Type I antifreeze proteins expressed in snailfish skin are identical to their plasma counterparts Robert P. Evans and Garth L. Fletcher Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada Teleost fish that inhabit icy seawater synthesize anti- freeze proteins ⁄ polypeptides (AFPs) or antifreeze glyco- proteins (AFGPs) for protection against freezing. Diverse species from numerous taxonomic groups pro- duce AFPs that are grouped into four distinct classes (types I, II, III and IV) based on their primary and secondary structural characteristics [1–3]. Regardless of protein structure, all fish AFPs lower the solution freezing point noncolligatively by binding to certain surfaces of ice crystals, modifying their structure and inhibiting further growth. The difference between the lowered freezing point and unaltered melting point is termed thermal hysteresis and is used as a measure of antifreeze activity [1,3,4]. Of the four classes of AFPs described thus far, the simplest is type I AFP found in right-eye flounders (Pleuronectes) and a few sculpin species (e.g. Myoxo- cephalus). These polypeptides have high alanine con- tent (> 60 mol%), have an amphipathic a-helical secondary structure, and are usually quite small (3.3– 4.5 kDa) [2,5]. Until the past decade, it was generally accepted that the synthesis of AFPs was confined solely to liver tissue (termed liver type) for secretion into blood for extracellular freeze protection. How- ever, more recently, a novel subclass of type I AFPs was isolated and characterized from the skin of winter flounder Pseudopleuronectes americanus (for- merly Pleuronectes americanus) [6]. These AFPs, which Keywords antifreeze; cDNA; protein expression; snailfish; type I Correspondence R. P. Evans, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Fax: +1 780 492 0886 Tel: +1 780 492 3481 E-mail: robert.evans@ualberta.ca (Received 13 June 2005, revised 8 August 2005, accepted 22 August 2005) doi:10.1111/j.1742-4658.2005.04929.x Type I antifreeze proteins (AFPs) are usually small, Ala-rich a-helical poly- peptides found in right-eyed flounders and certain species of sculpin. These proteins are divided into two distinct subclasses, liver type and skin type, which are encoded by separate gene families. Blood plasma from Atlantic (Liparis atlanticus) and dusky (Liparis gibbus) snailfish contain type I AFPs that are significantly larger than all previously described type I AFPs. In this study, full-length cDNA clones that encode snailfish type I AFPs expressed in skin tissues were generated using a combination of library screening and PCR-based methods. The skin clones, which lack both signal and pro-sequences, produce proteins that are identical to circulating plasma AFPs. Although all fish examined consistently express antifreeze mRNA in skin tissue, there is extreme individual variation in liver expression – an unusual phenomenon that has never been reported previously. Further- more, genomic Southern blot analysis revealed that snailfish AFPs are products of multigene families that consist of up to 10 gene copies per genome. The 113-residue snailfish AFPs do not contain any obvious amino acid repeats or continuous hydrophobic face which typify the structure of most other type I AFPs. These structural differences might have implica- tions for their ice-crystal binding properties. These results are the first to demonstrate a dual liver ⁄ skin role of identical type I AFP expression which may represent an evolutionary intermediate prior to divergence into distinct gene families. Abbreviations AFGPs, antifreeze glycoproteins; AFPs, antifreeze proteins ⁄ polypeptides; IBM, ice-binding motif; ORF, open reading frame; UTR, untranslated region. FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS 5327 are encoded by a separate subset of genes, were desig- nated as skin-type AFPs. They are synthesized as mature polypeptides that lack both signal and pro- sequences, which suggests that they remain intracellu- lar [6]. Recent publications of skin-type AFP isolation from shorthorn (Myoxocephalus scorpius) and long- horn (M. octodecemspinosus) sculpins indicate that the production of AFP in peripheral epithelial tissues may be a common trait in many fish species [7,8]. The char- acterization of known skin-type AFPs and the presence of antifreeze activity in skin tissues of other species has led to the hypothesis that skin-type AFPs are wide- spread ancestors of liver-type (plasma) AFPs [6,9]. Atlantic snailfish (Liparis atlanticus) and dusky snailfish (L. gibbus) belong to a large family (Cyclo- pteridae) of benthic and pelagic marine fishes that inhabit northern regions of the Atlantic Ocean. Snail- fish are closely related to sculpins, which belong to a different family of the same order Scorpaeniformes [10]. Both species spawn during the winter months in ice-laden inshore coastal regions around Newfound- land, which makes them prime candidates for produc- tion of AFPs. Type I AFPs were previously isolated and characterized from the blood plasma of both Atlantic and dusky snailfish which are the largest des- cribed to date (> 9.3 kDa) [11]. We have also shown recently that Atlantic snailfish skin tissues contain type I AFPs that have identical molecular mass and very similar amino acid content to their plasma counter- parts [12]. Further analysis of the snailfish AFPs would be helpful in determining the structure ⁄ function charac- teristics of these unusually large type I AFPs and to clarify the relationship between skin and plasma pro- teins. Pursuant to this, an Atlantic snailfish skin cDNA library was screened using a shorthorn sculpin skin-type AFP clone as a probe. Full-length cDNA sequences of both Atlantic and dusky snailfish skin type I AFPs were generated using a combination of library clones with RT-PCR and RACE techniques. Results from this study show that skin AFPs from both species are nearly identical to each other and their skin transcripts produce proteins that are identi- cal to their corresponding plasma proteins. Results cDNA library screening and cloning of snailfish skin AFP A cDNA library was constructed to investigate the presence of type I AFP mRNA in skin tissues. Two independent clones were identified from the library screen using the open reading frame (ORF) of a shorthorn sculpin skin cDNA as a probe. The  260 bp clones (clone-c1 and clone-c2) contained identical sequences, apart from a small difference in the length of poly(A) tail and a few nucleotides at their 5¢ ends. However, the clones appeared to be truncated versions of complete type I AFP messages. As indica- ted by the underline in Fig. 1, one reading frame gave an Ala-rich 26 amino acid peptide, that lacks an obligatory in-frame start codon. This sequence infor- mation was then used in 5¢-RACE reactions to ascer- tain the remainder of the skin AFP cDNA sequence. RNA ligase-mediated RACE was used to clone the remaining 5¢ portion of the snailfish AFP cDNA. The full L. atlanticus skin cDNA is 568 bp and contains a complete 342 bp ORF (Fig. 1). The ORF encodes an Ala-rich protein of 113 residues and was designated as Las-AFP (L. atlanticus skin AFP). The putative start and stop codons are underlined as well as three pos- sible polyadenylation signal sequences [13]. The Las-AFP sequence was utilized to design appro- priate RT-PCR and 3¢)5¢-RACE primers for de novo cloning of AFP sequence from dusky snailfish skin RNA. The 3¢-RACE procedure (primers indicated in Fig. 2) produced a single band that was  450 bp, whereas 5¢-RACE gave a 370 bp product. The overlap- ping sequences were combined into a 587 bp clone which contained a 342 bp ORF that encodes a 113 residue, Ala-rich, protein designated as Lgs-AFP (L. gibbus skin AFP). The putative start and stop codons are underlined along with the standard poly- adenylation signal sequence. The AFP cDNAs cloned from the skin tissues of Atlantic and dusky snailfish have strikingly similar nuc- leotide sequences that encode nearly identical proteins apart from a few minor differences. However, there is a 19 bp insertion in the Lgs-AFP 3¢-untranslated region (UTR) just before the poly(A) tail region. Snailfish AFP cDNA sequences are similar to skin-type AFPs from winter flounder and sculpins in that they do not appear to contain a signal sequence or pro-sequences [6–8]. Surprisingly, the amino acid composition of proteins purified from Atlantic snailfish skin tissue is quite sim- ilar to the AFP predicted from the cDNA sequence (Table 1). Any differences may be attributed to varia- tions in analytical procedures and the fact that mix- tures of AFPs were analyzed. Most importantly, the predicted molecular mass and N-terminal sequence for Las-AFP is identical to the isolated plasma proteins [11]. Dusky snailfish also express the same type I AFP in skin tissue that is circulating in their blood (Table 1). Expression of snailfish type I AFPs in skin tissue R. P. Evans and G. L. Fletcher 5328 FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS Analysis of snailfish AFP genes Total RNA from Atlantic snailfish tissues were probed with a section from the 3¢-UTR of Las-AFP to evalu- ate the distribution of snailfish AFP mRNA (Fig. 3A). A specific band was visible after a short exposure in skin tissue as well as a faint signal from gill is detect- able with longer exposures. No other tissues gave detectable signals on this northern blot. Similar results were observed in another fish, except that there was a Fig. 1. Nucleotide sequence and primary translation product of L. atlanticus skin AFP cDNA. The ORF is capitalized, whereas the 5¢- and 3¢-UTRs are in lower case letters. The putative start and stop codons are underlined in bold as are three possible polyadenylation signal sequences. The sequence obtained from the initial las-c1 and las-c2 cDNA clones are underlined. RT-PCR or RACE primer sequences are shown above (5¢fi3¢) or below (3¢fi5¢) the nucleotide sequence. GenBank Accession Number AY455862. Fig. 2. Nucleotide sequence and primary translation product of L. gibbus skin AFP cDNA. The ORF is capitalized, whereas the 5¢- and 3¢-UTRs are in lower case letters. The putative start and stop codons and the polyadenylation signal are underlined. RT-PCR or RACE primer sequences are shown above (5¢fi3¢) or below (3¢fi5¢) the nucleotide sequence. GenBank Accession Number AY455863. R. P. Evans and G. L. Fletcher Expression of snailfish type I AFPs in skin tissue FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS 5329 definite detectable signal in liver tissue RNA (Fig. 3B). RT-PCR performed using the same RNA (with ORF primer set) gave positive bands for skin, gill, blood cells, kidney, spleen and liver (Fig. 3C) and experi- ments using 3¢-UTR primers gave the same expression pattern (data not shown). Additional northern blot experiments with 3¢-UTR DNA probes gave very intense autoradiographic sig- nals in skin tissues from four Atlantic snailfish and a dusky snailfish (Fig. 4A), these were confirmed by RT-PCR analysis (Fig. 4B). Results of a northern blot using liver RNA from eight individual Atlantic snail- fish and a dusky snailfish showed that three of the Atlantic snailfish samples, but not the dusky snailfish, gave positive signals of varying intensities (Fig. 4C). RT-PCR analysis confirmed this result with the excep- tion of one liver sample (Fig. 4D). A recently pub- lished report of northern blots probed with shorthorn sculpin skin ORF cDNA, indicated that snailfish liver and skin tissues express type I AFP mRNA [11a]. To analyze snailfish genes, a Southern blot was probed with the same DNA probe applied to the pre- vious northern blots. At least nine individual frag- ments can be distinguished when Atlantic snailfish DNA is digested with HindIII (Fig. 5; lane 3), whereas up to 10 are visible for dusky snailfish and the same restriction enzyme (Fig. 5; lane 7). Results indicate that snailfish AFPs are expressed via a multigene fam- ily but the exact number gene copies cannot be deter- mined precisely here. Discussion Using a combination of cDNA library screening and 5¢-RACE, a complete cDNA corresponding to type I AFP was cloned from Atlantic snailfish skin tissue and Table 1. Amino acid composition (mol%) and molecular mass of snailfish type I AFPs. Amino acid LaP-AFP (protein) LaS-AFP (protein) Las-AFP (cDNA) LgP-AFP1 (protein) LgP-AFP2 (protein) Lgs-AFP (cDNA) Asx 3.6 5.5 3 5.4 5.5 3 Glx 3.0 4.9 2 2.6 2.6 2 Ser 2.8 4.7 5 2.0 2.0 5 Gly 4.6 3.7 2 3.9 3.9 2 Arg 1.6 2.4 1 1.8 0.9 3 Thr 10.3 10.8 15 8.9 9.0 15 Ala 58.8 45.9 69 51.2 51.7 66 Pro 2.5 2.9 2 4.2 4.2 3 Val 5.6 4.9 5 8.4 8.5 5 Ile 1.3 2.1 1 1.7 1.8 1 Leu 2.6 4.1 2 2.3 2.3 2 Lys 3.4 4.1 5 6.6 6.6 5 Mol mass (Da) 9344, 9344, 9415 a 9415 b 9646 a 9573, 9742 a 9742 b 9415 a 9457, 9387 9501 9514, 9814 a Based on ESI-MS analysis of HPLC peaks [11,12]. b Predicted from cDNA sequence excluding Met. LaP-AFP, L. atlanticus plasma AFP; LaS-AFP, L. atlanticus skin AFP; LgP-AFP, L. gibbus plasma AFP; LgS-AFP, L. gibbus skin AFP. A B C Fig. 3. Tissue distribution of Atlantic snailfish skin AFP mRNA. (A, B) Northern blots of samples from two individual fish with lanes labeled with RNA tissue source. Each lane contains 5 lg total RNA and blots were probed with a 175 bp fragment of the 3¢-UTR sequence of snailfish type I AFP cDNA. (C) A typical result of RT-PCR analysis. Numbers correspond with the tissue labels from the northern blots above. c1 is a water only control; c2 is no RT control. The lower panel shows RT-PCR products generated from b-actin primers used as a loading control. Expression of snailfish type I AFPs in skin tissue R. P. Evans and G. L. Fletcher 5330 FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS subsequently in closely related dusky snailfish. The nucleotide and protein sequences are almost identical, clearly suggesting that these AFPs shared a common ancestral gene prior to snailfish species divergence. This differs from taxonomically related shorthorn and longhorn sculpin skin AFPs which produce quite con- trasting proteins, whereas the UTRs of mRNA are nearly identical [8]. Based on the cDNA sequence, both snailfish species express 113 residue type I AFPs that are the largest described to date. The predicted proteins lack signal or pro-sequences, which indicates that the mature poly- peptides remain intracellular. This would imply that their location and function is analogous to the pre- sumptive intracellular skin AFPs of winter flounder [6] and sculpins [7,8]. However, the molecular mass of snailfish skin proteins predicted from cDNA and their N-terminal sequence are identical to the results deter- mined for their purified plasma AFPs [11,12]. Further- more, northern blots indicate that snailfish AFP mRNA has consistently significant expression only in skin tissue. Taken together, the evidence indicates that the circulating plasma AFPs and skin localized AFPs are identical proteins that are normally expressed by the same skin-specific gene. These results represent the first definitive report of fish that synthesize identical AFPs for protection in two different physiological locations. The assumption has been that skin-type AFPs are expressed via a different subset of genes from the liver multigene fam- ily [6–8]. The evidence from snailfish contradicts the A B C D Fig. 4. Distribution of type I AFP mRNA in skin and liver tissues from Atlantic and dusky snailfish. (A) Northern blot analysis of skin tissue RNA from four individual Atlantic snailfish and one dusky snailfish. Each lane contains 5 lg total RNA and blots were probed with a 175 bp fragment of the 3¢-UTR sequence of snailfish type I AFP cDNA. (B) The corresponding RT-PCR results from identical tissue samples. Numbers correspond with the tissue labels from the Northern blots. c1 is a water only control; c2 is no RT control. The lower panel shows RT-PCR products generated from b-actin primers used as a loading control. (C) Northern blot analysis of liver tissue RNA from eight individual Atlantic snailfish and one dusky snailfish. Blots were probed as indicated above. (D) The corres- ponding RT-PCR results from the same tissue samples as des- cribed above. Fig. 5. Southern blot analysis of Atlantic and dusky snailfish AFP genes. Ten micrograms of genomic DNA were digested with the indicated restriction enzymes and run in each gel lane. The blot was probed with the identical snailfish 3¢-UTR DNA fragment used in the northern blots. R. P. Evans and G. L. Fletcher Expression of snailfish type I AFPs in skin tissue FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS 5331 original hypothesis that separate sets of genes code for unique AFP isoforms to provide extracellular and intracellular antifreeze protection. Although the exact subcellular location has not yet been unequivocally established for skin-type AFPs, evidence from winter flounder indicates that skin AFPs are present in gill cell cytoplasm as well as in contact with the plasma membrane outside epithelial cells [14]. Clearly, snailfish AFPs produced in epithelial cells are secreted into blood to provide extracellular protec- tion but it is not clear whether some protein remains inside these cells. It is uncertain exactly how snailfish AFPs are secreted from the cells that expresses them if they do not contain the requisite signal sequences. There have been recent reports of mature type I AFPs being exported from cells in winter flounder epidermis despite the absence of a secretion signal or pro- sequence [14,15]. Furthermore, alternative pathways for protein export that circumvent the usual endo- plasmic reticulum–Golgi complex have been described previously [16,17]. The northern blot experiments exhibited unexpected variation in AFP expression patterns among individual fish. Whereas skin tissues consistently produced high levels of AFP mRNA, expression in liver ranged from undetectable to high levels. This extreme individual variation in mRNA expression has not been reported previously for any species producing antifreeze. How- ever, studies have shown geographic-dependent popu- lation differences in antifreeze gene copy number [18,19]. In fact, individual fish from one population of Newfoundland ocean pout had demonstrable differ- ences in antifreeze gene copies that indicate the malle- ability of antifreeze genes within a given fish genome [18]. Furthermore, there is a report in the literature of large variations in gene expression patterns in trans- genic rainbow trout [20]. It would be informative to determine if the diverse nature of the snailfish multi- gene family or if regulatory control regions within snailfish AFP gene(s) are responsible for the variation in observed tissue-specific gene expression. The physiological significance of the variegation in snailfish mRNA expression is not clear because all fish examined had significant levels of protein in blood and skin during the winter. It is possible that different phy- siological or environmental cues initiate expression in each tissue separately. Previous studies have shown that type I AFP expression in liver is seasonally adjus- ted from low in summer to high in winter based on environmental cues [1,3]. Moreover, skin AFP expres- sion is uniformly high in winter flounder but has an annual variation in shorthorn sculpin. It seems likely that skin AFP expression provides the primary source of AFP production in snailfish and the liver is an ancillary site of expression for contributing supple- mentary protection. Snailfish may rely more on skin (and its AFP content) to provide the primary barrier to ice crystal propagation. The primary structure of snailfish AFPs is unlike most other known type I AFPs. Although they are extremely a-helical proteins – determined experiment- ally by CD spectrometry – they possess only moderate thermal hysteresis activity compared with other type I AFPs [11]. Helical net and helical wheel representa- tions (Fig. 6) indicate that Las-AFP contain none of the ice-binding motifs (IBM) that were originally sug- gested as important for ice binding [21–23]. Recently, amino acid substitution experiments have determined that it is the conserved Ala-rich hydrophobic surface which is most important for ice-binding in type I AFPs [5,24–26]. Las-AFP contain no full-length hydrophobic surface is free from interfering polar residue side chains. Furthermore, snailfish AFPs do not contain Fig. 6. Schematic representations of Atlantic snailfish AFP secon- dary structure. (A) Helical net, (B) helical wheel diagrams were constructed by the EMBOSS package located on the Canadian Bioinformatics Resource web page. Hydrophilic residues DENQST are marked with diamonds. Positively charged residues HKR are marked with octagons. Aliphatic residues ILVM are marked with squares. Expression of snailfish type I AFPs in skin tissue R. P. Evans and G. L. Fletcher 5332 FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS the requisite hydrogen bonding amino acids necessary to create the elaborate terminal cap structures found in most type I AFPs [21]. The lack of complete hydro- phobic face and terminal caps might be responsible for the low activity of these AFPs. It should be noted, however, that the predicted structure of snailfish AFP may not exactly correspond with structural data provi- ded by experimental methods. It is possible that the protein contains kinks or bends in the backbone around the helix-breaking proline residues. Based on protein primary structure, most type I AFPs cluster into three distinct groups, depending on the nature of their highly conserved N-terminal sequences (Fig. 7). Two of the groups contain the clas- sic 11 residue (ThrX 10 ) repeat sequence, whereas the third group contains no such repeat structure. Although all polypeptides that fit in the three groups are small ( 3.3 to 4.5 kDa), the unusually large snail- fish and shorthorn sculpin skin AFPs are outliers that do not conform to either of the categories. Similarly, the novel hyperactive winter flounder type I AFP, which is unusually large (15 kDa) and without obvious amino acid repeats, would not fit into either major group [27]. Interestingly, there seems to be no connec- tion between the AFP structural groups and phylo- genic classification or tissue source of the proteins. With the discovery of snailfish skin proteins, it is apparent that type I AFPs can be divided into distinct structural subclasses based on size and the absence of amino acid repeat structure. This subclass could have unique evolutionary origins and a distinctive mechan- ism for ice-binding separate from the three groups mentioned above. Perhaps the fundamental property of a type I AFP, as represented by snailfish AFPs, is an Ala-rich protein with a-helical secondary structure that is capable of ice binding. Experimental procedures Tissue sample collection Twelve Atlantic snailfish (L. atlanticus) were collected by divers near Logy Bay, Newfoundland, in winter 2000. Two specimens of dusky snailfish (L. gibbus) were collected from Placentia Bay, Newfoundland during winter 1999. Tissues were removed from anesthetized fish, immediately frozen in liquid nitrogen and stored at )70 °C. Skin library construction and screening Total RNA from Atlantic snailfish skin tissue was isolated using TRIzolÒ reagent (Invitrogen Canada Inc, Burlington, ON, Canada) and poly(A) + mRNA was isolated from total RNA using an Oligotex mRNA Kit (Qiagen Inc, Mississ- auga, ON, Canada). A skin cDNA library was constructed, as described by the manufacturer, using Lambda ZAPÒ II library and ZAP-cDNAÒ Synthesis Kit and Gigapack Ò Gold III packaging extracts (Stratagene, La Jolla, CA, USA). The primary skin cDNA library contained around 5 · 10 5 clones. Normally,  50 000 plaques were grown on 15 cm NZYCM plates for primary screening; 9 cm plates were used in secondary and tertiary screens. Hybond-N nylon membranes (Amersham Biosciences, Piscataway, NJ, USA) were prepared and screened Fig. 7. Classification of known type I AFP sequences based on primary structural char- acteristics. Amino acid sequence alignments of the groups created by CLUSTALX analysis. Columns of identical amino acids are shown with black backgrounds, whereas those with a majority of identical amino acids are shaded gray. Abbreviations used: SS-3, shorthorn sculpin plasma AFP 3; AS-3, Arc- tic sculpin plasma AFP 3; GS-5, grubby scul- pin plasma AFP 5; lss-AFP, longhorn sculpin plasma AFP; wfs-AFP2, winter flounder skin AFP 2; wfl-HPLC6, winter flounder liver HPLC-6; AP-AFP, American plaice plasma AFP; wfl-AFP9, winter flounder liver AFP9; YT-AFP, yellowtail flounder AFP; sssAFP-2, shorthorn sculpin skin AFP 2; Las-AFP, L. atlanticus AFP. R. P. Evans and G. L. Fletcher Expression of snailfish type I AFPs in skin tissue FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS 5333 according to the manufacturer. Briefly, membranes were hybridized at 42 °C overnight in the following buffer: 5· NaCl ⁄ P i ,5· Denhardt’s, 0.5% SDS, 50% formamide and 100 lgÆmL )1 calf thymus DNA. Probe was labeled with [ 32 P]dCTP using an All-in-One Random-Primed Labeling Mix (Sigma-Aldrich, Oakville, ON, Canada) and purified prior to use with ProbeQuant G-50 Micro Columns (Amer- sham Biosciences). The final wash was performed in 1.0· NaCl ⁄ P i , and 0.1% SDS, at 52 °C for 20 min. A 300 bp DNA fragment corresponding to the ORF of shorthorn sculpin skin (s3–2) clone [7] was used as a probe to screen  2.0 · 10 5 clones of the primary cDNA library. Positive plaques were first isolated and then pBluescriptÒ phage- mids, to be used for sequencing inserts, were produced using an in vitro excision protocol (Stratagene). Northern blot analysis Total RNA from various tissues of Atlantic and dusky snailfish were isolated using TRIzolÒ reagent (Invitrogen Canada Inc) as described by the manufacturer. Five-micro- gram aliquots of total RNA were separated on 1% for- maldehyde gels and analyzed by a nonradioactive northern blotting procedure using positively charged nylon mem- branes (Roche Diagnostics Canada, Laval, QC, Canada). RNA was transferred to membranes using a VacuGene XL Vacuum Blotting System (Amersham Biosciences) and cross-linked with UV light. The membrane was hybridized at 50 °C overnight in DIG Easy Hyb buffer (Roche Diag- nostics). Probe was labeled with DIG-11–dUTP using a DIG-High Prime DNA Labeling kit or in some cases with a PCR DIG Probe Synthesis Kit (Roche Diagnostics) with chemiluminescent signal detection using CDP-StarÒ. The final wash was performed in 0.1· NaCl ⁄ P i , and 0.1% SDS, at 50 °C for 2 · 15 min. A 175 bp DNA fragment corres- ponding to the 3¢-UTR of the skin clone was used as a probe. Southern blot analysis Genomic DNA was isolated from liver of Atlantic and dusky snailfish using a WizardÒ Genomic DNA Purification Kit (Promega, Madison, WI, USA). Aliquots of RNAse A-treated genomic DNA were digested with various restric- tion endonucleases (Invitrogen). Five-microgram aliquots of the digestion products were separated in a 0.8% agarose gel, transferred to positively charged nylon membranes using a VacuGene XL Vacuum Blotting System (Amersham Bio- sciences) and cross-linked with UV light. A chemilumines- cent-based nonradioactive method was used to detect sequences on the membrane. Briefly, the membrane was hybridized at 42 °C overnight in DIG Easy Hyb buffer (Roche Diagnostics). Probe was labeled with DIG-11–dUTP using a PCR DIG Probe Synthesis Kit (Roche Diagnostics) with chemiluminescent signal detection using CDP-Star Ò . The final wash was performed in 0.5· NaCl ⁄ P i , and 0.1% SDS, at 65 °C for 2 · 15 min. A 175 bp DNA fragment cor- responding to the 3¢ UTR of the skin clone was used as a probe. RACE procedure Both 5¢- and 3¢-RACE reactions were performed using the RNA ligase-mediated GeneRacer TM Kit, as described by the manufacturer (Invitrogen Canada Inc). One microgram of DNase-treated total RNA combined with Thermo- script TM reverse transcriptase (Invitrogen Canada Inc) was used to generate adapter-linked first strand cDNA for 1 h in a 50 °C reaction. The first-strand cDNA was combined with the appropriate primers and touchdown PCR amplifi- cation was performed using DyNAzyme EXT TM DNA polymerase (Finnzymes, Oy, Finland) in an Eppendorf MastercyclerÒ. The touchdown cycling conditions consisted of an initial 95 °C denaturing step (2 min), followed by 10 cycles of 94 °C (15 s), 72 °C decreased to 60 °C (15 s), 72 °C (60 s) and 25 more cycles of 94 °C (15 s), 60 °C (15 s), and 72 °C (60 s). In order to obtain a product in most reactions, dimethylsulfoxide was added at 10% (v ⁄ v). RACE reaction products were separated on 1% agarose gels and then purified using spin columns provided in the kit GeneRacer TM Kit or by CONCERT TM Gel Extraction System (Invitrogen Canada Inc). A TOPO TA CloningÒ kit was used to clone the purified RACE products for sequencing into a pCRÒ4-TOPO cloning vector (Invitrogen Canada Inc). At least three independent clones were isola- ted and the purified plasmids sequenced. RT-PCR analysis One microgram of DNase-treated total RNA from each of the specified tissues was combined with 70 pmol of an anchored poly(T) primer. Thermoscript TM reverse transcrip- tase (Invitrogen Canada Inc) was then used to generate first-strand cDNA in a 1 h reaction at 50 °C, as described by the manufacturer. Normally, 1 ⁄ 10th of the RT reaction was combined with the appropriate primers and touchdown PCR amplification was performed using DyNAzyme EXT TM DNA polymerase in an Eppendorf MastercyclerÒ. The touchdown cycling conditions consisted of an initial 95 °C denaturing step (2 min), followed by 10 cycles of 94 °C (15 s), 72 °C decreased to 60 °C (15 s), 72 °C (60 s) and 25 more cycles of 94 °C (15 s), 60 °C (15 s), and 72 °C (60 s). RT-PCR products were separated on 1% agarose gels and visualized using ethidium bromide. DNA sequencing Sequencing was performed on the pBluescriptÒ phagemids or pCRÒ4-TOPO plasmids using T3 and T7 primers at the Expression of snailfish type I AFPs in skin tissue R. P. Evans and G. L. Fletcher 5334 FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS DNA sequencing facility in The Centre for Applied Genom- ics (Hospital for Sick Children, Toronto, ON, Canada). Bioinformatics programs Homologous nucleotide and protein sequences were searched through blast searches on the NCBI web server. The NCBI orf finder was utilized to identify putative open reading frames in the nucleotide sequences. Helical net and helical wheel diagrams were constructed using emboss package located on the Canadian Bioinformatics Resource web page (all located at http://www.ncbi.nlm.nih. gov/). Swiss PDB software used to generate a three-dimen- sional model of Las-AFP. clustalx and treeview (1.6.1) software were used to create an unrooted neighbor-joining tree. Acknowledgements We thank M. King and Dr M. Shears at the OSC for technical assistance and the OSC divers for sample col- lection. We also thank Dr Ming Kao for help with antifreeze activity measurements. This study was sup- ported by a grant from NSERC. References 1 Fletcher GL, Goddard SV, Davies PL, Gong Z, Ewart KV & Hew CL (1998) New insights into fish antifreeze proteins: physiological significance and molecular regu- lation. In Cold Ocean Physiology (Po ¨ rtner HO & Playle RC, eds), pp. 240–265. Cambridge University Press, New York. 2 Ewart KV, Lin Q & Hew CL (1999) Structure, func- tion and evolution of antifreeze proteins. 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Fletcher 5336 FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS . phylo- genic classification or tissue source of the proteins. With the discovery of snailfish skin proteins, it is apparent that type I AFPs can be divided into distinct structural. poly- peptides found in right-eyed flounders and certain species of sculpin. These proteins are divided into two distinct subclasses, liver type and skin type, which