AU696436B2

AU696436B2 - Polypeptide having cold-resistant pyruvate phosphate dikinase activity, DNA coding for the same, and recombinant vector and transformed plant both containing said DNA

Info

Publication number: AU696436B2
Application number: AU11203/95A
Authority: AU
Inventors: James Nigel Burnell; Shozo Ohta; Satoru Usami
Original assignee: Japan Tobacco Inc
Current assignee: Japan Tobacco Inc
Priority date: 1993-12-03
Filing date: 1994-12-01
Publication date: 1998-09-10
Anticipated expiration: 2014-12-01
Also published as: AU1120395A

Description

1

SPECIFICATION

Polypeptide Having Cold-stable Pyruvate, Orthophosphate Dikinase Activity, DNA Encoding the Same and Recombinant Vector and Transformed Plants Containing the DNA TECHNICAL FIELD The present invention relates to a novel polypeptide having cold-stable pyruvate, orthophosphate dikinase (hereinafter also referred to as "PPDK") activity, a cloned DNA encoding the same and a recombinant vector containing the DNA, as means for giving cold-stability to plants. The present invention also relates to plants transformed with the DNA according to the present invention.

BACKGROUND ART

C

4 plants have high abilities of photosynthesis under the conditions of strong light, high temperature or low

CO

z However, their photosynthesis abilities are largely reduced under low temperature except for those which are adapted to low temperature conditions. Although PPDK (EC 2.7.9.1, that catalyzes the reaction in which AMP, phosphoenol pyruvate and pyrophosphate are produced from ATP, pyruvate and orthophosphate) is one of the important enzymes in C 4 path, its activity is not sufficient with respect to the photosynthesis rate in leaf tissue, so that it is one of the enzymes which determine the rate of

CO

2 fixation in C 4 photosynthesis. Further, it has been 'AZ pointed out simultaneously with the discovery of this

-Y-

enzyme that this enzyme is cold-sensitive. In case of maize PPDK, the enzyme activity has a point of inflection at 11.7 0 C. This temperature is coincident with the limit temperature of growth of maize. From these, it is thought that PPDK is one of the causes which reduce the photosynthesis rate of C 4 plants at low temperature.

Therefore, by improving the cold-sensitivity of PPDK, the limit temperature of growth of maize may be lowered.

Flaveria brownii which is a plant belonging to the family Compositae is classified into C 3

/C

4 intermediate type, and it is known that its PPDK is not substantially inactivated by low temperature treatment at 0°C (Burnell JN: A comparative study of the cold-sensitivity of pyruvate, Pi dikinase in Flaveria species. Plant Cell Physiol. 31, 295-297 (1990)).

By cloning the gene encoding the cold-stable PPDK of Flaveria brownii, and by transforming a plant with the gene, it is expected that resistance to coldness can be given to the plant.

DISCLOSURE OF THE INVENTION Accordingly, an object of the present invention is to provide a novel polypeptide having cold-stable PPDK activity, a cloned DNA encoding the same and a recombinant vector containing the DNA, as means for giving cold-stability to plants. Another object of the present invention is to provide a plant transformed with q\ the above-mentioned DNA according to the present -ol L invention.

The present inventors intensively studied to succeed in cloning the complete PPDK gene of Flaveria brownii, determining the nucleotide sequence of the gene and the amino acid sequence encoded thereby, and in identifying the region in the PPDK gene, which gives cold-stability, thereby completing the present invention.

That is, the present invention provides a polypeptide having cold-stable PPDK activity, which has an amino acid sequence that is the same as the amino acid sequence of 1/6 region of the entire region from the C-terminal of the following polypeptide or except that at least one amino acid residues of said 1/6 region are substituted with other amino acid residues: a PPDK having an amino acid sequence shown in SEQ ID NO. 1 to 4 in Sequence Listing; and a polypeptide having an amino acid sequence which has a homology of not less than 50% with the amino acid sequence mentioned in said polypeptide having coldstable PPDK activity.

The present invention also provides a cloned DNA encoding the polypeptide having cold-stable PPDK activity according to the present invention. The present invention further provides a recombinant vector containing the DNA according to the present invention, which can express in a host a polypeptide having cold- ZX stable PPDK activity. The present invention still I ~a IP~i" I further provides a plant which is transformed with the DNA according to the present invention.

By the present invention, a gene encoding PPDK having cold-stability was cloned and sequenced. Further, the region in the gene, which gives cold-stability was also identified. Therefore, by transforming a plant having cold-sensitive PPDK with the gene according to the present invention, the cold-sensitive PPDK may be changed to cold-stable PPDK. Further, by incorporating the above-mentioned cold-stability-giving region into the corresponding region of a cold-sensitive PPDK, the coldsensitive PPDK may be changed to cold-stable PPDK. By this, the plant can be cultivated at a cold area in which the plant could not be hitherto cultivated. Therefore, it is expected that the present invention will much contribute to agriculture.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 schematically shows a construction method of an expression vector containing an example of the PPDK gene according tn the present invention; Fig. 2 shows the change in enzyme activity with time when PPDKs of Flaveria brownii, Flaveria bidentis and maize, which were expressed in Escherichia coli, were kept at 0°C; and Fig. 3 schematically shows a construction method of an expression vector containing an example of the PPDK NI. gene according to the present invention.

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BEST MODE FOR CARRYING OUT THE INVENTION By the present invention, the gene of PPDK having cold-stability of Flaveria brownii was cloned and its nucleotide sequence and the deduced amino acid sequence encoded thereby were determined. The nucleotide sequence and the amino acid sequence are shown in SEQ ID NO. 5 in Sequence Listing. As described in detail in the following examples, this sequence was determined by extracting the total RNAs from leaves of Flaveria brownii; preparing a cDNA library according to a conventional method; carrying out plaque hybridization method using a probe prepared by referring to the region of PPDK gene of Flaveria bidentis and the region of PPDK gene of maize, which regions have high homology; selecting and cloning positive clones; and sequencing the gene by dideoxy method. The sequence has high homology with the PPDK gene of Flaveria bidentis belonging to the same genus, and has a relatively high homology with the maize PPDK gene. Further, the sequences of the Nterminal region, C-terminal region and internal regions of the deduced amino acid sequence are completely the same as the corresponding sequences of the PPDK of Flaveria brownii directly purified from green leaves of this plant. Therefore, it is apparent that the cloned gene is the PPDK gene of Flaveria brownii. The PPDK gene of Flaveria bidentis was sequenced by carrying out plaque hybridization using maize cDNA as the probe; cloning the I g~ positive clones; and sequencing the gene by dideoxy method.

The amino acid sequence shown in SEQ ID NO. 5 is novel and 40 amino acid residues thereof are different from the amino acid sequence of the PPDK of Flaveria bidentis belonging to the same genus. About 180 amino acid residues thereof are different from the amino acid sequence of the maize PPDK. Thus, in spite of the fact that the amino acid sequence of PPDK of Flaveria brownii shown in SEQ. ID NO. 5 has a high homology with the amino acid sequence of the PPDK of Flave:ia bidentis belonging to the same genus, the PPDK of Flaveria bidentis is coldsensitive while that of Flaveria brownii is cold-stable.

Thus, the small difference in amino acid residues brings about the important difference of the character. The present invention provides a cloned PPDK gene encoding the amino acid sequence shown in SEQ ID NO. 5. As mentioned above, this amino acid sequence is novel and has a prominent effect that it has cold-stability. The gene according to the present invention is not restricted to that having the nucleotide sequence shown in SEQ ID NO. 5, but any nucleotide sequence which encodes this amino acid sequence is within the scope of the gene according to the present invention.

The present inventors tried to identify the region in the PPDK gene of Flaveria brownii shown in SEQ ID NO.

j\ 5, which region gives cold-stability. That is, as described in detail in the following examples, the PPDK gene of Flaveria brownii was divided into three regions having about the same size by restriction enzymes; each of the regions was exchanged with the corresponding region of maize PPDK gene to form chimeric PPiDK genes; and whether the PPDKs encoded by the obtained chimeric genes have cold-stability or not was determined. As a result, it was confirmed that the region giving coldstability exists in the last 1/3 region of the PPDK gene of Flaveria brownii. Further, this last 1/3 region was divided by a restriction enzyme into two regions having about the same size, and which region contains the region giving cold-stability was determined by the similar method. As a result, it was confirmed that the function tc give cold-stability is encoded in the region downstream of the Xho I site of the PPDK gene of Flaveria brownii shown in SEQ ID NO. 5. That is, it was confirmed that the function to give cold-stability is located within the amino acid sequence between the 832nd amino acid residue, arginine, and the 955th amino acid residue, valine, of the amino acid sequence shown in SEQ ID NO. (the amino acid sequence from the 832nd amino acid residue to the 955th amino acid residue may be hereinafter referred to as "cold-stability-giving sequence").

That is, it was proved that the region relating to cold-stability of PPDK is located in the 1/6 region of I-~-UI I the entire region from the C-terminal. On the other hand, in SEQ ID NO. 1 in Sequence Listing, the nucleotide sequence of the gene encoding PPDK of Flaveria bidentis and deduced amino acid sequence encoded thereby are shown, and in SEQ ID NO. 2, the nucleotide sequence of the gene encoding PPDK of maize and deduced amino acid sequence encoded thereby are shown (Journal of Biochemistry 263, 11080-11083 (1988)). In SEQ ID NO. 3, the nucleotide sequence of the gene encoding PPDK of Bacteroides symbiosus which is a bacterium, and deduced amino acid sequence encoded thereby are shown (Biochemistry 29, 10757-10765 (1990)). In SEQ ID NO. 4, the nucleotide sequence of the gene encoding PPDK of Entamoeba histolytica which is a bacterium, and deduced amino acid sequence encoded thereby are shown (Molecular and Biochemical Parasitology 62, 153-156 (1993)). As described above, it was proved by the present invention that the region relating to cold-stability of PPDK is located in the 1/6 region of the entire region from the C-terminal, it is possible to obtain cold-stable PPDK by substituting at least cne amino acid residue in the 1/6 region of the entire region from the C-terminal of the amino acid sequence shown in SEQ ID NO. 1, 2, 3 or 4.

Here, the term "cold-stable" means that activity of the enzyme after leaving the enzyme to stand at 0°C for minutes is not less than 60% of the original activity.

SAs mentioned above, since the region from the 832nd amino acid residue, arginine, to the 955th amino acid residue, valine, in the amino acid sequence shown in SEQ ID NO. 5 defines cold-stability, the cold-sensitive PPDKs having the amino acid sequence shown in SEQ ID NO. 1 4 can be converted to cold-stable PPDKs by substituting the corresponding regions of the cold-sensitive PPDKs with the amiro acid sequence from the 832nd amino acid residue, arginine, to the 955th amino acid residue, valine, of the amino acid sequence shown in SEQ ID NO. This finding is very important because by utilizing this, any desired cold-sensitive PPDK can be converted to a cold-stable PPDK, The method of giving cold-stability to a PPDK is not restricted to the method as described in the examples below, in which the cold-stability-giving sequence of PPDK of Flaveria brownii is exchanged with the corresponding region of a cold-sensitive PPDK to prepare a chimeric gene, but the cold-stability may be given by changing the corresponding region of a coldsensitive PPDK of a plant to the same sequence as the cold-stability-giving sequence of Flaveria brownii by site-directed mutagenesis. Therefore, any DNA which encodes a polypeptide having PPDK activity, which contains the above-described cold-stability-giving sequence is within the scope of the present invention.

In particular, the PPDK in which the 869th amino acid residue in the amino acid sequence shown in SEQ ID NO. 1 T is substituted by proline, and the PPDK in which the g 885th and 952nd amino acid residues in the amino acid sequence shown in SEQ ID NO. 1 are substituted by leucine and valine, respectively, have cold-stability.

The PPDKs to which cold-stability is to be given are not restricted to those shown in SEQ ID NOs. 1-4, but may be those having not less than 50% of homologies to the sequences shown in SEQ ID NOs. 1-4. Preferably, the nucleotide sequence of the gene encoding a PPDK to which cold-stability is to be given has a homology to the PPDK gene of Flaveria brownii in an amount of not less than 48.5%, more preferably, not less than It is well-known in the art that there are cases wherein the physiological activity of a physiologically active peptide is retained even if the amino acid sequence of the peptide is modified to a small extent, that is, even if one or more amino acids in the amino acid sequence are substituted or deleted, or even if one or more amino acids are added to the amino acid sequence.

Therefore, polypeptides having the same amino acid sequence as shown in SEQ ID NO. 5 except that the polypeptides have such modifications, which have coldstable PPDK activity, are included within the scope of the present invention. That is, the polypeptides having the same amino acid sequence as shown in SEQ ID NO. 5 except that one or more amino acids are added, deleted or substituted, which have cold-stable PPDK activity, are included within the scope of the present invention.

I il M Similarly, DNAs having the same nucleotide sequence as shown in SEQ ID NO. 5 except that one or more nucleotides are added, deleted or substituted, which encode polypeptides having cold-stable PPDK activity, are also within the scope of the present invention.

Modification of DNA which brings about addition, deletion or substitution of the amino acid sequence encoded thereby can be attained by the site-specificmutagenesis which is well-known in the art Nucleic Acid Research, Vol. 10, No, 20, p6487-6500, 1982). In the present specification, "one or more amino acids" means the number of amino acids which can be added, deleted or substituted by the site-specific mutagenesis.

Site-specific mutagenesis m7y be carried out by, for example, using a synthetic oligonucleotide primer complementary to a single-stranded phage DNA except that the desired mutation as follows. That is, using the above-mentioned synthetic oligonucleotide as a primer, a complementary chain is produced by a phage, and host bacterial cells are transformed with the obtained doublestranded DNA. The culture of the transformed bacterial cells is plated on agar and plaques are formed from a single cell containing the phage. Theoretically, 50% of the new colonies contain the phage having a singlestranded chain carrying the mutation and remaining 50% of the colonies contain the phage having the original Q\ sequence. The obtained plaques are then subjected to I PCI 12 hybridization with a kinase-treated synthetic probe at a temperature at which the probe is hybridized with the DNA having euactly the same sequence as the DNA having the desired mutation but not with the original DNA sequence that is not completely complementary with the probe.

Then the plaques in which the hybridization was observed are picked up, cultured and the DNA is collected, In addition to the above-mentioned site-specific mutagenesis, the methods for substituting, deleting or adding one or more amino acids without losing the enzyme activity include a method in which the gene is treated with a mutagen and a method in which the gene is selectively cleaved, a selected nucleotide is removed; added or substituted and then the gene is ligated.

By transforming a plant with PPDK gene of Flaveria brownii or with a DNA encoding a polypeptide containing the cold-stability-giving sequence, which has PPDK activity, a cold-resistant plant can be obtained.

Preferred examples of the plants to be transformed include maize, sugar cane, millet, barnyard grass and sorghum, although not restricted thereto.

Methods for transforming plants have already been established and the method utilizing Agrobacterium tumefaciens may preferably be employed. The method for transforming plants utilizing Agrobacterim tUmefaciens is well-known in the art. By this method, both dicotyledons Japanese Laid-open Patent Application 13 (Kokai) No. 4-330234) and monocotyledons (WO 94/00977) can be transformed. Alternatively, the DNA may be introduced into plant protoplasts by electroporation method or the like well-known in the art. Further, transformation may also be carried out by attaching the DNA to tungsten particles and the like and implanting the particles in embryos of a plant. Concrete methods of these transformation methods are described in the examples hereinbelow described.

ample The present invention will now be described more concretely by way of examples thereof. However, the present invention is not restricted to the examples below.

1. Cloning and Sequencing of PPDK Gene of Flaveria brownii Preparation of cDNA Library and Cloning of Fulllength cDNA Preparation of cDNA Library From green leaves (60 g) of F. brownii, total RNAs were isolated by the guanidine hydrochloride/phenol method. By this method, 26.5 mg of RNA after lithium precipitation was obtained. Then 118.9 pg of poly(A)+RNA was ybtained from 13.2 mg of the RNA according to a conventional method using a column containing Oligo dT cellulose Type 7 (commercially available from PHARMACIA).

For the preparation of the cDNA library, TimeSaver cDNA h Synthesis kit (commercially available from PHARMACIA), tit I IW E 14 Lambda ZAPII vector (commercially available from STRATAGENE) and the packaging reagent attached to cDNA cloning system XgtlO (commercially available from AMERSHAM) were used. Using an EcoRI/NotI linker, a cDNA library in which DNA fragments are inserted into the EcoRI site of Lambda ZAPII vectors was prepared. The size of the prepared cDNA library was 415,000 pfu. As the host cells, XL1-Blue cells were used.

(ii) Preparation of Probe Using a primer having the sequence of (designed based on the regions of PPDK of Flaveria bidentis and of maize PPDK, which have a high homology) and a R primer having the sequence of TATCGAGAAACCTTCTATAC (a part of the sequence of PPDK of Flaveria bidentis, complementary chain), a DNA fragment originated from RNA of F. brownii was amplified by reverse transcription PCR, and the amplified DNA fragment was inserted in pCRII vector (commercially available from INVITROGEN). Using the obtained vector as a template, and using the same primers as mentioned above, the DNA was amplified and the PCR product was subjected to electrophoresis, followed by recovering the DNA from the gel by using SUPREC-01 (commercially available from TAKARA SHUZO). By this process, a DNA fragment having a size of 428 bp starting from the 24th bp downstream of the N-terminal of the mature protein can be obtained. This fragment was labelled with 32 P using Multiprime DNA labelling system (commercially available from AMERSHAM) to prepare a probe.

(iii) Cloning of Full-length cDNA of Flaveria brownii The cDNA library was screened by the plaque hyb:idization method using the above-mentioned DNA fragment as a probe. As the hybridization filter, Hybond N (AMERSHAM) was used, and the hybridization was carried out in 6 x SSC containing 5 x Denhalt's solution, 0.1% SDS and 100 pg/ml of denatured salmon testis DNA at overnight. Washing was performed with 2 x SSC containing 0.1% SDS at room temperature for 5 minutes; with 2 x SSC containing 0.1% SDS at room temperature for 90 minutes; and 1 x SSC containing 0.1% SDS at 68°C for 90 minutes.

By this, 28 independent positive clones were obtained.

From these, 11 plaques which showed strong signals were selected and subjected to second screening. The second screening was carried out in the same manner as in the first screening except that the time of the second washing was 60 minutes. As a result, independent positive plaques originated from a single phage were obtained from 6 clones. To check the sizes of the inserted DNA fragments, PCR was carried out using the above-described R primer, Ml3PrimerM4 (GTTTTCCCAGTCACGAC, commercially available from TAKARA SHUZO) and Ml3PrimerRV (CAGGAAACAGCTATGAC, TAKARA SHUZO) as primers and using the phage as a template. As a result, 2 clones contained the full-length cDNA. Thereafter, by in vivo excision, the inserted DNA fragments were subcloned into a plasmid vector pBluescriptIISK(-) (commercially available from STRATAGENE). The recombinant plasmids obtained by the subcloning were named p411 and p631.

It was found by the above-described PCR that the library prepared as described above was a library containing sufficiently long inserts, which was suited for cDNA screening. Thus, for the isolation of mRNAs of Flaveria bro'nii, it is advantageous to employ the abovedescribed method and to obtain a large amount of mRNAs at once by treating a large amount of RNAs as described above.

Further, since the above-described cDNA library contains a number of inserts which are sufficiently long, screening of the full-length cDNA was able to be performed easily by preparing a probe prepared by using a primer which hybridizes with a region in the vicinity of the processing region of the desired protein.

Determination of Total Nucleotide Sequence of cDNA and Comparison of Deduced Amino Acid Sequence To determine the total nucleotide sequence of the inserted cDNA fragment of p631, deletion mutants were prepared. The deletion mutants were prepared by using Deletion Kit for Kilo-Sequence (commercially available from TAKARA SHUZO). However, the reaction by exonuclease III was stopped by transferring the reaction mixture to Mung Bean Nuclease buffer kept at 65 0 C. Determination of I I 1 I the nucleotide sequence was carried out using a plasmid purified by using Qiagen Plasmid Mini Kit (commercially available from Diagen), Taq DyeDeoxy Terminator Cycle Sequencing Kit (commercially available from ABI) and Applied Biosystems 373A DNA Sequencer (ABI). Both chains were sequenced except for some parts thereof. Based on the determined nucleotide sequence, amino acid sequence was determined. The determined nucleotide sequence and the amino acid sequence are shown in SEQ ID NO. 5 in the Sequence Listing.

It should be noted that in the preparation of the deletion clones for sequencing the isolated cDNA, it was important to preliminarily heat the Mung Bean Nuclease Buffer at 65*C because the reaction could not be stopped by merely transferring the reaction mixture to the Mung Bean Nuclease Buffer. Further, since the sequence at the region up to about 600 900 bp from the boarder between the vector and the insert could not be determined by deleting the insert from the upstream end alone, it was necessary to carry out the deletion from both ends.

On the other hand, PPDK was purified directly from Flaveria brownii and the amino acid sequences of its Nterminal region, C-terminal region and internal regions were determined. The PPDK was purified as follows. That is, green leaves were ground in triple volume of extraction buffer. After centrifugation, ammonium sulfate was added to the supernatant to 30% saturation I rs I I 18 and precipitated proteins were removed. Ammonium sulfate was further added to 70% saturation and precipitated proteins were recovered. The recovered proteins were applied to Sephadex G25 (commercially available from PHARMACIA) to remove salts. The resultant was applied to DEAE-Sepharose column (PHARMACIA) and the proteins adsorbed to the column were eluted by KC1 having a gradient of 50 400 mM. Fractions having PPDK activity were combined and concentrated by ammonium sulfate of saturation, followed by desalination by Sephadex The resultant was then applied to a hydroxyapatite column. The adsorbed proteins were eluted by phosphate buffer having a gradient of phosphate from 10 mM to mM. Fractions having PPDK activity were combined and concentrated by ammonium sulfate of 70% saturation, followed by desalination by Sephadex G25. The resultant was subjected to SDS-PAGE and the band of PPDK was cut out. Protein was recovered from the gel by electroelution. By this process, purified PPDK sample was obtained in an amount of about 5 10 nmol. The purified PPDK thus obtained showed a single band in SDS-

PAGE.

Then the amino acid sequences of N-terminal region, C-terminal region and internal regions of the thus obtained purified PPDK were determined. That is, the amino acid sequence of the N-terminal region was I determined by transferring the protein on a PVDF membrane

I

and then determining the sequence using a gas phase amino acid sequencer. The amino acid sequence of the C-terminal region was determined by digesting the purified PPDK with carboxypeptidase Y and estimating the amino acid sequence based on the relationship between the composition of the liberated amino acids and the digestion time. The amino acid sequences of the internal regions were determined by determining the amino acid sequence from the N-terminal of peptides generated by digesting the protein with a protease. The details of this method are as follows.

Firstly, as in the case of determining the amino acid sequence of the N-terminal region, PPDK from green leaves of Flaveria brownii was partially purified and subjected to an ordinary SDS-PAGE. The gel was stained with Coomasssie Brilliant Blue R250 and the band of PPDK was cut out. The cut out gel was equilibrated in an equilibrating buffer (Tris-HCl pH6.8, 125 mM, EDTA 1 mM, 0.1% SDS) and inserted in a well, followed by second SDS- PAGE. Here, an overlaying solution (Tris-HCl pH6.8, 125 mM, EDTA 1 mM, 0.1% SDS, 0.01% BPB, 20% glycerol) and an enzyme solution (Tris-HCl pH6.8, 125 mM, EDTA 1 mM, 0.1% SDS, 0.01% BPB, 10% glycerol, lysyl endopeptidase 1 ug or V8 protease 0.01 0.1 ug) were added together with the equilibrated gel. After carrying out electrophoresis for a while, digestion of the protein was carried out in the concentrated gel (the electric power was turned off and the gel was left to stand for 45 minutes). Then electrophoresis was restarted and the resultant was transferred to a PVDF membrane in the same manner as in the determination of the N-terminal sequence, and the amino acid sequences of the fragments obtained by the digestion were determined from the N-terminals thereof using the gas phase amino acid sequencer.

The determined sequences of N-terminal region, Cterminal region and internal regions are as follows: N-terminal Sequence: Asn Pro Val Ser Pro Pro Val (72-78) C-terminal Sequence: Leu Ala Ala Val Val (948-95r) Internal Sequence Lys Leu Tyr Glu Phe Leu Val Asn Ala Gin Gly Asp Val Val Ala (349-365) Internal Sequence Gin Leu Leu Ala Pro Pro Ala Met Ser Asn Ala Leu Thr (592-605) Internal Sequence Leu Thr Ala Asp Thr Gly Met Ser Lys Asp Glu Ile Tyr Ser Arg Ile Glu (721-738) Internal Sequence Ala Ser Phe Gly Thr Asn Asp Leu Cys Gln Met Val Phe Gly Ser (844-862) In the above-described amino acid sequences, means glatamine but could not be analyzed by the arnlyzer used, and means that the result of the analysis was unclear. The numbers in the parentheses indicate the amino acid number of the corresponding region in the amino acid sequence shown in SEQ ID NO. 5. Although parts of the Internal Sequences and are different from the amino acid sequence shown in SEQ ID s 7 Is I 1 s NO. 5, it is thought that this is due to errors of the amino acid sequencer. As is well-known, errors are caused by amino acid sequencers at considerable frequency while errors are not substantially caused by DNA sequencers.

Since the amino acid sequence shown in SEQ ID NO.

well corresponds with the partial amino acid sequences directly determined in the above-described purified PPDK, it was confirmed that the amino acid sequence shown in SEQ ID NO. 5 is the amino acid sequence of PPDK. The amino acid sequence shown in SEQ ID NO. 5 was compared with the amino acid sequences of known PPDKs of Flaveria bidentis and maize. As a result, 40 amino acid residues (Flaveria bidentis) and about 180 amino acid residues (maize) were different, respectively, in the mature protein region. Further, from the above-described results, it is thought that in the amino acid sequence shown in SEQ ID NO. 5, the amino acid sequence from 1st to 71st amino acid residue does not exist in the mature protein but a transit peptide necessary for passing through membranes, which is processed after passing through membranes. Differences between the amino acid sequences of mature proteins of Flaveria brownii and Flaveria bidentis are shown in Table 1 below.

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Table 1 b wnii ILbi dentis Amino Acid Numbers and Amino Acids Which are Different 5 10 28 41 48 49 52 57 61 62 66 81 92 97 114 140 150 154 170 201 205 263 265 323 356 374 Phe Pro Asn Thr Pro Ala Arg Arg Lys Ser Ile Thr Gly Leu Thr Asn Lys Ala Lys Leu, Gln Ala Asp Ala Ala Gin Cys Val Val 6 11 16 29 41 42 47 48 49 50 55 59 60 64 79 90 95 112 138 148 152 168 199 203 261 263 321 354 372 Ser Leu.

Arg Asn Ser Ser Leu Thr Pro Ala Pro Ser Ser Pro Ala Arg Arg Ser Ser Ser Asp Pro Ala Gly Val Lys Ser Ile Gly ?Wi-r Table 1 (continued) brownii bidentis Amino Acid Numbers and Amino 382 Glu 380 Asp Acids Which are Different 385 Arg 383 Lys 392 Val 390 Glu 396 Arg 394 Gly 465 Asn 463 Asp 490 Val 488 Cys 564 Val 562 Ile 583 Ser 581 Thr 605 Thr 603 Ile 672 Ala 670 Val 723 Thr 721 Ala 724 Ala 722 Val 730 Lys 728 Ala 736 Arg 734 Lys 739 Lys 737 Asn 777 Asn 775 Thr 803 Gly 801 Ser 818 Leu 816 Val 838 Asp 836 Glu 841 Ala 839 Gly 845 Glu 843 Asp 871 Pro 869 Gin 875 Ser 873 Ala 887 Leu 885 Ile 954 Val 952 Ile 2. Production of PPDK of Flaveria brownii in E. coli and Measurement of Cold-stability To confirm that a cold-stable enzyme is actually produced from the cDNA of PPDK of Flaveria brownii isolated as described above, expression in E. coli was carried out as follows: To remove the transit peptide and to ligate the DNA to an expression vector so that the reading frames are coincident, restriction sites were introduced as follows: That is, p631 was digested with SacI and recyclized to obtain p631Sac (a plasmid same as p631 except that downstream region of SacI site is deleted). PCR was performed using p631Sac as a template and using primer 4: GATATCAATCCGGTGTCTCCTCC containing EcoRV site, prepared based on the sequence in the vicinity of the processing region and primer M13 RV (commercially available from TAKARA SHUZO) complementary to the sequence of the vector, and the amplified fragment was subcloned into pCR II. A fragment containing the N-terminal region was cut out using restriction enzymes EcoRV and SacI. Three DNA fragments, that is, the thus cut out DNA fragment, the SacI-HindIII fragment from p631 (containing the remaining region of cDNA of PPDK) and a fragment obtained by digesting pKK233-2 with NcoIl blunting the ends with Klenow fragment and then digesting the resultant with HindIII, were ligated (Fig. E. coli MV1184 was i. transformed with the obtained plasmid and used for i expression experiments. One ml of a precultured medium was diluted with 9 ml of fresh LB medium (containing mg/1 of ampicillin) and the resultant was cultured with shaking for 3 hours at 37*C. Then IPTG was added to a concentration of 5 mM and culture was continued for another 3 hours, followed by collection of cells by centrifugation. The cells were suspended in 0.5 ml of an extraction buffer (50 mM Hepes-KOH pH7.5, 10 mM MgSO 4 1 mM EDTA, 5 mM DTT) and lysozyme was added to a final concentration of about 0.5 mg/ml. The suspension was left to stand in ice for 5 minutes and then treated by an ultrasonic disrupter (Model UCD-130T commercially available from COSMOBIO) f3.sa period of 30 seconds each, totally 5 minutes, thereby extracting the enzyme. The resultant was centrifuged by a microcentrifuge for minutes and the supernatant was applied to Sephadex column equilibrated with a column buffer (50 mM Hepes-KOH pH 7.0, 10 mM MgCl 2 2 mM EDTA, 10 mM DTT) to remove low molecular substances. The resultant was left to stand at 25*C for not less than 30 minutes thereby carrying out association to tetramer, which was then used for the measurement of activity.

The PPDKs produced in E. coli cells from PPDK cDNAs of Flaveria brownii, Flaveria bidentis and maize (Harvest Queen) exhibited substantially the same mobility as the enzymes originated from the plants in SDS-PAGE. Although the molecular weights of the mature enzymes, which are

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expected from the respective cDNAs are about the same, the apparent molecular weights on SDS-PAGE substantially differ. It was proved that this is due to the differences in amino acid compositions and not due to deletion processing of the proteins or modifications after translation, such as attaching of sugar chains.

The cold-stability of each PPDK produced in E. coli was identical to the corresponding enzyme originated from each plant. Thus, the cold-stability of the PPDK of Flaveria brownii is attained without an auxiliary factor specific to the plant or processing after translation, so that it is expected that cold-stable P!-DK is produced by introducing the cDNA into maize and expressing it. Fig.

2 shows the relationship between the time which lapsed after placing the enzyme at 0°C and relative activity of

PPDK.

In the above-described process for producing active PPDK in J. coli, the cDNA from which the region encoding the transit peptide was removed was inserted in the expression vector. For this, it was important to precisely match the site to be cut with the site corresponding to the N-terminal of the enzyme originated ftom the plant.

brownii) Amount of Enzyme Produced in E. coli Primer Used

MITAKKRVFTF...

MIPVSPPVTTTKKRVFTF...

MINPVSPPVTTTKKRVFTF...

NPVSPPVTTTKKRVFTF...

bidentis)

MITAKKRVFTF...

MIPVSPPVTTAKKRVFTF...

TAKKRVFTF...

2 3 4 (enzyme originated from leaves) (enzyme originated from leaves) (maize) MATKKRVFTF... TTKKRVFTF... (enzyme originated from leaves) More particularly, at the time point of carrying out the expression experiment of cDNA of Flaveria brownii, expression of PPDKs of maize and Flaveria bidentis had been succeeded, referring to the cutting site in maize in both cases. Thus, in expression of cDNA of Flaveria brownii too, firstly, the DNA was cut at the same site using primer 2 and it was tried to express the cDNA after constructing an expression vector containing the cDNA.

However, PPDK was not produced at all. Then, based on the sequence of the N-terminal region of the enzyme originated from leaves, an expression vector in which the inserted cDNA contains an additional sequence encoding the enzyme extended to the direction of N-terminal by 7 residues was constructed using the primer 3 and -1 wfli (TO;(wSi!tSg^[Tti^g^,^fflis^i['TOiTOll??Bj^ W5svra^'tfla".p.c¥ expression was carried out using the expression vector.

As a result, production of PPDK was confirmed. However, even with this expression vector, the amount of the produced enzyme was small, so that the enzyme was not obtained in an amount sufficient for measuring its activity. Thus, an expression vector in which the inserted cDNA contains a still additional sequence encoding the enzyme extended to the direction of Nterminal by 1 residue was constructed using the primer 4 and expression was carried out using the expression vector. As a result, a large amount of PPDK was produced and its cold-stability was confirmed. The nucleotide sequences of the primer 2 and the primer 3 were as follows: primer 2: CGGTGTCTCCTCCGGATATCACGGCTAAAAAGAG primer 3: TTGATATCCCGGTTGTCTCCTCCGGTA 3. Identification of Region Giving Cold-stability in PPDK Gene of Flaveria brownii Chimera of Flaveria brownii and Flaveria bidentis The expression vectors were recombined according to a conventional method using restriction enzymes. That is, the EcoRI-HindIiI fragment of pKK-brownii was exchanged with the corresponding fragment of pKK-bidentis (a plasmid obtained by inserting the cDNA of Flaveria bidentis into *KK 223-2 as in the construction of pKKbrownii) to obtain a plasmid pKK-011. On the other hand, Sthe EcoRI-Hindlil fragment of pKK-bidentis was exchanged with the corresponding fragment of pKK-brownii to obtain a plasmid pKK-100. Similarly, the NdeI-HindIII fragments were exchanged to obtain pKK-001 and pKK-110. Further, the Xho-HindIII fragment of pKK-110 was exchanged with the corresponding fragment of pKK-bidentis to obtain pKK- 1101, and the Xhol-HindIII fragment of pKK-bidentis was exchanged with the corresponding fragment of pKK-brownii to obtain pKK-1110. To further finely recombine the XhoI-HindIII fragments, the fragments were linked by PCR (linking PCR method). That is, primers complementary to the nucleotide sequence in the XhoI-HindIII fragment, which sequence is common in bidentis and brownii, that is, a primer link-F: GCAGAGATGATGTTGGCAAG and a primer link-R: CTTGCCAACATCATCTCTGC were prepared. Using the XhoI-HindIII fragment of brownii or bidentis subcloned into pBluescript as a template, and using combination of primers of link-F/RV or M4/link-R, the first PCR was carried out. The obtained fragments (totally 4 types) were purified by cutting out from the gel. Using the mixture of the fragment encoding the former half of brownii and the fragment encoding the latter half of bidentis, or the mixture of the fragment encoding the former half of bidentis and the fragment encoding the latter half of brownii as a template, and using primers M4/RV, the second PCR was performed. The amplified linked fragments were digested with XhoI and HindIII and the resultants were exchanged with the corresponding region of pKK-bidentis to obtain pKK-linkOl and pKK-linklD. Another pair of chimeric genes were prepared utilizing the PstI site between the recombination site in the linking PCR and the HindIII site. That is, the XhoI-PstI fragment of pKK-linklO and the PstI-HindIII fragment of pKK-bidentis were inserted into the XhoI-HindIII site of pKK-bidentis (three fragments-linking reaction) to obtain pKK-linklOl.

Similarly, the XhoI-PstI fragment of pKK-bidentis and the PstI-HindIII fragment of pKK-brownii were inserted into the XhoI-HindIII site of pKK-bidentis to obtain pKKlinkll0.

The 40 sites at which the amino acid residues are different between the mature PPDK proteins of Flaveria brownii and of Flaveria bidentis are mainly exist in the N-terminal and C-terminal regions, and do not exist so many in the center region, that is, in the active site.

Thus, the cDNAs were divided into three regions, that is, the front, middle and rear regions by utilizing EcoRI sites and Ndel sites which commonly exist in both of the genes, and these fragments were interchangeably exchanged to prepare chimeric genes. By checking these chimeric genes, it was determined which region is related to the cold-stability. As a result, cold-stability is acquired when the protein contains the rear 1/3 region of the cDNA of Flaveria brownii. In contrast, when the protein k contains the rear 1/3 region of the cDNA of Flaveria bidentis, the protein was cold-sensitive. Then the rear 1/3 region was divided into two regions by restriction enzyme XhoI, and the fragments were respectively introduced into the corresponding regions of pKK-bidentis and the cold-stability was checked. As a result, the region downstream of the XhoI site the 1/6 region from the C-terminal) was necessary and sufficient for attaining cold-stability. Then a chimeric gene containing the rear 1/6 region (XhoI-HindIII fragment, containing 7 substitutions of amino acid residues) was prepared by the linking PCR method and the amplified gene was introduced into pKK-bidentis, followed by measuring the cold-stability thereof. As a result, a chimeric enzyme pKK-linklO having the rear most region containing 4 substitutions was cold-stable while a chimeric enzyme pKK-linkOl having the former half of the rear region, which contains 3 substitutions was cold-sensitive. Then the rear region was recombined using a restriction enzyme PstI to prepare chimeric genes having two amino acid substitutions per gene, and the cold-stability of the obtained genes was checked. As a result, all of the chimeric genes exhibited cold-stability. Thus, it is assumed that there are two or more regions related to cold-stability.

Chimera of Maize and Flaveria brownii Using a primer PPDK-F: CTCACTGTTCGAAGAGAAGC and a primer mNdeI: CATATGCTCTGTCCGGCATAATC (complementary chain side) containing NdeI site as primers, and cDNA of maize PPDK as a template, PCR was performed and the amplified fragment was subcloned into pCR II. This fragment was cut out from pCR II by SacI and NdeI. This fragment, the SacI-Smal fragment (vector fragment) of pKK-PPDK and a fragment obtained by digesting cD~A of PPDK of F. brownii, blunting the resultant with Klenow fragment and then digesting the resultant with Ndel, were linked by three fragments-linking reaction (Fig. 3) to obtain pKK-mz/bro(Nde). TOR was performed using primer PPDK-F and a primer mXhol: CTCGAGGGATCTCAATCATTG (complementary chain), and maize PPDK as a template. The obtained fragment was subcloned into pCR II and the insert was cut out by SacI and XhoI from pCR II. The cut out fragment was ligated with the SacI-XhoI fragment (vector fragment) of pKK-mz/bro(Nde) to obtain pKKmz/bro(Xho Both of the chimeric enzyme containing the Cterminal 1/3 region (NdeI-HindIII fragment) of brownii PPDK in maize PPDK and the chimeric enzyme containing the C-terminal 1/6 region (XhoI-HindIII fragment) of brownii PPDK in maize PPDK exhibited cold-stability as strong as that of brownii. Thus, since cold-stability could be given to maize PPDK whose amino acid sequence is considerably different from that of F. brownii, it is thought that cold-stability can be given to PPDKs of various plants by introducing the rear 1/3 region or the

'I

C-terminal 1/6 region. Since the thus prepared maize/F.

brownii chimeric PPDK contains the transit peptide of maize PPDK as it is, if a transformed plant has a problem in transportation of the cold-stable PPDK to chloroplasts, in which the transit peptide is also originated from Flaveria brownii, this problem is thought to be overcome by introducing this chimeric gene in place of the gene originated from F. brownii.

Point-mutated Clones Amino acid residues of XhoI-HindIII fragment of pKKbrownii were changed one by one from brownii type to bidentis type. Similarly, amino acid residues of Xhol- HindIII fragment of pKK-bidentis were changed one by one from bidentis type to brownii type. Introduction of the mutation was carried out by subcloning the XhoI-HindIII fragments of PPDK cDNAs of F. brownii and of F. bidentis into pBluescript IISK(-), and then substituting nucleotides by Kunkel method using Megalabel kit (commercially available from TAKAR7 3HUZO) and Mutan-K kit (commercially available from TAKARA SHUZO). The sequence of the primers used for introducing mutations were as shown in Table 2. After confirming the mutated nucleotide sequence by a DNA sequencer, these fragments were inserted into the XhoI-HindIII site of pKK-bidentis.

Table 2 Primers Used for Preparation of Point-mutated Clones Introduction of Mutation from F. brownii type to F. bidentis type 836DE 839AG 5' GCTTCTTTTCCAATCTCATC 843ED 869PQ 873SA 885LI 5 TTCTGGTCAATAACCTCAATG 952V1 Introduction of Mutation from F. bidentis type to F. brownii type 836ED 839GA 843DE S'CGAAAAGAACTCAGCTTC 869QP 873AS 5 GAATGCCTPGAGAAAGATAAATC 885IL 952IV All of the enzymes in which one amino acid residue in the XhoI-HindIII region of pKK,-1l10, which amino acid residue is different between F, brownii and F. bidentis, was substituted to the corresponding amino acid residue of the bidentis type exhibited cold-stability. Thus, it 1- 1-4: 1 111~11~1 is thought that there are a plurality of mutations which give cold-stability (because the cold-stability is not lost by changing only one amino acid residue). Then enzymes in which one amino acid residue in the XhoI- HindIII region of pKK-bidentis, which amino acid residue is different between F. brownii and F. bidentis, was substituted to the corresponding amino acid residue of the brownii type were prepared, and it was checked whether the enzymes acquired cold-stability. That is, the enzyme activity after treatment at 0°C for 20 minutes was measured. As a result, by the mutation of 869Gln- Pro, the enzyme acquired cold-stability (the activity after the cold treatment is 60-70% of the original activity). With the mutation of 885Ile--Leu or 952Ile- Val, the loss of activity at low temperature was prevented a little. Taking these results and the results of chimeric enzyme pKK-linkllO described in into consideration, it is assumed that the enzyme acquires cold-stability when these mutations coexist. From these results, it was concluded that the three amino acid residues, that is, 869Pro, 885Leu and 952Val are related to cold-stability. However, among these residues relating to cold-stability of brownii, as for 869Pro and 885Leu, these residues are of brownii type in maize PPDK too. Therefore, cold-stability is not necessarily attained only by the fact that these residues are of k brownii type, but it is thought that these residues give complete cold-stability in the amino acid sequence of PPDK of brownii or bidentis. Therefore, in cases where cold-stability is to be given to a PPDK originated from a different species, whose amino acid sequence is considerably different from those of brownii and bidentis, it is preferred not to introduce point mutations, but to prepare a chimeric gene in which a region giving cold-stability is introduced as carried out for maize PPDK.

4. Transformation of Maize with PPDK Gene of Flaveria brownii In accordance with the method of Gordon-Kamm W.J. et al (The Plant Cell 2:603-618, 1990) or Koziel M.G. et al.(Bio/Technology 11: 194-200, 1993), gold or tungsten fine particles are coated with pKK-brownii, and the coated particles are implanted into maize immature embryos or suspended cultured cells. From the treated cells, transformants are selected, and obtained calli of transformants are cultured according to a conventional method, followed by regenerating plants from the calli.

The method of transformation is not restricted to the particle gun method, but the methods of transformation which may be employed include electroporation method (Rhodes C.A. et al., Science 240:204-207, 1988), PEG method (Armstrong C.L. et al., Plant Cell Reports 9:335- 339, 1990), tissue-electroporation method (D'Halluin K.

Q\ et al., The Plant Cell 4:1495-1505, 1992), Agrobacterium e I I method (Hiei Y. and Komari T. WO 9400977) and the like.

From the obtained plants, seeds are recovered and germinated to regenerate plants. PPDK is isolated from leaves of the obtained plants and its cold-stability is checked. For transformed plants and non-transformed plants, effect of temperature with respect to photosynthesis rate is checked. Stability of the transformation is assured by proliferating maize plants which exhibit high photosynthesis rates at low temperature for many generations, and measuring the photosynthesis rates at different temperatures and measuring the cold-stability of PPDKs isolated from the plants.

Transformation of Flaveria bidentis with PPDK gene of Flaveria brownii An intermediate vector containing the full-length cDNA shown in SEQ ID NO. 5 and a reporter gene is introduced into a disarmed Ti plasmid of Agrobacterium tumefaciens. This can be accomplished by the method described in Draper J et al. eds., Plant Genetic Transformation and Gene Expression a laboratory manual, Blackwell Scientific Publications (ISBN 0-632-02172-1).

On the other hand, leaf tissue or callus of Flaveria bidentis is infected with the above-mentioned Agrobacterium tumefaciens. This can be accomplished by culturing the tissue or callus together with Agrobacterium tumefaciens. Infected cells are selected -38based on the drug resistance. From the selected calli, whole plants are generated by a conventional methods.

.Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step 20 or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

I- SEQUENCE LISTING SEQ ID NO.: 1 SEQUENCE LENGTH: 2915 SEQUENCE TYPE: nucleic, acid S~EQUENCE DESCRIPTION CGATCTCCTT CTGCTATTGC TGATATCTC:,. ATTTCACAGG TGAAGAAGG ATG ATG AGT Met Met Ser 58 TCG TTG TCT GTT GAA GGT ATG Ser Leu Ser Val Glu Gly Met 10 TTA CCG GCG AGA GTG AAG CA Leu Pro Ala Arg Val Lys Gin CTT CTC Leu Leu CGG CGA Arg Arg

AAC

Asn 25 CAC CAC CGT CAA TCG TCG TTT GTC His His Arg Gin Ser Ser Plie Val AAG TCA GCC CGT GAG~ TCG TGC Lys Ser Ala Arg Glu Ser Cys AAC GGT GAT CTC CGG CGA TTG Asn Gly Asp Leu Arg Arg Leu 30 CGG TGT TTA ACT CCG GCG AGA Arg Cys Leu Thr Pro Ala Arg 45 GGC TTA ACT CCG CCG OGA GCA Gly Leu Thr Pro Pro Arg Ala ACG ACG GCT AAA AAG AGG GTT Thr Thr Ala Lys Lys Arg Val GTT AGC AGA CCA GAG TTG CGC AWX1 AGT Val Ser Arg Pro GlU LeU Arg Set~ Ser 60 GTT CTT AAT CCG GTG TCT CCT CCG GTG Val LeU Asn Pro Va2l Ser Pro Pro Val.

TTC ACT TTT Phe Thr Phe ,TG TTG GGA GGT AA.A GGA Gly Lys Gly AGA AGT GAA Arg Ser Gil GGC AAC, AGG GJly Asn Arg GAC- ATG AAA TCC Asp Met Lys Ser GGA AA GGA GCA PAT CTT GCT GAG ATG TCA AGC.ATT GGT mmomw Leu Gly Gly Lys TCA GTT CCT CCT Ser Val Pro Pro 120 CA CA AP.T GG Gin Gin Asn Gly Gly Ala Asn Leu Ala Glu 105 110 GGG CTC ACT ATT TCA ACT Gly Leu Thr Ile Ser Thr 125 AAG AGC OTA CCT CCA GGT Lys Ser Leu Pro Pro Gly Met Ser Ser Ile Gly 115 GA GCA TGT GAG GA Glu Ala Cys Glu Glu 130 TTG TGG GAT GAG ATT Leu Trp Asp Giu Ile

TAT

Tyr 135 TCA GA GGC TTA Ser Giu Giv Leu 140 145 GAT TAT GTC CAG AAA GAG ATG TCT GCA TCT CTC GGT Asp Tyr Val Gin Lys Giu Met Ser Ala Ser Leu Gly GAC CCG Asp Pro 165 AAA CCT Lys Pro CTC CTC Leu Leu 170 ATG GAC Met Asp 155 160 CTT TCC GTC CGT TCG GGT GCT GCC ATA Leu Ser Val Arg Ser Gly Ala Ala Ile TCT ATG CCT GGT Ser Met Pro Gly 180

ATG

Met ACT GTA TTG PAT Thr Val LeU Asn 185 GAG GTC GTA GCT GGT Glu Val Val Ala Gly 200 GAC TCG TAT AGA AGG Asp Set Tyr Arg Arg 215 ATC CCG CAT TCA TTA lie Pro His Ser Leu CTA GCT GGC AAA AGT Leu Ala Gly Lys Ser 205 TTT CTC GAT ATG TTT Phe Leu Asp Met Phe 175 CTC GGG OTT PAT GAT Leu Gly Leu Asn Asp 195 GCA CGG ?TT GCC TAT Ala Arg Phe Ala Tyr 210 AAC GTT GTA ATG GGT Asn Val Val Met GlY

GGC

Gly 225 GAG CAG ATG AA GCT GA Glu Gin Met Lys Ala G1U 230 AAA GGG ATT CTC TTT GAO GPA Phe Asp G1U 235 GAC ACC GAT

TTA

Leu 240 ACT GOT GOT GAT CAT CTC, CTT AAA GAT I Lys Gly 245 CTT GTT Leu Val Ile His Leu Asp Thr Asp Leu Thr Ala 260

TTT

Phe GAG AAA TAO AAG Glu Lys Tyr Lys 265 250 AAC GTG TAT Asn Val Tyr GTG GAA Val Glu 270 Ala Asp 255 GCA AAG Ala Lys Leu Lys Asp GGC GAA AAG Gly Giu Lys 275 ccC Pro ACA GAT Thr Asp CCA AAG AAA CAG CTA GAG Pro Lys Lys Gin Leu Glu 280 285 GAC AGC CCA AGG GCC AAT Asp Ser Pro Arg Ala Asn 300

TTA

Leu GCA GTG AAT GCT GTT Ala Val Asn Ala Val 290 TTT GAT TCT TGG Phe Asp Ser Trp 295 CAG ATA ACT Gin Lie Thr 310 TTT GGC AAC Phe Gly Asn 325 AAC CCA AGC Asn Pro Ser

GGA

Gly TTA AAG Leu Lys

GGG

Gly ACT GCA Thr Ala 315 TCA GGA Ser Gly

GTT

Val AAG TAC AGA AGT ATT AAC Lys Tyr Arg Ser Ile Asn 305 AAC ATT CAA AGC ATG GTG Asn Ile Gin Ser Met Val 320 GGT GTT CTT TTC ACT AGG Gly Val Leu Phe Thr Arg 874 922 970 1018 1066 1114 1162 1210 ATG GGA AAC ACT Met Gly Asn Thr 330 ACC GGT GAG AAG Thr Giy GlU Lys 345 GAG GAT GTT GTT Glu Asp Val Val 335

ACT

Thr 340

GCT

Ala AAG CTA TAC Lys Leu Tyr GCT GGG ATC Ala Gly Ile

GGG

Gly 350

GAG

GlU TTT TTA ATC AAT Phe Leu Ile Asn 355 CAG GGA Gin Giv 360 ACC ATG GAG ACT Thr Met Glu Thr

GGG

Gly 365 TGC ATG COT GAT GCA Cys Met Pro Asp Ala 380 AGA ACA OCA GAA GAT TTG Arg Thr Pro Glu Asp Leu 370 TAO AAA GAG OTT GTG GAG Tyr Lys Glu Leu Val Glu 385 375 AAC TGC GAG ATO TTA GAG GGA CAC TAO AAA GAT ATG ATG GAT ATT GAA 1258 1111-- Asn Cys Giu 390 TTC ACA GTT Phe Thr Val 405 Ile Leu Giu Gly His Tyr Lys Asp Met Met Asp Ile Giu CAA GAA AAC Gin Oiu Asn TGG ATG Trp Met TTG CAA TGC CGA ACA GGG Leu Gin Cys Arg Thr Gly 415 GCA GTA GAT ATG GTG AAC Ala Val Asp Met Val Asn

AAA

Lys CGT ACT GGT AAA GGT Arg Thr Giy Lys Gly 425 GGG CTA ATT GAT ACT Gly Leu Ile Asp Thr GTG AGA ATT Val Arg Ile AGA ACA GCA ATT Arg Thr Ala Ilie CAT CTA GAT CAG His Leu Asp Gin 455 AAA AGC CAT GTG Lys set His Val 470 440

CTT

Leu AGG GTT GAG ACT CAA Arg Val Giu Thr Gin 450 GAT CCG TCT GCT TAC Asp Pro Ser Ala Tyr CTT CAT CCA CAG Leu His Pro Gin 460 GCA ACC. GGT TTG Ala Thr Gly Leu 1306 1354 1402 1450 1498 TTT GAG Phe Glu 465 CCA GCA TCC CCC GGG Pro Ala Ser Pro Giy

GTA

Val GCA GCT Ala Ala 475 GTG GGA V'al GiV

CA

Gin 500

GTT

Val CAG GTT TGT TTT AGT Gin Vai Cys Phe Ser 490 AAG AGT O~CT ATC TTG Lys Ser Ala Ile Leu 505 GGT ATG CAT GCA GCA Gly Met His Ala Ala

GCA

Ala GTA AGG ACC Val Arg Thr GCT GGA ATC Ala Giy Ile 525 495 GPA ACA G1u Thr 510 TTA ACC Leu Thr GAG GAT GCA GAA ACA TGG CAT GCA Giu Asp Ala Giu Thr Trp, His Ala AGC CCA GAA GAT Set Pro Giu Asp 515 GCT AGA GGA GGC Ala Arg Gly Gly 1546 1594 1642

GGT

Gly 520 530 ATG ACA TCA CAT GCA GCG GTG GTG GOT CGC GGA TGG GGC AAA TGT TGT 19 1690 Met Thr Ser His Ala Ala Val Va A Arg Gly Trp Gly Lys Cys Cys GTT TCC GGT Val Ser Gly 535 TGT GCT Cys Ala GAT ATT CGT Asp lie Arg 555 AAC GAT Asn Asp 545 GAT ATG AAG ATT TTT Asp Met Lys Ile Phe 560 TGG CTT TCT CTT AAT Trp Leu Ser Leu Asn 550 ACG ATT GGC GAC Thr Ile Gly Asp 565 GGT ACA ACT GGT Gly Thr Thr Gly CGT GTG ATT Arg Val Ile 570 GAA GTC ATA Glu Val Ile

AAA

Lys GAA GGC GAC Glu Gly Asp 575 580 GCA ATG Ala Met TTG GGT AAA CAG CTA CTG Leu Gly Lys Gin Leu Leu 590 ATA TTC ATG TCA 1GG GCT Ile Phe Met Ser Trp Ala GCT CCA CCT Ala Pro Pro 595 GAT CAA GCA Asp Gin Ala 610 AGC AAT GAC Ser Asn Asp 600

GAA

Glu 605 GCA AAT GCA GAC ACA Ala Asn Ala Asp Thr AGG CGT CTC Arg Arg Leu ACA GCC AGA Thr Ala Ara 630 CAT ATG TTT His Met Phe 645 ATC ATG GCG Ile Met Ala 660 CTC CCA TAC

AAG

Lys 615

GTT

Val

ATG

Met CCT AAT GAT GCA TTA Pro Asn Asp Ala Leu 625 1738 1786 1834 1882 1930 1978 2026 2074 2122 AAC AAT GGT GCA CAA GGG Asn Asn Giy Ala Gin Gly 635 TTC GCT TCT GAT GAG AGG Phe Ala Ser Asp Giu Arg 650 GTC ACT CCA GAA CAA AGA.

Val Thr Pro Giu Gin Arg 665 CAA AGA TCC GAT TTT GAG ATC GGG CTC TGT AGA ACT GAA Ile Gly Leu Cys Arg Thr Glu 640 ATC AAA GCT GTA AGA AAG ATG lie Lys Ala Val Arg Lys Met 655

GCT

Ala

GTG

Val 670

ATT

OTA GAT Leu Asp CTC TTA Leu Leu 675 ATG GAT TTC CGA GCA Leu Pro Tyr Gin GGA CTT COT GTA Giy Leu Pro Val 695 TTA 000 GAA GGT Leu Pro Giu Gly 710 ACA GGO ATG AGT Thr Gly Met Ser Ser Asp Phe Giu Gly Ile Phe Arg Aia Met Asp 685 ATC OGO OTT OTA GAO Ile Arg Leti Leu Asp OCT OCA OTT OAT GAG TTT Pro Pro Leu His Giu Phe 705 AAO GAA OTT GOA GTO GAO Asn Giu Leu Ala Val Asp GAT CTA GAA CAC Asp Leu Giu His 715 GCA GAT GAA ATO Ala Asp Giu Ile 730 ATG OTT GGT TTO Met Leu Gly Phe ATA GtTG Ile Val 2170 2218 2266 720 TAT TCA AAA ATO GAA Tyr Ser Lys Ile Giu AAT OTA TOT Asn Leu Ser 725 GAA GTG Giu Val AAC COT Asn Pro CGT GGT TGC AGA Arg Gly Cys Arg 750 745 TTA GGG ATT TOA Leu Gly Ile Ser 755 TTT CAA GOT GOA Phe Gin Ala Ala CCC GAG OTA ACA Pro Giu Leu Thr 760

GAPA

GlU ATG CAA Met Gin GTT OGT Val Arg 765 GOG ATC Ala Ile GTG TOT ATG ACC Vai Ser Met Thr 775 COG TTA GTG GGG Pro Leu Val Gly 790 CGT GGA GTA GOT Arg Gly Val Ala 805 TAT AAA GTG GGPL AAT CAG GGG, GTG Asn Gin Giy Vai ACA OCT CAG GAA Thr Pro Gin Giu 795 GOA AAT GTG TTT Ala Asn Val Phe 810 AOG ATG ATT GAG ACT GTA ATA OCA GAG ATO ATG GTT Thr Vai Ile Pro Giu Ile Met Val 780 785 TTA OGT OAT CAA ATO AGT GTA ATT Leu Arg His Gin Ile Ser Vhl Ile 800 GOT GAA ATG GGG GTG ACA TTG GAA Ala GiU Met Giy Val Thr Leu GiU 815 ATT OCT CGA GOT GOT TTA ATA GOT 2314 2362 2410 2458 2506 2554 Tyr 820

GAA

Glu Lys Val Gly Thr Met 825 GAG ATT GGA AAA GAA Giu Ile Giy Lys GJlu 840 ACC CAG APTG ACA TTT Thr Gin Met Thr Phe Ile, Glu Ile Pro Arg Ala Ala Leu Ile Al1a GCT GAT TTC TTT Ala Asp Phe Phe 845 GGG TAC AGC AGA Giy Tyr Ser Arg TCG TTT GGA ACC Ser Phe Gly Thr GAT GAT GTT GGC Asp Asp Val Giy 865 AAT GAT Asn Asp 850 AAG TTT Lys Phe

CTG

TTG CAG ATT Leu Gin Ile 870 GTT ATT GAC Val Ile Asp 855 TAT CTT GCT CAA GGC Tyr Leu Ala Gin Gly 875 CAG AAA GGG GTG GGT Gin Lys Giy Vai Gly 890 GCA GCA AAT CCT AAC Ala Ala Asn Pro Asn ATT CTG CAG CAT GAT CCA TTT GAG Ile Leu Gin His Asp Pro Phe Glu 880 CAG TTG ATT AAG ATG GCT ACG GAG Gin Leu Ile Lys Met Ala Thr Giu 895 TTA AAG GTT GGG ATA TGT GGG GAG Leu Lys Val Gly Ile Cys Giy Glu 2602 2650 2698 2746 2794 2842 2890

AAA

Lys 885 GGT CGT Giv Arg 900

CAT

His 905 GGT GGG GAG CCT TCT Gly Gly Giu Pro Ser 910 TCT GTT GCA TTT Ser Val Ala Phe 925 CCA TTT AGG GTT Pro Phe Arq Val

TTT

Phe 915 GAT GGA GTT GGA CTA Asp Gly Vai Gly Leu 930 ATC GCA AGG TTG GCC Ile Ala Arg Leu Ala GAT TAT GTG TOG Asp Tyr Vai Ser 935

TCT

Ser

CCT

Pro GCT GCA CAA GTC ATT GTT TAAGCTT 2915 Ala Ala Gin Vai Ile Vai 950 SEQ ID NO.: 2 SEQUENCE LENGTH: 2880 SEQUENCE TYPE: nuclijc acid SEQUENCE DESCRIPTION CGGCGCAGTA GGGGATCGGA AGG ATG GCG GCA TCG GTT TCC AGG GCC ATC TGC Met Ala Ala Ser Val Ser Arg Ala Ile Cys GTA CAG AAG CCG GGC TCA .AAA TGC Val Gin Lys Pro Giy Set Lys Cys TTC GCC CGC CGA Phe Ala Arg Arg CGC CGG CGT CAT Arg Arg Arg His

TCG

Set ACC AGG GAC, AGG GAA GCG ACC TCC Thr Arg Asp Arg Giu Ala Thr Set 20 CCG AGG CCC CCG CAC GCC AAA GCC Pro Arg Pro Pro His Ala Lys Ala GTC GCA Val Ala 35 CCG CTC CGA CTC CGG Pro LeU Arg LeU Arg CGC GGG ACG GGG CCA CAT TGC Arg Giy Thr Gly Pro His Cys TCG CCG Set Pro AAG, AGG Lys Arg ATEG AAG Met Lys AGC ATO Set Ile CTG AGG GCC GTC GTT Leu Arg Ala Val Vai 65 GTG TTC CAC TTC GGC Val Phe His Phe Gly 80 GAC GCC GCG CCG ATA CAG ACG ACC AAA Asp Ala Ala Pro Ile Gin Thr Thr Lys AP.G GGC AAG AGC GAG GGC AAC AAG ACC Lys Giy Lys Set Giu Gly Asn Lys Thr 85 GAA CTG Giu LeU CTG GGC GGC AAG Leq Gly Gly Lys TCG GTG CCG OCA Ser Val Pro Pro GGC GCG Gly Ala 100 GGG TTC Gly Phe AAC CTG GCG GAG ATG GCG Xsn LeU Ala Giu Met Ala 105 ACG GTG TCG ACG GAG GCG Thr Val Set Thr G1u Ala GGG CTG Gly Leu TGC CAG CAG Cys Gin Gin 125 GCC GAG ATC Ala Glu Ile 115 CAG GAC GCC GGG TGC GCC Gin Asp Ala. Gly Cys Ala 130 GTC GAC GGC CTG CAG Val Asp Gly LeU Gi.n CTC CCC GCG Leu Pro Ala 135 GAG GAG TAC Giu Glu Tyr 120 GGG CTC TGG Gly Leu Trp ATG GGC GCC Met Gly Ala TGG GTG Trp Val 140 ACC CTG Thr Leu 155

GC

Ala GGC GAT CCG Gly Asp Pro GTG TCC ATG Val Ser Met

CAG

Gin 160 CCG CTC CTG CTC TCC GTC Pro Leu Leu Leu Ser Val 165 CGC TCC GGC Arg Ser Giy 170 AAC CTG GGG Asn LeV Gly 185

GCC

Ala 175 CCC GGC ATG ATG GAG ACG Pro Gly Met Met Asp Thr 180 GCC GCC GGG CTG GCG GCC Ala Ala Gly Leu Ala Ala GTG CTC Val Leu CTC AAC GAC GAA Leu Asn Asp Glu 190 TTC GCC TAC GAC Phe Ala Tyr Asp 205

GTG

Val AAG AGC GGG GAG CGC Lys Ser Gly GiU Arg 200 437 485 533 581 629 677 725 77$ 821 195 TCC TTC CGC CGC TTC set Phe Arg Arg Phe 210 CTC GAC ATG TTC GGC AAC GTC Leu Asp Met Phe Gly Asn Val 215 GAA GAG AAG CTT GAG CAC ATG Glu Glu Lvs Leu Giu His Met GTC ATG Val Met 220 AAG GAA Lvs Giu GAC ATC CCC CGC TCA Asp Ile Pro Arg Ser 225 TCC AAG GGG CTG AAG Ser Lys Gly Leu Lys 240 GAG CTC GTG GGT GAG AAC GAC ACC GAC Asn Asp Thr Asp 245 TAG AAG GAG GTC CTG TTC LeU Phe 235

CTC

CTC ACG GCC TCT GAG Leu Thr Ala Ser Asp 250 TAC CTC TCA GCC AAG AAA LeU Lys GlU LeU Val Giy Gin Tyr Lys Glu Val Tyir LeU Set Ala Lys GGA GAG CCA Gly GlU Pro CTG GCT GTG Leu Ala Val 285 AGC ATC AAC Ser Ile Asn 300

TTC

Phe 270 TCA GAC CCC AAG Ser Asp Pro Lys 275 260 265 AAG CAG CTG GAG CTA GCA GTG Lys Gin Leu Giu Leu Ala Vai 280 CCC AGG GCC AAG AAG TAC AGG Pro Arg Ala Lys Lys Tyr Arg TTC AAC TCG TGG GAG Phe Asn Ser Trp Giu 290 CAG ATC ACT GGC CTC Gin Ile Thr Gly Leu 305 TTC GGC AAC. ATG GGG Phe Gly Asn Met Gly

AGC

Ser AGG GGC ACC GCC GTG AAC GTG CAG Arg Gly Thr Ala Val Asn Val Gin 310 AAC ACT TCT GGC ACC GGC GTG CTC Asn Thr Ser Gly Thr Giy Vai Leu

TGC

Cys 315

TTC

Phe ATG GTG Met Val 320 ACC AGG AAC CCC AAC Thr Arg Asn Pro Asn 335 GTG AAC GCT CAG GGT Val Asn Ala Gin Gly 325

CTG

Leu ACC GGA GAG Thr Gly Glu GAG GAT GTG Giu Asp Val 355 AAG AAC CTC Lys Asn Leu

AAG

Lys 340 AAG CTG Lys Leu 330 TAT GGC GAG TTC Tyr Gly Giu Phe 345 350 GAG GAC GlU Asp CTT GTT leu Val

CTT

LeU 365

GAC

Asp GCC ATG Ala Met GTT GCC GGA ATA AGA ACC CCA Val Ala Gly Ile Arg Thr Pro 360 ATG CCA CAG GCC TAC GAC GAG Met Pro Gin Ala Tyr Asp Giu 375 AGC CAC TAC AAG GAA ATG CAG Ser His Tyr Lys Giu ,et Gin 965 1013 1061 1109 1157 1205 1253 370 380 GAT ATC Asp Ile GAG P.AC, TGC AAC ATC Glu Asn Cys Asn Ile 3985 GAG TTC ACT GTC CAG G.u, Phe Thr Val Gin CTG GAG LeU Giu 390 GAA AAC AGG CTG TGG ATG TTG CAG TGC GlU Asn Arg Leu Trp Met Leu Gin Cys 395

AGG

Arg 400 ACA GGG AAA CGT ACG GGC AAA Thr Gly Lys Arg Thr Gly Lys AGT GCC Ser Ala 420 415 ATG GTT AAC GAG GGC Met Val Asn Giu Gly 430 GAG CCA GGC CAC CTG Giu Pro Gly His Leu CTT GTT GAG CCC Lou Val Glu Pro 435 GAC CAG CTT CTT Asp Gin Leu Lou

CGC

Arg 4105 410 GTG AAG ATC GCC GTG GAO Val Lys Ile Ala Vai Asp 425 TCA GCG ATO AAG ATG GTA Ser Ala Ile Lys Met Val 440 OCT CAG TTT GAG AAO COG Pro Gin Phe Glu Asn Pro

OAT

His 445 TOG GOG TAO Ser Ala Tyr AAG GAT CAA GTC Lys Asp Gin Val 450 455 OCA GOC TOA OCT Pro Ala Ser Pro 460 GGG GOT Gly Ala GOT GTG GGO CAG Ala Val Gly Gin 480 475 ATT GOC ACT GGT OTG Ile Ala Thr Gly Lou 470 GTG TTO ACT GOT GAA Val Phe Thr Ala Giu 485 GOT ATT CTG GTA AGG, Ala Ile Leu Val Arg 500 CAC GOT GOT GTG GGG His Ala Ala Val Glv GAT GOT GAA GCA Asp Ala Giu Ala 490 1301 1349 1397 1445 1493 1541 1589 1637 1685 TGG CAT TOO CAA GGG AAA GOT Trp His Ser Gin Gly Lys Ala 495 COT GAG GAO GTT GGT GGO ATG Pro Giu Asp Val Gly Gly Met GOG GAG ACC AGO Ala Glu Thr Ser 505 ATT OTT ACA GAG Ile Leu Thr Giu 520 OGT TGG TGG GGG Arg Trp Trp Gly 515 AGG GGT GGC Arg Gly Gly 525 AAA TGC TGC ACT TOO CAC GOT Thr Ser His Ala 530 TOG GGA TGC TCA

GOT

Ala GTG GTO GCA Val Val Ala ATT CGO GTA 535 PAAC GTC GGC GAT GOG GAG Lys Cys Cys Val Ser Gly Cys Ser Gly Ile Arg Val Asn Asp Ala Giu AAG CTC GTG ACG ATC GGA Lys Leu Val Thr Ile Gly 555 560 TCG CTG AAT GGG TCG ACT Ser Leu Asn Gly Ser Thr

AGC

Ser

GGT

Gly CAT GTG CTG CGC His Val Leu Aixg 565 GAG GTG ATC CTT Glu Val Ile Leu 580 GAT CTG GGA ACT Asp Leu Gly Thr 550 GAA GGT GAG TGG CTG Giu Gly Giu Trp Leu 570 GGG AAG CAG CCG CTT Gly Lys Gin Pro Leu 585 TTC ATG GCC TGG GTG Phe Met Ala Trp Val 1733 1781 1829 1877 575 TCC OCA CCA GCC CTT AGT GGT Ser Pro Pro Ala Leu Ser Gly 590 GAT GAT GTT AGA AAG CTC, AAG Asp Asp Val Arg Lys Leu Lys 605 GAT GCA TTG ACT GCG CGA AAC Asn Ala Leu Thr Ala Ara Asn 595 GTC CTG Val Leu GCT AAC GCC GAT ACC CCT GAT Ala Asn Ala Asp Thr Pro Asp 620 CGG ACA Arai Thr GAG CAC Glu His 625 A'IG TTC TTT Met Phe Phe 640 AAT GGG Asn Gly GCT TCA Ala Ser 635

AGG

Arg GCA CAA GGPL Ala Gin Gly 6530 GAC GAG AGG Asp Glu Arg 645 GAG OTG AGG Glu Leu Arg 660 GAO TTO GAA Asp Phe Giu CAG ATG ATT ATG Gin Met Il~e Met 655

GCT

Ala CCC ACG CTT Pro Thr LeU ATT GGA TTA TGC Ile Gly Leu Cys ATT AAG GCT GTC Ile Lys Ala Val 650 CAG CAG GCG CTC Gin Gin Ala LeU 665 GGO ATT TTC CGT Gly Ile Phe Arg 680 CAC CAT CT TCT 1925 1973 2021 2069 2117 GAO CGT CTC TTG Asp Arg Leu Leu 670 GOT ATG GAT GGA ACG TAT GAG AGG Thr Tyr Gin Atg

TOT

Ser 675 OTC COG GTG AGO ATO OGA OTO CTG Met Asp Gly Leu Pro Val Thr Ile Arg Leu LeU Asp His Pro Ser TAC GAG Tyr Giu 700 TGT GCT Cys Ala CTT CCA GAA GGG Leu Pro GiU Giy 705 ATC GAG GAC Ile Giu Asp 695 ATT GTA AGT GAA TTA Ile Val Ser Giu Leu 710 CTC GCG CGA ATT GAA Leu Ala Arg Ile Glu.

730 GAG ACG GGA GCC AAC Giu Thr Gly Ala Asn 720 CAG GAG GAT Gin Giu Isp CTT TCA GAA GTA AAC CCG ATG CTT GGC Leu Set Giu Val Asn Pro Met Leu Giy 735 740 GGT ATA TCG TAG CCT Gly Ile Ser Tyr Pro 750 TTC CGT GGG TGC AGG CTT Phe Arg Gly Cys Arg Leu 745 CAA GCC CGG GCC ATT TTT Gin Ala Arg Ala Ile Phe 760 GAA TTG ACA GAG ATG Giu Leu Thr Giu Met 755 GJAA GCT GCT Giu Ala Ala 765 ATA ATG GTT Ile Met Val ATA GCA ATG ACC Ile Ala Met Thr CCT CTT GTT GGA Pro Leu Val Giy 785 CGC CAA GTT GCT Arg Gin Vai Ala 780 ACT CTT Thr Leu 795 AAC CAG GGT GTT CAA GTG TTC OCA GAG Asn Gin Gly Val. Gin Vai Phe Pro Giu 770 775 ACA CCA GAG GAA CTG GGG CAT CPA GTG Thr Pro Gin Giu LeU Giy His Gin Val 790 GAG AAA GTG TTC GCC PAT GTG GGC AAG Giu Lys Vai Phe Ala Asn Vai Gly Lys 805 81,0 AGA ATG ATT GAG ATC CCC, AGG GCA GCT Thr Met Ile Giu Ile Pro Axg Ala Ala 2165 2213 2261 2309 2357 2405 2453 2501 2549

ATC

Ile 800 ACT ATG, GGG TAG, AA Thr Ile Giy Tyr Lys 815 CTG GTG GCT GAT GAG ,b~eu Val. Ala Asp Glu

GTT

Val

GGA

Gly 825 GPA TTC TTC TGC TTC GGA Giu Phe Phe Ser Phe Gly ATA GCG GAG Ile Ala GiU GAG GGT Gln Ala ACG AAC Thr Asn

GAO

Asp 845

TTC

Phe 830 CTG ACG CAG Leu Thr Gin ATG ACC Met Thr 850 835 TTT GGG Phe Gly GCT CAG Ala Gin 840 TAC AGC AGG GAT GAT GTG Tyr Ser Arg Asp Asp Val 855 GGC ATC, CTC CAA CAT GAC Gly Ile Leu Gin His Asp GGA AAG Gly Lys 860 CCC TTC Pro Phe 875 GCT ACA Ala Thr ATT CCC GTT CAT Ile Pro Vai His 865

CTT

teu 870 GAG GTC- CTG Giu Val Leu GvAG AGG GGC Giu Arg Gly

GAC

Asp- 880

CAG

Gin AGG GGA GTG GGO Arg Gly Val Giy 885 GAG CTG GTG AAG TTT Giu Leu Val Lys iPhe 890 TTG )AAG GTG GGC ATT Leu Lys Val Giy Ile 905 2597 2645 2693 2741 27 89 2837 895

GGT

Gly CGC, AAA GOT AGG Arg Lys Ala Arg GGA GAG CCT TOG Gly Glu Pro Ser 915

OCT

Pro 900

AAO

Asn TGT GGA GAA Cys Gly Glu GCT GGG OTG Ala Gly Leu

CAC

His 910 925 AGG OTA GOT Arg LeU Ala 940 GAT TTC GTT 'iCT TGO TOO Asp Phe Vai Ser Cys Ser 930 GCA GOT CAG GTG OTT GTO Ala Ala Gin 'Val Leu Val 945 TOT GTG GOC TTO, TTO GCG AAG Ser Val Ala Phe Phe Ala Lyu 920 COT TTO AGG GTT COG ATT GOT Pro Phe Arg Val Pro Ile Ala 935 TGAGGOTGOO TOOTOG 2880 SEQ ID NO. 3 SEQUENCE LENGTH: 2610 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GAATTCTCAA TCCTTTGCTC ATCGCAGCAT ATCAATGTTA ACACATAAAC TT9'.GGAGGA AGAAAACTT ATG GCA A.AA TGG GTT TAT AAG TTC GAA GAA GGC AAT r.TCT Met Ala Lys Trp Val Tyr Lys Phe Giu Glu Gly Asn Ala Set ATG AGA AAC CTT CTT GGA GGC Met Arg Asn Leu Leu Gly Gly 20 ATC TTA GGA ATG COG ATT CCA Ile Leu Gly Met Pro Ile Pro TGT ACA GAG TAC TAO AAC AGT Cys Thr Giu Tgyr Tyr Asn Set AAA GGC TGC AAC Lys Gly Cys Asn 25 CTT GOA GAG ATG ACC Leu Ala Glu Met Thr CAG GGC TTT ACT GTA ACA, ACA GAA GCT Gin Gly Phe Thr Val Thr Thr Glu Ala 40 GGA AAA CAG ATC ACA CAG GAA ATT CAG Glv Lx's Gin Ile Thr G~n Glu Ile Gin GAT CAG Asp Gin 55 AAG TTC Lys Phe GCC CGC Ala Arg TTA AAO Leu Asn ATT TTC GAA GOT ATC ACA TGG TTA GAG GAA CTG AAC GGC AAG Ile Phe Giu Ala Ile Thr Trp, Leu Giu Giu Leu Asn Gly Lys 70 GGC GAC ACT GAA GAT CCG TTA TTA GTA TCT GTA CGT TCC GCG Gly Asp Thr Giu Asp Pro lieu Lou Val Ser Vai Arg Ser Ala 85 GOA TCC ATG CCG GGT ATG ATG GAT ACC ATC CTG AAC CTT GGT Ala Ser Met Pro Gly Met Met Asp Thr Ile Leu Asn Leu Gly 100 105 110 GAC GTT GCA GTA GAG GGC TTT GCA AAG AAA ACG GGA AAT CCA Asp Val Ala Vai Giu Gly Phe Ala Lys Lys Thr Gly Asn Pro 115 AGA TTT GCA TAT GAT Arg Phe Ala Tyr Asp 120 TCT TAC AGA AGA ATC CAG Set Tyr Atg Arg Phe Ile Gt 125 ATG TAT TC GAC Met Tyr Ser Asp GTA GTT ATS Val Val Met ATG AAA Met Lys 160 GAT TTA Asp Leu 145

GAA

GlU

AAA

Lys 130 GAA GTT CCG AAG TCC Giu Val Pro Lys Ser 150 GAA AAG GGC GTT CAC Glu Lys Gly Val His 165 GAG CTG GCT GAG AAG Glu Leu Ala Giu Lys 135 140 CAT TTC GAG AAA ATC ATC GAT GCG His Phe Giu lys Ie lie Asp Ala 155 TTC GAT Phe Asp ACA GAC Thr Asp 170 CTG ACT GCC GAT Leu Thr Ala Asp 175 180 TTC AAA GCT GTT Phe Lys Ala Val 185 TAC AAA Tyr Lys GAG GCT Giu Ala 190 ATG AAC GGC GAA GAG Met Asn Gly Giu GiU 195 GCT GTT AAA GCA GTT Ala Val Lys Ala Val 210 TAC CGC CGT ATG AAC Tyr Arg Arg Met Asn 225 GTT CAG ACC ATG GTA Val Gin Thr Met Val TTC CCA CAG GAG CCG Phe Pro Gin Glu Pro 200 TTC CGT TCC TGG GAC Phe Arg Set Trp Asp 215 GAT ATC CCT GGA GAC Asp Ile ?to Gly Asp 230 AAG GAT Lys Asp CAG TTA ATG GGC Gin Leu Met Gly 205 AAC CCT CGT GCA ATC GTA Asn Pro Arg Ala Ile Val 220 591 639 687 735 783 831 879 927 TGG GGT Trp Gly TTT GGT Phe Gly 240 GTT GCC TTC Val Ala Phe 245 ACA CGT AAC CCA Thr Arg Asn Pro 260 ATC AAT GCA CAG AAC AAG GGC GAG ACC Asn Lys Gly Glu Thr 250 TCC ACA GGT GAA AAA Ser Thr Gly Giu Lys ACT GCA GTT AAC Thr Ala Vai Asn 235 AGO GGT ACA GGC Ser Gly Thr Gly GGC ATC TAC GGT Gly Ile Tyr Gly 270 GCA GGT GTC CGC 255

GAG

265 GTA TAO CTG GGC OAG GAO GTT Glu Tyr laeu Ile Asn Ala Gin Gly Olu Asp Val Val Ala Gly Val Arg

I

275 280 285 ACA CCA GAG CGT ATC ACC GAG TTA GAG AAG GAT ATG GCT GAC TGC TAG Thr Pro Gin Pro Ile Thr Gin Leu Giu Asn Asp Met Pro Asp Cys Tyr 290 295 300 AAG GAG TTG ATG GAT CTG GCZ ATG AAG CG GAG AAA CAT TTC GGT GAC Lys Gin Phe Met Asp Leu Ala Met Lys Leu Glu Lys His Phe Arg Asp 975 1023 ATG GAG Met Gin 320 GAG ACA Gin Thr 3059

GAT

Asp ATG GAG Met GiU TTC ACA Phe Thr 325 AAG AGA Lys Arg 31i5 GAG GAA GGT AAA TTA TAG TTG TTA Glu Giu Gly Lys Leu Tyr Phe Leu 1071 GGT AAC Arg Asn

GGG

Gly ACA GCT CCG GGT Thr Ala Pro Ala 335

TGC

Gys 340 GAT TTA GTA GAG Asp Leu Val Asp 355

GAA

GiU 345 GGG ATG ATG ACA GAG Giy Met Ile Thr~ Glu 360 GCT GTT GAG ATT GCG Ala LeU Gin Ile Ala 350 GAA GAG GOT GTT GTA GiU Giu Ala Val Val 365 GAG CCG AGO TTC PAGC His Pro Thr Phe Asn AGA ATO GAP. GGA Arg Ile Glu Ala 370 COG GOT GGT TTA Pro Ala Ala Leu 385 TGT GGT GGG GGA Ser Pro Gly Ala AAA TOT CTT GAT GAG TTA OTT Lys Ser Leu Asp Gin Leu Leu 375 AAG GCC GGC GAA GTA ATG GGT Lys Ala Gly Giu Val Ile Gly 390 GCA GOA GGT AAA GTA TAG TTC Ala Ala Gly Lys Val. Tyr Phe 380 TOC GOT OTT Ser Ala Leu 395 AGO GGT GAT Thr Ala Asp 1119 1167 1215 1263 1311 1359 COG GCA Pro Ala GAG GOT Glu Ala 400 AAG GOT GOG GAG GAG AAC GAG AGA GTT ATG GTT OGT OTT GAG Lys Ala Ala His GlU Lys Gly GlU Arg Val Ile Leu Val Arg Leu Giu 415

ACA

Thr 425 430 TCT CCG GAA GAT Ser Pro Giu Asp 435 ACA GTG CGC GGC Thr Val. Arg Gly 450 ATG GGA ACA TGC Met Gly Thr Cys

GGT

Gly ATC GAA GGT ATG CAT GCA GCC GAA GGT ATC CTG Ile Glu G2ly Met His Ala Ala Giu Gly Ile Leu 440 445 ATG ACA AGC CAT GCA GCC GTA GTT GCA CGT GGT Met Thr Ser His Ala Ala Val Val Ala Arg Gly 455 460 1407 1455

TGC

Cys GTA TCC GGA Val Ser Gly 465

AAG

Lys GAA GCT Giu Ala 480 470 ACA TTC GAA CTT GGC Thr ?he Glu Leu Gly 485 TTA GAT GGT TCC ACA Leu Asp Gly Ser Thr

TAC

Tyr 495

GAG

GlU ATC TCC Ile Ser TGC GGT GAG ATC AAG ATC AAC GAA Cys Gly G1U Ile Ly's Ile Asn Glu 475 GGA CAC ACA TTT GCA GAG GGA GAT Gly His Thr Phe Ala Glu Gly Asp 490 GGT AAG ATT TAC AAG GGC GAC ATC Gly Lys Ile Tyr Lys Gly Asp le 505 510 GGA AGC TTC GAG CGT ATC ATG GTA Gly Ser Phe Giu Arg Ile Met Val.

520 525 PAG GTT CGT ACA AAT GCC GAC ACA L~ys Val Arg Thr Asn Ala Asp Thr ACT CAG GAA CGT Thr Gin Glu Arg 515 500 TCC GTA AGO Ser Val Ser AGA ACA TTA Arg Thr Leu PAT GCC GTT Asn Ala Val.

1503 1551 1599 1647 1695 1743 1791 TGG GCT GAC AAG Trp Ala Asp Lys 530 CCG GAA GAT ACA Pro GiU Asp Thr 545 CTT TGC CGT ACA Leu Cys Arg Thr

TTC

Phe 535

AA

Lhys 540 GCA GAG GGC ATC GGT Ala Giu Giy Ile Gly

CTC

ILeu CTG GGT LeU Gly GAG CAT ATG TTC Glu His Met Phe TTC GAG GCT Phe G1U Ala 555 GAO AGA ATC Asp Arg Ile ATG AAG Met Lys 560 ATC AGA AAG ATG ATC OTT Ile Arg Lys Met Ile Leu 565 TCC GAT Ser Asp 570 TCA GTG GAA GCA AGA GAA GAG GCT Ser Val Giu Ala Arg Giu Glu Ala 1839 AAC GAA TTA ATC Asn Giu Le Ile 595 580

CCG

Pro 585 TTC CAG AAG GGC GAT Phe Gin Lys Giy Asp 600 AAA GC-T CTG GAA Ti r 3XI 4 Leu Giu 610 CTG CAT GAG TTC Leu His Glu Phe 625 AAG PAC ATG GGC Lys Asn Met Giy GGC AGG CCA ATG ACG Giy Arg Pro Met Thr 615 GTT CCT CAT ACA GAA Val Pro His Thr Giu 630 CTT ACT TTA GCA GAA Leu Thr Leu Ala Giu 645 AAC CCA ATG ATG GGC Asn Pro Met Met Gly GTT CGC Vai Arg GAG GAG Giu Giu TTC AAG GCT ATG TAC Phe Ly's Ala Met Tyr 605 TAC. CTG GAT COG CCG Tyr Leu Asp Pro Pro 620 CAG GCT GAA CTG GCT Gin Ala Giu Leu Ala 635 GTA AAA GCA AAA GTT GAC GAA Val Lys Ala Lys Vai Asp GlU 1887 1935 1983 2031 2079 2127 2175 640 TTA CAC Leu His 650 CAT CGT GGC TGC His Arg Gly Cys GAG TTC Giu Phe 655

GTT

Val 660 TAT CCG GAA ATT GOA.AAG ATG Tyr Pro Giu Ile Ala Lys Met 665 CAG ACA CGT OTT GCA Arg Leu Ala 670 GTT ATG GAA ACC Thr AGA GC Gin Thr Axrg Ala Vai Met Giu 675 680 GGA ATC GAT ATT Gly Ile Asp Ile 685 GTT OCT GAG Val Pro Giu GCT GOT ATC Ala Ala Ile GAA GTG AAG GAA GAG GiU Vai Lys GiU GiU 690

ACA

Thr 695 700 ATO ATG ATT Ile Met Ile COG TTA GTT GGO GAG AAG AAA GAG CTT AAG Pro teu Vai Giy Giu Lys Lys Giu Leu Lys TTO GTT AAG Phe Vai Lys 2223 GAC GTA Asp Val 720 GAT ATG Asp Met

GTT

Val.

710 GTG GAA GTA GCT GAG CAG GTT AAG AAA Val. Giu Val Ala Glu Gin Val. Lys Lys 725 CAG TAC CAC ATC GGT Gin Tyr His Ile Gly ACC ATG ATC Thr Met Ile 735

CTC

Leu GAA ATT GlU Ile 745 GAG TTC Glu Phe GAG AAA GGT TCC Glu Lys Gly Set COT CGT GCA GOT Pro Arg Ala Ala 750 TTC TOO TTC GGT Phe Set Phe Giy 765 CGT GAC GAC GCC Arg Asp Asp Ala ACA GCA GAT GCC ATC GOT Thr Ala Asp Ala Ile Ala 755 GAG GAA GCA GlU Giu Ala 760 ACA AAC GAO TTA Thr Asn Asp Leu 770 GGC AAG, TTC CTG Gly Lys Phe Lea 785 ACA CAG ATG Thr Gin Met ACA TTC Thr Phe 775 GGC TTC TCO Gly Phe Ser 2271 2367 2415 2463 2511 2559 780 GAT TCC TAC TAT AAA Asp Ser Tyr Tyr Lys 790 OCA TTC Pro Phe 800 GCA GTT Ala Val.

GOA AGA CTT GAC CAG Ala Arg Lea~ Asp Gin 805 AAG AAA GGC CGT CAG Lys Lys Gly Arg Gin 820 GAG CAC GGC GAG A'rC Giu His Gly Giu Ile ACA GGC Thr Oly GCA AAA ATT Ala Lys Ile GTT GGC CAG Val Gly Gin 810 CCG GGC CTT Pro Gly Leu TAT GAG TCC GAT Tyr Glu Ser Asp 755 TTA GTA GAG ATG LeU Vai Giu Met AAG TGC GGO ATC Lys Cys Giy Ile ACA CGT Thr Arg 815

TGC

Cys

GGC

Gly CTT OTT CCG Leu Leu Pro 840 TAGAGTTCTG CCAOAAAGTA 2609 835 2610 SEQ ID NO.: 4 SEQUENCE LENGTH: 2722 SEQUENCE TYPE: nucleic acid SEQUENCE DES CR1IPTION GAACTATTTA AGGPATTTGT AAGAATTTAG AGTTCATTCA GATAATA ATG CAA AGA Met Gln Arg 1.

GTA TAT GCT TTC GAA GAT GGT GAT GGA ACC AAC AAG AAA CTC CTT GGA Val Tyr Ala Phe Glu Asp Gly Asp Gly Thr Asn Lys Lys Leu Leu Gly

GGA

Gi y

CCA

Pro AAG GGA GCT GGA CTT TGC ACA Lys Gly Ala Gly Leu Cys Thr 25 CPA GGA TTT GTT ATT ACA ACT Gin Gly Plie Val Ile Thr Thr ATG ACA AAA ATT Met Thr Lys Ile 30 GGA CTT CCA GTT Giy Leu Pro Val

GAA

Glu PAT GGA PAC AAA ATG CCA GPA GGA TTA Asn Gly Asn Lys Met Pro Glu Gly Leu 60 TAT CPA TTA GTT GPA PAG AAA TCA: GGA Tyr Gin Leu Val Glu Lys Lys ser Gly ATG TGT PAAA CAA TTC ATT GCT Met Cys Lys Gin Phe Ile Ala 45 ATG GPA GAA GTT AAA AAA GA Met Giu Giu Val Lys Lys Glu AAA GTC TTT GGA GGA GPA GA Lys Val Phe Gly Gly Glu Glu GGA GCT GCT ATG TOT ATG CCA Gly Ala Ala Met Ser Met Pro 152 200 248 296 344 PAT CCA OTT Asn Pro Leu GGT ATG ATG CTT GTT TCA GTC Leu Val. Set~ Val AGA TCA Arg Ser GAT ACT ATT CTT PAT CTT GGA OTT PAT GAT AAA ACT GTT Gly Met Met Asp Thr Ie Leu Asn LeU Gly Leu Asn Asp Lys Thr Val GCT CTT GCT AAA Val Ala Leu Ala Ly TAC AGA AGA TTT Tyr Arg Arg Phe 135 GAT GAA GTT TAT Avp Glu Val Tyr 150 105 TTA ACC AAC AAT Leu Thr Asn Asn TCC CTC TTC GGA Ser Leu Phe Gly 140 AAG ACT CTT GA Lys Thr Leu Glu GAA AGA TTT GCA TAT GAT TCA Glu Arg Phe Ala Tyr Asp Ser 125 130 AAG ATT GCT CTT AAT GCT TGT Lys Ile Ala Leu Asn Ala Cys 145 AAC AAA AAA GTT GA AAG GGA Asn Lys Lys Val Glu Lys Gly 160 AAT GAT ATG AAA GA CTT GCA Asn Asp Met Lys Glu Leu Ala

GAT

Asp 155 GTT AAA Val Lys 165 CAA GTC Gin Val TTA GAT ACT GAA TTA Leu Asp Thr Glu Leu 170 TTC ATT AAA AAG ACT Phe Ile Lys Lys Thr 185 CCA TAT GCT CA TTA Pro Tvr Ala Gin Leu

GAT

Asp 175

GCT

Ala 180

GTT

Val GA GAA TTC ACT Gl. G1U Phe Thr 190 GAA TTT GCC ATT Glu Phe Ala Ile

AAA

Lys

TGT

Cys CAA CCA TTC CCA Gin Pro Phe Pro 195 440 488 536 584 680 728 776 824

GAT

AsD 200 TGG ATG GGA AAG Trp Met Gly Lys 205 GCT GTT GAT TAC Ala Val Asp Tyr GCT GTA Ala Val GA TTC GlU Phe

TCA

Ser

AGA

Arg AGA AGA Arg Arg 215

CAA

Gln TTC AGA Phe Arg 210 AAG ATT Lys Ile ATG GTT Met Val ACT AGA Thr Arg

ACT

Thr CCA GA Pro GIu GCT GAT GGA ACT Ala Asp Gly Thr 235 230 TAT GGT AAT Tyr Gly Asn GCT GTT TC GTT GTT TCT Ala Val Ser Val Val Ser 240 GCT ACT GGT GTT TGT TTC Ala Thr Gly Val Cys Phe ATG GGT PAT GAT TCA Met Gly Asn Asp Ser 245 GAT CCA Asp Pro 260 GCA CAA Ala Gln GGA ACA GGA GAA Gly Thr Gly Glu 265 GGA GAA GAT GTT Gly Glu Asp Val 280 AAT ATG TTC TTC GGA Asn Met Phe Phe Gly 270 GTT GCT GGT ATT AGA Val Ala Gly Ile Arg 285 GAA TAT CTT AAG AAT GIU Tyr Leu Lys Asn 275 ACA CCA CAA ATT ATT Thr Pro Gin Ile lie 290 TGC TAT GAA CAA OTT Cys Tyr Giu Gin Len 305 TCA AAG Ser Lys ATG GCA Met Ala CTT GAT ATT Leu Asp lie 310 TTT GAA TTC Phe Giu Phe GAA GAT CGA GAT OTT Giu Asp Arg Asp Leu 300 AAG AAA TTA GAA GGA Lys Lys Leu Glu Gly 315 ATT GAA AGA AAG AAA Ile Glu Arg Lys Lys CCA GGT Pro Gly TAT TTC CAT GAA GTA CAA GAO Tyr Phe His Glu Vai Gin Asp 320 CTT TAC ATG CTC CAA ACT AGA Leu Tyr Met Leu Gin Thr Arg

ACT

Thr 872 920 968 1016 1064 1112 1160

I-RO

1256

AAT

Asn 340

GTT

Val 325

GGA

Giy ATG AAT GCA Met Asn Ala

AAG

Lys 330 ACT GOT ACT GTO AGA Thr Ala Thr Val Arg 350 ACA AAA GAA CAP GCC Thr Lys Giu Gin Al a 365 TTA OTT CAT AAG PAT Len Leu His Lys Asn GAA GAA GGA OTT Gin Giu Gly Len 360 ACA GGA GTT GAT ATG Thr Gly Val Asp Met 355 ATT ATG AGA ATT GCA Ile Met At le Ala 370 ATG CA GOT PAT TAT Met Pro Ala Asn Tyr 385 TCA CCA G=A GCT GOT Ser Pro Oly Ala Ala CCA CAA TCA GTT Pro Gin Ser Val 375 GCA GAA GOT OCA Ala Giu Ala Pro GAT CAA Asp G1i 380 TTA GTT AAA GGA OTT Leu Val Lys Gly LeU CCA GCA Pro Ala i~) ~I~LT-v 390 ACA GGA GCT GTT Thr Gly Ala Val 405 GGA AAG AAA GTT Gly Lys Lys Val 420 CAT GGA TTC TTT His Gly Phe Phe ACA TCA CAC GCA Thr Ser His Ala 455 TCA GGA GCT GAA Ser Gly Ala Glu 470 ATT GGA AGC CTT Ile Gly Ser Leu 395 GTT TTT GAT GCC Val Phe Asp Ala 410 CTT CTT CTT AGA Leu Leu Leu Arg 400 GAT GAT GCA GTT GAA CAA GCT AAA Asp Asp Ala Val Glu Gin Ala Lys 415 GAA GAA ACT AAA CCA GAA GAT ATT Giu Giu Thr Lys Pro Giu Asp Ile 425 430 GTT GCT GAA GGT ATT TTA ACC Val Ala Giu Gly Ile Leu Thr TGC AGA GGA GGA AAA Cys Arg Giy Gly Lys 450 GTC GTT GCT Val Val Ala AGA GGT ATG GGT AAA CCA TGT GTT Arg Gly Met Gly Lys Pro Cys Val.

460 465 GGA ATT AAA GTT Gly Ile Lys Val 475 GAT GTT GCT Asp Val Ala GAA GTT Glu Val.

485 CAT GAA GGA GAT ATT His Giu Gly Asp lIle 490 AAG GGA GAA GTT CCA Lys Giy Gu Val Pro AAG AAA ATT GCT AAG Lys Lys Ile Ala Lys 480 TTA ACT ATT GAT GGA Leu Thr Ile Asp Gly 495 TTA GAA GAA CCA CAA Leu GlU GlU Pro Gln 515 TGG GCC AAT GAA ATT Trp Ala Asn Giu lie 1304 1352 1400 1448 1496 1544 1592 1640 1688

TCA

Ser Soo

GTT

Val ACT GGA TGT GTC TAT Thr Giy Cys Val Tyr 505 GGA TCA GGA TAT TTC Gly Ser Gly Tyr Phe 510

AAA

Lys

TTT

GGA ACC ATC TTA Gly Thr Ile Leu 525 GCT GCT GGA GAT 530 GCT AAG AAA AAG ATT GGA OTT CCA TOA GOT Lys Lys Ile Gly Val Phe Ala Ala Gly Asp Leu Pro Ser Ala Ala Lys I- AAA GCC CTT Lys Ala Leu 550 CGT ATG TTC Arg Met Phe 565 CTT TCA AAT Leu Ser Asn 535 GAA TTT Giu Phe 540 GGA GOT GAA GGT Gly Ala Giu Gly 545 ATT GGA CTT TGC AGA Ile Gly Leu Cys Arg ACT GAA Thr Giu 1736 560 AAT GCA GTT Asn Ala Val ACC OTT GAA Thr Leu Giu 585 AAA CAA GAT Lys Gin Asp

GAA

Giu 570 CTT CCA ATT GTT GTC AAG ATG ATT Letu Pro Ile Val Val Lys Met Ile 1784 580

CCA

Pro CTT CAA Leu Gin GAA AGA AAG AAA TAT Giu Arg Lys Lys Tyr 590 TTC ATT GGA TTA TTG Plie Ile Gly Leu Leu 605 OTT CTT GAT OCA OCA LeU Lau~ Asp Pro Pro 600 CTT CCA GTC ACT GTC AGA Leu Pro Val Thr Vai Arg 615 CCA ACT OTT GAA GAG TTA Pro Thr Leu Glu GiU LeU OTT AAT GAA OTT ATG Leu Asn Giu Leu Met 595 AAG ACT ATG AAT GGA Lys Thr Met Asn Giy 610 TTA OAT GA, TTC CTC Leu His Giu Phe Leu 625 GAA ATG AAA OTT TCA Glu Met Lys Leu Ser

ATG

met 620 AGA GAPL ATC TTT Arg Giu Ile Phe 1832 1880 1928 1976 2024 2072 2120 630 GGT AAG ACT GAA Giv Lys Thr GlU 635 GAA AAA GAA GTT GTT Giu Lys Giu Val Val 645 AA~A GAA Lys Giu GGA OTT GCA Gly Leu Ala 650 640 CTT AAG AAA GTT LeU Lys Lys Val AGA GGA ATT AGA Arg Gly Ile Arg 675 ATT AGA GCA TTC 660

OTT

CTT ATG GAA GTT AAT CCA Lev Met GlU Val Asn Pro 665 ACT ACT AAT CCA GAA ATT ATG ATT GGA Met Ile GJly 670 TAT GA. ATG

GGA

CA

Gly Thr Thr Asn Pro GIU Ile Tyr GiU Met Gin Ile Arg Ala Phe TTA GAA GOT Leu Glu Ala GAA ATT ATG Giu Ile Met 710 AGA AAG AAT Arci Lys Asn GAA GTT GiU Val

ATT

Ile ATT CCA MAT GTT ACA Ile Pro Asri Val Thr 715 GTT OTT GAA CCA GTT Val Leu G1U Pro Val 730 GTA COA TTC TCG TAT Val Pro Phe Ser Tyr 685 690 AAG GMA GGA ATT AAO GAT CAT CGA Lys GiU Gly Ile Asn Asp His Arg 700 705 GAA GTT MAT GAA CTT ATT AAC TTA Giu Val Asn Glu Leu Ile Asn Leu 720 OAT GAA GAA GTT GAA AAG AAM TAT His Giu Glu Vai GlU Lys Lys Tyr 725 GGT ATT Giv Ile

AAA

Lys 740

GCA

Ala TTA ACA GOT Lea Thr Ala 745 GGT ACT ATG Gly Thr Met 750 GMA TGT GTT AGA GiU Cys Val Arg 755

GCA.

Ala TTC GGA ACT MAT GAT Phe Gly Thr Ashi Asp 775 GAT TCA GAA AC AMA Asp Ser Glu Ash Lys 790 CCA GOT AAT CCA TTT Pro Ala Ash Pro Phe 805 ATG AGA ATT GOT GTT GAT AAG ATT GOT ACA Asp Lys Ile Ala Thr 765 OTT ACA CAA~ GGA ACA Leu Thr Gin O4y Thr 780 TTO, ATT CCA AMA TAT Phe Ile Pro Lys Tyr 1 95 GJAA ATT CTT GA~T AGA GlU Ile Leu Asp Arg 810 ACT AAM GG3A AGA CA GAA GOT TCA TTC TTO TCA G1U Ala Ser Phe Phe Ser 770 TTC TCA TAO TCA CGT GAA Phe Ser Tyr Ser Arq GiU 785 GTT GMA OTT AAG ATT CT Val GlU Leu Lys Ile Lea 800 OCA GGT GTT GGA GMA GTT Pro Gly Val. Gly Gin Val.

2168 2216 2264 2312 2360 2408 2456 2504 2552 815 ACA AGA CCA GAA TTA OTT Met Arg Ile Ala.Val Thr Ly's G3.Y Arg Gin Thr Arg Pro Gin LeU Leu 825 GGT ATT TGT GGA GAA Gly Ile Cys Gly Glu 840 CAC ATG ATT GGA TTG His Met Ile Giy Leu

TGC

Cys CAC GGA GGA His Giy Giy AAC TAT GTT Asn Tyr Val 860 GCT GCT CAA Ala Ala Gin 875 830 GAA CCA TCA TCA GiU Pro Ser Ser 845 TCA TGT TCT TCA Ser Cys Ser Ser GCC CAA ATT AGA Ala Gin lie Arg 880 835 ATT GAA TGG 2600 Ile Giu Trp 850 TAC AGA ATT 2648 Tyr Arg lie 855 CCA GTT GCT AGA Pro Vai Ala Arg 870 ATT GCT Ile Ala CCA AGA 2696 Pro Arg GAA AAT TAAATTAACT TTTTTGGTTT Glu Asn 885 2722 SEQ ID NO.: SEQUENCE LENGTH: 3180 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION CTGAAATTCC CGTAATCTAT CATCATTTAC ACCACAAATC AGAAATCAAL TATCATTTAC TCCATCTCAC GATCTCCTTT TTTCGCAGGT GAPAGGCGGAC G ATG AGT TCG TTG TTT Met Ser Ser Leu Phe GATTCACATC CTCACCGAAT TGCTATTGCT GATACCTCA 120 GTT GAA GOT ATG COT 171 Val Glu Gly Met Pro 1 5 CTG AAG TCA GCC AAT GAG TOG TGC TTA CCG GCG AGO GTG AAG CAA CGG LeU Lys Set Ala Ash GiU Ser Cys Leu Pro Ala Set Val Lys Gin Arg 20 CGA ACC GGT GAT CTC AGG CGA TTG AAC CAC CAC CGT CAA COG GCG TTT Arg Thr Gly Asp GTC CGG GGG ATT Val Arg Gly Ile TTG CGC ACC GGT Leu Arg Thr Gly Leu Arg Arg Leu TGC CGT CGG AAG Cys Arg Arg Lys His His Arg Gin AGT GGA.

Ser Gly GTT AGC Vai Ser GTG CTT Val. Leu Pro Ala Phe AGA ATA GAG Arg Ile GJlu AAT CCG GTG Asn Pro Val.

GGT TTA ACT Gly Leu Thr CTG CCA CGA GCG Leu Pro Arg Ala

TCT

Ser

GGA

Gly CCT CCG GTA ACG ACG Pro Pro Val Thr Thr 80 AAC AGT GAA GGC AAC Asn Ser Glu Gly Asn ACT AAA AAG AGG GTT Thr Lys Lys Arg Vai 85 AAG GAC ATG AAA TCC Lys Asp Met Lys Ser 100 ATG GCA A(3C ATT GGC Met Ala Ser le Gly TTC ACT TTT GGT AAA Phe Thr Phe Gly Lys TTG TTG GGA GGA AAA Leu Leu Gly Gly Lys 105 CTA TCA GTT CCT OCT Leu Ser Val Pro Pro 120 TAT CAA CAA AAT GGA Tyr Gln Gin Asn Gly GGT GCA AAT OTT Giy Ala Asn Leu 110

GAG

Glu 411 459 507 555 603 651 GGG CTC Gly Leu AAA AAA Lys Lys 140 TAT GTC Tyr Val 155 ACT ATT TCA ACT GAA GCA Thr Ile Ser Thr Giu Ala 125 130 CTG COT CCA GGT TTA TGG Leu Pro Pro Gly Leu Trp 145 CAG AAA~ GAG ATG TOT GOCA Gin Lys GlU Met Set Ala 160 TGT GAG GAA Cys GlU GlU 135 GAT GAG ATT CTG GAA GGC TTA CAG Asp GlU Ile LeU GiU Gly Leu Gin TOT OTC GGT Ser Leu Gly 165 COG TOT AAA GOT Pro ser Lys Ala 170 CTC CTO OTT TOO GTO OGT TOG GGT GOT GOC ATA TOG ATG COT GGT ATG Leu Leu Leu Ser Val Arg Ser Gly Ala Ala Ile Ser Met Pro 175 ATG GAC ACT GTA TTG Met Asp Thr Val Leu Gly Met GAT GGT Asp Gly AAT CTC GGG CTT AAT GAT GAG GTC Asn Asp Glu Val Asn Leu Gly Lcu 195 CGC TTT Arg Phe CTA GCT GCC Leu Ala Ala 205 TTT CTA GAT Phe Leu Asp AGT GGA GCT Ser Giy Ala 210 ATG TTT GGC AAC GTT Met Phe Gly Asn Val GCC TAT GAC TCG TAT AGG AGG Ala Tyr Asp Ser Tyr Arg Arg 215 ATG GGT ATC CCA CAT TCG TTA Met Gly Ile Pro His Ser Leu 230

GTA.

Val 220 TTT GAT Phe Asp 235

GAC

Asp GAA AAG TTA GAG Glu Lys Leu Giu 240 GAT CTC ACT GCT Asp Leu Thr Ala 225

CAG

Gin ATG AAA GCT Met Lys Ala GAA AAA GGG ATT Giu Lys Gly Ile CAT CTC His Leu 250

ACT

Thr 255 GCT GAT CTT AAA Ala Asp Lsu Lys 260 245

GAT

Asp CTT GCT GAG CAA TAC LeU Ala Glu Gin Tyr 265 747 795 843 891 939 987 1035 1083 1131 AAG AAC GTG TAT Lys Asn Val Tyr 270 AAG AAA CAG CTA Lys Lys Gin Leu

GTG

Val GPA GCA AAG GGC G1u Ala Lys Gly 275 TTA GCA GTG AAT Leu Ala Val Asn

GA

Glu AAG TTT CCC ACA GAT CCA Lys Phe Pro Thr Asp Pro 280

GAG

GiU 285 AGC CCA AGG GC Ser Pro Arg Ala 290 AAT AAG TAC AGG AGT Asn Lys Tyr Arg Ser GCG GTT TTT GAT TOT TGG GAO Ala Val Phe Asp Ser Trp Asp 295 ATT AAC CAG ATA ACT GGG TTA Ile Asn Gin Ile Thr Gly Leu 300 GGG ACC GCG GTT AAC 305

ATT

310 CAA T ATG TG G ACAT G CAA TGC ATG GTG GGC AAC ATG GGG Lys Gly Thr Ala 315 AAC ACT TCA GGA Asn Thr Ser Gly Val Ile Gin Cys Met Val 325 TTC ACT AGG Phe Thr Arg Phe

AAC

ARsn ACC GGT GTT CTT Thr Gly Val Leu 335 TAT GGG GAG TTT Tyr Gly Glu Phe Gly Asn Met CCA AGC ACT Pro Ser Thr 345 CAG GGA GAG Gin Gly Glu 360 Gly 330

GGT

Gly

GAT

Asp GAG AAG Glu Lys AAG CTG Lys Leu 350

ATC

Ile GTT GTT GCT Val Val Ala 365

GGG

Gly AGA ACA CCA Arg Thr Pro 370 TAC AGA GAG Tyr Arg Giu 385 340 TTA GTC PAT GCT Leu Val Asn Ala 355 GAA GAT TTG GTG Glu Asp Leu Val CTT GTG GAG AAC Leu Val Glu Psn 390 ACC ATG GAG ACT Thr Met Glu Thr 375 TGT GTG ATT TTA Cys Val Ile Leu TGC ATG CCT GAA GCA Cys Met Pro Giu A.,a 380 GAG AGA CAC TAO APA Glu Arg His Tyr Lys 1179 1227 1275 1323 1371 1419 1467 1515 395 AAC AGA CTT TGG ATG Asn Arg Leu Trp Met 415 GGT GCG GTG AGA ATT Gly Ala Val Arg Tie 430 ACT AGA ACA GCA ATT Thr Arg Thr Ala Ile 445 CTT CAT CCA CAG TTT GAT ATG ATG GAT ATT GAA Asp Met Met Asp Lie Glu 400 405 CTG CAA TGC CGA ACA GGG Leu Gin Cys Arg Thr jly 420 GCA GTA GAT ATG GTG AAC Ala Vai Asp Met Val Asn 435 AAG AGG GTT GAG ACT CAA Lys Arg Val Glu Thr Gin 450 TTC ACA GTT CAA GAA Phe Thr Val Gin Glu 410 AAA CGT ACT GGG AAA ILys Arg Thr Gly Lys 425 GAA GGG CTA ATT GAT Glu Gly Leu Ile Asp 440 CAT CTA GAT CAG CTT His Leu Asp GIn LeU GAG AAT CCG TCT GCT TAC AAA AGC CAT GTG GTA 1563 I I Leu His Pro Gin Phe Giu 460 GCA ACC GGT TTG CCA GCA Ala Thr Gly Leu Pro Ala Asn Pro 465 TCC CCT Ser Pro ser Ala Tyr Lys Ser His Val Val 470 GGG GCA GCC GTG GGG CAG GTT GTG Gly Ala Ala Val Gly Gin Val Val TTC AGC GCA GAG GAT GCT Phe Ser Ala Glu Asp Ala 495 ATC TTG GTA AGG ACT GAA Ile Leu Val Arg Thr Giu 510 GCA GCA GCT GGA ATC TTA Ala Ala Ala Gly Ile Leu 485 GAA ACA TGG CAT GCA G2lu Thr Trp His Ala 500 ACA AGC CCA GAA GAT Thr Ser Pro Glu Asp CAA GGA Gin Gly 490 AAG AGT GCT Lys Ser Ala 1705 GTT GGT GGT ATG CAT Val Gly Gly Met His 520 515 ACC GCT AGA Thr Ala Arg 525 GCA GTG GTG Ala Val Val 530

GGC

Gly GGA GGA AT"G ACA TCA CAT GCA Gly Gly Met Thr Ser His Ala 535 TGT TGT GTT TOT GGT TGT GCT Cys Cys Val. set Gly Cys Ala 1611 1659 1707 1755 1803 1851 1899 1941 540 GAT ATT Asp Ie GCT CGC GGA TGG Ala Arg Gly Ttp 545 GTG P.AC GAT CAT Val Asn Asp Asp

AA

Lys 550

CGT

Arg 560 ATG AAG GTT TTT Met Lys Vai Phe 565 ACG ATA Thr Ile GGT GAC CGT Gly Asp Arg 570 ATT AAA GAA GGT Ile Lys Glu Gly 515 ATA TTG GGT AAA Ile Leu Gly Lys GAC TGG CTT TCA CTT Asp Trp Leu Ser Leu 580 CAG CTA CTG GCT OCA Gin Leu Leu Ala Pro 595 AAT GGT TCA ACT GGC GAT4 Asn Gly Set Thr Gly Giu 585 OCT GCA ATG AGC PAT GAT Pro Ala Met Ser Asn Asp 600

GTC

Val 590 TTA GAA ACA TTC ATG TCA TGG GOT GAT CAA GCA AGG CGT CTC AAG GTT 1 1995 Leu Giu Thr Phe 605 ATG GCA AAT GCA Met Ala Asn Ala 620 GGT GCA CAA GGG Gly Ala Gin Gly 635 TCT GAC GAG AGG Ser Asp Giu Arg CCA GAA CAA AGA Pro Giu Gin Arg 670 TCC GAT TTT GAG Ser Asp Phe Giu 685 ATC CGC CTT CTA Ile Ara Leu Leu Met Ser Trp Ala Asp 610 AAT GAT Asa Asp Gin Ala Arg Arg 615 GCA TTA ACA GCC Ala Leu Thr Ala Leu L~ys Val AGA AAC AAT Arg Asa Asn

GAC

Asp

ATC

Ile

ATC

Ile 655

AAA

Lys ACA CCT Thr Pro 625 GGA CTC Gly Leu 2043 TGT AGA Cys Arg ACT GAA CAT ATG TTT TTC GCT Thr Giu His Met Phe Phe Ala 645 650 AAG ATG ATC ATG GCG GTC ACT Lys Met Ile Met Ala Vai Thr AAA GCT GTA AGA Lys Ala Val Arg GCG GCT CTA GAO Ala Ala Leu Asp 660 665 GGC ATT TTC CGA Giy Ile Phe Arg 690 GAC CCT CCA CTT Asp Pro Pro Leu CTC TTA CTC CCA TAC CAA AGA Leu Leu Leu Pro Tyr Gin Arg 680 ATG GAT GGA CTT CCT GTA ACA Met Asp Gly Leu Pro Val Thr 695 GAG TTT CTA CCC GAA GGT GAT Giu Phe Leu Pro Giu Gly Asp 2091 2139 2187 2235 2283 2331 2379 2427

CAT

His 700 710 OTT ACA GCG GAT ACA Leu Thr Ala Asp Thr CTA GAA CAC Leu Giu His 715 ATA GTG le Val GGC ATG AGO Gly Met Ser

GAT

Asp GAA ATC TAT TOA Glu Ile Tyr Ser 735 GGT TTC OGT GGT AGA ATC GAA AAA Arg Ile Giu Lys GAA GTG AAC OCT ATG Giu Val Asa Pro Met 7145 TAO 000 GAG OTA ACA O A1

TT

TO OGA TTA GGG ATT TCA Leu Gly Phe Arg 750 Gly Cys Arg GAA ATG CPA GTT Giu Met Gin Val 765 CAG, GGG GTG ACT Gin Gly Val Thr CGT GCG ATC Arg Ala Ile Leu Gly Ile 755 TTT CAA GCT Phe Gin Ala 770 GCA GTG TCT ATG AAC AAT Ala Val Ser Met Asn Asn 775 GTT CCG TTA GTC GGA ACA Val Pro Leu Val Gly Thr GTA ATA Val Ile Ser Tyr Pro Giu Lau Thr 760 GAG ATC Giu Ile

ATG

Met 780 CCT CAG GAA Pro Gin Glu 790 TTA CGG CAT Leu Arg His 800 CAA ATC GGC GTA ATT Gin Ile Giy Vai Ile 805 GGG CTG ACG TTG GAG Gly Leu Thr Leu Giu 820

CGT

Arg GGT GTA GCT GCA Gly Vai Ala Ala 810 GTT TTT GCT GAA Val Phe Ala GiU 815

ATG

Met TAT AAA GTG GGA ACG Tyr Lys Val Giy Thr 825 2475 2523 2571 2619 2667 2715 2763 2811 ATG ATT GAG ATT Met Ile Giu le 830 GAA GCC GAG TTC Giu Ala GiU Phe 845 TTT GGG TAC AGC Phe Gly Tyr Ser CCT CGA GCT GCT TTG Pro Arg Ala. Ala Leu 835 TTT TCG TTT GGA ACC Phe Ser Phe Giy Thr 850 AGA GAT GAT GTT GGC Ara Aso Aso Val Giv ATT GCT GAT GAG ATT GCA AAA Ile Ala Asp Giu Ile Ala Lys 840 AAT GAT TTG ACC CAG ATG ACA Asn Asp Leu Thr Gin Met Thr AAG TTT TTG Lys Phe Leu 860 TOT CAA Ser Gin 875 865 GGC ATT CTG CAG CAT Gly Ile Lau Gin His 880 870 GAT CCA TTT GAG GTT Asp Pro Phe Giu Val 885 855 CCG ATT TAT CTT Pro Ile Tyr Lau OTT GAC CAG AAA Leu Asp Gin Lys 890 GGG GTG GGT CAA TTG ATO AAG, ATG GCC ACG GAG AAA GGT CGT GCA GCC~5 2859 Gly Val Gly Gin Leu 895 AAT CCT AAC TTA AAG Asn Pro Asn Leu Lys 910 TCT TCT GTT GCA TTT Ser Ser Vai Ala Phe 925 TCT CCA TTC AGG GTT Ser Pro Phe Arg Val 940 Ile Lys Met Ala Thr Glu Lys Gly Arg Ala Ala 900 905 GTT GGG ATA TGT GGG GAG CAT GGT GGA GAA CCT Val Gly Ile Cys Gly Giu His Gly Gly Giu Pro 915 920 TTT GAC GGA GTT GGA CTA GAT TAT GTG TCG TGC Phe Asp Giy Val Gly Leu Asp Tyr Val Ser Cys 930 935 CCT ATC GC1A AGG, TTG GCC GCT GCA CA.A GTC GTT Pro Ile Ala Arg Leu Ala Ala Ala Gin Val Val 2907 2955 3003 GTT TAAGCTTTGA AAGGAGGATG GCTTATTTGC TTCATGTTTT CCGCCATTGT 3056 Val 955 ATATTATTTT GGTTTCATCC TTATTGTAAT GGTGAAAATG AACGATGTTT AAACAAAACA 3116 ACCCATTATA TTTTGGTTTG GTATGCAPLTA ATCTACTTTT CAAACAAAAA AAAAAAAAAA 3176 AAAA 3180 SEQ ID NO.: 6 SEQUENCE LENGTH: 18 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GACGGCTAAA AAGAGGGT 18 SEQ ID NO.: 7 SEQUENCE LENGTH: SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION TATCGAGAAA CCTTCTATAC SEQ ID NO.: 8 SEQUENCE LENGTH: 17 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GTTTTCCCAG TCACGAC 17 SEQ ID NO.: 9 SEQUENCE LENGTH: 17 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION CAGGAAACAG CTATGAC 17 SEQ ID NO.: SEQUENCE LENGTH: 23 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GATATCAATC CGGTGTCTCC TCC 23 SEQ ID NO.: 11 SEQUENCE LENGTH: 134 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION CGGTGTCTCC TCCGGATATC ACGGCTAAAA AGAG 74 SEQ ID NO.: 12 SEQUENCE LENGTH: 27 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION TTGATATCCC GGTTGTCTCC TCCGGTA 27 SEQ ID NO.: 13 SEQUENCE LENGTH: SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GCAGAGATGA TGTTGGCAAG SEQ ID NO.: 14 SEQUENCE LENGTH: SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION CTTGCCAACA TCATCTCTGC SEQ ID NO.: SEQUENCE LENGTH: SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION CTCACTGTTC GAAGAGAAGC SEQ ID NO.: 16 SEQUENCE LENGTH: 23 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION CATATGCTCT GTCCGGCATA ATC 23 SEQ ID NO.: 17 SEQUENCE LENGTH: 21 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION CTCGAGGGAT CTCAATCATT G 21 SEQ ID NO.: 18 SEQUENCE LENGTH: 18 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GCAATCTCTT CAGCAATC 18 SEQ ID NO.: 19 SEQUENCE LENGTH: SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GCTTCTTTTC CAATCTCATC SEQ ID NO.: SEQUENCE LENGTH: 18 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION CGAAAAGAAA TCGGCTTC 18 76 SEQ ID NO.: 21 SEQUENCE LENGTH: 24 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GAAAGATAAA TCTGCAAAAA CTTG 24 SEQ ID NO.: 22 SEQUENCE LENGTH: 19 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GCCTTGAGCA AGATAAATC 19 SEQ ID NO.: 23 SEQUENCE LENGTH: 21 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION TTCTGGTCAA TAACCTCAAT G 21 SEQ ID NO.: 24 SEQUENCE LENGTH: SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GCTTAAACAA TGACTTGTGC SEQ ID NO.: SEQUENCE LENGTH: 22 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION CCAATCTCAT CAGCTATTAA AG 22 SEQ ID NO.: 26 SEQUENCE LENGTH: SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GCTTCTTTTG CAATCTCTTC SEQ ID NO.: 27 SEQUENCE LENGTH: 18 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION CGAAAAGAAC TCAGCTTC 18 SEQ ID NO.: 28 SEQUENCE LENGTH: 23 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION CAAGATAAAT CGGCAAAAAC TTG 23 SEQ ID NO.: 29 SEQUENCE LENGTH: 23 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GAATGCCTTG AGAAAGATAA ATC 23 ~--_1111~111111 1 C SEQ ID NO.: SEQUENCE LENGTH: 24 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION CTTTCTGGTC AAGAACCTCA AATG 24 SEQ ID NO.: 31 SEQUENCE LENGTH: SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION GCTTAAACAA CGACTTGTGC

Claims

1. An isolated or recombinant polypeptide having cold-stable pyruvate orthophosphate dikinase activity wherein said polypeptide comprises an amino acid sequence which is at least about 50% similar to SEQ ID NO:5 or a homologue, analogue or derivative of said isolated or recombinant polypeptide having cold-stable pyruvate orthophosphate dikinase activity.

2. An isolated or recombinant polypeptide having cold-stable pyruvate orthophosphate dikinase activity wherein said polypeptide comprises an amino acid sequence which is at least about 50% similar to any one of SEQ ID NOS: 1-5, subject to the proviso that said amino acid sequence further includes: the amino acid sequence from position 832 to position 955 of SEQ ID NO: or a homologue, analogue or derivative thereof capable of conferring cold-stability on a pyruvate orthophosphate dikinase polypeptide; or (ii) one or more amino acid residues of the Flaveria brownii pyruvate orthophosphate dikinase polypeptide as set forth in Table 1; or (iii) one or more amino acid residues of SEQ ID NO:5 selected from the list comprising 838Asp, 841Ala, 845Glu, 871Pro, 875Ser, 887Leu and 954Val; wherein the amino acid sequence or amino acid residue defined by sub-paragraphs or (ii) or (iii) are further located at a position in said polypeptide such as to confer cold-stability on said isolated or recombinant polypeptide.

3. The isolated or recombinant polypeptide of claim 1 or 2, wherein said polypeptide S. comprises an amino acid sequence which is at least 50% similar to SEQ ID NO:5 and at least includes the amino acid sequence from position 832 to position 955 of SEQ ID NO:

4. The isolated or recombinant polypeptide according to any one of claims 1 to 3, further comprising one or more amino acid residues of SEQ ID NO:5 selected from the list comprising 838Asp, 841Ala, 845Glu, 871Pro, S75Ser, 887Leu and 954Val located at a position in said polypeptide such as to confer cold-stability on said isolated or recombinant I I I i~i r Ut clr4i4( I c N INO polypeptide. The isolated or recombinant polypeptide according to any one of claims 1 to 4, comprising the amino acid sequence set forth in SEQ ID

6. The isolated or recombinant polypeptide according to any one of claims 1 to comprising a homologue, analogue or derivative of the amino acid sequence set forth in SEQ ID NO:2 having cold-stable pyruvate orthophosphate dikinase activity.

7. The isolated or recombinant polypeptide of claim 6, at least comprising a proline residue located at a position in said polypeptide such as to confer cold-stability on said isolated or recombinant polypeptide.

8. The isolated or recombinant polypeptide according to claim 7, wherein the proline residue is located at position 869 in said polypeptide.

9. The isolated or recombinant polypeptide according to any one of claims 6 to 8, further comprising a leucine residue located at a position in said polypeptide such as to confer cold- stability on said isolated or recombinant polypeptide. e The isolated or recombinant polypeptide according to claim 9, wherein the leucine So residue is located at position 885 in said polypeptide.

11. The isolated or recombinant polypeptide according to any one of claims 6 to further comprising a valine residue located at a position in said polypeptide such as to confer cold-stability on said isolated or recombinant polypeptide.

12. The isolated or recombinant polypeptide according to claim 11, wherein the valine residue is located at position 952 in said polypeptide. ppl-~F- -sQR- Ilr0flilMR0\ 12W .CLM 1913/98 -81-

13. An isolated nucleic acid molecule which comprises a nucleotide sequence which is capable of encoding the isolated or recombinant polypeptide according to any one of claims 1 to 12 or a complementary nucleotide sequence thereto or a homologue, analogue or derivative thereof which is capable of encoding a polypeptide having cold-stable pyruvate orthophosphate dikinase activity.

14. The isolated nucleic acid molecule according to claim 13 comprising DNA. The isolated nucleic acid molecule according to claims 13 or 14, comprising the nucleotide sequence set forth in SEQ ID NO:5 or a complementary nucleotide sequence thereto or a homologue, analogue or derivative thereo' wi uch is capable of encoding a polypeptide having cold-stable pyruvate orthophosphate dikinase activity.

16. An isolated nucleic acid molecule comprising a nucleotide sequence which is capable of encoding a polypeptide comprising an amino acid sequence which is at least about 96% identical to the amino acid sequence set forth in SEQ ID

17. The isolated nucleic acid molecule according to claim 16, comprising the nucleotide sequence set forti in SEQ ID

18. A vector which comprises the isolated nucleic acid molecule according to any one of claims 13 to 17. oo

19. The vector according to claim 18, wherein said vector is capable of expressing a recombinant polypeptide having cold-stable pyruvate orthophosphate dikinase activity when S introduced into a cell. The vector according to claim 19 wherein the cell is a bacterial cell.

21. The vector according to claim 20, wherein the bacterial cell is an Escherichia coli cell. n r~ g C- ~p1 u l r f I I 82

22. The vector according to claim 19 wherein the cell is a plant cell.

23. The vector according to claim 22 wherein the plant cell is derived from maize or Flaveria bidentis.

24. A cell which has been transformed with the isolated nucleic acid molecule according to any one of claims 13 to 17 or the vector according to any one of claims 18 to 23. The cell according to claim 24, further defined as a bacterial cell.

26. The cell of claim 25, further defined as an Escherichia coli cell.

27. The cell according to claim 24, derived from a plant.

28. The cell according to claim 27 wherein the plant is maize or Flaveria bidentis.

29. A plant which has been transformed with the isolated nucleic acid molecule according to any one of claims 13 to 17 or the vector according to any one of claims 18 to 23. r r o r a a a plant. The plant according to claim 29, further defined as a maize plant or Flaveria bidentis

31. The isolated or recombinant polypeptide according to any one of claims 1 to 12 substantially as hereinbefore described with reference to the Figures and/or Examples.

32. The isolated nucleic acid molecule according to any one of claims 13 to 17 or the vector according to any one of claims 18 to 23 substantially as hereinbefore described with reference to the Figures and/or Examples.

33. The cell according to any one of claims 24 to 28 substantially as hereinbefore urrs*~uli~aaa~drr -u ~a~r~l yyP n~~~r~AULWk*eY14WldDr~,~*~~ uhunanmnm Il'o)rllMItOil 120l.9ill OWlr -83- described with reference to the Figures and/or Examples.

34. The plant according to any one of claims 29 to 30 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this 30TH day of July, 1998 Japan Tobacco Inc. by DAVIES COLLISON CAVE Patent Attorneys for the Applicants sees e e S* Se ,~c~smranaPcnR1*?rrrans~.Rncl~amr~~~~nn ;ri~;~~kau~.lnmnl Rl)~rr nIF-r rncr~n i illYi ABSTRACT As means for giving cold-stability to plants, a novel polypeptide having cold-stable pyruvate, orthophosphate dikinase activity, a cloned DNA encoding the same, and a recombinant vector containing the DNA are disclosed. The polypeptide according to the present invention has cold-stable pyruvate, orthophosphate dikinase activity, which has an amino acid sequence that is the same as the amino acid sequence of 1/6 region of the entire region from the C-terminal of the following polypeptide or except that at least one amino acid residues of said 1/6 region are substituted with other amino acid residues: a pyruvate, orthophosphate dikinase having an amino acid sequence shown in SEQ ID NO. 1 to 4 in Sequence Listing; and a polypeptide having an amino acid sequence which has a homology of not less than 50% with the amino acid sequence mentioned in said polypeptide having cold- stable pyruvate, orthophosphate dikinase activity. I