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[54] NUCLEIC ACID SEQUENCES ENCODING LG. Duman et al.. ‘Thermal hysteresis proteins”. Advances DENDROIDES ANTIFREEZE PROTEINS in Low Temperature Biology, vol. 2. pp. 131-182, (1993). [75] Inventor: John G. Duman. South Bend. Ind. D. Tursman et al., “Cryoprotective eifects of Thermal Hys teresis Protein on Survivorship of Frozen Gut Cells from the [73] Assignee: University of Notre Dame du Lac, Freeze Tolerant Centipede Lithobius for?catus”, Journal of Notre Dame, Ind. Experimental Zoology. vol. 272, pp. 249-257 (1995). D.W. Wu et al., “Activation of antifreeze proteins from [21] Appl. No.: 485,359 larvae of the beetle Dendroides canadensis”, Comp. Physiol. [22] Filed: Jun. 7, 1995 B, vol. 161, pp. 279-283 (1991). [51] Int. Cl.6 ...... C12P 21/02; C07H 21/04 D.W. Wu et al., “Puri?cation and characterization of anti [52] US. Cl. .. 435/69.1; 536/235; 536/2431 freeze proteins from larvae of the beetle Dendroides [58] Field of Search ...... 435/3201, 69.1; canadensis”, Comp. Physiol. B, vol. 161, pp. 271-278 536/235. 24.31 (1991). [56] References Cited Primary Examiner—Dian C. Jacobson PUBLICATIONS Assistant Examiner-Kawai Lau J.G. Duman et al., “Hemolymph proteins involved in insect Attorney, Agent, or Firm—Barnes & Thomburg subzero tolerance: Ice nucleators and antifreeze proteins”, In Insects at Low Temperatures, Chapman and Hall (N .Y.), pp. [57] ABSTRACT 94-127 (1991). The present invention is directed to nucleic acid sequences D.W. Wu et al., “Enhancement of insect antifreeze protein encoding Dendroides canadensis thermal hysteresis pro activity by antibodies”, Biochem. Biophys. Acta 1076, teins. The THPs of Dendroides have signi?cantly greater 416-420 (1991). thermal hysteresis activity than any other known anti-freeze LG. Duman et al., “Adaptations of insects to subzero temperatures”, Quart. Rev. Biol. 66, (4) pp. 387-410 (1991). protein. The thermal hysteresis activity of the puri?ed THPs LG. Duman et al., “Hemolymph Proteins involved in the can be further enhanced by combining the THPs with cold tolerance of terrestrial arthropods: Antifreeze and Ice various “activating” compounds. Nucleator Proteins”. In Water and Life, Springer-Verlag (Berlin). Chapters 17 & 18 pp. 282-315, (1992). 4 Claims, 5 Drawing Sheets US. Patent May 6, 1997 Sheet 1 of 5 5,627,051
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PUP POP <90 U00 U00 *** axx OOH P<< U<< F<< O<< < U.S. Patent May 6, 1997 Sheet 4 0f 5 5,627,051 Percent - Survival 40: l -30 -25 ~20 -15 -1O -5 0 Temperature (C) F16 5m 80: Percent 60': Survival 40: 20" 0‘ ' ‘ -3o -25 -2o -15 -1o -5 0 Temperature (C) FIE 5% Percent 60 Survival 40-_ 20" -30 -25 ~20 --15 -10 -5 0 Temperature (C) F170;‘. 54: U-S. Patent May 6, 1997 Sheet 5 of 5 80" Percent 60' Survival 40"‘ 0 -30 -_25 -20 -15 -10 -5 0 Temperature (C) F16 5d 80' Percent ~ _ - Survival 40'. ' 20 -30 -25 -20 -15 -1o -5 0 Temperature (C) F16. 5e 5 ,627,051 1 2 NUCLEIC ACID SEQUENCES ENCODING DENDROIDES ANTIFREEZE PROTEINS TABLE 1 Amino acid compositions (mol %) of representative THPs. Species from a broad phylogenetic range of insects have been reported to produce thermal-hysteresis proteins Amino 1 2 2 (THPs). THPs are believed to play an important role in many acid (H-l) T-4 T-3 3 4 plant and animal species’ ability to survive exposure to Asx 14.3 7.3 5.3 9.5 7.1 subzero temperatures. By de?nition. the equilibrium melting Thr 17.2 i 6.6 2.3 6.0 2.7 Ser 10.3 7.4 11.1 13.0 30.5 and freezing points of water are identical. However. the Glx 5.2 8.9 12.4 11.0 12.3 presence of thermal-hysteresis proteins lowers the non Pro 2.6 5.9 0.0 5.0 0.0 equilibrium freezing point of water without lowering the Gly 6.6 8.3 11.4 15.0 20.0 Ala 8.4 14.3 5.0 8.0 6.8 melting point (equilibrium freezing point). Thus when THPs I/2Cys 15.9 0.0 28.0 6.0 0.0 are added to a solution they produce a diiference between the Val 1.7 11.5 2.3 3.0 3.0 freezing and melting temperatures of the solution. and this Met 0.2 4.8 0.0 0.0 0.0 difference has been termed “thermal-hysteresis”. 116 1.5 7.1 1.0 1.2 1.9 1.611 1.9 0.0 2.2 6.5 3.1 In the absence of THPs a small (about 0.25 mm diameter) Lys 3.4 6.8 15.4 3.1 7.5 ice crystal that is about to melt at the melting point tem Arg 4.8 2.6 0.0 8.0 0.0 perature will normally grow noticeably if the temperature is 'Iyr 3.9 2.3 0.0 1.0 2.0 Phe 0.0 3.9 0.0 2.2 1.1 lowered by 0.0l° to 0.02° C. However. if THPs are present, 20 H15 1.9 1.9 3.1 0.0 2.3 the temperature may be lowered as much as 5° to 6° C. below the melting point (depending upon the speci?c activ l = Dendroides canadensis; 2 = Tenebn'o malitar; 3 = Choristoneura ity and concentration of the proteins present) before notice fumg'ferana; 4 = Oncopeltus fasciatu: able crystal growth occurs. Consequently, because of THPs Several of the insect THPs have signi?cant amounts of ability to lower the freezing point of aqueous solutions. they 25 cysteine. In particular 16 mole % of the amino acid residues are commonly referred to as antifreeze proteins. THPs lower of the THPs of D. canadensis are cysteine, approximately the freezing point of aqueous solutions via a non-colligative half of which are involved in disul?de bridges. Treatment with dithiothreitol, which reduces these disul?de bonds, or mechanism that does not depress the vapor pressure or raise alkylation of free sulfhydryls, results in complete loss of the osmotic pressure of water, as is the case with colligative 30 thermal-hysteresis activity. type antifreezes such as glycerol. The thermal-hysteresis activity present in the Anti-freeze proteins are believed to exert their eifect by hemolymph (the circulatory ?uid of insects) of Dendroides adsorbing onto the surface of potential seed crystals via canadensis insects in mid-winter averages 3°—6° C. with hydrogen bonding. When anti-freeze proteins adsorb onto some individuals having as much as 8°-9° C. This activity the ice surface they interfere with the addition of water 35 is signi?cantly greater than that which is present in the blood molecules and force growth of the crystal into many highly of polar ?sh; the maximal activity achievable with very high curved fronts with high surface free energy. Consequently, concentrations of the ?sh THPs is about 1.7° C. FIG. 1 growth of the crystal requires the temperature to be further compares the activities of puri?ed THPs from two species of lowered to allow crystal growth to proceed ?sh and two species of insects: l=Dendr0ides canadensis Thennal-hysteresis proteins were ?rst discovered, and 40 THP, the most active THP currently known (solid squares); have been best studied, in marine teleost ?shes inhabiting 2=Tenebrio molitor THP (solid circles); 3: activity of the seas where subzero temperatures occur, at least seasonally. most active ?sh THP, i.e. Antarctic eelpout and THP of In these THP-producing Antarctic ?sh an ice crystal present Antarctic nototheniids (open squares); 4: activity of the in the blood serum that melts at -—1.1° C., will fail to grow least active ?sh THP, from cod Gadus morhua (open in size until the temperature is lowered to —2.5° C. (thermal 45 circles). The maximal activity of the most active ?sh and T hysteresis=l.4° C.). Thus the ?sh is protected from freezing molitor THPs are similar, however, the THPs of D. canaden in its ice-laden —1.86° C. seawater environment. sis have a greater speci?c activity and a much greater Over the last 15 years it has been demonstrated that many maximal activity than any other known THP. In addition as insects and other terrestrial arthropods (including certain Will be described later, the Dendroides canadensis THPs can spiders, mites, and centipedes) also produce thermal 50 be activated by the presence of certain other proteins to hysteresis proteins. Low levels of thermal hysteresis activity produce even greater levels of thermal hysteresis activity. are also quite common in overwintering plants and are The present invention is directed to nucleic acid present in certain fungi and bacteria. sequences encoding peptides having antifreeze properties, Thermal-hysteresis proteins (THPs) have been isolated wherein the nucleic acid sequence comprising sequences ?'om four species of insects: Tenebrio molitor, the milkweed 55 from a species selected from the genus Dendroides. The bug, Oncopeltus fasciatus, the spruce budworm, Choristo activity of Dendroides antifreeze proteins is three to six neurafumiferina, and D. canadensis. The molecular masses times greater than those of the ?sh anti-freeze proteins. of these THPs range from approximately 14 to 20 kDa. The Isolation of the nucleic acid sequences encoding these insect THPs characterized to date do not have a carbohydrate proteins allows the synthesis of the THP proteins in large component, nor do they have high percentages of alanine amounts. Advantageously, these proteins can be used to like the type-I ?sh (?ounder) THPs. enhance the supercooling properties of a ?uid to prevent the The amino acid compositions of representative insect freezing of ?uids at temperatures below their equilibrium THPs are shown in Table 1, and can be generally charac melting temperature. The proteins can also be used to terized as having higher percentages of hydrophilic amino prevent or limit ice growth or recrystalization of frozen acids (i.e. Thr, Ser, Asx, Glx. Lys, Arg) than the ?sh THPs, 65 goods, and provide protection from damage that normally with generally 40-50 mol % of the residues being capable of would result from freezing biological materials. These forming hydrogen bonds. effects can be mediated by adding puri?ed THPs alone, or 5 ,627,051 3 4 alternatively the THPs can be combined with various “acti vating” compounds that are known to enhance THP activity. TABLE 2-continued BRIEF DESCRIPTION OF THE DRAWINGS Amino acid compositions of four Dendroides THPs. Values are in mole percent. (Tryptophan was destroyed FIG. 1 is a graph illustrating the relative thermal during acid hydrolysis) hysteresis activities of two ?sh and two insect THPs. H1 H2 H3 H4 FIG. 2 is a graph illustrating the enhanced thermal hysteresis e?‘ect of combining different Dendroides Phe 0.0 0.0 0.5 0.6 Ice 1.5 2.3 1.2 1.9 canadensis THPs at various concentrations. 10 Leu 1.9 2.5 2.1 2.2 FIG. 3 depicts an alignment of the seven repeat nucleic Lys 3.4 3.2 3.6 2.9 acid sequences of Seq. 1]) No.1 1. Pro 2.6 3.5 6.7 4.0 FIG. 4 depicts an alignment of the seven repeat amino acid sequences of Seq. ID No.: 2 (using IUPAC-IUB single Even at very high concentrations. the maximal activity of letter amino acid designations). Because the NH3-terminus puri?ed THPs (about 2.7° C.) is signi?cantly less than that of the THPs is blocked. the exact starting position of the seen in the hemolymph of D. canadensis species in mid mature protein has not been con?rmed. winter. The population mean thermal hysteresis (freezing FIG. 5 is a graph illustrating the protective eifect of point-melting point) activity of Dendroides hemolymph Dendroides canadensis THPs to cells frozen under condi ranges from 3° to 6° C.. with some individuals having tions designed to promote recrystalization. 20 hysteresis activities as high as 8° to 9° C. Running the assay at the optimal pH (~7. 8) and addition of inorganic ions or DETAILED DESCRIPTION OF THE polyols increases activity slightly. however. the activity is INVENTION still considerably less than that seen in winter hemolymph. A clue to this dilemma was provided by noting the e?ect The THPs of Dendroides canadensis have signi?cantly 25 speci?c antibodies raised against THPs had on thermal greater thermal hysteresis activity than any other known hysteresis activity. anti-freeze proteins. The maximal activity of puri?ed THPs alone is approximately 2.7° C.. whereas the maximal activ Surprisingly, the addition of speci?c polyclonal antibod ity of the most active ?sh anti-freeze protein is only approxi ies to the puri?ed D. canadensis THPs was found to sub stantially increase thermal-hysteresis activity of the THPs. mately 1.5° C. In addition. certain activator proteins can be 30 combined with the Dendroides canadensis THPS to increase Addition of 0.5% (v/v) antiserum to aqueous solutions of the activity of Dendroides THPs to 5°-6° C. Activation of THP above about 3 mg/ml resulted in a doubling of activity. ?sh THPs has not been demonstrated. At slightly lower THP concentrations where thermal hysteresis activity was not detectable. activities of 1.5? to Four THPs have been puri?ed from D. canadensis and the 25° C. were achieved with antibody addition. Similar amino acid composition of each of the THPs has been 35 increases in activity were produced with T molitor THPs determined according to the procedure of Example 1. Table upon addition of a speci?c antibody. 2 shows that the amino acid compositions (in mole percent) of the four THPs are similar. All four of the proteins have One explanation for this enhanced THP activity upon high Cys contents (i.e.. ~16 mol %) and approximately half addition of speci?c antibodies is based on the assumption that THPs bound to IgGs are still able to hydrogen bond to of the Cys residues are involved in disul?de bridges. Hydro 40 philic amino acids. such as Asx. Glx, Ser, His, Thr. Arg. and ice crystals. If this is the case, then a THP-IgG complex will Lys comprise 45-55 mol % of the amino acid residues. block a larger surface area of the seed crystal than a single Dendroides canadensis THPs separated by electrophoresis THP molecule alone (the molecular mass of IgG is about on native PAGE and stained with Periodic Acid-Schiif (PAS) 150 kDa while and that of the THPs is about 8 kDa). If this explanation for the enhancing effect of the antibody addition revealed that these THPs are not glycosylated (i.e. the PAS 45 stained gels were negative). In addition, the amino acid is correct. then one would predict that addition of goat-anti analyses did not indicate the presence of amino sugars in any rabbit IgG to the solution of the THPs plus rabbit anti-THP of the Dendroides THPs. IgG should increase activity even more, because the com plex of THP plus rabbit anti-THP IgG plus goat anti-rabbit IgG antibody should block an even larger surface area of the TABLE 2 50 ice crystal. Experimental data has shown that the addition of Amino acid compositions of four Dendroides THPs. goat anti-rabbit IgG antibody to the solution of the THPs Values are in mole percent. (Tryptophan was destroyed plus rabbit anti-THP IgG did result in an additional enhance during acid hydrolysis) ment of thermal-hysteresis activity. H1 H2 H3 H4 55 Thus the activity of the D. canadensis THPs can be enhanced or “activated” by another protein present in the Asx 14.3 13.3 12.8 15.0 Total Cys 15.9 15.8 16.5 15.7 insect hemolymph that is capable of binding to the THP. To (Cystine) (3.7) (3.6) (4.2) (3.4) determine whether a particular protein or other macromol Glx 5.2 5.6 4.4 7.6 ecule could activate the Dendroides anti-freeze protein, the Ser 10.3 10.0 6.5 9.5 thermal hysteresis activity of an anti-freeze preparation was His 1.9 2.5 0.7 2.2 Gly 6.6 7.1 6.4 9.3 measured with and without the potential activator as Thr 17.2 14.9 16.5 13.9 described in Example 2. Arg 4.8 4.9 1.6 1.8 To isolate additional factors that enhance thermal hyster Ala 8.4 7.8 11.6 7.9 'Iyr 39 3.8 4.4 2.1 esis activity in the hemolymph, hemolymph protein frac Met 0.2 0.5 0.2 0.2 65 tions (from a gel ?ltration column) that lacked thermal Val 1.7 2.3 4.3 3.4 hysteresis activity were systematically added to an aqueous solution containing a low concentration of THPs (4 mg/ml). 5,627,051 5 6 Most fractions had no eifect on activity (i.e. LIB). but one other comprising THPs H3 and H4 are combined. the result particular fraction greatly enhanced activity to 524° C. (See ing mixture has greatly increased thermal-hysteresis activity. Table 3, #2). The activating protein present in this fraction FIG. 2 summarizes data showing the effect of combining was puri?ed. It has a molecular mass of 70 kDa and had been four different Dendroides canadensis THPs at various con designated as the endogenous THP-activator protein. centrations on thermal-hysteresis activity: (open circles rep resent THP H3 alone; X’s represent THP H4 alone; solid TABLE 3 squares represent a combination of H3 and H4; and open triangles represent a combination of H1.H2.H3 and H4). Eli‘ects of addition of various proteins on D. canadensis THP fractions 6A and 6B (representing activity of a solution of THPs (4 mg/ml) from D. canadensir“ 10 H1/I-I2 and H3/H4, respectively) separately have thermal hysteresis activity of 1.60° C. when present at a concentra Thermal Thermal Hysteresis tion of 2 mg/ml. However when these two fractions are Hysteresis Activity mixed together the resulting mixture has a 330° C. thermal Addition (°C.) increase (°C.) hysteresis value. This level of thermal hysteresis activity is not achieved by doubling the concentration of either fraction (1) None 1.60 O 15 (2) Other D. canadenris hemolymph alone. protein (non-THP) fractions; In summary. it appears that the high levels of thermal hysteresis in D. canadensis hemolymph in winter result LIB 1.52 0 THP-Activator 5.24 3.64 from (a) increased concentrations of THPs, (b) the combi (3) Gelatin (5 mg/ml) 3.13 1.53 20 nation of the four, perhaps more, THPs. and (c) the enhance (4) Tipula ice-nucleator ment of activity by the endogenous activator protein. proteins: Genes encoding D. canadensis THPs have now been LPlN (1 mg/ml) 3.49 1.89 isolated and characterized. The DNA sequences encoding PIN (1 mg/ml) 3.98 2.38 the dendroides THPs were isolated from a D. canadensis (5) 'Iipula PlN (1 mg/ml) + 5.80 4.20 25 expression cDNA library using techniques familiar to those gelatin (5 mg/ml) skilled in the art. The THP encoding genes were identi?ed by (6) Antiserum (0.5%) 3.18 1.58 antibody screening using anti-Dendroides THP rabbit Serum Interestingly, the endogenous THP-activator protein has These cloned Dendroides THP genes can be inserted into ice-nucleator activity (as determined by the assay described commercially available DNA vectors (expression vectors) to in Example 2). Other insect ice-nucleator proteins and a express the encoded gene protein product. These expression lipoprotein ice-nucleator (from the freeze-tolerant crane?y vectors have promoter sequences and other regulatory lipula trivitta) were also assayed for the ability to activate sequences necessary for expression in host cells. The tech the D. canadensis THPs (See Table 3, #4). Although these nique of using expression vectors to introduce exogenous proteins were not as e?icient as the endogenous THP 35 genes and express their protein products in a host cell is well activator protein, they did provide signi?cant enhancement, known to those familiar with the art. For example the increasing activity from 1.60° C. in their absence to expression vector pEI‘2 la is commercially available and can 3.0°—4.0° C. when present. Thus it is anticipated that all be used to express proteins in E. coli. Alternatively the ice-nucleator proteins will function as effective enhancers of protein can be expressed in a eukaryotic cell, such as yeast, THP thermal-hysteresis activity. Low concentrations of using Pichia expression vectors (i.e. PHIL-D2) commer gelatin or agar (below that needed for a gel state) also were cially available from Invitrogen. The Baculovirus system is activators. also commercially available and can be used to express the A modi?ed Western blotting procedure was used to dem THP genes in insect cultures. onstrate that the activator proteins bind to the THPs. The Once the THP gene or fragment thereof has been sub activator was run on an electrophoresis gel and transblotted 45 cloned into an expression vector, the resulting vector can be to nitrocellulose paper (or spotted on nitrocellulose paper used to transform a host cell, using procedures known to directly), and the paper incubated in a solution containing those familiar with the art. Such transformation procedures the THPs, to allow the THPs the opportunity to complex to include but are not limited to microinjection. mircoproj ectile the activator. which was immobilized on the nitrocellulose. bombardment, electroporation, calcium chloride The solution of THPs was removed, the nitrocellulose paper premeablization, polyethylene glycol permeabilization, pro washed, and then incubated with anti-THP rabbit antiserum, toplast fusion or bacterial mediated mechanisms such as followed by goat-anti-rabbit IgG labeled with horseradish Agrobacterium tumafaciens or Agrobacterium rhizogenes. peroxidase antiserum to detect the presence of THPs. In Host cells may be selected from any cell in which every instance, the activators were shown to bind to the expression of modi?ed proteins can be made compatible, THPs or vice versa. 55 including bacteria, fungus, yeast, plant cells and animal The enhancement of thermal-hysteresis activity by ice cells. Suitable host cells include prokaryotes selected ?om nucleator proteins, including the endogenous activator the genus Escherichia and eukaryotes selected from the protein, and the demonstration that these activators complex genus Pichia. The transformed host cells will synthesize the with the D. canadensis THPs suggested that in the process modi?ed protein which can be isolated and puri?ed using of binding to these ice-nucleators the THPs may inactivate standard methods known to those familiar with the art. One the ice nucleators. This has now been demonstrated. embodiment of the present invention comprises a method for In addition to the enhanced activity induced by the using a host cell transformant, having heterologous DNA presence of ice-nucleator proteins, the combination of the sequences encoding a Dendroides antifreeze protein, to different THPs present in D. canadensis hemolymph pro produce a protein having antifreeze properties. The method vides a synergistic eifect. At least four similar THPs are 65 comprises the step of culturing the cell transforrnant under present in the winter hemolymph of D. canadensis. When conditions conducive to the expression of the antifreeze two THP fractions, one comprising THPs H1 and H2 and the protein. 5,627,051 7 8 In accordance with one embodiment of the present genes that has not yet been isolated. However. using tech invention. THP genes are inserted into vectors and used to niques known to those skilled in the art. knowledge of the transform plant cells using techniques known to those amino acid sequence of the 28 amino acid peptide enables sln'lled in the art. These transformed plant cells can then be the construction of probes for isolating the gene encoding used to regenerate an entire plant or seed that expresses the TIIP that correlates to the peptide fragment. Dendroides THP proteins. Thus a plant entity consisting Examination of the derived protein sequences of Seq ID essentially of a plant cell. seed or plant can be produced from No: 3 and Seq ID No: 4 reveal an amino acid repeat the in vitro introduction of DNA sequences encoding one or sequence in the Dendroides anti-freeze proteins. Based on more Dendroides antifreeze proteins into plant cells. It is these two sequences an amino acid consensus sequence. as anticipated that the expression of the anti-freeze proteins in represented by Seq ID No: 8. can be derived that is repre the cells of transgenic plants will enhance their resistance to sentative of the thirteen amino acid repeat sequence. The freezing temperatures and decrease frost damage to plants repeat sequences of the two amino acid sequences (Seq ID exposed to freezing temperatures. Similar techniques known No: 2 and Seq ID No: 4) has been aligned as shown in FIG. to those skilled in the art can be used to generate other 4 to indicate the thirteen amino acid consensus sequence. Positions 1. 8, and 9 are completely conserved throughout transgenic animals. 15 Two THP genes have been sequenced and the nucleic acid all the repeat sequences and represent a cysteine, cysteine and threonine amino acids respectively. Positions 4. 6, and sequence of these two genes is represented by Seq ID No: 1 11 are also highly conserved and are generally alanine. and Seq ID No: 3 respectively. Initiation codons for the two threonine and serine, respectively. The thirteenth position is sequenced THP genes are located at positions 16 and 22 and generally either aspartic acid or asparagine. Throughout stop codons are located at positions 340 and 349 of Seq ID 20 much of the TI-IP proteins every sixth amino acid is cysteine. No: 1 and Seq ID No: 3, respectively. These two genes are Three of the repeat sequences (repeats D. E and F) of very similar in sequence to one another; there are only a total Dendroides anti-freeze proteins Seq ID No: 2 and Seq ID of 8 single base pair di?erences and a single 3 base pair No: 4 comprise a repeat sequence of only 12 amino acids. deletion in the coding sequence of Seq ID No: 1 relative to Based on these three repeat sequences an amino acid con Seq ID No: 3 making the two sequences over 95% identical 25 sensus sequence. as represented by Seq ID No: 9. can be in sequence. derived that is representative of the twelve amino acid repeat Each of the two Dendroides THP genes (Seq. ID No: l sequence. and Seq ID No: 3) contain a 39 base pair sequence and a 36 It is anticipated that an arti?cial gene could be synthesized base pair sequence that are repeated four times and three that would encode a protein having either the 13 amino acid times. respectively. within the coding portion of the THP consensus repeat (Seq ID No: 8) or the 12 amino acid gene. When the repeat sequences are aligned. as indicated in consensus repeat (Seq 1]) No: 9) or a combination of the two. FIG. 3. a consensus sequence can be generated that is The protein encoded by this gene would be expected to have representative of the repeat portions. The consensus nucleic anti-freeze properties similar to the natural Dendroides acid sequence for the 39 base pair repeat is: THPs. TGYYNYRll/INK SNNNWRNNNN NTGTACNNRN 35 Advantageously. information generated from the TSNWMNRWY (Seq ID No: 5) sequencing of the THP proteins and their respective genes and the consensus nucleic acid sequence for the 36 base pair allows portions of the genes to be used as probes for repeat is: detecting additional TI-IP genes. In one embodiment the TGYNNNAANG CNNNAACNTG TACNNRNTCA probe comprises nucleic acid sequences at least ten nucle WMNRWY (Seq ID No: 6) otides long. more particularly, longer than 20 nucleotides. wherein A is adenine, C is cytosine, G is guanine, T is The portion of the THP gene utilized for a probe is selected thymidine. Y is cytosine or thymidine, R is adenine or from a region of the THP gene that is highly conserved, to guanine, M is adenine or cytosine, K is guanine or ensure detection of all THP genes. Nucleic acid probes thyrnidine, S is guanine or cytosine, W is adenine or derived from Dendroides THPs can also be used to identify thymidine and N is adenine, cytosine, guanine or thymidine. 45 additional THPs from other organisms that have similar high The protein sequence of the two sequenced Dendroides therrnal-hystersis properties. Isolation of additional THP THP genes has been determined empirically and is repre genes from Dendroides, or other freeze avoiding or freeze sented by Seq ID No: 2 and Seq ID No: 4 respectively. The tolerant species, can be accomplished by labelling the ?rst 19 amino acids of the deduced amino acid sequence nucleic acid probe and hybridizing the probe with a genomic represent a signal peptide that is cleaved from the mature 50 or cDNA library using techniques know to the skilled protein. The two protein sequences are very similar; the practitioner. mature proteins have only two di?ierences. An Asparagine THPs are also believed to play an important role in (Asn) is substituted for an Isoleucine (He) at amino acid 60 allowing organisms to survive freezing of their extracellular and an additional Threonine (Thr) is inserted in Seq ID No: ?uids. Organisms that can survive freezing are referred 4 relative to Seq ID No: 2 at amino acid 100. 55 herein as “freeze tolerant” organisms to distinguish them Direct sequencing of protein fragments has also been from “freeze avoiding” organisms (which survive subzero conducted. 'I‘wo fragments have been sequenced. The ?rst temperatures by preventing freezing). Some freeze tolerant fragment was a 57 amino acid sequence that matched arthropods and plants produce THPs in winter but usually identically to a 57 residue region from the derived protein the level of thermal hysteresis activity is lower in freeze sequence of the nucleic acid Seq ID No: 1. A second peptide tolerant species than that typically seen in freeze avoiding of 28 amino acids has also been sequenced (Seq ID No: 7). species. For example, the centipede Lithobius for?catus and compared to the 57 amino acid sequence. Direct com produces THPs, but too little thermal hysteresis activity is parison of the 28 amino acid sequence to the amino acid present to provide signi?cant antifreeze protection. sequences of Seq ID No: 2 and Seq ID No: 4 revealed a total However, L. for?catus are freeze tolerant, and thus even if of seven differences over the 28 residue region relative to 65 freezing is initiated across the cuticle from external ice, the either Seq ]D No: 2 or Seq ID No: 4. Thus the 28 amino acid organism can still survive temperatures at least as low as —6° sequence is likely encoded by one of the Dendroides THP C. 5,627,051 9 10 To test THPs’ ability to protect cells from freeze damage, L. for?catus cells were frozen at various temperatures and TABLE 4-continued then thawed, either with or without the addition of Dendroi Survivorship of L. for?catus gut cells frozen des canadensis’ THPs, and the survival of the cells evalu under conditions designed to promote recrystallization ated. The cells tested were isolated from the rnidgut of both winter and summer collected L. for?catus. (See example 3) Freezing Freezing Season of Temperature Time % Survivorship The addition of THPs increased the survival of cells from freeze damage and in particular a THP concentration Collection (°C.) (Min) (—)THPs (+)THPs between 0.02 mg/ml (2.4><10'6M) and 0.20 mg/ml (2.4><10_ E) Summer —14 10 s) was determined to be optimal. It is noted that high i l concentrations of THP actually reduce cell survival upon —6 30 7.4 36.7 freezing; a THP concentration of 2 mg/ml results in zero F) Winter —14 10 percent survivorship. The presence of THPs (0.02 mg/ml) in l l the bathing medium produced a statistically signi?cant —6 30 15.4 41.7 increase in cell survival, indicating a cryoprotective function 15 for the THPs. The LTS0 (temperature of 50% survival) of Cell preparations were also pretreated with thermal hys cells isolated from centipedes collected in the summer was teresis protein and then the protein was removed from the shifted from —8.2° C. to —15.0° C., and the LT5O of cells bathing medium prior to freezing to determine if the THP isolated from centipedes collected in the winter was shifted antifreeze properties can be mediated through binding to cell from —l2.1° C. to —15.1° C. by addition of (0.02 mg/ml) membranes or internalization of the THPs. L. forn'catus cells THPs. Also, cells from summer collected centipedes which pretreated with THPs and washed prior to freezing showed had been incubated in media containing THPs. then washed enhanced survival after freezing. in butter to remove the protein from the media prior to Immuno?uorescent photomicrographs were prepared for freezing, demonstrated signi?cantly increased survivorship cell preparations collected from both winter and summer 25 centipedes and either treated, or left untreated, with THPs. after freezing (LT5O=—14.5). Untreated cells (not exposed to THPs) from animals col The presence of THPs in the medium also provided some lected in summer show very little ?uorescence (presumably protection to cells frozen under conditions designed to auto?uorescence) while those treated with Dendroides promote the potentially damaging process of recrystalliza THPs, and then rinsed, immuno?uoresce strongly, indicating tion (See FIG. 5). To induce recrystalization, preparations of that the protein has adhered to the surface of the cells and/or cells were frozen in the presence of 2 mg/ml THP (fraction has possibly been incorporated into the cells. Cells harvested 6B, see example two) at —14° C., held for ten minutes, then from species collected in the winter, and not treated with raised to —6° C. and held at this higher temperature for an THPs, still immuno?uoresced, suggesting the natural occur additional 30 minutes before being thawed and their survival rence of THPs in and/or on the centipede cells in winter. The evaluated. A control freezing treatment without thermal trypsin treatment used in the isolation of these cells could be hysteresis protein was conducted simultaneously with the expected to cleave exposed proteins on the surface of test sample to allow direct comparison of THP induced cell membranes. Yet su?icient THP remains on the winter cells freeze resistance. This procedure is similar to that used to interact with the anti-THP antibody. previously to demonstrate that winter centipede hemolyrnph Thus immuno?uorescence studies of cells treated with contains recrystallization inhibition activity (Tursman et a1, TI-IPs indicated that the THPs were present in the cells ’94) The survival rates of cells frozen without THPs under and/or on the cell membrane. This implies that membrane conditions'designed to promote recrystalization are lower bound, or possibly intracellular, THPs provide protection. than the survival rates of cells frozen and held at a constant temperature of either —6° or —14° C. and then quickly EXAMPLE 1 thawed (See Table 4). L. for?catus gut cells, either with or 45 Puri?cation of Dendroides canadensis THPs without THPs, were frozen under conditions designed to Collection of larvae. Larvae of Dendroides canadensis promote recrystallization (treatments E and F), freezing were collected periodically from late fall to early spring initially at —14° C. for 10 minutes and then raising the from under the loose bark of dead trees near South Bend, temperature to —6° C. for an additional 30 minutes. Control Ind, USA. Larvae were held at 4° C. in containers with cells (treatments A-D) were held at either —14° or —6° C. for 50 partially decomposed wood from oak logs, until they were ‘ either 10 or 40 minutes. As the data in Table 4 indicate, the processed. presence of THPs in low concentrations greatly enhanced PUIi?C?tiOIl. Both hemolyrnph and whole bodies were the survival of cells frozen under conditions that promote used as the starting material. Hemolymph was obtained from recrystallization. the Dendroides larvae by punctln‘ing the larvae in the dorsal 55 midline with a 28-gauge needle and collecting the TABLE 4 hemolyrnph (approximately 5 p1 per larva) in a 10 ul glass survivorship of L. far?catus gut cells ?ozen microcapillary via capillary action. The pooled hemolyrnph under conditions designed to promote recrystallization was frozen at —30° C. After bleeding, the larvae were frozen at —30° C. until processed. Puri?cation from hemolyrnph. Freezing Freezing Season of Temperature Time % survivorship Step 1: Ultrogel AcA-54 gel ?ltration chromatography. Dendroides hemolyrnph (2-3 ml) was applied to an Ultrogel Collection ("C.) (Min) (—)THPs (+)Tl-lPs AcA-54 (IBF Biotechnics, Inc.) column (1.9><120 cm.). The eluting bu?er was 0.025M TRIS (pH 7.5), 0.1N NaCl at a A) Summer —14 10 18.3 48.0 ?ow rate of 5 ml/hr (tube volumes collected were 2.2 ml). B) Summer —14 40 18.9 45.1 C) Summer —6 10 68.3 87.9 Elution of protein fractions was monitored at an absorbance D) Summer ——6 40 65.9 -— of 230 nm. Tubes representing protein peaks were pooled, concentrated by freeze drying. redissolved in water, dialyzed 5,627,051 11 12 exhaustively against distilled water to remove salts. freeze Measurement of Ice Nucleator Activity dried a second time and redissolved. Each protein fraction To determine whether certain proteins have ice nucleator was then assayed for antifreeze activity according to the melting point-freezing point di?erence (thermal hysteresis) activity the effect of addition of the protein on the super method of DeVries [Methods in Enzymology. Vol. 127. p. cooling point (nucleation temperature) of a bu?er solution was determined by a droplet technique [Zachariassen et al., 293-303(1986)]. The active fractions were usually rechro matographed through a second. smaller (1.4Xll0 cm) Ultro Cryobiology. 192180-184 (1982)]. using droplets of 1 pl gel AcA-54 column. volume. Addition of a protein with ice nucleator activity Step 2: Reversed-phase HPLC ultrasphere ODS column. causes a statistically signi?cant increase in supercooling point of the buffer solution. Two active fractions were separated from the AcA-54 col 10 umn. designed 6A and 6B. These were further separated on EXAMPLE 3 reversed-phase high pressure liquid chromatography (HPLC). using an ultrasphere C18 column (Rainin). 5 pm. Dendroides THP Cryoprotective Capabilities 4.6 mm (ID) X25 cm (L) equipped with a guard column of Cell Preparation. the same stationary phase. The columns were equilibrated with 0.1% tri?uoracetic acid (TFA) in dHZO. The proteins L. forticatus centipedes were collected from woodland were eluted over a 60-min period with a 20-26% linear habitats in Porter County. Ind. (northwestern Indiana) in gradient of acetonitrile with 0.1% TFA at a ?ow rate of 1 both winter and summer. Cells were isolated from the gut in ml/min. Elution of protein peaks was monitored at an the following manner. Guts were dissected free from other tissues through a dorsal midline incision in the cuticle. The absorbance of 220 nm. Peak‘eluant fractions were freeze 20 dried. redissolved in distilled H20 and checked for thermal middle portion of the gut was used. after the gut contents and hystersis activity. the Malpighian tubules were removed. To remove the gut Puri?cation from whole larvae. Whole larvae were contents. the gut tube was grasped at one end with ?ne homogenized in 50% ethanol at 4° C. and centrifuged at forceps. wrapped several times about a 20 gauge hypoder mic needle shaft. and slowly pulled perpendicular to the 11.220Xg at 4° C. for 15 min. The supernatant was dialyzed 25 (Spectrapor. 2500 MW cuto?') against 25 mM TRIS-Cl needle. squeezing ‘out the contents. After rinsing in phos buffer (pH 9.0) and chromatographed on a DEAE-Sepharose phate butfered saline (PBS. 0.9% NaCl. w/v; pH=7.4) the CL-6B (Phannacia) (2.5><20 cm) ion-exchange column. tissue was immersed in 0.5 ml of a trypsin solution (15-20 Fractions were eluted using stepwise increases in NaCl (tube units of activity/ml) in a small centrifuge tube and minced volumes were 6 m1). Active fractions were subsequently run with ?ne scissors. This preparation was held at room tem on gel ?ltration (Ultragel AcA-54). followed by HPLC as perature for a period of 20 minutes with occasional gentle described above for puri?cation from the hemolymph. agitation. Polyacrylamide gel electrophoresis (PAGE). Native and The minced sample was centrifuged at 1000 g for one SDS-PAGE were performed as criteria of purity using the minute. and the supernatant discarded. The remaining cel technique of Ornstein (1964) and Laemmli (1970). BIO 35 lular pellet was resuspended in PBS. respun and the super RAD low range molecular weight markers were used in the natant was decanted. The pellet of cells were then resus SDS-PAGE. The gels were usually stained with Coomassie pended in 100 pl of PBS containing 0.2% Trypan Blue. This blue (Fairbanks et al. 1971). Periodic acid-Schiff stain was stock suspension was held in an ice bath at 0° C. Aliquots of used to determine whether the proteins might contain car 15 pls were removed and the cells either counted directly in bohydrate (Kapitany and Zebrowski 1973). a hemocytometer chamber as controls. or subjected to the Amino acid analysis. Puri?ed THP (0.1 mg) was hydro various freezing treatments described below and then lyzed in 5.7N HCl for 24 h at 110° C. Amino acid analyses counted. and their viability determined (see below) using a were done on a Beckman Model 117 amino acid analyzer microscope at 440> SEQUH‘ICE LISTING ( 1 )GENEZAL INFORMATION: ( i i i )NUMIBER OF SEQUENCES: 9 ( 2 ) INFORMATION FOR SEQ ID NOzl: ( i )SEQUENCE CI-IARAC'I‘ERIS'I‘ICS: ( A )LENGTH: 55 base pairs ( B )TYPE: nucleic acid ( C )STRANDEDNESS: double ( D )TOPOLOGY: linear ( i i )MOLECULE TYPE: cDNA to mRNA ( i i i )HYPUI‘HE'I‘ICAL:NO ( i v )ANTl-SENSEzNO ( v i )ORIGINAL SOURCE: ( A ) ORGANISM: Dendroides canadensis ( x i )SEQUENCE DESCRIPTION: SEQ ID N011: GTTGAGTTGA ACAAAATGGT TTGGGTTTGC AAAAATTCGA TATTAGTAAT TAGTGTAGTG 60 CTCATGTACG TATGTCATGA GTGTTATGGC CAATGTACTG GTGGTTCCGA TTGTCGCTCG 120 TGTACAGTGT CTTGTACTGA CTGCCAGAAC TGTCCAAATG CACGTACAGC ATGTACTCGC 1 80 TCTTCAAACT GCATTAACGC GTTAACCTGT ACGGATTCAT ATGATTGCCA CAATGCCGAA 240 ACCTGTACTA GATCAACCAA TTGTTATAAG GCTAAAACCT GTACTGGATC AACCAACTGT 3 00 TACGAAGCTA CAGCCTGTAC CGATTCCACG GGAVTGTCCAT GATCTTATTC TATTCAGAGG 3 60 ATGAAGTACA GGAGCGATAA TAATTATTTT ACTCATCAGA TATATATGTT TATTAATATT 420 AGAAATTATA TGGAGTTAAA TTAAACATGT TTGATATCAT GTTTGATATC ATGTATTACG 480 GAACTCTAAA TCAATACATA TAAATAAAAT AACATGTCAG TAATA 5 25 ( 2 ) INFORMATION FOR SEQ ID N012: ( i ) SEQUENCE mamcrmusncs; ( A ) LENGTH: 108 amino acids (B )TYPEzaminoaeid (D)'IDPOLOGY:linear ( i i )MOLECULE TYPE: protein ( iii )HYPOI‘HETICAL:YES ( i v )AN'II-SENSEzNO ( v i )ORIGINAL SOURCE: ( A )ORGANISM: Dendroides canadcnsis ( x i ) SEQUENCE DESCRIPTION: SEQ ID N022: 5,627,051 -continued Met Val Trp Val Cys Lys Asn Ser Ile Lou Val 110 Set ValVVal Lou 1 5 10 15 Met Tyr Val Cys His 6111 Cys Tyr Gly Gln Cys Thr Gly Gly Sc: Asp 2O 25 3O Cys Arg Ser Cys Thr Val Se: Cys Thr Asp Cys Gln Asn Cys Pro Asn 35 4O 45 Ala Arg Thr Ala Cys Th1’ Arg Set Set Asn Cys 11: Asn Ala Leu Th: 50 55 0 Cys Thr Asp Sc! Tyr Asp Cys His Asn Ala Glu Th: Cys Thr Arg Se: 65 70 75 80 Thr Asn Cys Tyr Lys Ala Lys Thr Cys Thr Gly Se: Thr Asn Cys Tyr 85 90 95 Glu Ala Th: Ala Cys Th1- Asp Scr Th: Gly Cys Pro 100 105 (2 )INFORMATIONFORSEQIDNO?: ( i )SEQUENCE CHARACTERISTICS: (A )LENG'I‘H:487bascpairs (B )TYPEznucleicacid ( c )STRAN'DEDN'ESS: double (D)TOPOLOGY:lincar (ii)MOLECULE TYPE: cDNAtomRNA (iii)HYPOTHETICAL:NO (iv_)ANTI-SENSE:NO (v i )ORIGINAL SOURCE: ( A ) ORGANISM: Dendroirks canadensis ( x i ) SEQUENCE DESCRIPTION: SEQ ID N05: ; CGAGCAGTTG AGTTGAACAA AATGGTTTGG GTTTGCAAAA GTTCGATATT AGTAATTAGT 6O GTAGTTCTCA TGTACGTATG TCATGAGTGT TATGGCCAAT GTACTGGTGG TTCCGATTGT 120 CGCTCGTGTA CAGTGTCTTG TACTGACTGC CAGAACTGTC CAAATGCACG TACAGCATGT 180 ACTCGCTCCT CAAACTGCAA CAACGCGTTA ACTTGTACGG ATTCATATGA TTGCCACAAT 240 GCCGAAACC-T GTACTAGATC AACCAATTGC TATAAGGCCA AAACCTGTAC TGGATCAACC 300 AACTGTTACG AAGCTACTAC AGCCTGTACC GATTCCACGG GATGTCCATG ATCTTATTCT 360 I \ ATTCAGAGGA TGAAGTACAG GAGCGATGAT AATAATTATT TTACTCATCA CATATATATG 420 TTTATTAATA TTAGAAATTA TATGGAGTTA AATTAAACAT 'GTATAACATG TTTGATATCA 480 TATATAC 487 (2 )INFORMATIONFORSEQIDNOt4: ( i )SEQUENCE CHARACI‘EZISTICS: (A)I_.ENG'IH:l09aminoacids (B)TYPE:aminoacid (D)TOPOLOGY:li.near (ii)MOI_.ECULETYPE:pmtcin (iii)HYPOTI-1EI'ICAL:YES (iv )ANTI-SENSEzNO (vi )ORIGB‘IALSOURCE: (A )ORGANISMzDcnrh-oides canarknsis ( X i ) SEQUH‘ICE DESCRH’TION: SEQ ID NO:4: Met Val Trp Val Cys Lys Set Set 11c Lou Val Ile Se: Val Val Leu 1 5 1"0 15 5,627,051 20 -continued Me t Ty :- Val Cy 5 H1 s Glu Cy s Ty 1- G1 y G 1 n Cy s Th 1' Gly G l y S e 1' As p 20 25 30 Cys Arg Sex Cys Th: Val Ser Cys Thr Asp Cys Gln Asn Cys Pro Asn 35 4o 45 Ala Arg Th! Ala Cys Thr Arg Set Set Asn Cys Asn Asn Ala Leu Thr 50 55 60 Cys Thi- Asp Sc! Tyr Asp Cys His Asn Ala Glu Th: Cys Thr Axg Ser 65 7O 75 80 Thr Asn Cys Tyr Lys Ala Lys Thr Cys Th: Gly Sex Th: Asn Cys Tyr 85 90 95 Glu Ala Thr Thr Ala Cys Th: Asp Set Th: Gly Cys Pro 100 105 ( 2 ) INFORMATION FOR SEQ ID N05: ( i )SEQUENCE CHARACTERISTICS: (A )LE‘IGTl-h39basepairs ( B )TYPE: nucleic acid ( C )STRANDEDNESS: double ( D )TOPOLOGY: linear ( i i )MOLECU'LE TYPE: cDNA to mRNA ( i i i )HYPOTHEI'ICALzYES ( i v )ANTl-SENSEtNO ( v i )ORIGINAL SOURCE: ( A ) ORGANISM: Dcndroidcs canadensis ( X i ) SEQUH‘ICE DESCRIPTION: SEQ ID N05: TGYYNYRMNK SNNNWRNNNN NTGTACNNRN TSNWMNRWY 39 ( 2 ) INR)RM.ATION FOR SEQ ID No.6: ( i )SEQUENCE CHARACTERISTICS: (A )LENG'I‘HzSGbasepairs ( B )TYPE: nuclcic acid ( C ) STRANDEDNESS: double ( D )TOPOLOGY: linear ( i i ) MOLECULE TYPE: cDNA to mRNA ( i i i ) HYPOTHETICAL: YE ( i v )ANTISENSE: N0 ( v i )ORIGINAL SOURCE: ( A ) ORGANISM: Dendmides canadcnsis ( x i ) SEQUBQCE DESCRIPTION: SEQ ID NO:6: TGYNNNAANG CNNNAACNTG TACNNRNTCA WMNRWY 36 ( 2 ) INFORMATION FOR SEQ ID N027: ( i )SEQUBJCE CHARACTERISTICS: (A )IENGTHzZSaminoacids ( B )TYPE: amino acid ( D )TOPOLOGY: linear ( i i )MOLECULETYPE:pcptide ( i i i )HYPUI'HETICALzNO ( i v )ANTI-SE‘ISEzNO ( v ) FRAGMENT TYPE: inlmnal ( v i )ORIGINAL SOURCE: 5,627,051 21 22 -continued ( A ) ORGANISM: Dendroidies canadensis ( X i ) SEQUENCE DESCRIP'HON: SEQ ID N027: Asp Cys Val Asn Cys Pro Asn Ala Leu Thr Ala Cys Thr Arg Ser Thr 1 5 10 Asn Cys Tyr Lys Ala Val Thr Cys Thr Lys Ser Tyr 20 25 ( 2 ) INFORMATION FOR SEQ ID NO:8: ( i )SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 13 amino acids ( B )TYPE: amino acid ( D )TOPOLOGY: linear ( i i )MOLECULE TYPE: peptide ( i i i )HYPOI'I-l'EI'ICALzYES ( i v )AN'II-SENSE: NO ( v )FRAGMENT TYPE: internal ( v i )ORIGINAL SOURCE: ( A ) ORGANISM: Dendroides canadcnsis ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:8: Cys Xaa Xaa Ala Xaa Thr Xaa Cys Thr Xaa Ser Xaa 1 5 ( 2 ) INFORMATION FOR SEQ ID NO:9: ( i )SEQUENCE CHARACTERISTICS: (A )LENG'I'H:12aminoacids ( B ) TYPE: amino acid ( D )TOPODOGY: linear ( i i )MOLECULE TYPEzpeptide ( i i i )HYPOTl-IEI‘ICALzYES ( i v )ANTI-SENSEzNO ( v )FRAGMENT TYPE?mmxal ( v i )ORIGINAL SOURCE: ( A ) ORGANISM: Dendroides canadensis ( x i ) SEQUH‘ICE DESCRIPTION: SEQ ID N09: Cys Xaa Xaa Ala Xaa Thr Cys Thr Xaa Ser Xaa Asx 1 5 10 50 I claim: 2. A isolated nucleic acid sequence comprising the 1. A method for using a cell transformant comprising a sequence as set forth in Seq ID NO:1. heterologous DNA Sequence selected from the group con 3. An isolated nucleic acid sequence comprising the sisting of Seq ID NO: land Seq ID N023 to produce a protein sequence as set forth in Seq ID N023. having antifreeze properties, said method comprising the 55 4. A polynucleotide sequence having a 20 base pair nucleotide portion identical in sequence to a consecutive 20 step of base pair portion of the sequence as set forth in Seq 1D NO:1. culturing said cell transformant under conditions condu cive to the expression of the antifreeze protein.