# Patent

## Title
Insect desiccation resistance genes and uses thereof

## Abstract

An objective of the present invention is to provide polynucleotides encoding insect desiccation resistance proteins, and uses thereof. cDNA libraries were produced from Polypedilum vanderplanki larvae in a desiccated state, a P. vanderplanki EST database was constructed, and genes encoding LEA proteins were isolated. This resulted in the successful isolation of three types of novel gene encoding LEA-like proteins. When secondary structure predictions and motif searches were performed on the proteins deduced from each of the genes, all three proteins had α-helix-rich structures and LEA_4 motifs, which are characteristic of LEA proteins. Moreover, the recombinant proteins synthesized from these genes were heat soluble even when boiling, so that these proteins have hydrophilic properties as high as plant LEA proteins. Therefore, the three isolated genes were found to be novel P. vanderplanki-derived LEA genes. Furthermore, introduction of these genes into animal cells successfully conferred desiccation resistance to the cells.

## Background
RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 11/222,641, (now U.S. Pat. No. 7,473,769), filed Sep. 8, 2005, which claims priority under 35 U.S.C. §119 or 365 to Japanese Application No. 2004-261412, filed Sep. 8, 2004.
The entire teachings of the above applications are incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to polynucleotides encoding insect-derived desiccation resistance proteins, and to uses thereof.
BACKGROUND OF THE INVENTION
Polypedilum vanderplanki only inhabits semi-arid regions of Africa, and is the only insect whose larvae can resume normal growth within one hour of being placed in water for absorption even after 48 hours of complete dehydration (Watanabe, M., Kikawada, T., Minagawa, N., Yukuhiro, F., and Okuda, T. (2002) J Exp Biol 205, 2799-2802). This state, in which the organism can recover even after complete desiccation, is called cryptobiosis. After entering a cryptobiotic state, P. vanderplanki can survive at temperatures from −270° C. to +102° C., or in 100% ethanol (Hinton, H. E. (1960) J Insect Phys 5, 286-300; and Hinton, H. E. (1960) Nature 188, 336-337). Trehalose has been said to be essential to inducing and maintaining the cryptobiotic state. However, even when desiccation occurs after accumulating a high concentration of trehalose in the body, there are some cases when the cryptobiotic state is not achieved (Watanabe, M., Kikawada, T., and Okuda, T. (2003) J Exp Biol 206, 2281-2286). Since trehalose accumulation alone is insufficient to explain the mechanism of cryptobiosis induction and maintenance, factors other than trehalose should be necessary for cryptobiosis.
The dormancy of plant seeds is a type of cryptobiosis. Seed dormancy occurs in late embryogenesis, and for approximately 20 years, proteins called late embryogenesis abundant (LEA) proteins have been known to accumulate specifically during this period (Dure, L., 3rd, Greenway, S. C., and Galau, G. A. (1981) Biochemistry 20, 4162-4168; Grzelczak, Z. F., Sattolo, M. H., Hanley-Bowdoin, L. K., Kennedy, T. D., and Lane, B. G (1982) Can J Biochem 60, 389-397). Under desiccation stimulus, expression of these proteins is increased not only in seeds, but also in pollen and in plant bodies (Ingram, J., and Bartels, D. (1996) Annu Rev Plant Physiol Plant Mol Biol 47, 377-403). LEA proteins have characteristic secondary structures and share an a-helix-rich structure (Goyal, K., Tisi, L., Basran, A., Browne, J., Burnell, A., Zurdo, J., and Tunnacliffe, A. (2003) J Biol Chem 278, 12977-12984). The results from transgenic yeast and rice have revealed that these proteins show stress-resistant functions such as desiccation resistance, cold resistance, and salinity resistance; however, the detailed biochemical functions (activities) of the proteins themselves are still unknown (Wise, M. J., and Tunnacliffe, A. (2004) Trends Plant Sci 9, 13-17). These proteins were thought to exist exclusively in plants. However, following their discovery in nematodes in 2002 (Browne, J., Tunnacliffe, A., and Burnell, A. (2002) Nature 416, 38), the existence of LEA proteins in organisms other than plants has been strongly suggested.
SUMMARY OF THE INVENTION
The present invention was made in view of such a situation, and an objective of the present invention is to provide polynucleotides encoding desiccation-resistance proteins of insects and uses thereof. More specifically, the present invention provides polynucleotides encoding P. vanderplanki-derived desiccation-resistance proteins, vectors carrying the polynucleotides, and uses thereof.
Since the LEA proteins of nematodes and plants are not highly homologous, RT-PCR and library screening are likely to be unavailable for isolating LEA genes of P. vanderplanki. Therefore, to solve the above-mentioned problems, the present inventors originally constructed EST database of P. vanderplanki using cDNA libraries from the larvae at 0, 12 and 36 hours after desiccation, so that they progressed isolation of genes for LEA proteins.
Thereby, the present inventors succeeded in isolating 3 types of novel genes (PvLEA1, PvLEA2, and PvLEA3) that encode LEA-like proteins. When secondary structure prediction and motif search were performed on the proteins deduced from the respective genes, these 3 proteins were found to have an α-helix-rich structure and LEA—4 motifs, which are characteristics of Group 3 LEA proteins. Consequently, the 3 genes isolated this time were suggested to be novel P. vanderplanki-derived LEA genes.
Next, when variations in expressions of the isolated LEA genes due to desiccation were investigated, the expression levels started to increase 1 hour after desiccation treatment, reached maximum values 6 hours after the treatment, and remained constant thereafter. Such behavior is similar to those reported so far for LEA genes, and therefore, PvLEA1, 2, and 3 genes were confirmed to be desiccation inducible.
Furthermore, the recombinant proteins synthesized from the three types of LEA genes isolated in the present invention were heat-treated, and the hydrophilicities of these proteins were examined. The results showed that the recombinant proteins do not aggregate at all, regardless of the heat treatment. This finding suggested that the translation products of the LEA genes of the present invention are highly hydrophilic proteins, exhibiting the activity of synonymous substitution for water; more specifically, it suggested that they function as desiccation resistance proteins.
Furthermore, cellular expression systems were used for examination to determine whether the LEA proteins of P. vanderplanki have desiccation-protective function in organisms. PvLEA1, 2, and 3 genes were introduced into CHO-K1 cells, and the number of cell colonies formed after desiccation treatment was determined. As a result, among cells that expressed the PvLEA1, 2, and 3 genes, only those cultured in a medium containing trehalose formed 33 to 55 colonies after desiccation treatment. On the other hand, cells that expressed only the vector formed hardly any colonies regardless of the presence of trehalose. Therefore, the coexistence of trehalose and expressed PvLEA1, 2, and 3 genes was found to confer desiccation resistance to animal cells.
To date, isolation of LEA genes from insects has never been reported, and the present invention provides the first example of isolating LEA genes from insects.
That is, the present invention relates to polynucleotides encoding the following insect-derived desiccation resistance proteins and uses thereof. More specifically, the present invention provides:
[1] an insect-derived polynucleotide of any one of (a) to (d),

    
    
        (a) a polynucleotide encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 2, 4, and 6,
        (b) a polynucleotide comprising a coding region of the nucleotide sequence described in any one of SEQ ID NOs: 1, 3, and 5,
        (c) a polynucleotide encoding a protein comprising an amino acid sequence with one or more amino acid substitutions, deletions, additions, and/or insertions in the amino acid sequence of any one of SEQ ID NOs: 2, 4, and 6, and
        (d) a polynucleotide that hybridizes under stringent conditions with the polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5;
    
    


[2] the polynucleotide of [1], which is derived from Polypedilum vanderplanki; 
[3] a vector comprising the polynucleotide of [1] or [2];
[4] a host cell carrying the polynucleotide of [1] or [2], or retaining the vector of [3];
[5] a pharmaceutical agent for conferring desiccation resistance to a cell, wherein the agent comprises the polynucleotide of [1] or [2], or the vector of [3];
[6] a method for conferring desiccation resistance to a cell, wherein the method comprises expressing in the cell a protein encoded by the polynucleotide of [1] or [2];
[7] a polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5, or comprising at least 15 nucleotides complementary to a complementary strand thereof;
[8] a method for determining whether a test cell has desiccation resistance, wherein the method comprises measuring the polynucleotide of [1] or an expression level of a protein encoded by the polynucleotide of [1] in the cell; and
[9] a method for determining a desiccation state of a test cell, wherein the method comprises measuring the polynucleotide of [1] or an expression level of a protein encoded by the polynucleotide of [1] in the cell.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 compares the amino acid sequences of the desiccation-resistant nematode LEA (AavLEA1) and plant LEAs. The amino acid sequences of LEA proteins derived from a nematode (Aphelenchus avenae), European white birch (Betula pendula), soybean (Glycine max), corn (Zea mays), and Arabidopsis thaliana are shown in SEQ ID NOs: 7 to 11, respectively. Conserved regions could not be found on comparison of the LEA protein of the desiccation-resistant nematode with the LEA proteins of plants (Betula pendula, Glycine max, Zea mays, and Arabidopsis thaliana),
FIG. 2 compares the amino acid sequences of P. vanderplanki LEA proteins (PvLEA1, set forth in SEQ ID NO:2, PvLEA2, set forth in SEQ ID NO:4, and PvLEA3set forth in SEQ ID NO:6) and that of the nematode LEA protein (AavLEA1set forth in SEQ ID NO:7). All three P. vanderplanki LEAs had low homology (30% or less) with AavLEA1.
FIG. 3 shows the conserved domains of the PvLEA proteins and their secondary structures. A motif search using HMMER showed that the three PvLEA proteins comprised LEA—4 domains. Furthermore, the predicted secondary structures were rich in α-helix.
FIG. 4 is a set of photographs showing variations in PvLEA gene expression after desiccation treatment. Expression levels of the three PvLEA genes began to increase one hour after desiccation treatment, reaching maximum values six hours after treatment, and remaining constant thereafter until individual larvae were completely desiccated, or more specifically, until they reached a cryptobiotic state. The PvLEA genes were not expressed tissue-specifically.
FIG. 5 is a photograph showing the results of studying the high hydrophilicity of the LEA proteins. PvLEA1, 2, and 3 proteins expressed using baculoviruses (M) were subjected to 100° C. for 15 minutes, and then fractionated into supernatant (S) and precipitate (P) by centrifugation. It should be noted that the molecular weights are increased by 2 kDa since the recombinant proteins are His-tagged (recombinant PvLEA1: about 100 kDa; recombinant PvLEA2: about 28 kDa; recombinant PvLEA3: about 55 kDa).
FIG. 6 shows the result of colony counting after introducing the PvLEA genes into CHO-K1 cells followed by desiccation treatment. Among the cells expressing the PvLEA1, 2, and 3 genes, only those cultured in the medium containing trehalose formed 30 to 55 colonies after desiccation treatment. On the other hand, cells that only expressed the vector hardly formed any colonies regardless of the presence of trehalose.



DETAILED DESCRIPTION OF THE INVENTION
A description of example embodiments of the invention follows.
The present inventors succeeded in isolating three distinct genes encoding late embryogenesis abundant (LEA) proteins from P. vanderplanki. The present invention is based on these findings, and provides polynucleotides that encode the insect-derived LEA proteins and uses thereof. The nucleotide sequences of the genes encoding the three LEA proteins (PvLEA1, PvLEA2, and PvLEA3) derived from P. vanderplanki are shown in SEQ ID NOs: 1, 3, and 5, and the amino acid sequences of these proteins are shown in SEQ ID NOs: 2, 4, and 6, respectively.
The present invention provides insect-derived polynucleotides comprising a coding region of the nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5. The polynucleotides of the present invention comprise polynucleotides encoding proteins that are functionally equivalent to those comprising the amino acid sequence of SEQ ID NO: 2, 4, or 6. Such polynucleotides include polynucleotides encoding proteins comprising an amino acid sequence with one or more amino acid substitutions, deletions, additions, and/or insertions in the amino acid sequence of SEQ ID NO: 2, 4, or 6 (such as mutants, derivatives, alleles, variants, or homologs).
Herein, a “functionally equivalent protein” is a protein which makes cells desiccation resistant, and that has an a-helix-rich structure and at least one or more LEA—4 motifs, which are characteristic of LEA proteins. Proteins that do not aggregate after treatment with high temperature, and are highly hydrophilic can be included in the “functionally equivalent” proteins of the present invention. Whether a certain protein is functionally equivalent to the proteins of the present invention can be confirmed by predicting its secondary structure from the amino acid sequence of the test protein, and by conducting motif analysis. Alternatively, it can be judged by actually expressing the test protein in cells, and observing whether the cells become desiccation. Whether a certain protein is functionally equivalent to the proteins of the present invention can also be confirmed by expressing the test proteins, subjecting them to high temperature, and examining whether they are highly hydrophilic.
Proteins that are functionally equivalent to the insect-derived LEA proteins of the present invention can be prepared by those skilled in the art, for example, by using methods for introducing mutations to amino acid sequences in proteins (such as site-directed mutagenesis (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 8.1-8.5)). Mutations in protein amino acid sequences due to mutations in the nucleotide sequences that encode the proteins may also occur in nature. Polynucleotides encoding such proteins that comprise an amino acid sequence with one or more amino acid substitutions, deletions, or additions to the naturally-occurring insect-derived LEA protein (SEQ ID NO: 2, 4, or 6) are comprised in the polynucleotides of the present invention, as long as they encode proteins functionally equivalent to the naturally occurring proteins. The number of modified amino acids is not particularly limited as long as the modified protein has functions equivalent to the LEA proteins of the present invention. However, the modified amino acids are generally 50 amino acids or less, preferably 30 amino acids or less, and more preferably 10 amino acids or less (for example, 5 amino acids or less, and 3 amino acids or less).
To retain the function of the protein, the amino acids used for substitution are preferably those comprising similar properties to the original amino acids prior to substitution. Examples of properties of amino acid side chains include: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids comprising the following side chains: aliphatic side-chains (G, A, V, L, I, P); hydroxyl group-containing side-chains (S, T, Y); sulfur atom-containing side-chains (C, M); carboxylic acid- and amide-containing side-chains (D, N, E, Q); base-containing side-chains (R, K, H); and aromatic-containing side-chains (H, F, Y, W) (The letters within parentheses indicate the one-letter amino acid codes).
The hydropathic index (Kyte and Doolitte, (1982) J Mol Biol. 1982 May 5; 157(1):105-32) or hydrophilicity value (U.S. Pat. No. 4,554,101) for each of the amino acids before and after modification is preferably within ±2, more preferably within ±1, and most preferably within ±0.5. The amino acid regions to be modified are not particularly limited, but regions other than those with characteristic motifs are preferred. Such regions other than those with characteristic motifs can be determined by referring to FIG. 3.
Proteins functionally equivalent to the insect-derived LEA proteins of the present invention can be isolated using hybridization techniques or gene amplification techniques well known to those skilled in the art. More specifically, using the nucleotide sequences of polynucleotides encoding the proteins of the present invention, or portions thereof, those skilled in the art can routinely perform hybridization (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publisher: John Wiley & Sons Section 6.3-6.4) to isolate DNAs that are highly homologous to these nucleotide sequences, and to obtain functionally equivalent proteins from these DNAs. The present invention also comprises polynucleotides that hybridize under stringent conditions with the polynucleotides encoding the insect-derived LEA proteins of the present invention. The insects from which functionally equivalent proteins are isolated are preferably insects that are highly resistant to desiccation. Proteins that confer desiccation resistance to an organism may be present in such insects. Most preferably, such insects are P. vanderplanki, which are insects with the ability to recover from even complete desiccation (cryptobiosis).
Hybridization conditions for isolating DNAs encoding the functionally equivalent proteins can be appropriately selected by those skilled in the art. Conditions for hybridization may be stringent conditions. Herein, the phrase “stringent conditions” means conditions in which specific hybrids are formed, while non-specific hybrids are not formed. An embodiment of the stringent conditions of the present invention includes low stringency conditions. Low stringency conditions mean that the washing conditions after hybridization are, for example, 42° C., 5×SSPE, and 0.1% SDS, or preferably 50° C., 5×SSPE, and 0.1% SDS. Examples of hybridization conditions that are more preferable are highly stringent conditions. An example of highly stringent conditions is 65° C., 0.1×SSPE, and 0.1% SDS. Under such conditions, higher temperatures enable DNAs with higher homology to be obtained more efficiently. However, the combinations of SSPE, SDS, and temperature conditions mentioned above are only examples, and those skilled in the art can appropriately combine the above-mentioned or other factors (for example, probe concentration, probe length, and hybridization reaction lime) that determine hybridization stringency to accomplish similar stringencies to those described above.
Alternatively, when using gene amplification techniques (PCR) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1-6.4), one can design primers based on portions of the nucleotide sequences of polynucleotides encoding the insect-derived LEA proteins of the present invention, then isolate polynucleotide fragments that are highly homologous to these nucleotide sequences or portions thereof, and obtain proteins that are functionally equivalent to the proteins of the present invention based on these fragments.
Proteins isolated using such hybridization techniques and gene amplification techniques ordinarily have amino acid sequences with high homology to the LEA proteins of the present invention. The present invention encompasses polynucleotides comprising nucleotide sequences with high homology to the nucleotide sequences of any one of SEQ ID NOs: 1, 3, and 5. Furthermore, the present invention encompasses proteins or peptides comprising amino acid sequences with high homology to the amino acid sequences of any one of SEQ ID NOs: 2, 4, and 6. “High homology” refers to sequence identity of at least 50% or more, preferably 75% or more, and more preferably 85% or more. More preferably, it means an identity of 90% or more, or 95% or more (such as 96% or more, 97% or more, 98% or more, or 99% or more). Identity can be determined using the BLAST algorithm.
The homologies of amino acid sequences and nucleotide sequences of the present invention can be determined using the BLAST algorithm according to Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Programs called blastn and blastx have been developed based on this algorithm (Altschul et al., J. Mol. Biol. 215:403-410, 1990). When a nucleotide sequence is analyzed using blastn, based on BLAST, the parameters are set, for example, at score=100 and wordlength=12. Also, when an amino acid sequence is analyzed using blastx, based on BLAST, the parameters are set, for example, at score=50 and wordlength=3. When using the BLAST and Gapped BLAST programs, default parameters for each of the programs are used. Specific procedures for these analysis methods are known (www.ncbi.nlm.nih.gov/).
The polynucleotides of the present invention may be in any form as long as they encode the proteins of the present invention. More specifically, the polynucleotides may be cDNAs synthesized from mRNAs, genomic DNAs, chemically synthesized DNAs or such. Furthermore, polynucleotides with an arbitrary nucleotide sequence based on genetic code degeneracy are encompassed, as long as they encode the proteins of the present invention.
The polynucleotides of the present invention can be prepared by methods known to those skilled in the art. For example, cDNA libraries are constructed from insect larvae, and hybridization is conducted using DNA segments that encode insect-derived LEA proteins of the present invention as probes, thus preparing DNAs derived from nature. Furthermore, the polynucleotides of the present invention can be produced by preparing RNAs from insect larvae, synthesizing cDNAs using reverse transcriptase, synthesizing oligonucleotide DNAs based on the DNAs encoding the proteins of this invention, and then conducting PCR using the oligonucleotide DNAs as primers to amplify the cDNAs encoding the proteins of the present invention.
Known methods can be used to isolate mRNAs. For example, total RNA is prepared by guanidine ultracentrifugation (Chirgwin J. M. et al. Biochemistry 18:5294-5299 (1979)), AGPC methods (Chomczynski R and Sacchi N. Anal. Biochem. 162:156-159 (1987)), and so on, and mRNAs are purified from total RNA using mRNA Purification Kit (Amersham Biosciences) and such. Alternatively, mRNAs can be directly prepared using a QuickPrep mRNA Purification Kit (Amersham Biosciences).
The obtained mRNAs are used to synthesize cDNAs using reverse transcriptase. cDNAs can be synthesized using a kit such as SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Alternatively, cDNAs can be synthesized and amplified by the 5′-RACE method (Frohman M. A. et al., Proc. Natl. Acad. Sci. U.S.A. 85: 8998-9002 (1988); Belyaysky A. et al., Nucleic Acids Res. 17: 2919-2932 (1989)) using 5′-Ampli FINDER RACE Kit (Clontech), and polymerase chain reaction (PCR).
The polynucleotides encoding the insect-derived LEA proteins of the present invention can be used to confer desiccation resistance to vertebrate cells, insect cells, or individual insects. More specifically, the present invention provides methods for conferring desiccation resistance to cells, in which the methods comprise expressing the insect-derived LEA proteins in the cells. Herein, the phrase “confer desiccation resistance (become desiccation resistant)” means that there is greater desiccation resistance than when an insect-derived LEA protein is not expressed. More preferably, this phrase means that expression of an insect-derived LEA protein enables acquisition of the ability to resuscitate after return to water, even when moisture was lost (cryptobiosis).
To express insect-derived LEA proteins in cells, the polynucleotides encoding the proteins must be introduced into cells. Genes are generally introduced into cells by incorporating the polynucleotides into appropriate vectors. The vectors that are used are not particularly limited, as long as the inserted polynucleotides are stably retained, and the vectors are selected appropriately according to the type of cells to be conferred with desiccation resistance. The present invention comprises vectors that comprise these polynucleotides that encode the insect-derived LEA proteins, and host cells that retain these vectors.
Host cells that express the insect-derived LEA proteins of the present invention include insect cells or vertebrate cells. Examples of insect cultured cells include Sf9 and Sf21 (both from Invitrogen), and examples of vertebrate cells include NIH/3T3, CHO, HepG2, and Jurkat. The vectors that enable genes to be expressed in these cells include the pIZT/V5-His vector (Invitrogen) for insect cultured cells. For vertebrate cells (in particular mammalian cells), the pGeneV5-His vector of the GeneSwitch system (Invitrogen) can be used for various cells such as NIH/3T3, CHO, HepG2, and Jurkat.
Vectors can be introduced into each of the host cells by appropriately using known gene introduction methods, according to the type of host cell. For methods involving transfection, methods such as calcium phosphate coprecipitation, electroporation, and complex formation with DEAE-dextran or with cationic lipids are used. For example, vectors are introduced into insect cultured cells using the cationic lipid for gene introduction, Cellfectin (Invitrogen®). Furthermore, for introduction of vectors into most vertebrate cells (in particular, mammalian cells), the cationic lipid for gene introduction, Lipofectamine2000 (Invitrogen®), can be used. For floating cells such as Jurkat cells, the cationic lipid for gene introduction reagent, DMRIE-C (Invitrogen®), may be used. When the insect-derived LEA proteins of the present invention are introduced and expressed in cells, trehalose may be added.
The polynucleotides encoding the insect-derived LEA proteins of the present invention, which can be utilized to confer desiccation resistance to vertebrate cells, insect cells, or individual insects, may be used in the form of pharmaceutical agents. More specifically, the present invention provides pharmaceutical agents for conferring desiccation resistance to cells, which comprise polynucleotides encoding the insect-derived LEA proteins, or vectors carrying these polynucleotides. In addition to the above-mentioned polynucleotides and vectors, the pharmaceutical agents of the present invention may appropriately comprise buffers such as TE buffer (10 mM Tris-Cl pH7.5, 1 mM EDTA), preservative solutions, or trehalose.
The present invention also provides oligonucleotides comprising at least 15 nucleotides that are complementary to the DNAs of the present invention, or to complementary strands thereof.
Herein, the term “complementary strand” refers to one strand of a double-stranded nucleic acid comprising A:T (U for RNA) and G:C base pairings to the other strand. In addition, “complementary” is defined as not only completely complementary within a continuous region of at least 15 nucleotides, but also having a homology of at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95% or more (for example, 96%, 97%, 98%, 99%, or higher) in a nucleotide sequence. Homology may be determined using an algorithm described herein. Furthermore, the term “oligonucleotide” includes polynucleotides.
The oligonucleotides of the present invention can be used as probes or primers for detecting and amplifying DNAs encoding the proteins of the present invention, and for detecting the expression of these DNAs. Furthermore, the oligonucleotides of the present invention can be used in the form of a DNA array platform.
When such oligonucleotides are used as primers, their lengths are normally 15 bp to 100 bp, and preferably 17 bp to 30 bp. The primers are not particularly limited as long as at least a portion of the DNAs of the present invention, or complementary strands thereof can be amplified. Furthermore, when using such oligonucleotides as primers, their 3′ end regions can be designed to be complementary, and restriction enzyme recognition sequences or tags can be added to their 5′ ends.
When using these oligonucleotides as probes, the probes are not particularly limited, as long as they specifically hybridize to at least a portion of the DNAs of the present invention, or complementary strands thereof. The probes may be synthetic oligonucleotides, and are normally at least 15 bp or longer.
When the oligonucleotides of the present invention are used as probes, they are preferably labeled as necessary. Examples of labeling methods include methods that use T4 polynucleotide kinase to phosphorylate the 5′ ends of oligonucleotides with 32P, and methods that incorporate a substrate nucleotide, labeled with an isotope such as 32P, a fluorescent dye, or biotin, using a DNA polymerase such as Klenow enzyme, and using a random hexamer oligonucleotide or such as a primer (random priming methods and so on).
The oligonucleotides of the present invention can be produced using, for example, a commercially available oligonucleotide synthesizer. The probes may be produced as double-stranded DNA fragments obtained by restriction enzyme treatment.
The polynucleotides or the oligonucleotides of the present invention can be used to confirm whether the LEA proteins of the present invention are expressed in test cells. Furthermore, since the LEA proteins of the present invention are specifically expressed only in cells with desiccation resistance, the expression of the LEA proteins in these cells can be confirmed using the polynucleotides or oligonucleotides of the present invention to determine whether the test cells show desiccation resistance. Therefore, when the expressions of the LEA proteins are confirmed, the test cells can be determined to have desiccation resistance, and when the expressions of the LEA proteins are not confirmed, the test cells can be determined to have no desiccation resistance.
Furthermore, since the expression levels of the LEA proteins of the present invention increase as cell desiccation progresses, the expression levels of the LEA proteins can be examined to determine whether the test cells are in a desiccated state, or to confirm the degree of test cell desiccation. Therefore, when the expression levels of the LEA proteins are high, the desiccation state in the test cells can be determined to be progressing, and when the expression levels of the LEA proteins are low, the desiccation state in the test cells can be determined not to be progressing.
Methods well known to those skilled in the art may be used for quantifying the expression levels of the LEA proteins in the test cells. For example, the transcriptional levels of the LEA genes can be measured by extracting test cell mRNAs according to standard methods, and using these mRNAs as templates to perform Northern hybridization or RT-PCR. Furthermore, the expression levels of the LEA proteins can be measured using DNA array technology.
The translational levels of the genes can also be measured by detecting the expressions of the LEA proteins by electrophoresis, such as by SDS-PAGE.
Alternatively, as described in Example 2, the expressions of the LEA proteins can be confirmed by producing 32P radiolabeled probes from full length LEA cDNAs using the Strip-EZ Kit (Ambion), and hybridizing the probes to a nylon membrane to which the RNAs of the test cells have been transferred.
Furthermore, the translational levels of the genes can be measured by performing Western blotting using antibodies against the LEA proteins, and detecting the expressions of the LEA proteins. The antibodies used for detecting the LEA proteins are not particularly limited, as long as they are detectable antibodies. However, for example, either monoclonal antibodies or polyclonal antibodies, or both may be used. The antibodies can be prepared by methods well known to those skilled in the art. For example, the polyclonal antibodies can be obtained as follows: An LEA protein or a recombinant protein expressed in a microorganism such as E. coli as a fusion protein with GST, or a partial peptide thereof, is used to immunize small animals such as rabbits to obtain their serums. The antibodies are prepared by purifying this serum using methods such as ammonium sulfate precipitation, protein A or protein G column chromatography, DEAE ion exchange chromatography, or chromatography through an affinity column coupled to the LEA protein or to a synthetic peptide. For monoclonal antibodies, for example, the LEA protein or a partial peptide thereof is used to immunize small animals such as mice, the spleens are removed from these mice and ground to separate the cells. These cells are fused to murine myeloma cells using reagents such as polyethylene glycol, and from the fused cells (hybridomas) thus obtained, clones that produce antibodies binding to the LEA protein are selected. Next, the obtained hybridomas are implanted intraperitoneally to mice, and their ascites are collected and purified by methods such as ammonium sulfate precipitation, protein A or protein G column chromatography, DEAE ion exchange chromatography, or chromatography through an affinity column coupled to the LEA protein or to a synthetic peptide to prepare monoclonal antibodies.
Expression of LEA proteins encoded by the genes of the present invention (PvLEA1, PvLEA2, and PvLEA3) that were isolated from P. vanderplanki is considered to enable organisms whose cells have been desiccated to recover from their desiccated state. Therefore, whether test cells have desiccation resistance can be determined by measuring the polynucleotides of the present invention or the expressions of the proteins encoded by these polynucleotides in these cells.
Furthermore, since the expression levels of the LEA proteins of the present invention increase as cell desiccation progresses, the polynucleotides of the present invention or the expression levels of the proteins encoded by these polynucleotides in test cells can be examined to determine whether the test cells are in a desiccated state, or to confirm the degree of test cell desiccation. In addition, expression of these genes in organisms other than P. vanderplanki enables these organisms (or tissues or cells) to be desiccated and preserved. For example, introduction of PvLEA1, PvLEA2, and PvLEA3 genes to natural enemy insects used in agriculture to eliminate insect pests, beneficial insects such as silkworms and honeybees, and laboratory insects such as fruit flies, allows preservation of lineages in the desiccated state without successive breeding. Furthermore, if insects to which these genes have been introduced are transported in a cryptobiotic state, insect death due to accidents during transport (damages due to warming, low temperature, and such) can be prevented, and the cost of transportation can be reduced due to reductions in weight. In addition, introducing these genes into vertebrates, if possible, will clearly contribute to desiccation and preservation technologies for these cells.
Therefore, the genes of the present invention (PvLEA1, PvLEA2, and PvLEA3) appear to be highly useful.
Any patents, published patent applications, and publications cited herein are incorporated by reference.
EXAMPLES
Hereinafter, the present invention will be specifically described using examples, however, it is not to be construed as being limited thereto.
Example 1
Isolation of Polypedilum vanderplanki Homologs of the LEA Genes
Since nematode and plant LEA proteins are not highly homologous (FIG. 1), the present inventors presumed that amino acid homologies between the LEA genes of P. vanderplanki and known LEA genes were also low, and that RT-PCR and library screening were unlikely to be effective in isolating the P. vanderplanki LEA genes. Therefore, the inventors prepared P. vanderplanki cDNA libraries 0, 12, and 36 hours after desiccation, constructed their own unique P. vanderplanki EST database, and then proceeded to isolate genes encoding LEA proteins with low homology.
ISOGEN (Nippon Gene) was used to extract total RNAs from P. vanderplanki larvae 0, 12, and 36 hours after desiccation. cDNAs were then synthesized using oligo dT primers, and these cDNAs were cloned into pBlueScript II KS+vectors (Stratagene) to produce cDNA libraries. Clones were randomly extracted from the three types of constructed libraries, and their nucleotide sequences were determined using T7 and T3 primers. An EST database was then constructed using the obtained data. As a result of BLAST searches (www.ncbi.nlm.nih.gov:80/BLAST) and annotations, clones that appeared to encode LEA proteins were identified. Clone contigs were constructed by aligning the identified clones, and the existence of the contigs thus-obtained was confirmed by RT-PCR. Finally, the full-length nucleotide sequences of the cDNAs were determined using the SMART-RACE method (Clontech).
As a result, three genes encoding distinct LEA-like proteins were successfully isolated, and they were named PvLEA1 (RNA: approximately 2500 nt, Protein: 742 AA), PvLEA2 (RNA: approximately 740 nt, Protein: 180 AA), and PvLEA3 (RNA: approximately 1560 nt, Protein: 484 AA). The nucleotide and amino acid sequences of PvLEA1 are shown in SEQ ID NOs: 1 and 2 respectively; the nucleotide and amino acid sequences of PvLEA2 are shown in SEQ ID NOs: 3 and 4 respectively; and the nucleotide and amino acid sequences of PvLEA3 are shown in SEQ ID NOs: 5 and 6 respectively.
The coding regions were identified from the obtained nucleotide sequence data using Genetyx-Mac (SDC), the amino acid sequences of the putative translation products were determined, and their secondary structures were then predicted. Further, motif analysis using HMMER (motif.genome.adjp) was carried out to predict the function of these translation products. The results showed that these proteins all had low homology with the nematode LEA (AavLEA 1) protein: 24.6% for PvLEA1 protein, 28.0% for PvLEA2 protein, and 24.4% for PvLEA3 protein (FIG. 2).
Furthermore, according to Chou-Fasman secondary structure prediction, the proteins deduced from all three genes were predicted to have α-helix-rich structures, characteristic of LEA proteins. The results of motif searches using HMMER showed that all three isolated genes carried at least one or more LEA—4 motifs (FIG. 3). Therefore, the three genes isolated herein seemed to be novel insect-derived LEA genes.
Example 2
Expression Analysis of the LEA Genes
To investigate variations in the expression levels of the isolated LEA genes accompanying desiccation, total RNAs were extracted from P. vanderplanki larvae using ISOGEN (Nippon Gene) 0, 1, 3, 6, 24, and 48 hours after desiccation treatment. The obtained RNAs were subjected to electrophoresis using guanidine-denatured agarose gel, and were transferred to a nylon membrane using a vacuum blotter. Strip-EZ kit (Ambion) was used to produce 32P radiolabeled probes from the obtained full-length LEA cDNAs, and they were used in hybridization with the aforementioned nylon membrane. After washing so that only the specific bands remained, image analysis was performed using LAS-2500 (Fuji film). The results showed that the expression levels of the three isolated genes started to increase one hour after desiccation treatment, reached a maximum value six hours after treatment, and remained constant thereafter (FIG. 4). Therefore, PvLEA1, 2, and 3 genes are desiccation inducible. This finding is similar to the LEA genes reported so far.
Furthermore, to investigate the tissue specificity of LEA gene expression, the midguts were removed from larvae twelve hours after desiccation treatment, and tissues containing large amounts of fat body were separated. Image analysis of gene expression was carried out using RNA isolation, electrophoresis, and hybridization, as described above. Expression levels of PvLEA 1, 2 and 3 genes in the midgut were comparable to those in the tissue containing large amounts of fat body (FIG. 4). This therefore suggested that these genes are expressed and exert their functions in all tissues and cells.
Example 3
Study of the Hythophilicity of LEA Proteins
Since plant desiccation resistance proteins are highly hydrophilic, they are considered to have activities that allow synonymous substitution for water, and more specifically, they have been shown to function as ion scavengers and chaperonins (Dure, L., 3rd (1993) The American Society of Plant Physiologist Vol. 10, pp. 91-103; Ingram, J. and Bartels, D. (1996) Annu Rev Plant Physiol Plant Mol Biol 47, 377-403). Accordingly, if the LEA genes isolated in the present invention are similarly hydrophilic proteins, they may also have the functions proposed for hydrophilic proteins in plants. It is known that highly hydrophilic proteins do not aggregate when treated with boiling water (Dure, L., 3rd (1993) The American Society of Plant Physiologist Vol. 10, pp. 91-103).
Thus, using the baculovirus protein expression system, recombinant His-tagged PvLEA1, PvLEA2, and PvLEA3 proteins were synthesized from LEA genes (M). These proteins were treated at 100° C. for 15 minutes, and then fractionated into supernatant (S) and precipitate (P) by centrifugation. Western blot analysis was performed on the obtained fractions using anti-His-tag antibody to determine whether or not the recombinant proteins were aggregated (FIG. 5). The results showed that the recombinant proteins synthesized from the three types of LEA genes isolated in the present invention did not aggregate at all, even though they were heat-treated. Therefore, the translation products of the LEA genes of the present invention were considered to be highly hydrophilic proteins, which have the activity of synonymous substitution for water, and more specifically, which function as desiccation resistance proteins.
Example 4
Desiccation-protective Function of Insect LEA Proteins
Next, cellular expression systems were used for examination to determine whether the LEA proteins of P. vanderplanki have desiccation-protective function in organisms.
The PvLEA1, 2, and 3 genes were respectively subcloned into the EcoRV/BamHI sites of pIRESneo3, pIRESbleo3, and pIRESpuro3 vectors (Clontech) to construct pPvLEA1-IRES-neo3, pPvLEA2-IRES-bleo3, and pPvLEA3-IRES-puro3 plasmids. These plasmids were introduced into 50% confluent CHO-KI cells (a cell line derived from Chinese hamster ovary cells) using FuGene6 (Roche), and the media were exchanged 24 hours later with either 0.1 M trehalose-containing medium-or trehalose-free medium. Cells to which the genes were introduced were cultured continuously for two days, followed by complete removal of the medium, and the cells were left to stand for 4 hours in a sealed container at a relative humidity of 95%. The cells were suspended in Ham's F-12 medium for rehydration, and plated on a 12-well culture plate (5×104 cell/well). After culturing for six days, the number of cell colonies that formed was determined. The CHO-K1 cells were cultured in 10% fetal calf serum-containing Ham's F-12 medium (Sigma) at 95% humidity, 5% carbon dioxide and 37° C.
As a result of the above examination, among the cells expressing the PvLEA1, 2, and 3 genes, only those cultured in the medium containing trehalose formed 30 to 55 colonies after desiccation treatment. On the other hand, cells that expressed only the vector formed hardly any colonies regardless of the presence of trehalose. Therefore, the coexistence of trehalose and expressed PvLEA 1, 2, and 3 genes was found to confer desiccation resistance to animal cells.

## Claims

1. An isolated insect-derived polynucleotide, the polynucleotide is selected from the group consisting of: (a) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4; and (b) a polynucleotide comprising a coding region for encoding the amino acid sequence of SEQ ID NO:4 in the nucleotide sequence of SEQ ID NO: 3.

2. The polynucleotide of claim 1, which is derived from Polypedilum vanderplanki.

3. A vector comprising the polynucleotide of claim 1.

4. A host cell transformed with polynucleotide of claim 1.

5. A host cell containing the vector of claim 3.

6. An agent for conferring desiccation resistance to a cell, wherein the agent comprises an isolated insect-derived polynucleotide of (a) or (b):
(a) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4; or
(b) a polynucleotide comprising a coding region for encoding the amino acid sequence of SEQ ID NO:4 in the nucleotide sequence of SEQ ID NO:3.

7. An agent for conferring desiccation resistance to a cell, wherein the agent comprises a vector comprising an isolated insect-derived polynucleotide of (a) or (b):
(a) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4; or
(b) a polynucleotide comprising a coding region for encoding the amino acid sequence of SEQ ID NO:4 in the nucleotide sequence of SEQ ID NO:3.

8. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:3.