Abstract:
The present invention relates to a novel carboxypeptidase gene and the polypeptide encoded thereby. In particular, the present invention relates to the use of the present carboxypeptidase and polypeptide in the manufacture of cocoa flavor and/or chocolate.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application is a continuation-in-part of the US national phase designation of International application PCT/EP02/07162 filed Jun. 28, 2002, the content of which is expressly incorporated herein by reference thereto. 
     
    
     
       BACKGROUND ART  
         [0002]    The present invention relates to a novel carboxypeptidase gene and the polypeptide encoded thereby. In particular, the present invention relates to the use of the present carboxypeptidase in the manufacture of cocoa flavor and/or chocolate.  
           [0003]    It is known that in processing cacao beans the generation of the typical cocoa flavor requires two steps—a fermentation step, which includes air-drying of the fermented material, and a roasting step.  
           [0004]    During fermentation two major activities may be observed. First, the pulp surrounding the beans is degraded by micro-organisms with the sugars contained in the pulp being largely transformed to acids, especially acetic acid (Quesnel et al., J. Sci. Food. Agric. 16 (1965), 441-447; Ostovar and Keeney, J. Food. Sci. 39 (1973), 611-617). The acids then slowly diffuse into the beans and eventually cause an acidification of the cellular material. Second, fermentation also results in a release of peptides exhibiting differing sizes and a generation of a high level of hydrophobic free amino acids. This latter finding led to the hypothesis that proteolysis occurring during the fermentation step is not due to a random protein hydrolysis but seems to be rather based on the activity of specific endoproteinases (Kirchhoff et al., Food Chem 31 (1989), 295-311). This specific mixture of peptides and hydrophobic amino acids is deemed to represent cocoa-specific flavor precursors.  
           [0005]    Until now several proteolytic enzyme activities have been investigated in cacao beans and studied for their putative role in the generation of cocoa flavor precursors during fermentation.  
           [0006]    An aspartic endoproteinase activity, which is optimal at a very low pH (pH 3.5) and inhibited by pepstatin A has been identified. A polypeptide described to have this activity has been isolated and is described to consist of two peptides (29 and 13 kDa) which are deemed to be derived by self-digestion from a 42 kDa pro-peptide (Voigt et al., J. Plant Physiol. 145 (1995), 299-307). The enzyme cleaves protein substrates between hydrophobic amino acid residues to produce oligopeptides with hydrophobic amino acid residues at the ends (Voigt et al., Food Chem. 49 (1994), 173-180). The enzyme accumulates with the vicilin-class (7S) globulin during bean ripening. Its activity remains constant during the first days of germination and does not decrease before the onset of globulin degradation (Voigt et al., J. Plant Physiol. 145 (1995), 299-307).  
           [0007]    Also a cysteine endoproteinase activity had been isolated which is optimal at a pH of about 5. This enzymatic activity is believed not to split native storage proteins in ungerminated seeds. Cysteine endoproteinase activity increases during the germination process when degradation of globular storage protein occurs. To date, no significant role for this enzyme in the generation of cocoa flavor has been reported (Biehl et al., Cocoa Research Conference, Salvador, Bahia, Brasil, Nov. 17-23, 1996).  
           [0008]    Moreover, a carboxypeptidase activity has been identified which is inhibited by PMSF and thus belongs to the class of serine proteases. It is stable over a broad pH range with a maximum activity at pH 5.8. This enzyme does not degrade native proteins but preferentially splits hydrophobic amino acids from the carboxy-terminus of peptides (Bytof et al., Food Chem. 54 (1995), 15-21).  
           [0009]    During the second step of cocoa flavor production—the roasting step—the oligopeptides and amino acids generated at the stage of fermentation are obviously subjected to a Maillard reaction with reducing sugars present in fermented beans eventually yielding substances responsible for the cocoa flavor as such.  
           [0010]    In the art there have been many attempts to artificially produce cocoa flavor.  
           [0011]    Cocoa-specific aroma has been obtained in experiments wherein acetone dry powder (AcDP) prepared from unfermented ripe cacao beans was subjected to autolysis at a pH of 5.2 followed by roasting in the presence of reducing sugars. It was conceived that under these conditions preferentially free hydrophobic amino acids and hydrophilic peptides should be generated and the peptide pattern thus obtained was found to be similar to that of extracts from fermented cacao beans. An analysis of free amino acids revealed that Leu, Ala, Phe and Val were the predominant amino acids liberated in fermented beans or autolysis (Voigt et al., Food Chem. 49 (1994), 173-180). In contrast to these findings, no cocoa-specific aroma could be detected when AcDP was subjected to autolysis at a pH of as low as 3.5 (optimum pH for the aspartic endoproteinase). Only few free amino acids were found to be released but a large number of hydrophobic peptides were formed. When peptides obtained after the autolysis of AcDP at a pH of 3.5 were treated with carboxypeptidase A from porcine pancreas at pH 7.5, hydrophobic amino acids were preferentially released. The pattern of free amino acids and peptides was similar to that found in fermented cacao beans and to the proteolysis products obtained by autolysis of AcDP at pH 5.2. After roasting of the amino acids and peptides mixture as above, a cocoa aroma could be generated.  
           [0012]    It has also been shown that, a synthetic mixture of free amino acids alone with a similar composition to that of the spectrum found in fermented beans, was incapable of generating cocoa aroma after roasting, indicating that both the peptides and the amino acids are important for this purpose (Voigt et al., Food Chem. 49 (1994), 173-180.  
           [0013]    In view of the above data a hypothetical model for the generation, during fermentation, of the said mixture of peptides and amino acids, i.e. the cocoa flavor precursors, had been devised (FIG. 1), where in a first step peptides having a hydrophobic amino acid at their end, are formed from storage proteins, which peptides are then further degraded to smaller peptides and free amino acids. To produce the said peptides having C-terminal hydrophobic amino acids, an aspartic endoproteinase activity related to that mentioned above seems to be involved. Yet, for splitting off hydrophobic amino acids from peptides formed in the preceding step the only known enzymatic activity, which might be considered in this respect, is that of a carboxypeptidase. However, such enzyme has not been isolated and studied in detail in cacao and it is therefore still questionable, which cacao enzyme might be responsible for the generation of hydrophobic amino acids required for cocoa flavor.  
           [0014]    Though some aspects of cocoa flavor production have been elucidated so far there is still a need in the art to fully understand the processes involved, so that the manufacture of cocoa flavor may eventually be optimized.  
         SUMMARY OF THE INVENTION  
         [0015]    The present invention provides means for further elucidating the processes involved in the formation of cocoa-specific aroma precursors during the fermentation of cacao seeds, to improve the formation of cocoa flavor during processing and manufacturing and eventually providing means assisting in the artificial production of cocoa flavor.  
           [0016]    This problem has been solved by providing a nucleotide sequence encoding a novel carboxypeptidase from cacao beans (termed cacao CP-III), which is identified by SEQ. ID. No. 1, or functional derivatives thereof having a degree of homology that is greater than 80%, preferably greater than 90% and more preferably greater than 95%.  
           [0017]    It will be appreciated by the skilled person that a gene encoding a specific polypeptide may differ from a given sequence according to the Wobble hypothesis, in that nucleotides are exchanged that do not lead to an alteration in the amino acid sequence. Yet, according to the present invention also nucleotide sequences shall be embraced, which exhibit a nucleotide exchange leading to an alteration of the amino acid sequence, such that the functionality of the resulting polypeptide is not essentially disturbed.  
           [0018]    This nucleotide sequence may be used to synthesise a corresponding polypeptide by means of recombinant gene technology, in particular a polypeptide as identified by SEQ. ID. No. 2.  
           [0019]    As has been shown in a comparison with other carboxypeptidases from other plants the present enzyme does not show a substantial homology to any of the carboxypeptidases known so far. Since it is assumed, that cocoa may furthermore contain additional carboxypeptidases that might exhibit a higher homology to the carboxypeptidases known so far it must be considered as a surprising fact that this very enzyme has been detected.  
           [0020]    For producing the polypeptide by recombinant means, the nucleotide of the present invention is included in an expression vector downstream of a suitable promoter and is subsequently incorporated into a suitable cell, which may be cultured to yield the polypeptide of interest. Suitable cells for expressing the present polypeptide include bacterial cells, such as e.g.  E. coli,  or yeast, insect, mammalian or plant cells.  
           [0021]    The present DNA sequence may also be incorporated directly into the genome of the corresponding cell by techniques well known in the art, such as e.g. homologous recombination. Proceeding accordingly will provide a higher stability of the system and may include integration of a number of said DNA-sequences into a cell&#39;s genome.  
           [0022]    The cells thus obtained may in consequence be utilized to produce the polypeptide in batch culture or using continuous procedures, with the resulting polypeptide being isolated according to conventional methods.  
           [0023]    The recombinant carboxypeptidase obtained may be used for the manufacture of cocoa flavor. To this end, the enzyme described herein may be utilized in an artificial trial run, wherein a mixture of different proteins, such as cacao storage proteins, or protein hydrolysates of other resources, are subjected to enzymatic degradation by means of enzymes, known to be involved in proteolytic degradation to eventually assist in the production of flavor precursors. The enzyme may likewise also be utilized in the production of cocoa liquor, and in the manufacture of chocolate.  
           [0024]    Yet, the present invention also provides plants, in particular cacao plants, comprising a recombinant cell, containing one or more additional copies of the carboxypeptidase of the present invention. Such a cacao plant will produce beans, which will exhibit a modified degradation of storage proteins when subjected to the fermentation process, allowing a more rapid degradation or a pattern of hydrolysis that yields a higher level of cocoa flavor precursor, since a higher amount of carboxypeptidase will be present.  
           [0025]    The carboxypeptidase of the present invention may also be used to produce other transgenic plants such as soybean and rice, producing seeds with this new protein modifying enzyme. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWING FIGURES  
       [0026]    In the figures,  
         [0027]    [0027]FIG. 1 shows a scheme illustrating a potential process for the proteolytic formation of cocoa-specific aroma;  
         [0028]    [0028]FIG. 2 shows the cloning strategy used for the isolation of a cDNA encoding a carboxypeptidase from  Theobroma cacao;    
         [0029]    [0029]FIG. 3 shows a comparison of the hydrophilicity Plot-Kyte-Doolittle for the cacao CP-III sequence with Barley CP-MI, CP-MII and CP-MIII;  
         [0030]    [0030]FIG. 4 shows a Northern blot analysis of cacao CP-III. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0031]    As described above, it was suggested that a carboxypeptidase could be involved in the production of cocoa flavor precursors during cacao fermentation. However it was not known in the art which cacao carboxypeptidase carried out this function considering that five classes of carboxypeptidases (Type I-V) have been identified in different plants by references to differences in substrate specificities, molecular weights and chromatographic profiles. Furthermore 50 sequences having homologies with serine carboxypeptidases exist in the completed Arabidopsis genome.  
         [0032]    The following examples illustrate the invention further without limiting it thereto. In the examples the following abbreviations have been used:  
         [0033]    PCR: Polymerase Chain Reaction  
         [0034]    RACE: Rapid Amplification cDNA Ends  
         [0035]    cDNA: complementary deoxyribonucleic acid  
         [0036]    mRNA: messenger ribonucleic acid  
         [0037]    DEPC: Diethyl pyrocarbonate  
         [0038]    3,4-DCI: 3,4-dichloroisocoumarin  
       EXAMPLES  
       [0039]    Materials  
         [0040]    Cacao ( Theobroma cacao L. ) seeds (male parent unknown) from ripe pods of clone ICS 95 were provided by Nestle ex-R&amp;D Center Quito (Ecuador). The seeds were taken from the pods immediately after arrival at Nestle Research Center Tours (4-5 days after harvesting). The pulp and the seed coat were eliminated and the cotyledons were frozen in liquid nitrogen and stored at −80° C. until use.  
         [0041]    Preparation of mRNA  
         [0042]    Total RNA was prepared using the following method. Two seeds were ground in liquid nitrogen to a fine powder and extraction was directly performed with a lysis buffer containing 25 mM Tris HCl pH8, 25 mM EDTA, 75 mM NaCl, 1% SDS and 1M P-mercaptoethanol. RNA was extracted with one volume of phenol/chloroform/isoamylalcohol (25/24/1) and centrifuged at 8000 rpm, 10 min at 4° C. The aqueous phase was extracted a second time with one volume of phenol/chloroform/isoamylalcohol (25/24/1). RNA was precipitated with 2M lithium chloride at 4° C. overnight. The RNA pellet obtained after centrifugation was resuspended in DEPC treated water and a second precipitation with 3M sodium acetate pH 5.2 was performed in presence of two volumes of ethanol. The RNA pellet was washed with 70% ethanol and resuspended in DEPC treated water. Total RNA was further purified using the Rneasy Mini kit from Qiagen®.  
         [0043]    Cloning of a carboxypeptidase cDNA  
         [0044]    Cloning Strategy  
         [0045]    A 1.5 kb 5′ end fragment of a carboxypeptidase from cacao seed was amplified by RT-PCR using a degenerate oligonucleotide. Based on the sequence of this fragment, a primer was designed to amplify a 3′-end fragment. Finally, a full-length cDNA (cacao CP-III) was amplified using primers specific to both extremities (FIG. 2).  
         [0046]    Primer Design  
         [0047]    A search for carboxypeptidase sequences in the GenBank database lead to the identification of several plant sequences. A multiple alignment of these sequences revealed the presence of conserved regions. The conserved sequence MVPMDQP located near the histidine catalytic site has been used to design a degenerate oligonucleotide in the antisense orientation: pCP2r (5′-GGYTGRTCCATNGGNACCAT) (SEQ ID No. 3).  
         [0048]    Synthesis of cDNA  
         [0049]    Total RNA (see above) was used to synthesise first strand 3′ and 5′ cDNAs with the SMART RACE cDNA Amplification Kit (Clontech, USA). Synthesis has been performed exactly as described in the kit instructions using 1 μg of total RNA and the Superscript™ MMLV reverse transcriptase (Gibco BRL, USA). After synthesis, cDNAs were used directly for PCR or kept at −20° C.  
         [0050]    5′ RACE Amplification  
         [0051]    Specific cDNA amplification was performed with 2.5 μl of the first strand 5′ cDNA in 50 μl buffer containing: 40 mM Tricine-KOH pH 8.7, 15 mM KOAc, 3.5 mMMg(OAc) 2 , 3.75 μg/ml BSA, 0.005% Tween-20, 0.005% Noninet-P40, 0.2 mM dNTP&#39;s, 14 pmoles of pCP2r primer, 5 μl of 1OX Universal primer Mix (UPM) and 1 μl 5OX Advantage 2 polymerase Mix (Clontech, USA). Amplification was performed in a Bio-med thermocycler 60 (B. Braun). A first denaturation step (94° C., 2 min) was followed by 35 cycles of denaturation (94° C., 1 min), primer annealing (55° C., 1.5 min) and extension (72° C., 2 min). The extension time was increased by 3 sec at each cycle. Amplification was ended by a final extension step (72° C., 10 min). The amplified fragment was cloned in pGEMO-T vector and sequenced.  
         [0052]    3′ RACE PCR  
         [0053]    The sequence information obtained after the sequencing of the 5′ end fragment was used to design a specific oligonucleotide pCP5 (5′-GCTTTTGCTGCCCGAGTCCACC) (SEQ ID No. 4), which was used for 3′-RACE amplification. 3′-RACE PCR was performed with 2.5 gl of SMART single strand 3′ cDNA in 50 μl buffer containing 40 mM Tricine-KOH pH 8.7, 15 mM KOAc, 3.5 mM Mg(OAc) 2 , 3.75 μg/ml BSA, 0.005% Tween-20, 0.005% Nonidet-P40, 0.2 mM dNTP&#39;s, 10 pmoles of pCP5 primer,  101 t of 1OX Universal primer Mix (UPM) and 1 μl 5OX Advantage 2 polymerase Mix (Clontech, USA). Amplification was performed via touchdown PCR, in a Bio-med thermocycler 60 (B. Braun).  
         [0054]    A first denaturation step (94° C., 1 min) was followed by:  
         [0055]    5 cycles including denaturation at 94° C. for 30 sec and annealing/extension at 72° C. for 3 mm  
         [0056]    5 cycles including denaturation at 94° C. for 30 sec and annealing/extension at 70° C. for 30 sec and 72° C. for 3 min  
         [0057]    30 cycles including denaturation at 94° C. for 30 sec and annealing/extension at 68° C. for 30 sec and 72° C. for 3 min.  
         [0058]    The amplified fragment was cloned in PGEMO-T vector and sequenced.  
         [0059]    Full Length cDNA  
         [0060]    The sequence information obtained after the sequencing of 5′-and 3′-RACE fragments was used to design two specific oligonucleotides.  
         [0061]    pCP8: A sense primer (5′-CAAAGAGAAAAAGAAAAGATGGC) (SEQ ID No. 5)  
         [0062]    pCP7r: A reverse primer (5′-CCCCAGAGCTTTACGATACGG) (SEQ ID No. 6).  
         [0063]    PCR reaction was performed with 2.5 gl first strand cDNA in 50 μl buffer containing: 40 mM Tricine-KOH pH 8.7, 15 mM KOAc, 3.5 mM Mg(OAc) 2 , 3.75 μg/ml BSA, 0.005% Tween-20, 0.005% Noninet-P40, 0.2 mM dNTP&#39;s, 10 pmoles of pCP8 primer, 10 pmoles of pCP7r primer and 1 gl 5OX Advantage 2 polymerase Mix (Clontech, USA). Amplification was performed in a Bio-med thermocycler 60 (B. Braun). A first denaturation step (94° C., 1 min) was followed by 35 cycles of denaturation (94° C., 30 sec), primer annealing (63° C., 1 min) and extension (72° C., 2 min). The extension time was increased by 3 see at each cycle. Amplification was ended by a final extension step (72° C., 10 min). The amplified fragment was cloned in pGEMO-T Easy vector and sequenced.  
         [0064]    Sequencing and Analysis of DNA Sequences  
         [0065]    cDNA sequencing has been performed by Eurogentech (Belgium) and ESGS (France). Sequence analysis and comparison were performed with Lion&#39;s software bioScout, Lasergene software (DNAStar) and Genedoc programme.  
         [0066]    The cacao CP-III cDNA sequence is 1768 bp long. A putative initiation start codon was assigned by comparison with other carboxypeptidase sequences. It is located 25 bp from the 5′ end. The open reading frame is broken by a stop codon (TGA) at position 1549, followed by a putative polyadenylation signal (TATAAA) at position 1725.  
         [0067]    Cacao CP-III encodes a 508 amino acid type III carboxypeptidase C with a predicted molecular weight of 56 kDa and a μl of 5.04. The catalytic amino acids are present at position Ser 2 , Asp and His 473 . Hydrophilicity analysis (FIG. 3) reveals that cacao CP-III encodes a hydrophilic protein with a very hydrophobic N-terminal end, indicating the presence of a signal peptide.  
         [0068]    Northern Blot Analysis  
         [0069]    Total RNA samples were separated on 1.5% agarose gel containing 6% formaldehyde (FIG. 4). After electrophoresis, RNA was blotted onto nylon membranes (Appligene) and hybridized with  32 P-labeled cacao CP-III probe at 65° C. in 250 mM Na-phosphate buffer pH 7.2, 6.6% SDS, 1 mM EDTA and 1% BSA. Cacao CP-III cDNA fragment was amplified by PCR using pCP8 and pCP7R primers and labelled by the random priming procedure (rediprime II, Amersham Pharmacia Biotech). Membranes were washed three times at 65° C. for 30 min in 2×SSC, 0.1% SDS, in 1×SSC, 0.1% SDS and in 0.5×SSC, 0.1% SDS.  
     
       
       
         1 
         
           
             6  
           
           
             1  
             1768  
             DNA  
             Theobroma cacao  
           
            1 

gactctcaaa gagaaaaaga aaagatggca aatccgaaaa tcttataccc gttttctgtt     60 

tcccttctct tcctcatttc catctcctcc gcggccgctt cctccttctt agacgagcgg    120 

cgactcggag gatcaagttt cccctcgata catgcgaaga agttgataag ggagttgaat    180 

ttgtttccta aggaggaagt caacgtcgtt gatggaggcc aggtttcctt accggaggat    240 

tcgaggttgg tggagaagcg gttcaagttc ccgaatttgg cggtgcctgg tggggtttcc    300 

gttgaggatt tgggtcatca tgctggttat tacaagctag ctaattctca tgatgccaga    360 

atgttctatt tcttctttga atcacgaaat agcaaaaagg accctgttgt aatctggttg    420 

actggagggc cagggtgtag tagtgaattg gctttgtttt atgaaaatgg tccttttacc    480 

attgctgaga acatgtctct tatttggaat cagtatggtt gggacatggc atcaaacctt    540 

ctgtatgtgg accaacccat tggtaccggc tttagttata gttctgatag aagggacatt    600 

cgtcataatg aagatgaagt tagcaacgac ctatatgact tcttacaggc attctttgct    660 

gaacaccctg agtttgaaaa gaatgacttt tatataactg gagaatcata tgctgggcac    720 

tacattccag cttttgctgc ccgagtccac caaggaaaca aagctaaaga tggaattcat    780 

ataaacctaa agggatttgc tattggtaat ggcctgactg accctgcaat ccagtataaa    840 

gcttacacag attatgcttt ggacatgggg gtaattaaga agtctgacta caatcgtatc    900 

aacaagctgg ttccagtttg tgaaatggca ataaagcttt gtggcactga tggcacaatc    960 

tcttgcatgg cttcatattt tgtctgcaat gccatattca ctggcatcat ggcacttgct   1020 

ggcgatacaa attactacga cattagaacg aaatgtgaag ggagcctttg ctatgacttc   1080 

tcaaacatgg agacatttct gaaccaggaa tctgttaggg atgcccttgg agttgggagt   1140 

attgactttg tgtcctgcag tcctacagtg tatcaggcca tgctggttga ctggatgagg   1200 

aatcttgaag ttggcattcc tgctctcctt gaggatggtg tcaagcttct tgtatatgct   1260 

ggagaatatg atctcatctg caactggctt ggcaattcga gatgggtcca tgcaatggaa   1320 

tggtctggtc agaaggagtt tgtagcatct cctgaggttc cttttgtcgt tgatggctca   1380 

gaagcaggag tcttgagaac tcatggacct cttggtttcc taaaggttca cgatgcaggt   1440 

cacatggttc ctatggacca gccaaaggca gcattggaga tgctgaagcg gtggactaag   1500 

ggtacattat ctgaagctgc cgattcagag aaattggttg ctgaaatatg atttccatca   1560 

ttgcactgct tgcatacaat ttagttggca ttagaatggg aatagccgta tcgtaaagct   1620 

ctggggtttc tatgtatgcc tgtaaataat tgcatgttaa tgctagtaca atggtatctt   1680 

tgttttttga agatcaccta ctgaacttat atgaatcaag gacttataaa aatcttctaa   1740 

aaaaaaaaaa aaaaaaaaaa aaaaaaaa                                      1768 

 
           
             2  
             508  
             PRT  
             Theobroma cacao  
           
            2 

Met Ala Asn Pro Lys Ile Leu Tyr Pro Phe Ser Val Ser Leu Leu Phe 
1                5                  10                  15 

Leu Ile Ser Ile Ser Ser Ala Ala Ala Ser Ser Phe Leu Asp Glu Arg 
            20                  25                  30 

Arg Leu Gly Gly Ser Ser Phe Pro Ser Ile His Ala Lys Lys Leu Ile 
        35                  40                  45 

Arg Glu Leu Asn Leu Phe Pro Lys Glu Glu Val Asn Val Val Asp Gly 
     50                  55                 60 

Gly Gln Val Ser Leu Pro Glu Asp Ser Arg Leu Val Glu Lys Arg Phe 
65                  70                   75                  80 

Lys Phe Pro Asn Leu Ala Val Pro Gly Gly Val Ser Val Glu Asp Leu 
                85                  90                  95 

Gly His His Ala Gly Tyr Tyr Lys Leu Ala Asn Ser His Asp Ala Arg 
            100                 105                 110 

Met Phe Tyr Phe Phe Phe Glu Ser Arg Asn Ser Lys Lys Asp Pro Val 
        115                 120                125 

Val Ile Trp Leu Thr Gly Gly Pro Gly Cys Ser Ser Glu Leu Ala Leu 
    130                 135                 140 

Phe Tyr Glu Asn Gly Pro Phe Thr Ile Ala Glu Asn Met Ser Leu Ile 
145                 150                 155                 160 

Trp Asn Gln Tyr Gly Trp Asp Met Ala Ser Asn Leu Leu Tyr Val Asp 
                165                 170                175 

Gln Pro Ile Gly Thr Gly Phe Ser Tyr Ser Ser Asp Arg Arg Asp Ile 
           180                  185                 190 

Arg His Asn Glu Asp Glu Val Ser Asn Asp Leu Tyr Asp Phe Leu Gln 
        195                 200                 205 

Ala Phe Phe Ala Glu His Pro Glu Phe Glu Lys Asn Asp Phe Tyr Ile 
    210                  215                220 

Thr Gly Glu Ser Tyr Ala Gly His Tyr Ile Pro Ala Phe Ala Ala Arg 
225                 230                 235                 240 

Val His Gln Gly Asn Lys Ala Lys Asp Gly Ile His Ile Asn Leu Lys 
                245                 250                 255 

Gly Phe Ala Ile Gly Asn Gly Leu Thr Asp Pro Ala Ile Gln Tyr Lys 
            260                 265                 270 

Ala Tyr Thr Asp Tyr Ala Leu Asp Met Gly Val Ile Lys Lys Ser Asp 
        275                 280                 285 

Tyr Asn Arg Ile Asn Lys Leu Val Pro Val Cys Glu Met Ala Ile Lys 
    290                 295                 300 

Leu Cys Gly Thr Asp Gly Thr Ile Ser Cys Met Ala Ser Tyr Phe Val 
305                 310                 315                 320 

Cys Asn Ala Ile Phe Thr Gly Ile Met Ala Leu Ala Gly Asp Thr Asn 
                325                 330                 335 

Tyr Tyr Asp Ile Arg Thr Lys Cys Glu Gly Ser Leu Cys Tyr Asp Phe 
            340                 345                 350 

Ser Asn Met Glu Thr Phe Leu Asn Gln Glu Ser Val Arg Asp Ala Leu 
        355                 360                 365 

Gly Val Gly Ser Ile Asp Phe Val Ser Cys Ser Pro Thr Val Tyr Gln 
    370                 375                 380 

Ala Met Leu Val Asp Trp Met Arg Asn Leu Glu Val Gly Ile Pro Ala 
385                 390                 395                 400 

Leu Leu Glu Asp Gly Val Lys Leu Leu Val Tyr Ala Gly Glu Tyr Asp 
                405                 410                 415 

Leu Ile Cys Asn Trp Leu Gly Asn Ser Arg Trp Val His Ala Met Glu 
            420                 425                 430 

Trp Ser Gly Gln Lys Glu Phe Val Ala Ser Pro Glu Val Pro Phe Val 
        435                 440                 445 

Val Asp Gly Ser Glu Ala Gly Val Leu Arg Thr His Gly Pro Leu Gly 
    450                 455                 460 

Phe Leu Lys Val His Asp Ala Gly His Met Val Pro Met Asp Gln Pro 
465                 470                 475                 480 

Lys Ala Ala Leu Glu Met Leu Lys Arg Trp Thr Lys Gly Thr Leu Ser 
                485                 490                 495 

Gln Ala Ala Asp Ser Glu Lys Leu Val Ala Glu Ile 
            500                 505 

 
           
             3  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer  
             
           
            3 

ggytgrtcca tnggnaccat                                                 20 

 
           
             4  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer  
             
           
            4 

gcttttgctg cccgagtcca cc                                              22 

 
           
             5  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer  
             
           
            5 

caaagagaaa aagaaaagat ggc                                             23 

 
           
             6  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer  
             
           
            6 

ccccagagct ttacgatacg g                                               21