insect. As w ell a s revealing additional i soform
variation, the isoforms show an unexpected differential
transcript accumulation pattern, especially during d iapause.
MATERIALS AND METHODS
Nucleic acid probes
DNA probes for the detection and isolation of speci®c
members of the CfAFP gene family were generated from
cDNA clones 10 and 337 [16] or genomic sequences.
A cDNA 10-speci®c probe encompassed the entire 414-bp
coding region of the isoform. A probe speci®c for the 337
cDNA isoform was generated b y PCR with SPF and SPR
primers as previously described [16]. A sequence 2.7-speci®c
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. fu miferan a were obtained from t he Canadian Forest
Service ( Sault Ste-Marie, O N, Canada). Genomic DNA was
puri®ed from a single sixth instar larva by proteinase K

digestion and phenol/chloroform extraction using the
method of Blin & Stafford [17]. DNA samples (20 lg) were
digested with the restriction enzymes HindIII, EcoRI, SstI,
and BamHI (Gibco BRL, Burlington ON, USA), electro-
phoresed in a 0.9% a garose gel, and subsequently trans-
ferred to Hybond N nylon membrane (Pharmacia, Baie
d'Urfe
Â
, Quebec, Canada), following the manufacturer's
protocol. Hybridization was carried in 0.25
M
Na
2
HPO
4
/
7% SDS [18] or 5 ´ NaCl/Cit/0.5% SDS/5 ´ 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 u nder low stringency ( 0.1
M
Na
2
HPO
4
/5% SDS, 65 °C, 2 ´ 15 min). To detect
speci®c 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 ´ NaCl/Cit/0.1% SDS, 65 °C 15 min). After washing,

blots w ere exposed to a Phosphorimager screen (Pharmacia)
at room temperature or to Biomax MS ®lm (Eastman
Kodak, Rochester, NY, USA) at )80 °C.
Genomic DNA library screening
A C. fumiferana genomic library constructed in kDASH II
(Stratagene, LaJolla, CA, USA) was plated, and 6.5 ´ 10
5
plaques were s creen ed using the 337 cDNA fragment probe.
Filters were washed using the low stringency conditions
described above. Positive clones were randomly picked, a nd
their DNA isolated and d igested with HindIII, SstIand
XhoI restriction enzyme s to generate restriction enzyme
maps. DNA fragments, obtained by restriction digests or
long PCR (Expandä 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 s trands. Noncoding DNA
was sequenced in one direction only.
RNA isolation and Northern blots
C. fu miferan a used for developmental studies were reared
on an arti®cial 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 ®rst instar
(L1) samples w ere s acri®ced 48±51 h after egg hat ching. L2s
were collected from che esecloth at various times a fter
hibernaculum (cocoon) spinning and molting: after 1 and
2 w eeks at 2 3 °C and 1, 5, 10, 15 a nd 3 0 weeks af ter storage

at 2 °C. Post-diapausing instars (L3±L6) were sacri®ced
24±48 h after molting. Animals were frozen in liquid n itrogen
andstoredat)80 °C. Total RNA was isolated with Trizol
(Gibco BRL) using a modi®cation of t he manufacturer's
protocol [21]. RNA (10 lg) was loaded onto a 1.2%
agarose/formaldehyde gel and transferred to Hybond N
according t o established metho ds [ 19]. Blots w ere h ybridized
successively with the appropriate AFP probes in 50%
formamide/5 ´ NaCl/Cit/5 ´ Denhardt's solution/0.5%
SDS and 100±150 lg of sheared salmon sperm DNA
(Gibco BRL) for a minimum o f 12 h. T he blots were a lso
probed with a n a-tubulin fragment from Drosophila mela-
nogaster as a RNA loading c ontrol. High stringency washes
were carried out in 0.1 ´ NaCl/Cit/0.5% S DS at 65 °Cfora
minimum of 20 min. Detection of signal on X-ray ®lm 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 identi®ed. Of these, two were chosen and
mapped using restriction enzymes (Fig. 1). One of the two,
2.7, h ad a 13.9-kb insert that contained the complete coding,
upstream and downstream nucleotide sequence c orrespond-
ing to a member of the long 3¢ UTR class of isoform. This
gene, designated Afp-Lu1 (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-Lu1 is an allelic variant of
either of these  9-kDa isoform proteins, or represents a
closely related isoform. A single 3.6-kb intron, identi®ed by
comparison with the cDNA sequence, is positioned after the
®rst nucleotide of t he Val16 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. AF32 5859).
Afp-Lu1 contains several kb o f 5¢ ¯anking 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 cont ained 6 kb of 3¢ ¯anking DNA, of
which 1.4 kb contiguous to Afp-Lu1, were sequenced. This
section was very similar to the 3¢ UTR of isoform 337,
except for an insertion of an additional 517 nucleotides. No
Ó FEBS 2002 Antifreeze protein genes in Choristoneura (Eur. J. Biochem. 269)39
splice jun ctions were found within this 3¢ UTR insertion
indicating that it is not an intron. Sequencing also allowed
the identi®cation of putative polyadenylation signals at
positions 4548, 4650 and 4785. Upstream of the Afp-Lu1
gene a t )641 t here was a large i nverted repeat spanning
about 500 bp (Fig. 1). Approximately 200 nonoverlapping
nucleotides of this palindrome w ere s equenced 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-Lu1. This sequence, tentatively desig-
nated 2.7a (as i t should not be designated a formal gene
symbol without the corresponding cDNA), was located
 3.7 kb upstream from the translation start of Afp-Lu1.
The sequence does not have the obvious features of a
pseudogene such as stop codons or frameshifts. It has
conserved C ys residues and putative ÔThr buttonÕ ice-
binding motifs (Thr-Xaa-Thr; where X aa is any a mino acid)
every 15±17 residues (Fig. 2A) a nd therefore probably
encodes a functional CfAFP. With a predicted mass of
9320 Da, i t would be placed either in the long or short
3¢ UTR isoform class ( 1000 and  200 nucleotides,
respectively) a nd 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,
it has been provisionally placed in the long UTR isoform
class b ased on its nucleic acid sequence and the potential size
of its mRNA. Molecular modeling of t his new, putative
isoform, based on the NMR-derived structure of the 337
isoform, is consistent with the 2.7 a sequence e ncoding a
functional AFP (Fig. 2B). In deed, this gene would e ncode
an isoform containing all ÔperfectÕ Thr-Xaa-Thr repeats. Of
a dozen previously identi®ed 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 p laque characterized from the g enomic
library, clone 2.26, had a 13.9-kb insert which included the
complete sequence for a gene of the intermediate size 3¢

UTR c lass of AFP ( Fig. 1). With t he exception of the longer
coding region, the overall structure of the AF P-Iu1 (inter-
mediate UTR 1) gene is very similar to Afp-Lu1. The open
reading f rame is interrupted by a s ingle phase 1 intron at t he
conserved V al codon (Val15 in Af pIu-1). At 3 kb, this intron
is only 600 bp sm aller t han t hat o f Afp-Lu1 .Theinterme-
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-Iu1 (GenBank accession
no. AF325857) matched the larger 12-kDa isoforms with
10 nucleotide differences when compared to cDNA 10
(four resulted in amino-acid changes: Asp13 ® 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-Iu1 had the same
polyadenylation signal, AATATA, found in the cDNA 10
isoform.
Southern analysis
In order to estimate the copy number of the cloned genes,
probes representing cDNA 337 and 10 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 i s presen t in
multiple copies. Washing conditions were chosen to detect
hybridization equivalent to a T
m
 20 °C below that of
routine washes with the 337 probe. Assuming a 1%

mismatch per degree of difference [24], it 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, 10, could also be seen in 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 SstI, HindIII and EcoRI digests of the DNA from
individual animals. Reprobing the same blots with sequenc-
es speci®c 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 ®ve 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-Lu1) and two copies hybridizing t o the
Fig. 1. Restriction maps of C. fumiferana genomic clones encoding the
AFP genes Afp-Lu1 and 2.7a (A) and Afp- Iu1 (B). Horizontal black
boxes below the long ho riz ontal lines indicate the co ding regions of the
AFP genes, and horizontal thin lines, r egions sharing homology with
cDNA untranslated regions. The dashed horizontal line for the gene
2.7a ind icate s the putative 3¢ UTR o f the ge ne, b ased on the t ranscript
size estimation. The white box in the 3¢ end of Afp-Lu1 is a 517-
nucleotide nonint ronic sequence that is not present in the 3¢ UTR of
cDNA 337. Ve rtical arrows point to p utative polyadenylation sites and
the s tars indi cate those that a re used based again on transcript size

estimation. V-shape d t hin lines joining exons represe nt s pliced regions
of the AFP gene s, and the inward pointing triangles i n clone 2.7 rep-
resent a  50 0 bp inverted repeat. The restriction enzymes used were:
H, HindIII; S, SstI; X, XhoI.
40 D. Doucet et al. (Eur. J. Biochem. 269) Ó FEBS 2002
2.7a gene probe. Hybridization to the cDNA 10 probe
(corresponding to Afp-Iu1) shows that there are at least ®ve
copies (Fig. 3A,B).
Northern analysis
The probes used f or South ern hybridizations were also us ed
to study the pattern of expression of each isoform
throughout C. fumiferana development. The sizes of tran-
scripts corresponding to Afp-Lu1 and Afp-Iu1 are consistent
with the size class of the previously cloned cDNA homo-
logues, 337 and 10, respe ctively ( Fig. 4). However, North-
ern blots probed w ith the 337 probe showed an additional,
smaller mRNA of 1 kb as well as the expected transcript at
1.4 kb. There was a single 1-kb transcript detected with the
cDNA 10 probe, although a faint band representing
Fig. 2. Compendium of CfAFP sequences. (A) Amino-acid alignment of sequences retrieved from clones 2.7 (Afp- Lu1 and 2.7a)andclone2.26
(Afp-Iu1) as well as representatives of t he intermediate (cDNAs 10, 105 and 501), long (cDNAs 4, 337 and 333), and short (cDNA 339)UTRclasses
of CfAFP c DNAs. Amino acids identical to Afp-I u1 a re r epresented by dots whil e d ashes indicate gaps in the alignment. Afp-Iu1 and c DNAs 10 ,
104 and 501 each encode a longer ( 12-kDa) isoform than the other cDNA or gene s e quences, which encode  9-kDa AFPs. The shaded b oxes in
the alignments in dicate the conserved putative ice-binding Ôthreonine buttonsÕ 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.
Ó FEBS 2002 Antifreeze protein genes in Choristoneura (Eur. J. Biochem. 269)41
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
30weeksaftertransferto2°C. Unexpectedly, howeve r, the
transcripts that correspond to the Afp-Lu1 and Afp-Iu1
genes also accumulated to relatively high levels in ®rst
instars and in diapausing second instars maintained at
23 °C. A faint Afp-Lu1 signal could even be detected in egg
mRNA. No Afp-Iu1 signals were seen in eggs but low
transcript levels were seen in the ®fth and sixth larval instars
kept at 23 °C.
In order to examine the expression of 2.7a,forwhichno
cDNA has been previously described, a probe was s ynthe-
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
1.4 k b and 1 kb (Fig. 4). In contrast to the expression
Low
stringency
337 10
23.1
9.4
6.6
4.4
2.3
2.0
123 45 12 345 12 345 12 345
B
Low
stringency
337 10
ESH

A
2.7a
BESHB ESHB ESHB
2.7a
23.1
9.4
6.6
4.4
2.3
2.0
Fig. 3. Southern hybridization of three CfAFP sequences to restriction-digested C. fumiferana DNA. (A) DNA from a single s ixth instar larva w as
digested with four restriction enzymes. The ®rst panel shows low stringency washes of the CfAFP 337 isoform probe after hybridization.
Hybridization and high stringency wash es for probes of isoforms 337 (corresponding t o Afp-Lu1), 10 (to identify Afp-Iu1 )and2.7a are s hown in the
second, third and fourth panels, respectively. DNA was digested with: S, SstI; E, EcoRI ; H, Hin dIII and B, BamH1. Molecular ma ss markers ( in kb)
for the restriction fragments are indicated on the left. (B) Southern hybridization o f ®ve dierent individua l sixth instar DNA under the same
conditions as in (A) above. Genomic DNAs were digested with the restriction enzyme SstI.
42 D. Doucet et al. (Eur. J. Biochem. 269) Ó FEBS 2002
pattern of the previous described genes, however, the two
2.7a mRNAs accumulated to higher levels in the ®rst 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 p reviously mentioned t here were four
potential polyadenylation sites identi®ed in 2.7a. Assuming
thatthegenehasa5¢ exon and 5¢ UTR of roughly t he 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 b e expected, suggesting that t he ®r st and third sites
are recognized.
DISCUSSION
AFP gene families

Based on the discovery of several CfAFP isoforms differing
in sequence, it was postulated i n an earlier s tudy [16] that
members o f a multigene family would encode them. H ere w e
have addressed this hypothesis b y cloning genomic frag-
ments containing insect AFP genes. There are  17 unique
loci, making t his a low abundance g ene f amily, a nd 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 g ene copies also appear t o be selected
in response to environmental stress [28]. Even in Lepi-
dopteran (moths) and Dipteran (¯ies) orders that have a
compact genome [29], gene ampli®cation can occur if the
selection p ressure is strong, such as i n the ampli®cation of
esterase genes in the mosquito organophosphate insecticide
resistance phenotype [30], the metallothionein gene dupli-
cation in metal resistant Drosophila [31] and the magni®ca-
tion of ribosomal DNA in bb
±
mutants [32].
In ®sh genomes there may be even less constraint to
increasing gene copy number by selection. Indeed, AFPs in
®sh a re 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 mgámL
)1

) during the winter [33]. Analogously,
multiple gene copies may have been sele cted in this
C. fu miferan a population, collected from the northern
boreal forest, to satisfy a similar demand for elevated levels
Egg L1
Alpha-
tubulin
2.7a
10
337
Probe
1w 2w 1w 5w 10w 15w 30w L3 L4 L5 L6 Pu Ad
1.5
Size
(kb)
1.1
1.0
1.4
1.0
1.4
23°C2°C
L2 (Diapause)
Fig. 4. Expression of CfAFP isoforms 337, 10 and 2.7 a and a control gene (a-tubulin) during d evelopment. Eggs, all six larval instars ( indicated w ith
the ÔLÕ p re®x a nd a number), p upal (Pu) a nd adult ( Ad) stages of the insects w ere tested f or e xpression by northern hybridization. S izes o f the AFP
mRNAs (in kb ) are indicated at t he right. Second instars in diapause, 1 and 2 weeks (w) after hibernaculum spinning (L2, 23 °C) as well as 1, 5, 10,
15 and 3 0 weeks after transfer to cold storage (L2, 2 °C) were sa mpled. The arrow on t he is oform 1 0 p anel i ndicates a 1.4-kb transcript seen later
during diapause. The 337 probe hybridizes with transcripts c orrespondin g to the Afp-Lu1 gene and the 10 probe hybridizes w ith transcripts
corresponding to the Afp-Iu1 gene.
Ó FEBS 2002 Antifreeze protein genes in Choristoneura (Eur. J. Biochem. 269)43
of AFP in the hemolymph of overwintering larvae. I nsect

AFP is hyperactive compared to ®sh 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, T. molitor with at least
twice the number of A FP genes i s a domestic s pecies 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 w ell as synthesizing A FPs, 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 °Corlower.
The existence of a C. fumiferana AFP s equence upstream
of the gene Afp-Lu1 on clone 2.7 provides the ®rst direct
evidence that some AFP genes are tightly linked in this
insect. However, v ery l arge arr ays of tandem genes a s f ound
in ®sh type I and III AFPs [11,13], and hypothesized for
Tenebrio AFP [ 14], are unlikely. Southern blots indicate a
somewhat smaller A FP gene family (Fig. 3A,B), and
because the hybridization signals were distributed between
several, nonidentical large f ragments of DNA, it is likely
that at least some of the  17 genes of the family are spaced
several kb ap art (as fo r Afp-Lu1 and 2 .7a), e ven though they
may be linked.

In both Afp-Lu1 an d Afp-Iu1, 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 d evelopmentally regulated g enes
[34,35]. In addition, a large palindrome of  500 bp (of
which  200 b p at each extremity was sequenced) was
found 641 bp upstream of Afp-Lu1 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-Lu1 and Afp-Iu1 accu-
mulate in the second larval instar. This is consistent with
the detection of CfAFP isoforms in larval extracts of the
same stage, 1 2 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-speci®c
because approximately equivalent levels were detected in
®rst instars, which in the wild, are exposed to the high
temperatures of mid and late summer. Temperature, and
constant darkness, did not affect tran script accumulation
of Afp-Lu1 or Afp-Iu1 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, kep t at 2 °C for 10 weeks, to 15 °C for 1 w eek;
this treatment had no effect on transcript accumulation
(not shown). Expression in other stages was low and
isoform-speci®c, as exempli®ed by the transcript corre-
sponding to Afp-Lu1 found in eggs and the Afp-Iu1
transcripts found in third, ®fth 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 Ôstress genesÕ such as the
AFP genes from T. molitor [39], the heat shock protein
gene, hsp70, from the ¯ esh ¯y, 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-
Lu1 con®rmed that it was transcribed a nd therefore not a
pseudogene but transcript accumulation was different than
for Afp-Lu1 and Afp-Iu1. As transcript levels for 2.7a were
low in L2s, it is not surprising that an isoform correspond-
ing to this gene w as not re covered in plaque lifts of a cDNA
library made from s econd 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 f or extended periods after
hatching from the egg in late summer [41]. I t i s possible t hen,
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-Lu1 and 2.7a. As Souther n blots showed that there were
two d ifferent gene copies, t hese different transcript lengths
could b e 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-Lu1
and 2.7a genes, followed within 30 bp by the ÔCAÕ 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 a ttractive,
as Afp-Lu1 and Afp-Iu1 appear to be similarly r egulated, but
these A FP gene family members have no obvious regulato ry
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 t itre of hemolymph AFP. I t 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 modelin g the 2.7a protein.
One of two genomic libraries used was a gift from Dr Donal Hickey
(University of Ottawa). The r esearch w as supported by s cholarships

from the Fonds pour la Formation des Chercheurs et l'Aide a
Á
la
Recherche and t he Ontario Gov ernment to D. D. and a grant from the
44 D. Doucet et al. (Eur. J. Biochem. 269) Ó FEBS 2002
National Science and Engineering R esearch Council of Canada to
V. K. W. P. L. D. is funded by Canadian Institutes of Health
Research.
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