Patent Application: US-44306700-A

Abstract:
the present invention provides genetic sequences encoding polyphenol oxidase enzymes of lettuce , banana , tobacco and pineapple plants , and recombinant vectors comprising same , and methods of identifying related sequences using said nucleic acid molecules . the invention further provides methods of modifying ppo expression in plants using the inventive nucleic acid molecules .

Description:
in a first aspect of the present invention there is provided a method for preparing nucleic acid encoding ppo , fragments and derivatives thereof , which method includes a source of a polypeptide having ppo activity , a first primer having a sequence corresponding to a first conserved a second primer having a sequence corresponding to a second conserved region of ppo in antisense orientation ; applicant has found that the method of the present invention , which involves the use of a second primer based on ppo , means that there is less likelihood that other ( non - ppo ) genes are amplified . furthermore , the method of the present invention dramatically increases the amount of genuine product formed in most cases . moreover , the added specificity provided by the second ppo - based primer makes it possible to clone ppo more readily from certain plants in which it was difficult to obtain a clone using one primer and oligo - dt . for example , with lettuce cdna the applicant saw only a faint smear of a range of products with gen3 / gen8 and oligo - dt but strong bands of the predicted size with gen3 / gen8 and rev1 . in a preferred aspect of the present invention there is provided a method for preparing nucleic acid encoding banana , lettuce , tobacco or pineapple ppo , fragments and derivatives thereof , which method includes a source of a polypeptide having banana , lettuce , tobacco or pineapple ppo activity , a first primer having a sequence corresponding to a first conserved region of banana , lettuce , tobacco or pineapple ppo in sense orientation , and a second primer having a sequence corresponding to a second conserved region of banana , lettuce , tobacco or pineapple ppo in antisense orientation ; isolating rna from the source of polypeptide having banana , lettuce , tobacco or pineapple ppo activity ; the terms “ nucleic acid encoding banana / lettuce / tobacco / pineapple ppo ” and “ banana / lettuce / tobacco / pineapple ppo gene ” as used herein should be understood to refer to a banana / lettuce / tobacco / pineapple ppo gene or a sequence substantially homologous therewith . for example , these terms include sequences which differ from the specific sequences given in the examples hereto but which , because of the degeneracy of the genetic code , encode the same protein . applicants have found that there are families of ppo genes in most plants . thus , there are likely to be other ppo genes in lettuce , banana , tobacco and pineapple in addition to those which have been isolated . these could be clones using the methods of the present invention . thus , the terms “ nucleic acid encoding banana / lettuce / tobacco / pineapple ppo ” and “ banana / lettuce / tobacco / pineapple ppo gene ” should be understood to include banana / lettuce / tobacco / pineapple ppo genes other than those specific genes that have been isolated . the terms may also include presequences such as chloroplast transit sequence as well as sequences encoding mature ppo protein . the term “ derivative ” as used herein includes nucleic acids that have been chemically or otherwise modified , for example mutated , or labelled , or nucleic acids incorporating a catalytic cleavage site . the term “ fragment ” includes functionally active fragments of a ppo gene which encode a polypeptide or peptide having ppo activity or are capable of altering expression of the ppo genes . examples of alteration of the gene may include up - regulation or down - regulation of the gene , coding of the gene , transcription of the gene , binding of the gene or stability of the gene sequence . the source of polypeptide having ppo activity is preferably a source of polypeptide having banana , lettuce , tobacco or pineapple ppo activity . the source of polypeptide having banana ppo activity may be banana fruit , preferably young banana fruit , more preferably the flesh of young banana fruit . the source of polypeptide having banana ppo activity may be banana peel , preferably young banana peel , more preferably the peel of young banana fruit . the source of polypeptide having lettuce ppo activity may be lettuce leaves , preferably young lettuce leaves . the source of polypeptide having tobacco ppo activity may be tobacco leaves , preferably young tobacco leaves . the source of polypeptide having pineapple ppo activity may be pineapple fruit , preferably the flesh of the pineapple fruit , more preferably the flesh of pineapple fruit exhibiting blackheart disorder . the rna may be isolated by any suitable method including extraction for example with a detergent such as ctab , use of an oligo - dt spun column as described in pct / au92 / 00356 the entire disclosure of which is incorporated herein by reference , or use of a commercially available kit such as the polyattract 1000 system from promega corporation . the step of treating the rna to construct cdna according to this aspect of the present invention may include treating the rna with reverse transcriptase and an adapter primer to form cdna . the adapter primer may be an oligonucleotide adapter primer including the following sequence or part thereof : the step of treating the rna to construct cdna according to this aspect of the present invention may include treating the rna with reverse transcriptase and reverse primer to form cdna . the adapter primer may be replaced with a reverse primer having a sequence corresponding to a conserved region of ppo genes including the following sequence of part thereof : the first primer has a sequence corresponding to a first conserved region of ppo . preferably the first primer has a sequence corresponding to at least a portion of or in close proximity to a first copper binding site of ppo . the second primer has a sequence corresponding to a second conserved region of ppo . preferably the second primer has a sequence corresponding to at least a portion of or in close proximity to a second copper binding site of ppo . more preferably the first primer has a sequence corresponding to at least a portion of or in close proximity to one of the cua or cub binding sites of ppo , and the second primer has a sequence corresponding to at least a portion of or in close proximity to the other of the cua or cub binding sites of ppo . the first and second primers may be degenerate . the first primer may include one of the following sequences or part thereof : preferably , the second primer comprises a nucleotide sequence selected from the group consisting of : ( ii ) 5 ′- gcctgcagtt [ tc ] tc [ ag ] tc [ ag ] tagaa - 3 ′( seq id no : 37 ) the cdna may be amplified using the polymerase chain reaction ( pcr ). those skilled in the art will appreciate that if the cu binding sites are internal , the nucleic acid isolated will be a fragment of the ppo gene lacking 3 ′ and 5 ′ termini . however , it is possible to determine the complete nucleic acid sequence of the ppo gene and to prepare or isolate nucleic acid encoding such ppo or antisense to such ppo . accordingly , in a further aspect of the present invention , there is provided a method for preparing nucleic acid encoding the c - terminus of ppo ( i . e . comprising the 3 ′- end of the ppo gene ), which method includes : there is also provided a method for preparing nucleic acid encoding the n - terminus of ppo ( i . e . comprising the 5 ′- end of the ppo gene ), including : attaching the anchor to the 5 ′ end of the cdna so formed ; and the source of polypeptide having ppo activity is preferably a source of polypeptide having banana , lettuce , tobacco or pineapple ppo activity . the source of polypeptide having banana ppo activity may be banana fruit , preferably young banana fruit , more preferably the flesh of young banana fruit . the source of polypeptide having banana ppo activity may be banana peel , preferably young banana peel , more preferably the peel of young banana fruit . the source of polypeptide having lettuce ppo activity may be lettuce leaves , preferably young lettuce leaves . the source of polypeptide having tobacco ppo activity may be tobacco leaves , preferably young tobacco leaves . the source of polypeptide having pineapple ppo activity may be pineapple fruit , preferably the flesh of the pineapple fruit , more preferably the flesh of pineapple fruit exhibiting blackheart disorder . the rna may be isolated by any suitable method including extraction for example with a detergent such as ctab , use of an oligo - dt spun column as described in pct / au92 / 00356 the entire disclosure of which is incorporated herein by reference , or use of a commercially available kit such as the polyattract 1000 system from promega corporation . the step of treating the rna to construct cdna according to this aspect of the present invention may include treating the rna with reverse transcriptase and an adapter primer to form cdna . the adapter primer may be an oligonucleotide adapter primer including the following sequence or part thereof : the adapter primer may be replaced with a reverse primer having a sequence corresponding to a conserved region of ppo genes including the following sequence of part thereof : the primer in sense orientation may be a lettuce ppo specific primer . the primer in sense orientation may include the following sequence or part thereof : the primer in sense orientation may be a banana ppo specific primer . the primer in sense orientation may include the following sequence or part thereof : the primer in sense orientation may be a tobacco or pineapple ppo specific primer . the adapter primer may include the following sequence or part thereof : the primers in antisense orientation may be lettuce ppo specific primers . the primers in antisense orientation may include the following sequences or part thereof : the primers in antisense orientation may be banana ppo specific primers . the primers in antisense orientation may include the following sequences or part thereof : the primers in antisense orientation may be tobacco ppo specific primers . the primers in antisense orientation may be pineapple ppo specific primers . the primers in antisense orientation may include the following sequences or part thereof : the anchor may be of any suitable type . the anchor may be attached by ligation for example using t4 rna ligase . the anchor primer should be capable of hybridizing with the anchor . those skilled in the art will appreciate that using the methods of the present invention it is possible to determine the complete nucleic acid sequence of the ppo gene of interest and to prepare or isolate nucleic acid encoding such ppo or antisense to such ppo . in a further aspect of the present invention , there is provided a nucleic acid encoding banana ppo or antisense to banana ppo , fragments and derivatives thereof . preferably the nucleic acid has the sequence shown in fig1 a - 4c , 11 a - 11 g or 12 a - 12 c fragments and derivatives thereof , and substantially homologous sequences . in a further aspect of the present invention , there is provided in nucleic acid encoding lettuce ppo or antisense to lettuce ppo , fragments and derivatives thereof . preferably the nucleic acid has the sequence shown in fig1 a - 15g fragments and derivatives thereof , and substantially homologous sequences . in a further aspect of the present invention , there is provided a nucleic acid encoding tobacco ppo or antisense to tobacco ppo , fragments and derivatives thereof . preferably the nucleic acid has the sequence shown in fig5 a - b , 6 a - c , and 7 a - c , fragments and derivatives thereof , and substantially homologous sequences . in a further aspect of the present invention , there is provided a nucleic acid encoding pineapple ppo or antisense to pineapple ppo , fragments and derivatives thereof . preferably the nucleic acid has the sequence shown in fig8 a - 10h , 13 a - 13 e and 14 a - 14 c and derivatives thereof , and substantially homologous sequence . the nucleic acid may be prepared by a method as hereinbefore described . the nucleic acid may be modified , for example by inclusion of a catalytic cleavage site . in a further aspect of the present invention there is provided a method for preparing a recombinant vector including a nucleic acid encoding banana ppo or antisense to banana ppo , fragments and derivatives thereof , which method includes nucleic acid encoding banana ppo or antisense to banana ppo , fragments and derivatives thereof ; and in a further aspect of the present invention there is provided a method for preparing a recombinant vector including a nucleic acid encoding lettuce ppo or antisense to lettuce ppo , fragments and derivatives thereof , which method includes nucleic acid encoding lettuce ppo or antisense to lettuce ppo , fragments and derivatives thereof ; and in a further aspect of the present invention there is provided method for preparing a recombinant vector including a nucleic acid encoding tobacco ppo or antisense to tobacco ppo , fragments and derivatives thereof , which method includes nucleic acid encoding tobacco ppo or antisense to tobacco ppo , fragments and derivatives thereof ; and in a further aspect of the present invention there is provided a method for preparing a recombinant vector including a nucleic acid encoding pineapple ppo or antisense to pineapple ppo , fragments and derivatives thereof , which method includes nucleic acid encoding pineapple ppo or antisense to pineapple ppo , fragments and derivatives thereof ; and the nucleic acid may be prepared by a method as hereinbefore described . the nucleic acid may be modified , for example by inclusion of a catalytic cleavage site . the vector may be plasmid expression vector . for example bluescript sk + has been found to be suitable . alternatively , the vector may be a binary vector . the recombinant vector may contain a promoter , preferably a constitutive promoter upstream of the nucleic acid . the cloning step may take any suitable form . a preferred form may include isolating a fragment of the expected size , for example from the column or gel ; and ligating said fragment into a suitable restriction enzyme site of the vector , for example the eco rv site of a bluescript sk + vector . in order to test the clones so formed , a suitable microorganism may be transformed with the vector , the microorganism cultured and the polypeptide encoded therein expressed . the microorganism may be a strain of escherichia coli , for example e . coli dh5 has been found to be suitable . alternatively , appropriate vectors may be used to transform plants . in a further aspect of the present invention there is provided a recombinant vector including a nucleic acid encoding banana ppo or antisense to banana ppo , fragments and derivatives thereof , which vector is capable of being replicated , transcribed and translated in a unicellular organism or alternatively in a plant . in a further aspect of the present invention there is provided a recombinant vector including a nucleic acid encoding lettuce ppo or antisense to lettuce ppo , fragments and derivatives thereof , which vector is capable of being replicated , transcribed and translated in a unicellular organism or alternatively in a plant . in a further aspect of the present invention there is provided a recombinant vector including a nucleic acid encoding tobacco ppo or antisense to tobacco ppo , fragments and derivatives thereof , which vector is capable of being replicated , transcribed and translated in a unicellular organism or alternatively in a plant . in a further aspect of the present invention there is provided a recombinant vector including a nucleic acid encoding pineapple ppo or antisense to pineapple ppo , fragments and derivatives thereof , which vector is capable of being replicated , transcribed and translated in a unicellular organism or alternatively in a plant . the nucleic acid may be prepared by a method as hereinbefore described . the nucleic acid may be modified , for example by inclusion of a catalytic cleavage site . the vector may be a plasmid expression vector . for example bluescript sk + has been found to be suitable . alternatively , the vector may be a binary vector . the recombinant vector may contain a promoter , preferably a constitutive promoter upstream of the nucleic acid encoding banana , lettuce , tobacco or pineapple ppo or antisense to banana , lettuce , tobacco or pineapple ppo , fragments and derivatives thereof . the microorganism may be a strain of escherichia coli , for example e . coli dh5 has been found to be suitable . in a further aspect of the present invention there is provided a method of decreasing the level of ppo activity in a plant tissue , which method includes a nucleic acid encoding banana ppo , a modified nucleic acid encoding banana ppo , or a nucleic acid antisense to banana ppo , fragments and derivatives thereof ; and introducing said nucleic acid into said plant sample to produce a transgenic plant . in a further aspect of the present invention there is provided a method of decreasing the level of ppo activity in a plant tissue , which method includes a nucleic acid encoding lettuce ppo , a modified nucleic acid encoding lettuce ppo , or a nucleic acid antisense to lettuce ppo , fragments and derivatives thereof ; and introducing said nucleic acid into said plant sample to produce a transgenic plant . in a further aspect of the present invention there is provided a method of decreasing the level of ppo activity in a plant tissue , which method includes a nucleic acid encoding tobacco ppo , a modified nucleic acid encoding tobacco ppo , or a nucleic acid antisense to tobacco ppo , fragments and derivatives thereof ; an d introducing said nucleic acid into said plant sample to produce a transgenic plant . in a further aspect of the present invention there is provided a method of decreasing the level of ppo activity in a plant tissue , which method includes a nucleic acid encoding pineapple ppo , a modified nucleic acid encoding pineapple ppo , or a nucleic acid antisense to pineapple ppo , fragments and derivatives thereof ; and introducing said nucleic acid into said plant sample to produce a transgenic plant . ppo activity may be decreased by the use of sense constructs ( cosuppression ). alternatively the nucleic acid may include a sequence encoding antisense mrna to banana , lettuce , tobacco or pineapple ppo or a functionally active fragment thereof . alternatively the nucleic acid may encode banana , lettuce , tobacco or pineapple ppo or a functionally active fragment thereof and incorporate a catalytic cleavage site ( ribozyme ). the nucleic acid may be included in a recombinant vector as hereinbefore described . in a preferred aspect , the nucleic acid may be included in a binary vector . in a further preferred aspect , the introduction of a binary vector into the plant may be by infection of the plant with an agrobacterium containing the binary vector or by bombardment with nucleic acid coated microprojectiles . methods for transforming banana , lettuce , tobacco or pineapple with agrobacterium are known to those skilled in the art and are described in , for example , may et al ., bio / technology ( 1995 ) 13 : 486 - 492 , michelmore et al ., plant cell reports ( 1987 ) 6 : 439 - 442 , and curtis et al ., journal of experimental botany ( 1994 ) 45 : 1141 - 1149 , the entire disclosure of which are incorporated herein by reference . in a further aspect of the present invention there is provided a method of increasing the level of ppo activity in a plant tissue , which method includes introducing said nucleic acid into said plant sample to produce a transgenic plant . in a further aspect of the present invention there is provided a method of increasing the level of ppo activity in a plant tissue , which method includes introducing said nucleic acid into said plant sample to produce a transgenic plant . in a further aspect of the present invention there is provided a method of increasing the level of ppo activity in a plant tissue , which method includes introducing said nucleic acid into said plant sample to produce a transgenic plant . in a further aspect of the present invention there is provided a method of increasing the level of ppo activity in a plant tissue , which method includes introducing said nucleic acid into said plant sample to produce a transgenic plant . the nucleic acid may be included in a recombinant vector as hereinbefore described . in a preferred aspect , the nucleic acid may be included in a binary vector . in a further preferred aspect , the introduction of the binary vector into the plant may be by infection of the plant with an agrobacterium containing the binary vector or by bombardment with nucleic acid coated microprojectiles . the plant may be of any suitable type . however the method is particularly applicable to banana , lettuce , tobacco or pineapple . in a further aspect of the present invention there is provided a transgenic plant , which plant contains nucleic acid capable of modifying expression of the normal banana ppo gene . in a further aspect of the present invention there is provided a transgenic plant , which plant contains nucleic acid capable of modifying expression of the normal lettuce ppo gene . the plant may be of any suitable type . preferably , the plant is banana . in a further aspect of the present invention there is provided a transgenic plant , which plant contains nucleic acid capable of modifying expression of the normal tobacco ppo gene . the plant may be of any suitable type . preferably , the plant is tobacco . in a further aspect of the present invention there is provided a transgenic plant , which plant contains nucleic acid capable of modifying expression of the normal pineapple ppo gene . the plant may be of any suitable type . preferably , the plant is pineapple . messenger rna ( mrna ) was isolated directly from young leaves of lettuce using the polyttract 1000 system from promega corporation . first strand cdna was synthesised with reverse transcriptase using a timesaver cdna synthesis kit ( pharmacia biotech ) utilising an oligo - dt primer adapter as described in frohman , mass . ( 1990 ) in “ pcr protocols : a guide to methods and applications ” ( m a inris , d h gelfrand , j j sninsky and t j white , eds ) academic press , new york pp 28 - 38 , the entire disclosure of which is incorporated herein by reference : oligonucleotide primers were designed based on known plant ppo dna sequences in the conserved regions of the gene which encode the copper binding sites , cua and cub as described in dry , i b and robinson , s p ( 1994 ) “ molecular cloning and characterisation of grape berry polyphenol oxidase ”, plant molecular biology 26 : 495 - 502 , the entire disclosure of which is incorporated herein by reference . two forward primers designed around the cua site ( gen3 and gen8 ) and one reverse primer designed around the cub site ( rev1 ) were synthesised : although the primers are in the region of the cu binding sites , one of them ( gen8 ) is just outside of what is traditionally accepted to be a cu binding site of the enzyme . the first strain cdna was amplified by the polymerase chain reaction ( pcr ) essentially according to the method of frohman using gen3 and rev1 or gen8 and rev1 primers , each at a final concentration of 1 μm ( dry et al .). amplification involved an initial program of 2 cycles of denaturation at 94 ° c . for 1 min , annealing at 37 ° c . for 2 min , a slow ramp to 72 ° c . over 2 min and elongation at 72 ° c . for 3 min , followed by 25 cycles of denaturation at 94 ° c . for 1 min , annealing at 55 ° c . for 1 min , and elongation at 72 ° c . for 3 min . a sample of the amplified dna was run on an agarose gel and stained with ethidium bromide to determine the size of the pcr products and the remainder was purified and concentrated using pcr wizard prep columns ( promega corporation ). the purified dna was cloned into eco rv - cut bluescript sk + vector ( stratagene ) which had been t - tailed with taq polymerase and the ligated dna was introduced into e . coli dh5α by electroporation . recombinant clones which had an insert of the predicted size were selected and their dna sequence was determined by automated sequencing . three putative lettuce ppo clones ( lpo316 , lpo812 and lpo813 ) were identified based on their homology to known plant ppo genes . using this sequence information a specific forward primer ( let3p ) and two reverse primers ( let5p1 and let5p2 ) were synthesised : to obtain the 3 ′- end of the lettuce ppo gene , the first strand cdna described above was amplified by the same pcr procedure using 1 μm let3p primer and 100 nm adapter primer : the amplified cdna was purified as described above and run on a 2 % nusieve gtg ( fmc bioproducts ) agarose gel . a 1000 bp fragment was excised from the gel and the dna was cloned into t - tailed , eco rv - cut bluescript sk + to yield the 3 ′- end clones lpo9 and lpo10 , which were sequenced . the 5 ′- end of the lettuce ppo gene was cloned by a modification of the 5 ′- race procedure originally described by frohman using a 5 ′- amplifinder race kit ( clonetech laboratories ). first strand cdna was synthesised from mrna with reverse transcriptase using the let5p2 primer and an amplifinder anchor was ligated onto the 5 ′- end of the cdna . the cdna was amplified by pcr with let5p1 primer and the amplifinder anchor primer . the amplified cdna was purified as described above and run on a 2 % nusieve gtg ( fmc bioproducts ) agarose gel . an 850 bp fragment was excised from the gel and the dna was cloned into t - tailed eco rv - cut bluescript sk + to give the 5 ′- end clones lpo4 , lpo5 , lpo6 , and lpo7 , which were sequenced . the 5 ′- and 3 ′- clones were found to have the predicted overlapping sequences with the original clone and the complete sequence of lettuce ppo ( lpo1 ) was derived by combining the sequences from the various clones ( fig1 ). total rna was isolated from young banana fruit . fruit tissue ( 3 g ) was frozen and ground to a fine powder in liquid nitrogen with a coffee grinder then added to 20 ml of extraction buffer ( 2 % hexadecyltrimethylammonium bromide ( ctab ), 2 % polyvinyl pyrolidone , 100 mm tris - hcl , ph 8 . 0 , 25 mm edta , 2 m nacl , 0 . 05 % spermidine , 2 % β - mercaptoethanol ) at 65 ° c . the extract was mixed with 20 ml of chloroform / iaa then centrifuged for 20 minutes at 5 , 000 rpm and the aqueous phase was re - extracted with chloroform / iaa . the aqueous phase was filtered through miracloth and 0 . 25 volumes of 10 m licl were added then the sample was incubated overnight at 4 ° c . before centrifuging for 20 minutes at 8 , 000 rpm . the supernatant was removed and the pellet was resuspended in 0 . 5 ml of 1 m nacl , 0 . 5 % sds , 10 mm tris , ph 8 . 0 , 1 mm edta . the rna was extracted once with an equal volume of chloroform / iaa and 2 volumes of ethanol was added . after incubation for 40 mins at − 70 ° c . the solution was centrifuged for 15 minutes at 10 , 000 rpm . the supernatant was removed and the pellet was rinsed with 80 % ethanol , drained , and dried . the pellet was resuspended in 50 μl of sterile water . first strand cdna was synthesised from 10 μg total rna with reverse transcriptase as described in dry , i . b . and robinson , s . p . ( 1994 ) “ molecular cloning and characterisation of grape berry polyphenol oxidase ”, plant molecular biology 26 : 495 - 502 , the entire disclosure of which is incorporated herein by reference , utilising an oligo - dt primer adapter ( frohman , m . a . ( 1990 ) in “ pcr protocols : a guide to methods and applications ” ( m . a . innis , d . h . gelfrand , j . j . sninsky and t . j . white , eds .) academic press , new york pp 28 - 38 , the entire disclosure of which is incorporated herein by reference ): oligonucleotide primers were designed based on known plant ppo dna sequences in the conserved regions of the gene which encode the copper binding sites , cua and cub ( dry et al .). a forward primer designed around the cua site ( gen3 ) and a reverse primer designed around the cub site ( rev1 ) were synthesised : the first strand reaction was amplified by the polymerase chain reaction ( prc ) essentially according to the method of frohmen using gen3 and rev1 primers , each at a final concentration of 1 μm ( dry et al .). amplification involved an initial program of 2 cycles of denaturation at 94 ° c . for 1 min , annealing at 37 ° c . for 2 min , a slow ramp to 72 ° c . over 2 min and elongation at 72 ° c . for 3 min , followed by 25 cycles of denaturation at 94 ° c . for 1 min , annealing at 55 ° c . for 3 min , and elongation at 72 ° c . for 3 min . a sample of the amplified dna was run on an agarose gel and stained with ethidium bromide to determine the size of the pcr products and the remainder was purified and concentrated using pcr wizard prep columns ( promega corporation ). the purified dna was cloned into eco rv - cut bluescript sk + vector ( stratagene ) which had been t - tailed with taq polymerase and the ligated dna was introduced into e . coli dh5α by electroporation . recombinant clones which had an insert of the predicted size were selected and their dna sequence was determined by automated sequencing . a putative banana ppo clone ( bpo3 ) was identified based on its homology to known plant ppo genes . using this sequence information a specific forward primer ( ban1 ) and two specific reverse primers ( ban2r and ban3r ) were synthesised : to obtain the 3 ′- end of this banana ppo gene , the first strand cdna described above was amplified by the same pcr procedure using 1 μm ban1 primer and 100 nm adapter primer : the dna was amplified using 25 cycles of denaturation at 94 ° c . for 1 min , annealing at 55 ° c . for 1 min , and elongation at 72 ° c . for 3 min . the amplified dna was purified using a qiaquick spin pcr purification kit ( qiagen ) and run on a 2 % nusieve gtg ( fmc bioproducts ) agarose gel . a 1000 bp fragment was excised from the gel and the dna was cloned into t - tailed eco rv - cut bluescript sk + to yield the 3 ′- end clone bpo17 , which was sequenced and shown to encode the 3 ′- end of bpo3 . the 5 ′- end of bpo3 was cloned by a modification of the 5 ′- race procedure originally described by frohmann . first strand cdna was synthesised from banana fruit rna as described above but utilising the banana ppo specific primer ban2r . the dna was tailed with terminal transferase as described in frohmann and amplified by pcr with ban3r and b26 primers , each at a final concentration of 1 μm . the dna was amplified using 30 cycles of denaturation at 94 ° c . for 1 min , annealing at 55 ° c . for 1 min , and elongation at 72 ° c . for 3 min . the amplified dna was run on a 1 . 8 % nusieve gtg ( fmc bioproducts ) agarose gel and a 700 bp fragment was excised from the gel . the dna was extracted with a qiaquick gel extraction kit and cloned into t - tailed eco rv - cut bluescript sk + yield the 5 ′- end clone bpo26 which was sequenced and shown to encode the 5 ′- end of bpo3 . the overlapping clones bpo3 , bpo17 and bpo26 were fully sequenced in both directions and the sequence of this banana ppo gene ( banppo1 ) was derived by combining the sequences ( fig1 a - g ). in the course of these experiments a number of clones were obtained from the banana fruit cdna by pcr amplification using the b25 primer with one of the degenerate primers based on conserved sequences in other plant ppo genes : using the methods described above . most of these clones were identical to banppo1 but one clone , designated banppo11 , was found to be distinctly different and its sequence is shown in fig1 a - 12c . total rna was isolated from the peel of young banana fruit . fruit tissue ( 3 g ) was frozen and ground to a fine powder in liquid nitrogen with a coffee grinder then added to 20 ml of extraction buffer ( 2 % hexadecyltrimethylammonium bromide ( ctab ), 2 % polyvinyl pyrrolidone , 100 mm tris - chl , ph 8 . 0 , 25 mm edta , 2 m nacl , 0 . 05 % spermidine , 2 % β - mercaptoethanol ) at 65 ° c . the extract was mixed with 20 ml of chloroform / iaa then centrifuged for 20 minutes at 5 , 000 rpm and the aqueous phase was re - extracted with chloroform / iaa . the aqueous phase was filtered through miracloth and 0 . 25 volumes of 10 m licl were added then the sample was incubated overnight at 4 ° c . before centrifuging for 20 minutes at 8 , 000 rpm . the supernatant was removed and the pellet was resuspended in 0 . 5 ml of 1 m nacl , 0 . 5 % sds , 10 mm tris , ph 8 . 0 , 1 mm edta . the rna was extracted once with an equal volume of chloroform / iaa and 2 volumes of ethanol was added . after incubation for 40 mins at − 70 ° c . the solution was centrifuged for 15 minutes at 10 , 000 rpm . the supernatant was removed and the pellet was rinsed with 80 % ethanol , drained and dried . the pellet was resuspended in 50 μl of sterile water . first strand cdna was synthesised from 10 μg total rna with reverse transcriptase as described in ref . 2 , utilising an oligosaccharide - dt primer adapter ( ref 1 ): oligonucleotide primers were designed based on known plant ppo dna sequences . comparison of a number of ppo sequences from a range of different plants allowed identification of the conserved regions of the gene , which are mostly in or near the regions which encode the two copper binding sites . cua and cub ( 2 ). forward primers designed around the cua site ( gen8 , gen9 and gen 10 ) and reverse primers designed around the cub site ( rev1 and rev2 ) were synthesised : the first strand reaction was amplified by the polymerase chain reaction ( pcr ) essentially according to the method of frohman ( 1 ) using gen and rev primers , each at a final concentration of 1 μm ( 2 ). amplification involved an initial program of 2 cycles of denaturation at 94 ° c . for 1 min , annealing at 37 ° c . for 2 min , a slow ramp to 72 ° c . for 3 min , followed by 33 cycles of denaturation at 94 ° c . for 1 min , annealing at 55 ° c . for 1 min , and elongation at 72 ° c . for 3 min . a sample of the amplified dna was run on an agarose gel and stained with ethidium bromide to determine the size of the pcr products . the remainder was run on a low melting point agarose gel and the bands of interest were excised . dna was purified from the agarose with a qiaquick pcr purification kit ( qiagen ). the purified dna was cloned into eco - rv - cut bluescript sk + vector ( stratagene ) which had been t - tailed with taq polymerase and the ligated dna was introduced into e . coli dh5α by electroporation . recombinant clones which had an insert of the predicted size were selected and their dna sequence was determined by automated sequencing . two putative banana ppo clones ( bppo2 , fig1 a - 1b ; and bppo8 , fig2 a - 2b ) were identified by their homology to other plant ppo genes . the 3 ′- end of bppo2 was cloned using a primer designed to the sequence of bppo2 : at a final concentration of 1 μm and a b25 adaptor primer : at a final concentration of 1 μm ( ref 1 ). amplification involved 35 cycles of denaturation at 94 ° c . for 1 min , annealing at 55 ° c . for 1 min , and elongation at 72 ° c . for 3 min . a sample of the amplified dna was run on an agarose gel and stained with ethidium bromide to determine the size of the pcr products . the remainder was run on a low melting point agarose gel and the bands of interest were excised . dna was purified from the agarose with a qiaquick pcr purification kit ( qiagen ). the purified dna was cloned into eco rv - cut bluescript sk + vector ( stratagene ) which has been t - tailed with taq polymerase and the ligated dna was introduced into e . coli dh5α by electroporation . recombinant clones which had an insert of the predicted size ( 1150 bp ) were selected and their dna sequence was determined by automated sequencing . two putative banana ppo clones ( banppo34 , fig3 a - c ; and banppo35 , fig4 a - c ); were identified based on their homology to known plant ppo genes . the sequences of banppo34 and bppo2 were identical . total rna was isolated from young leaves ( 1 - 3 cm long ) of glasshouse grown plants . approximately 2 g of frozen leaf material was ground to a fine powder in liquid nitrogen then extracted in 15 ml of extraction buffer ( 50 mm tris - hcl , ph 9 . 0 , 150 mm licl , 5 mm edta , 5 % sds and 0 . 6 % β - mercaptoethanol ) by shaking vigorously in a 50 ml screw cap tube for 1 - 2 minutes . approximately 15 ml of phenol / chloroform / iaa ( 25 : 24 : 1 ) was added and the homogenate mixed then centrifuged for 15 minutes at 5 , 000 rpm , 4 ° c . the upper aqueous phase was removed and re - extracted twice with phenol / chloroform / iaa and then once with chloroform / iaa and then centrifuged for 10 minutes at 5 , 000 rpm , 4 ° c . the supernatant was removed , licl was added to a final concentration of 2 m and the mixture was incubated overnight at 4 ° c . after centrifuging for 10 minutes at 8 , 000 rpm , 4 ° c . the supernatant was removed and the pellet was resuspended in 6 ml of 0 . 4 m licl then 2 ml of 8m licl was added and the mixture was incubated overnight at 4 ° c . the mixture was centrifuged for 10 minuters at 8 , 000 rpm , 4 ° c ., the supernatant was removed and the pellet was resuspended in 0 . 5 ml of sterile water and centrifuged briefly to remove any insoluble material . mrna was isolated from the total rna using a polyattract kit ( promega ). first strand cdna was synthesised from 10 μg total rna or 2 μg mrna with reverse transcriptase as described in ref 2 , utilising an oligo - dt primer adapter ( ref 1 ): the first strand reaction was amplified by the polymerase chain reaction ( pcr ) essentially according to the method of frohman ( 1 ) using gen and rev primers described in example 1 , each at a final concentration of 1 μm ( 2 ). amplification involved an initial program of 2 cycles of denaturation at 94 ° c . for 1 min , annealing at 37 ° c . for 2 min , a slow ramp to 72 ° c . over 2 min and elongation at 72 ° c . for 3 min , followed by 28 cycles of denaturation at 94 ° c . for 1 min , annealing at 55 ° c . for 1 min , and elongation at 72 ° c . for 3 min . a sample of the amplified dna was run on an agarose gel and stained with ethidium bromide to determine the size of the pcr products . the remainder was run on a low melting point agarose gel and the bands of interest were excised . dna was purified from the agarose with a qiaquick pcr purification kit ( qiagen ). the purified dna was cloned into eco rv - cut bluescript sk + vector ( stratagene ) which had been t - tailed with taq polymerase and the ligated dna was introduced into e . coli dh5α by electroporation . recombinant clones which had an insert of the predicted size were selected and their dna sequence was determined by automated sequencing . three putative tobacco ppo clones ( tobppo6 , fig5 a - b ; tobppo25 , fig6 a - c ; and tobppo26 , fig7 a - c ) were identified based on their homologies to known ppo genes . mature pineapple fruit were treated to induce blackheart disorder by holding the fruit for 17 days at 12 ° c . then for 4 days at 25 ° c . flesh showing blackheart symptoms was dissected from the fruit , frozen in liquid nitrogen and ground to a fine powder in a pre - cooled coffee grinder . to isolate total rna 10 g of the powder was ground in a mortar and pestle then extracted with 30 ml of homogenisation buffer ( 100 mm tris - hcl , ph9 . 0 , 200 mm nacl , 15 mm edta , 0 . 5 % sarkosyl and 1 % β - mercaptoethanol ), 30 ml of phenol and 6 ml of chloroform / iaa . the mixture was stirred in a beaker , 2 . 1 ml of 3m naac ( ph 5 . 2 ) was added and the mixture was kept on ice for 15 minutes then centrifuged for 15 minutes at 8 , 000 rpm , 4 ° c . the upper aqueous phase was removed and equal volume of isopropanol was added . the mixture was incubated for 30 minutes at − 70 ° c . then centrifuged for 20 minutes at 8 , 000 rpm , 4 ° c . in corex tubes . the supernatant was removed and the pellet was rinsed with 70 % ethanol and centrifuged for 5 minutes at 8 , 000 rpm , 4 ° c . the ethanol was removed and the pellet was air dried then resuspended in 0 . 75 ml sterile water and centrifuged to removed any insoluble material . licl was added to a final concentration of 3 m and the mixture was incubated overnight at − 20 ° c . then centrifuged for 30 minutes at 15 , 000 rpm , 4 ° c . the pellet was rinsed with 70 % ethanol , centrifuged briefly , drained and air dried . the pellet was resuspended in 75 μl sterile water and centrifuged to remove any insoluble material . oligonucleotide primers were designed based on known plant ppo dna sequences . comparison of a number of ppo sequences from a range of different plants allowed identification of the conserved regions of the gene , which are mostly in or near the regions which encode the two copper binding sites , cua and cub . forward primers designed around the cua site ( gen8 , gen9 and gen 10 ) and reverse primers designed around the cub site ( rev1 and rev2 ) were synthesised : first strand cdna was synthesized from 10 μg total rna with reverse transcriptase as described in ref 2 , utilising the rev2 primer : the first strand reaction was amplified by the polymerase chain reaction ( pcr ) essentially according to the method of frohman ( 1 ) using the gen and rev primers described in example 1 , each at a final concentration of 1 μm ( 2 ). amplification involved an initial program of 2 cycles of denaturation at 94 ° c . for 1 min , annealing at 37 ° c . for 2 min , a slow ramp to 72 ° c . over 2 min and elongation at 72 ° c . for 3 min , followed by 33 cycles of denaturation at 94 ° c . for 1 min , annealing at 55 ° c . for 1 min , and elongation at 72 ° c . for 3 min . a sample of the amplified dna was run on an agarose gel and stained with ethidium bromide to determine the size of the pcr products . the remainder was run on a low melting point agarose gel and the bands of interest were excised . dna was purified from the agarose with a qiaquick pcr purification kit ( qiagen ). the purified dna was cloned into eco rv - cut bluescript sk + vector ( stratagene ) which had been t - tailed with taq polymerase and the ligated dna was introduced into e . coli dh5α by electroporation . recombinant clones which had an insert of the predicted size were selected and their dna sequence was determined by automated sequencing . a putative pineapple ppo clone ( pinpp020 ; fig8 a - c ) was identified based on its homology to known ppo genes . first strand cdna was also synthesised from 10 μg total rna with reverse transcriptase as described in dry , i . b . and robinson , s . p ( 1994 ), utilising an oligo - dt primer adapter ( ref 1 ): this first strand reaction was amplified by the polymerase chain reaction ( pcr ) essentially according to the method of frohman , m . a . ( 1990 ) using gen9 and gen10 primers : at a final concentration of 1 μm and a b25 adaptor primer : at a final concentration of 0 . 1 μm ( frohman , m . a . ( 1990 ); dry , i . b . and robinson , s . p . ( 1994 )) amplification involved a program of 33 cycles of denaturation at 94 ° c . for 1 min , annealing at 55 ° c . for 1 min , and elongation at 72 ° c . for 3 min . a sample of the amplified dna was run on an agarose gel and stained with ethidium bromide to determine the size of the pcr products . the remainder was run on a low melting point agarose gel and the bands of interest were excised . dna was purified from the agarose with a qiaquick pcr purification kit ( qiagen ). the purified dna was cloned into eco rv - cut bluescript sk + vector ( stratagene ) which had been t - tailed with taq polymerase and the ligated dna was introduced into e . coli dh5α by electroporation . recombinant clones which had an insert of the predicted size were selected and their dna sequence was determined by automated sequencing . two putative pineapple ppo clones ( pinpp01 ; fig1 a - e ; and pinpp02 , fig9 a - e ) were identified based on their homologies to known ppo genes . the sequence of pinppo1 was nearly identical to that of pinppo20 . the 5 ′- end of pinppo1 was obtained using a 5 ′- race system for rapid amplification of cdna ends , version 2 . 0 , from gibco - brl , according to the manufacturer &# 39 ; s instructions . specific oligonucleotide primers based on the sequences of pinppo1 and pinppo2 were used : a number of clones were sequenced and one , 5pina ( fig1 a - c ), was found to be nearly identical to pinppo1 ( fig1 a - e ) in the overlapping regions . a full - length pineapple cdna clone was isolated using a primer designed to the 5 ′- end sequence of 5pina : primers designed to the 5 ′- end of the pineapple ppo gene was used with the b25 adaptor primer as described above to amplify cdna prepared from blackheart - induced pineapple fruit rna . amplification involved a program of 33 cycles of denaturation at 94 ° c . for 1 min , annealing at 55 ° c . for 1 min , and elongation at 72 ° c . for 3 min . a sample of the amplified dna was run on an agarose gel and stained with ethidium bromide to determine the size of the pcr products . the remainder was run on a low melting point agarose gen and the bands of interest were excised . dna was purified from the agarose with a qiaquick pcr purification kit ( qiagen ). the purified dna was cloned into eco rv - cut bluescript sk + vector ( stratagene ) which had been t - tailed with taq polymerase and the ligated dna was introduced into e . coli dh5α by electroporation . recombinant clones which had an insert of the predicted size ( 2 . 2 kbp ) were selected and their dna sequence was determined by automated sequencing . a pineapple ppo clone ( pinppofl ; fig1 a - h ) was identified based on its homology to the pinppo20 ( fig8 a - c ), pinppo1 ( fig1 a - e ) and 5 pina ( fig1 a - c ) clones . the sequence of pinppofl was found to be nearly identical to that of pinppo20 , pinppo1 and 5pina in the overlapping regions . 1 . frohman , m a ( 1990 ) in “ pcr protocols : a guide to methods and applications ” ( m a innis , d h gelfrand , j j sninsky and t j white , eds ) academic press , new york , pp 28 - 38 . 2 . dry , i b and robinson , s p ( 1994 ) “ molecular cloning and characterisation of grape berry polyphenol oxidase ”. plant mol . biol . 26 , 495 - 502 . cac tgt gcg tat tgt gat ggc gcc tac gac cag atc ggc ttc ccc aac 48 his cys ala tyr cys asp gly ala tyr asp gln ile gly phe pro asn ctc gag ctc caa gtc cac aac tcc tgg ctc ttc ttc cct tgg cac cgc 96 leu glu leu gln val his asn ser trp leu phe phe pro trp his arg ttc tac ctc tac ttc cac gag agg atc ctc gga aag ctc ata ggc gac 144 gac act ttc gcc ctc cct ttc tgg aac tgg gac gcg ccc ggc ggc atg 192 aag ctg ccg tcg atc tac gcc gac cct tcg tcc tcg ctc tat gac aag 240 ttt cgc gac gcc aag cac cag ccg cca gtc ctc gtc gac ctc gac tac 288 phe arg asp ala lys his gln pro pro val leu val asp leu asp tyr aac gga acc gac cct agt ttc acc gac gca gag cag atc gat cag aac 336 asn gly thr asp pro ser phe thr asp ala glu gln ile asp gln asn ctc aag atc atg tac cgg cag gtg atc tcc aac ggc aag acg ccg ttg 384 leu lys ile met tyr arg gln val ile ser asn gly lys thr pro leu ctc ttc tta ggc tcg gct tac cgt gcc ggc gac aac cca aac ccc ggc 432 gcg ggc tcg ctc gag aac ata cca cac ggc ccc gtc cac ggg tgg act 480 ala gly ser leu glu asn ile pro his gly pro val his gly trp thr ggc gac aga agc caa ccc aat ctc gag gac atg ggc aac ttc tac tcc 528 gly asp arg ser gln pro asn leu glu asp met gly asn phe tyr ser gcg ggg cgc gac cct atc ttc ttc gcc cac cat tca aat gtc gat cgc 576 ala gly arg asp pro ile phe phe ala his his ser asn val asp arg his cys ala tyr cys asp gly ala tyr asp gln ile gly phe pro asn leu glu leu gln val his asn ser trp leu phe phe pro trp his arg phe arg asp ala lys his gln pro pro val leu val asp leu asp tyr asn gly thr asp pro ser phe thr asp ala glu gln ile asp gln asn leu lys ile met tyr arg gln val ile ser asn gly lys thr pro leu ala gly ser leu glu asn ile pro his gly pro val his gly trp thr gly asp arg ser gln pro asn leu glu asp met gly asn phe tyr ser ala gly arg asp pro ile phe phe ala his his ser asn val asp arg ttg ccg ttt tgg aat tgg gac gcg ccc ggc ggc atg aag ctg ccg tcg 48 leu pro phe trp asn trp asp ala pro gly gly met lys leu pro ser atc tac gcc gac cct tcg tcc tcg ctc tat gac aag ttt cgc gac gcc 96 aag cac cag ccg ccg gtc ctc gtc gac ctc gac tac aac gga acc gac 144 lys his gln pro pro val leu val asp leu asp tyr asn gly thr asp cct agt ttc acc gac gca gag cag atc gat cag aac ctc aag atc atg 192 pro ser phe thr asp ala glu gln ile asp gln asn leu lys ile met tac cgg cag gtg atc tcc aac ggc aag acg ccg ttg ctc ttc tta ggc 240 tyr arg gln val ile ser asn gly lys thr pro leu leu phe leu gly tcg gct tac cgt gcc ggc gac aac cca aac ccc ggc gcg ggc tcg ctc 288 gag aac ata cca cac ggc ccc gtc cac ggg tgg act ggc gac aga agc 336 glu asn ile pro his gly pro val his gly trp thr gly asp arg ser caa ccc aat ctc gag gac atg ggc aac ttc tac tcc gcg ggg cgc gac 384 gln pro asn leu glu asp met gly asn phe tyr ser ala gly arg asp cct atc ttc ttc gcc cac cat tca aat gtc gat agc atg tgg 426 pro ile phe phe ala his his ser asn val asp ser met trp leu pro phe trp asn trp asp ala pro gly gly met lys leu pro ser lys his gln pro pro val leu val asp leu asp tyr asn gly thr asp pro ser phe thr asp ala glu gln ile asp gln asn leu lys ile met tyr arg gln val ile ser asn gly lys thr pro leu leu phe leu gly glu asn ile pro his gly pro val his gly trp thr gly asp arg ser gln pro asn leu glu asp met gly asn phe tyr ser ala gly arg asp pro ile phe phe ala his his ser asn val asp ser met trp g ttg ctc ttc tta ggc tcg gct tac cgt gcc ggc gac aac cca aac ccc 49 ggc gcg ggc tcg ctc gag aac ata cca cac ggc ccc gtc cac ggg tgg 97 gly ala gly ser leu glu asn ile pro his gly pro val his gly trp act ggc gac aga aac caa ccc aat ctc gag gac atg ggc aac ttc tac 145 thr gly asp arg asn gln pro asn leu glu asp met gly asn phe tyr tcc gcg ggg cgc gac cct atc ttc ttc gcc cac cat tca aac gtc gac 193 ser ala gly arg asp pro ile phe phe ala his his ser asn val asp cgc atg tgg tac ttg tgg aag aag ctc ggc ggg aag cat cag gac ttt 241 arg met trp tyr leu trp lys lys leu gly gly lys his gln asp phe aac gat aag gac tgg ctc aac acc acc ttc ctc ttc tac gac gag aat 289 gct gac tta gtt cga gtc acc ctc aag gac tgc ttg cag ccg gag tgg 337 ala asp leu val arg val thr leu lys asp cys leu gln pro glu trp ctt cgt tac gat tac caa gac gtc gag atc ccg tgg ctg aag acc cgg 385 leu arg tyr asp tyr gln asp val glu ile pro trp leu lys thr arg ccg act ccc aaa gcc ttg aag gcg cag aaa acc gca gcg aaa aca ctg 433 aaa gct aca gca gag acg ccg ttc ccg gtg acg ctg caa tcc gcg gtg 481 agc acg acg gtg agg agg ccc aag gta tcg agg agc ggc aag gag aag 529 gaa gag gaa gag gag gtc ctc atc gtg gag ggg atc gag ttc gac cgc 577 gac tac ttc ata aag ttc gac gtc ttc gtg aac gcc acc gag ggt gag 625 asp tyr phe ile lys phe asp val phe val asn ala thr glu gly glu ggc atc acg ccg ggc gcc agc gag ttc gcg ggc agc ttc gtc aac gtc 673 ctc tgc ctg ggg atc act gac ctg ctc gag gac atc ggg gcg gag gac 769 gac gac agc gtg ctc gtc acc atc gtc ccg aaa gcc gga aag ggc aag 817 gtg tcg gtc gcc ggc ctc cgc atc gat ttc cca aat tgaagtaata 863 val ser val ala gly leu arg ile asp phe pro asn gly ala gly ser leu glu asn ile pro his gly pro val his gly trp thr gly asp arg asn gln pro asn leu glu asp met gly asn phe tyr ser ala gly arg asp pro ile phe phe ala his his ser asn val asp arg met trp tyr leu trp lys lys leu gly gly lys his gln asp phe ala asp leu val arg val thr leu lys asp cys leu gln pro glu trp leu arg tyr asp tyr gln asp val glu ile pro trp leu lys thr arg asp tyr phe ile lys phe asp val phe val asn ala thr glu gly glu val ser val ala gly leu arg ile asp phe pro asn g ttg ctc ttc tta ggc tcg gct tac cgt gcc ggt gac cag cct aac ccc 49 leu leu phe leu gly ser ala tyr arg ala gly asp gln pro asn pro ggc gcg gga tcc atc gag aac atg ccg cac aac aac gtg cac ttg tgg 97 gly ala gly ser ile glu asn met pro his asn asn val his leu trp acc ggc gac cgc acc cag ccc aac ttc gag aac atg ggc acc ttc tac 145 thr gly asp arg thr gln pro asn phe glu asn met gly thr phe tyr gcg gcg gcg cgc gac ccc atc ttc ttc gcc cac cac gcc aac atc gac 193 cga atg tgg tac ctg tgg aag aag ctc agc agg aag cac cag gac ttc 241 arg met trp tyr leu trp lys lys leu ser arg lys his gln asp phe aat gac tcg gac tgg ctc aaa gct tcc ttc ctc ttc tac gac gag aac 289 gcc gac tta gtt cgg gtc acg gtc aag gac tgc ttg gag acc gag tgg 337 ctg cgc tac acg tac caa gac gtg aag atc cca tgg gcg aac acc cga 385 leu arg tyr thr tyr gln asp val lys ile pro trp ala asn thr arg ccg act ccc aag ctc gcc aag gcg agg aaa gcc ggc agc aga tcg ctg 433 aaa gcc acc gcg gag gtg cag ttc cct gtg acg ctg gaa tcc ccg gtc 481 aaa gtg acg gtg aag agg ccc aag gtg ggg agg agc ggc aag gag aag 529 gaa gat gag gag gag ata ctc ata gtg gag ggg atc gag ttc gac cgc 577 gac tac ttc atc aag ttc gac gtc ttc gtg aac gcg acg gag ggc gac 625 asp tyr phe ile lys phe asp val phe val asn ala thr glu gly asp ggc atc acg gcc ggg gcc agt gag ttc gcc ggc agc ttc gtg aac gtc 673 ccg cac aag cac aag cac cgc aag gat gag aat aag ctg aag acg agg 721 ctg tgt ctg gga atc acc gac ctg ctc gag gac atc ggc gcg gag gac 769 gac gac agc gtg ctc gtc acc atc gtg ccg aag gca ggc aaa gga aag 817 gtg tcc gtc ggc ggt ctt cgg att gac ttt tcc aag tgaggaaata 863 leu leu phe leu gly ser ala tyr arg ala gly asp gln pro asn pro gly ala gly ser ile glu asn met pro his asn asn val his leu trp thr gly asp arg thr gln pro asn phe glu asn met gly thr phe tyr arg met trp tyr leu trp lys lys leu ser arg lys his gln asp phe leu arg tyr thr tyr gln asp val lys ile pro trp ala asn thr arg asp tyr phe ile lys phe asp val phe val asn ala thr glu gly asp gat ccg acg ttt gcg ttg cca tat tgg aac tgg gat cat cca aag ggc 48 asp pro thr phe ala leu pro tyr trp asn trp asp his pro lys gly atg cgt ttg cca cac atg ttt gat caa cca aac gtg tac cct gat ctt 96 met arg leu pro his met phe asp gln pro asn val tyr pro asp leu tac gat cca aga cgt aac caa gaa cac cgc ggt tct gta atc atg gac 144 tyr asp pro arg arg asn gln glu his arg gly ser val ile met asp ctt ggt cat ttt ggt caa gac gtg aaa gga act gac ttg caa atg atg 192 agc aat aac ctt act cta atg tat cgt caa atg att acc aat tca cca 240 tgt cca caa ctc ttt ttc ggt aag cca tat tgt acg gaa gtt gga ccc 288 cys pro gln leu phe phe gly lys pro tyr cys thr glu val gly pro aaa cca ggg cag gga gct att gaa aac atc cct cat act cct gtc cac 336 lys pro gly gln gly ala ile glu asn ile pro his thr pro val his att tgg gtt ggt agt aag cct aat gag aat aac tgt aaa aac ggt gaa 384 gat atg gga aat ttc tat tca gct ggt aag gat cct gct ttc tat agt 432 cac cat gca aat gta gat cgc atg tgg aca ata tgg aaa aca tta gga 480 his his ala asn val asp arg met trp thr ile trp lys thr leu gly gga aaa cgc aag gac atc aac aag cca gat tat ttg aac act gag ttc 528 gly lys arg lys asp ile asn lys pro asp tyr leu asn thr glu phe asp pro thr phe ala leu pro tyr trp asn trp asp his pro lys gly met arg leu pro his met phe asp gln pro asn val tyr pro asp leu tyr asp pro arg arg asn gln glu his arg gly ser val ile met asp cys pro gln leu phe phe gly lys pro tyr cys thr glu val gly pro lys pro gly gln gly ala ile glu asn ile pro his thr pro val his his his ala asn val asp arg met trp thr ile trp lys thr leu gly gly lys arg lys asp ile asn lys pro asp tyr leu asn thr glu phe tg cac tgt gcg tat tgc aac ggt gct tac aaa att ggt ggc aaa gag 47 tta caa gtc cat ttc tcg tgg ctt ttt ttc cct ttt cat aga tgg tac 95 ttg tac ttc tat gaa aga atc ttg ggc tct tta att aat gat cct act 143 leu tyr phe tyr glu arg ile leu gly ser leu ile asn asp pro thr ttt ggt ttg cca tat tgg aac tgg gac cat cca aag ggc atg cgt ata 191 phe gly leu pro tyr trp asn trp asp his pro lys gly met arg ile cct ccc atg ttc gat cgt gaa ggg tct tcc ctt tac gac gaa aaa cgt 239 pro pro met phe asp arg glu gly ser ser leu tyr asp glu lys arg aac caa agt cac cgt aat gga acc ata att gat ctt ggt cat ttc ggt 287 asn gln ser his arg asn gly thr ile ile asp leu gly his phe gly caa gaa gtc caa aca act caa ctg cag cag atg act aat aac tta act 335 ata atg tat cgt caa atg ata act aat gct cct tgc ccc ttg ctc ttc 383 ile met tyr arg gln met ile thr asn ala pro cys pro leu leu phe ttt ggt cag cct tac cct cta gga act gat ccc agt cca ggg atg ggc 431 act att gaa aac atc cct cat act cct gtc cac att tgg gtt ggt agt 479 agg ctt gat gag aat aat acg aaa cac ggt gag gat atg ggt aat ttt 527 arg leu asp glu asn asn thr lys his gly glu asp met gly asn phe tac tcg gcc ggt tta gac ccg ctt ttc tat tcc cat cac gcc aat gtg 575 tyr ser ala gly leu asp pro leu phe tyr ser his his ala asn val gac cgg atg tgg tcc gag tgg aaa gcc tta gga ggg aaa aga agg gat 623 ctc acg cac aaa gat tgg ttg aac tcc gag ttc ttt ttc tac gat gaa 671 leu thr his lys asp trp leu asn ser glu phe phe phe tyr asp glu tyr phe tyr glu arg ile leu gly ser leu ile asn asp pro thr phe gly leu pro tyr trp asn trp asp his pro lys gly met arg ile pro pro met phe asp arg glu gly ser ser leu tyr asp glu lys arg asn gln ser his arg asn gly thr ile ile asp leu gly his phe gly gln met tyr arg gln met ile thr asn ala pro cys pro leu leu phe phe ile glu asn ile pro his thr pro val his ile trp val gly ser arg leu asp glu asn asn thr lys his gly glu asp met gly asn phe tyr ser ala gly leu asp pro leu phe tyr ser his his ala asn val asp thr his lys asp trp leu asn ser glu phe phe phe tyr asp glu tg cat tgt gcg tat tgc aac gat gct tac aca atg ggt gac caa aag 47 his cys ala tyr cys asn asp ala tyr thr met gly asp gln lys tta caa gtt cac caa tcg tgg ctt ttc ttc ccg ttt cat aga tgg tac 95 ttg tac ttc tac gag aga atc ttg ggc tcc ctc atc gat gat cca act 143 leu tyr phe tyr glu arg ile leu gly ser leu ile asp asp pro thr ttt gct ctg cca tat tgg aac tgg gac cat cca agc ggc atg cgt ttg 191 phe ala leu pro tyr trp asn trp asp his pro ser gly met arg leu cct gct atg ttc gat gtc gaa ggt tct tcc ctc tac gat gca aga cgt 239 pro ala met phe asp val glu gly ser ser leu tyr asp ala arg arg aat cca cat gtc cgt aat gga acc ata atc gat ctt ggt ttt ttc ggt 287 asn pro his val arg asn gly thr ile ile asp leu gly phe phe gly gat gaa gtc aaa act aat gaa ata cag atg ata act aac aac tta att 335 cta atg tat cgt caa atg ata act aat gct cca tgc ccg ctg ttg ttc 383 leu met tyr arg gln met ile thr asn ala pro cys pro leu leu phe ttc gga gag cct tac aga ttc gga tct aaa ccc aat ccg ggg cag gga 431 acc att gaa aac att cct cat act ccg gtt cac att tgg act ggt act 479 gtg cgg tgt acg gat ttg ggt aat tgt gtg cca tca tac ggt gag gat 527 val arg cys thr asp leu gly asn cys val pro ser tyr gly glu asp atg ggt aat ttc tac tca gct ggt tta gac cca gtt ttt tac agc cac 575 met gly asn phe tyr ser ala gly leu asp pro val phe tyr ser his cac gcc aat gtg gac cgc atg tgg aat gaa tgg aaa gca cta gga ggg 623 his ala asn val asp arg met trp asn glu trp lys ala leu gly gly aaa aga agg gat ctc aca gac aat gat tgg tta aac tcg gag ttc ttt 671 his cys ala tyr cys asn asp ala tyr thr met gly asp gln lys leu tyr phe tyr glu arg ile leu gly ser leu ile asp asp pro thr phe ala leu pro tyr trp asn trp asp his pro ser gly met arg leu pro ala met phe asp val glu gly ser ser leu tyr asp ala arg arg asn pro his val arg asn gly thr ile ile asp leu gly phe phe gly asp met tyr arg gln met ile thr asn ala pro cys pro leu leu phe phe gly glu pro tyr arg phe gly ser lys pro asn pro gly gln gly thr arg cys thr asp leu gly asn cys val pro ser tyr gly glu asp met gly asn phe tyr ser ala gly leu asp pro val phe tyr ser his his ala asn val asp arg met trp asn glu trp lys ala leu gly gly lys tg cat tgt gcg tac tgc gac ggc gcg tat gac caa atc ggc ttc ccc 47 gat ctc gag atc cag atc cac aac tcg tgg ctc ttc ttt cct tgg cac 95 asp leu glu ile gln ile his asn ser trp leu phe phe pro trp his cgg ttc tac ctc tac ttc aac gag cgc ata ctc ggg aaa ctt atc ggc 143 gac gac acg ttc gcg ctg cct ttc tgg aac tgg gac gcg ccg ggg ggc 191 atg cag ttc ccg tct atc tac acg gac cct tca tcc tcg cta tat gac 239 aag ctg cgt gat gcg aag cac cag ccg ccg act ttg att gac ctc gac 287 tac aat ggc acc gat cct acc ttc tcc cct gaa gaa cag att aac cac 335 tyr asn gly thr asp pro thr phe ser pro glu glu gln ile asn his aac ctc gcc gtc atg tac cga cag gtg ata tcc agt gga aag aca cca 383 asn leu ala val met tyr arg gln val ile ser ser gly lys thr pro gag ctg ttt atg ggc tca gcg tac cgc gcc ggt gac cag cct gac ccc 431 glu leu phe met gly ser ala tyr arg ala gly asp gln pro asp pro ggc gca ggt tct gta gag cag aag ccg cac ggc ccg gtg cat gtg tgg 479 aca ggt gat cgc aac cag ccc aat cgc gaa gac atg ggc acg ctc tac 527 thr gly asp arg asn gln pro asn arg glu asp met gly thr leu tyr tcg gcg gcg tgg gac ccc gtt ttt ttc gca cac cac ggc aac atc gac 575 ser ala ala trp asp pro val phe phe ala his his gly asn ile asp cgc atg tgg tac gtg tgg agg aac ctt ggc ggc aag cac cgc aac ttc 623 arg met trp tyr val trp arg asn leu gly gly lys his arg asn phe acc gac ccc gac tgg ctc aac gcg tcc ttc ctg ttc tac gac gaa aa 670 thr asp pro asp trp leu asn ala ser phe leu phe tyr asp glu leu glu ile gln ile his asn ser trp leu phe phe pro trp his arg leu arg asp ala lys his gln pro pro thr leu ile asp leu asp tyr asn gly thr asp pro thr phe ser pro glu glu gln ile asn his asn leu ala val met tyr arg gln val ile ser ser gly lys thr pro glu leu phe met gly ser ala tyr arg ala gly asp gln pro asp pro gly ala gly ser val glu gln lys pro his gly pro val his val trp thr gly asp arg asn gln pro asn arg glu asp met gly thr leu tyr ser ala ala trp asp pro val phe phe ala his his gly asn ile asp arg met trp tyr val trp arg asn leu gly gly lys his arg asn phe thr ttg ccg ttt tgg aat tgg gac gcg ccg ggg ggc atg cag atc ccg gcc 48 leu pro phe trp asn trp asp ala pro gly gly met gln ile pro ala atc tac gcc gac gct tcg tcc ccg ctc tac gac aag ctg cgc aat gcg 96 aag cac cag ccg ccg act ttg gtc gac ctc gac tac aac ggc acc gac 144 lys his gln pro pro thr leu val asp leu asp tyr asn gly thr asp ccg acc ttc acc cct gag cag cag atc gcc cac aac ctc acc atc atg 192 pro thr phe thr pro glu gln gln ile ala his asn leu thr ile met tac cga cag gtg ata tcc ggc ggg aag acg ccg gag ttg ttt atg ggc 240 tyr arg gln val ile ser gly gly lys thr pro glu leu phe met gly gcg gcg tac cgc gcg ggc gac gcg cca gac ccg ggc gca ggc act cta 288 gag ctc gtg ccg cac aac acg atg cat ttg tgg acc ggc gac ccc aac 336 glu leu val pro his asn thr met his leu trp thr gly asp pro asn caa ccc aac gac gaa gac atg ggc acg ttc tac gcg gcg gcg cgg gac 384 gln pro asn asp glu asp met gly thr phe tyr ala ala ala arg asp ccc atc ttc ttc gcc cac cac ggc aac gtc gac cgc atg tgg tac gtg 432 pro ile phe phe ala his his gly asn val asp arg met trp tyr val tgg cgg aaa ctc ggg ggc acg cac cgc gat ttc acc gac ccc gac tgg 480 ctc aac gcg tcc ttc ctc ttc tac gac gag aac gcg cag ctc gtc cgc 528 leu asn ala ser phe leu phe tyr asp glu asn ala gln leu val arg gtc aaa gta aag gac tgc ttg agc gcc gac gcg ctg cgg tac acg tac 576 cag gac gtc gac atc ccg tgg atc agt gcg aag ccg acg ccg aag aaa 624 aca ccg ggg ggc gct gcg cct tcc acg aca gag gct ata ttt ccg gtg 672 gtg ctg gat aag ccg gtg agc tct acg gtg gcg agg ccg aag acg ggg 720 agg agt act ggg gag gag gag gtg ttg gtg gtg gag gga atc gag ctg 768 gac aag gac gtg gcc gtg aag ttc gac gtg tat ata aac gcg ccg gac 816 aac gaa ggg gtg ggg ccg gag gcg agc gag ttc gca ggg agc ttc gtc 864 cag gtg ccg cac aag cac aag aag ggg aag aag gag aag gcg agg att 912 aaa acg acg ctc agg ctc ggg ata acg gac ctg ctc gag gac atc ggc 960 gcc gag gac gac gag agc gtg ctc gtc acg ctc gtg ccg agg ata ggc 1008 ala glu asp asp glu ser val leu val thr leu val pro arg ile gly gag ggg ttg gtc aag gtt ggt ggg cta agg atc gat ttc tcc aag 1053 leu pro phe trp asn trp asp ala pro gly gly met gln ile pro ala lys his gln pro pro thr leu val asp leu asp tyr asn gly thr asp pro thr phe thr pro glu gln gln ile ala his asn leu thr ile met tyr arg gln val ile ser gly gly lys thr pro glu leu phe met gly glu leu val pro his asn thr met his leu trp thr gly asp pro asn gln pro asn asp glu asp met gly thr phe tyr ala ala ala arg asp pro ile phe phe ala his his gly asn val asp arg met trp tyr val leu asn ala ser phe leu phe tyr asp glu asn ala gln leu val arg ala glu asp asp glu ser val leu val thr leu val pro arg ile gly c ggt atc gat aag ctt gat cca gtg cct ggt tta ggt gta ttc act atg 49 gcc acc ctc tct aaa cta gct tcc caa cca ata aca cct cca ctc tcc 97 ccg ctc cct cct ttg cat gct cct tct ctc acc aaa agc ttc acc acc 145 acc ttc ctc tcc cct gta ggg gtc cca aac cac ccc gtc ata aga tct 193 thr phe leu ser pro val gly val pro asn his pro val ile arg ser cat gca aat cta agg agc aac aag aga atg ccg aca agc ctg cgg gcc 241 gca tcg ccc gcc gcg acc tac tcc tgg gcc ctc ggc ggg ctt tac ggt 289 gcc acc act ggg ctc ggc ctc aac cgt cga gcg gcc gcc gcc cct atc 337 ctg gct ccc gac ctc tca act tgt ggg ccg cct gcc gac ctc cct gcc 385 tcc gcc cga ccg aca gtt tgc tgc ccg cca tac caa tcc acc atc atc 433 gac ttc aag ctc ccc ccg cga tct gct ccg ctt cgc gtc cgg cct gcg 481 gcc cac ttg gtt gac gcc gac tac ctg gcc aag tat aag aag gcg gtc 529 gag ctc atg agg gcc ctg ccg gcc gac gac ccg cgc aac ttc gta cag 577 glu leu met arg ala leu pro ala asp asp pro arg asn phe val gln caa gcg aaa gtg cac tgt gcg tat tgc gac ggc gcg tat gac caa atc 625 ggc ttc ccc gat ctc gag atc cag atc cac aac tcg tgg ctc ttc ttt 673 gly phe pro asp leu glu ile gln ile his asn ser trp leu phe phe cct tgg cac cgg ttc tac ctc tac tcc aac gag cgc ata ctc ggg aaa 721 pro trp his arg phe tyr leu tyr ser asn glu arg ile leu gly lys ctt atc ggc gac gac acg ttc gcg ctg cct ttc tgg aac tgg gac gcg 769 ccg ggg ggc atg cag ttc ccg tct atc tac aca gac cct tca tcc tcg 817 cta tat gac aag ctg cgt gat gcg aag cac cag ccg ccg act ttg att 865 leu tyr asp lys leu arg asp ala lys his gln pro pro thr leu ile gac ctc gac tac aat ggc acc gat cct acc ttc tcc cct gaa gaa cag 913 asp leu asp tyr asn gly thr asp pro thr phe ser pro glu glu gln att aac cac aac ctc gcc gtc atg tac cga cag gtg ata tcc agt gga 961 ile asn his asn leu ala val met tyr arg gln val ile ser ser gly aag acg cca gag ctg ttt atg ggc tca gcg tac cgc gcc ggt gac cag 1009 lys thr pro glu leu phe met gly ser ala tyr arg ala gly asp gln cct gac ccc ggc gca ggc tct gta gag cag aag ccg cac ggc ccg gtg 1057 cat gtg tgg aca ggt gat cgc aac cag ccc aat cgc gaa gac atg ggc 1105 his val trp thr gly asp arg asn gln pro asn arg glu asp met gly acg ctc tac tcg gcg gcg tgg gac ccc gtc ttc ttc gca cac cac ggc 1153 thr leu tyr ser ala ala trp asp pro val phe phe ala his his gly aac atc gac cgc atg tgg tac gtg tgg agg aac ctt ggc ggc aag cac 1201 asn ile asp arg met trp tyr val trp arg asn leu gly gly lys his cgc aac ttc acc gac ccc gac tgg ctc aac gcg tcc ttc ctg ttc tat 1249 arg asn phe thr asp pro asp trp leu asn ala ser phe leu phe tyr gat gag aat gcg cag ctc gtc cgt gtt aaa gta aaa gac tgc tta gag 1297 gcc gac gca atg cgg tac aca tac cag gat gta gag atc ccg tgg ctc 1345 ala asp ala met arg tyr thr tyr gln asp val glu ile pro trp leu aaa gca aag ccg acg cca aag agc gcc cta cag aag ata aag agc aag 1393 gta tcg acg ctg aag gca aca cca agg ggg acg acg act acc aca gca 1441 gag act aca ttt ccg gtg gtg ctg gat aag ccg gtg agt gca aca gtg 1489 gct aga ccg aag gcc agg agg agt ggg aag gag aag gaa gaa gag gag 1537 aag ttt gat gtg tat ata aac tcg ccg gag cac gaa ggg gtg ggg ccg 1633 lys phe asp val tyr ile asn ser pro glu his glu gly val gly pro gag gcg agt gag ttc gca ggg agc ttc gtc cac gtg cca cac aag cac 1681 aag aag gcg aag aag ggg aag gag atg gcc agg atg aac aca agg ctt 1729 aag ctc ggg ata acg gac ctg ctc gag gac atc ggc gct gag gac gac 1777 gag agc gtg ctc atc acg ctc gtg ccc agg agc ggc aag gga atg gtg 1825 glu ser val leu ile thr leu val pro arg ser gly lys gly met val aag gtt gga ggg cta agg att gat ttc tcc aag tgatgagcat attgtgaaga 1878 thr phe leu ser pro val gly val pro asn his pro val ile arg ser glu leu met arg ala leu pro ala asp asp pro arg asn phe val gln gly phe pro asp leu glu ile gln ile his asn ser trp leu phe phe pro trp his arg phe tyr leu tyr ser asn glu arg ile leu gly lys leu tyr asp lys leu arg asp ala lys his gln pro pro thr leu ile asp leu asp tyr asn gly thr asp pro thr phe ser pro glu glu gln ile asn his asn leu ala val met tyr arg gln val ile ser ser gly lys thr pro glu leu phe met gly ser ala tyr arg ala gly asp gln his val trp thr gly asp arg asn gln pro asn arg glu asp met gly thr leu tyr ser ala ala trp asp pro val phe phe ala his his gly asn ile asp arg met trp tyr val trp arg asn leu gly gly lys his arg asn phe thr asp pro asp trp leu asn ala ser phe leu phe tyr ala asp ala met arg tyr thr tyr gln asp val glu ile pro trp leu lys phe asp val tyr ile asn ser pro glu his glu gly val gly pro glu ser val leu ile thr leu val pro arg ser gly lys gly met val ctg ctg ttg aac tct agc ttc acc ggt gct tcc tct gca tgc ctc ctc 106 caa cgg gaa agg tcc cgc cgc cgc cgc ctc cac gtc cct ggc gtg aca 154 tgc cgc cag ggc agt aat ggt gac cgc aga gat gcc gcc ccc cag cag 202 cag tcg ccg ccg ctg ctg gat cgg cgc gac atg ctg ttg ggt tta gga 250 ggg ctt tac ggc gtg acc gca gga ccc aag gtt ctg gcg gcg ccg ata 298 atg ccg ccg gat ctg tcc aag tgc tac cct gcc acc gca cct gcc ctc 346 gac aac aaa tgc tgc ccg cct tac gac ccc ggc gag acg atc tcg gag 394 asp asn lys cys cys pro pro tyr asp pro gly glu thr ile ser glu tac agc ttc cct gct acg ccc ctc cgg gtg cgg cgg ccg gcc cat atc 442 tyr ser phe pro ala thr pro leu arg val arg arg pro ala his ile gtg aag gac gat cag gag tat atg gac aag tac aag gag gca gtg agg 490 agg atg aag aat ctg ccg gca gac cac cct tgg aac tac tac cag cag 538 arg met lys asn leu pro ala asp his pro trp asn tyr tyr gln gln gcg aac atc cac tgc cag tat tgc aac tac gcc tac cac cag caa aat 586 acc gac gac gtg ccc atc cag gtc cac ttc agc tgg atc ttc ctc cca 634 thr asp asp val pro ile gln val his phe ser trp ile phe leu pro tgg cac cgc tac tac ctc cac ttc tac gaa agg atc ctc ggc aag ctc 682 gac ggg atg acg ttc ccc gcc atc ttc cag gat gcg gca tcc ccg ctg 778 asp gly met thr phe pro ala ile phe gln asp ala ala ser pro leu tac gac ccg aga cgc gac caa cgc cac gtc aag gac ggc aag atc ctc 826 tyr asp pro arg arg asp gln arg his val lys asp gly lys ile leu gac ctc aag tac gcc tac acc gaa aac act gca tcc gac agc gag atc 874 ata cgg gag aac ctc tgc ttc ata cag aag acg ttc aag cac agc ctg 922 ile arg glu asn leu cys phe ile gln lys thr phe lys his ser leu tcg ctg gcg gaa ctg ttc atg ggg gat ccc gtg cgc gcg ggg gag aag 970 ser leu ala glu leu phe met gly asp pro val arg ala gly glu lys gag atc cag gag gct aat ggg cag atg gaa gtc atc cac aat gcg gcg 1018 cac atg tgg gtc gga gag ccg gac gga tac aag gaa aac atg ggg gac 1066 his met trp val gly glu pro asp gly tyr lys glu asn met gly asp ttc tcc acc gcc gcc cgc gat tct gtt ttc ttc tgc cac cat tcc aat 1114 gtc gac cgc atg tgg gac atc tac cgc aac ctc cgc ggc aac cgc gtc 1162 gag ttc gaa gac aac gac tgg ttg gac agc acc ttc ctc ttc cac gac 1210 gag aac gaa cag ctc gtc aaa gtc aag atg agc gac tgc ctc aac ccg 1258 glu asn glu gln leu val lys val lys met ser asp cys leu asn pro acc aag ctt cgg tac acg ttc gag caa gtt ccc ctc cca tgg ctg ggc 1306 thr lys leu arg tyr thr phe glu gln val pro leu pro trp leu gly aaa atc aat tgc cag aag acg gca gag acg aag tcc aag gcc acg acg 1354 gag ctg tcg ctg acg cgc gtg aac gaa ttc ggg acg acg gcc cag gca 1402 glu leu ser leu thr arg val asn glu phe gly thr thr ala gln ala ctc gac gcg agc aac ccg ctg cgg gtg atc gtg gca agg ccg aag aag 1450 aac cgc aag aag aag gag aag caa gag aag gtg ggg gtg att cag atc 1498 aag gat att aag gtg acc acc aac gag aca gct cgc ttc gac gtc tat 1546 lys asp ile lys val thr thr asn glu thr ala arg phe asp val tyr gtc gcg gtt cct tac ggt gac ctc gcc gga ccc gac tac ggc gag ttc 1594 gcg ggc agc tac gtg agg ctg gcg cat agg atg aag gga agc gac ggg 1642 ala gly ser tyr val arg leu ala his arg met lys gly ser asp gly acc gaa aag cag ggc ccc aag aag aag gga aaa ctc aag ctg ggt att 1690 acg ccg ctg ctc gag gac atc gat gct gag gac gcc gac aag ttg gtg 1738 gtc acc ctg gtt ctc cgc act ggg agc gtc acc gtg ggg gga gtt tcc 1786 atc aat ctc ctg cag aca gat tct acc gcc gcc atc taaatgatgg 1832 val thr cys arg gln gly ser asn gly asp arg arg asp ala ala pro pro ile met pro pro asp leu ser lys cys tyr pro ala thr ala pro ala leu asp asn lys cys cys pro pro tyr asp pro gly glu thr ile val arg arg met lys asn leu pro ala asp his pro trp asn tyr tyr gln asn thr asp asp val pro ile gln val his phe ser trp ile phe thr lys asp gly met thr phe pro ala ile phe gln asp ala ala ser glu ile ile arg glu asn leu cys phe ile gln lys thr phe lys his ser leu ser leu ala glu leu phe met gly asp pro val arg ala gly ala ala his met trp val gly glu pro asp gly tyr lys glu asn met ser asn val asp arg met trp asp ile tyr arg asn leu arg gly asn his asp glu asn glu gln leu val lys val lys met ser asp cys leu asn pro thr lys leu arg tyr thr phe glu gln val pro leu pro trp leu gly lys ile asn cys gln lys thr ala glu thr lys ser lys ala gln ile lys asp ile lys val thr thr asn glu thr ala arg phe asp glu phe ala gly ser tyr val arg leu ala his arg met lys gly ser aatgtggatc gg atg tgg acg gtg tgg aag aag ctg cac ggc gac aag ccg 51 gag ttc gtc gac cag gag tgg ctc gag tct gaa ttc acc ttc tac gac 99 gag aat gtg cgc ctg cgc agg atc aag gtg cgc gac gtg ttg aac ata 147 gac aaa ctc agg tac cgg tac gaa gac atc gac atg cca tgg ctc gct 195 asp lys leu arg tyr arg tyr glu asp ile asp met pro trp leu ala gca cgt ccc aag cct tcc gtt cac cct aag atc gcg cgc gac ata ttg 243 aag aag cgt aat ggc gaa ggc gta ctg aga atg ccc ggc gaa acg gat 291 lys lys arg asn gly glu gly val leu arg met pro gly glu thr asp cgt tca caa ctc tcc gaa gat ggt agc tgg aca ctg gac aag agc atc 339 arg ser gln leu ser glu asp gly ser trp thr leu asp lys ser ile acc gtg agg gtt gac agg cca agg atc aac agg aca ggg caa gaa aaa 387 thr val arg val asp arg pro arg ile asn arg thr gly gln glu lys gag gaa gaa gag gag atc tta ttg gtc tac gga atc gat act aag aga 435 agc aga ttc gtc aaa ttc gat gtg ttc atc aac gtc gtc gac gaa acc 483 gtg ctg aac cca aag tcg agg gag ttc gca ggg acc ttc gtc aat ctc 531 val leu asn pro lys ser arg glu phe ala gly thr phe val asn leu cac cac gtc tcg agg acg aaa agc cat gag gat ggc ggc gtg ggt tcg 579 aag atg aaa agc cac ctt aag ctc ggt ata tcg gaa ctt ttg gaa gac 627 ctc gag gca gac gaa gat gat tgc atc tgg gtg aca ctg gtg cca aga 675 leu glu ala asp glu asp asp cys ile trp val thr leu val pro arg ggc ggc acg ggg gtc aac acc acc gta gac ggc gtc cgg atc gac tac 723 met trp thr val trp lys lys leu his gly asp lys pro glu phe val asp gln glu trp leu glu ser glu phe thr phe tyr asp glu asn val asn gly glu gly val leu arg met pro gly glu thr asp arg ser gln leu ser glu asp gly ser trp thr leu asp lys ser ile thr val arg val asp arg pro arg ile asn arg thr gly gln glu lys glu glu glu glu glu ile leu leu val tyr gly ile asp thr lys arg ser arg phe pro lys ser arg glu phe ala gly thr phe val asn leu his his val asp glu asp asp cys ile trp val thr leu val pro arg gly gly thr tg cac tgt gcg tat tgc gac ggc gcg tat gac caa atc ggc ttc ccc 47 gat ctc gag atc cag atc cac aac tcg tgg ctc ttc ttt cct tgg cac 95 asp leu glu ile gln ile his asn ser trp leu phe phe pro trp his cgg ttc tac ctc tac tcc aac gag cgc ata ctc ggg aaa ctt atc ggc 143 gac gac acg ttc gcg ctg cct ttc tgg aac tgg gac gcg ccg ggg ggc 191 atg cag ttc ccg tct atc tac acg gac cct tca tcc tcg cta tat gac 239 aag ctg cgt gat gcg aag cac cag ccg ccg act ttg att gac ctc gac 287 tac aat ggc acc gat cct acc ttc tcc cct gaa gaa cag att aac cac 335 tyr asn gly thr asp pro thr phe ser pro glu glu gln ile asn his aac ctc gcc gtc atg tac cga cag gtg ata tcc agt gga aag aca cca 383 asn leu ala val met tyr arg gln val ile ser ser gly lys thr pro gag ctg ttt atg ggc tca gcg tac cgc gcc ggt gac cag cct gac ccc 431 glu leu phe met gly ser ala tyr arg ala gly asp gln pro asp pro ggc gca ggc tct gta gag cag aag ccg cac ggc ccg gtg cat gtg tgg 479 aca ggt gat cgc aac cag ccc aat cgc gaa gac atg ggc acg ctc tac 527 thr gly asp arg asn gln pro asn arg glu asp met gly thr leu tyr tcg gcg gcg tgg gac ccc gtc ttc ttc gca cac cac ggc aac atc gac 575 ser ala ala trp asp pro val phe phe ala his his gly asn ile asp cgc atg tgg tac gtg tgg agg aac ctt ggc ggc aag cac cgc aac ttc 623 arg met trp tyr val trp arg asn leu gly gly lys his arg asn phe acc gac ccc gac tgg ctc aac gcg tcc ttc ctg ttc tat gat gag aat 671 thr asp pro asp trp leu asn ala ser phe leu phe tyr asp glu asn gcg cag ctc gtc cgt gtt aaa gta aaa gac tgc tta gag gcc gac gca 719 atg cgg tac aca tac cag gat gta gag atc ccg tgg ctc aaa gca aag 767 met arg tyr thr tyr gln asp val glu ile pro trp leu lys ala lys ccg acg cca aag agc gcc cta cag aag ata aag agc aag gta tcg acg 815 ctg aag gca aca cca agg ggg acg acg act acc aca gca gag act aca 863 ttt ccg gtg gtg ctg gat aag ccg gtg agt gca aca gtg gct aga ccg 911 aag gcc agg agg agt ggg aag gag aag gaa gaa gag gag gag gtg ttg 959 gtg gtg gag gga atc gag ttg gag aag gac gtg ttc gtg aag ttt gat 1007 gtg tat ata aac tcg ccg gag cac gaa ggg gtg ggg ccg gag gcg agt 1055 gag ttc gca ggg agc ttc gtc cac gtg cca cac aag cac aag aag gcg 1103 aag aag ggg aag gag atg gcc agg atg aac aca agg ctt aag ctc ggg 1151 ata acg gac ctg ctc gag gac atc ggc gct gag gac gac gag agc gtg 1199 ctc atc acg ctc gtg ccc agg agc ggc aag gga atg gtg aag gtt gga 1247 ggg cta agg att gat ttc tcc aag tgatgagcat attgtgaaga gaaaatttgc 1301 leu glu ile gln ile his asn ser trp leu phe phe pro trp his arg phe tyr leu tyr ser asn glu arg ile leu gly lys leu ile gly asp leu arg asp ala lys his gln pro pro thr leu ile asp leu asp tyr asn gly thr asp pro thr phe ser pro glu glu gln ile asn his asn leu ala val met tyr arg gln val ile ser ser gly lys thr pro glu leu phe met gly ser ala tyr arg ala gly asp gln pro asp pro gly ala gly ser val glu gln lys pro his gly pro val his val trp thr gly asp arg asn gln pro asn arg glu asp met gly thr leu tyr ser ala ala trp asp pro val phe phe ala his his gly asn ile asp arg met trp tyr val trp arg asn leu gly gly lys his arg asn phe thr arg tyr thr tyr gln asp val glu ile pro trp leu lys ala lys pro ac aac aaa cca gtg cct ggt tta ggt gta ttc act atg gcc acc ctc 47 tct aaa cta gct tcc cca acc aat aac acc tcc act ctc ccc gct ccc 95 tcc ttt gca tgc tcc ttc tct cac caa aag ctt cac cac cac ctt cct 143 ctc ccc tgt agg ggt ccc aaa cca ccc cgt cat aag atc tca tgc aaa 191 tct aag gag caa caa gag aat gcc gac aag cct gcg ggc cgc atc gac 239 ser lys glu gln gln glu asn ala asp lys pro ala gly arg ile asp cgc cgc gac cta ctc ctg ggc ctc ggc ggg ctt tac ggt gcc acc act 287 ggg ctc ggc ctc aac cgt cga gcg gcc gcc gcc cct atc ctg gct ccc 335 gac ctc tca act tgt ggg ccg cct gcc gac ctc cct gcc tcc gcc cga 383 ccg aca gtt tgc tgc ccg cca tac caa tcc acc atc atc gtc ttc aag 431 ctc ccc ccg cga tct gct ccg ctt cgc gtc cgg cct gcg gcc cac ttg 479 gtt gac gcc gac tac ctg gcc aag tat aag aag gcg gtc gag ctc atg 527 agg gcc ctg ccg gcc gac gac ccg cgc aac ttc gta cag caa gcg aaa 575 gtg cac tgt gcg tac tgc gac ggc gcg tac gac caa atc ggc ttc ccc 623 val his cys ala tyr cys asp gly ala tyr asp gln ile gly phe pro gat ctc gag atc cag atc cac aac tcg tgg ctc ttc ttt cct tgg cac 671 asp leu glu ile gln ile his asn ser trp leu phe phe pro trp his cgg ttc tac ctc tac ttc aac gag cgc ata ctc ggg aaa ctt atc ggt 719 gac gac acg ttc gcg ctg cct ttc tgg aac tgg gac gcg ccg ggg ggc 767 atg cag ttc ccg tct atc tac aca gac cct tca tcc tcg cta tat gac 815 aag ctg cgt gat gcg aag cac cag ccg ccg act ttg att gac ctc gac 863 asn lys pro val pro gly leu gly val phe thr met ala thr leu ser thr val cys cys pro pro tyr gln ser thr ile ile val phe lys leu leu glu ile gln ile his asn ser trp leu phe phe pro trp his arg leu arg asp ala lys his gln pro pro thr leu ile asp leu asp tyr gaccacccat agatg atg gct tct ctc gcc ttg tct agt ctt ccc acc tcc 51 acc aca acc aaa aaa ccc tta ttt tcc aaa aca tcc tcg cat gtt aag 99 cca ttc cat cgc ttc aaa gtt tca tgc aat gca ccc gct gat aac aat 147 pro phe his arg phe lys val ser cys asn ala pro ala asp asn asn gac aaa acc gtc aat aat tct gat acc cca aag ctc ata cta ccc aaa 195 aca cca ctt gaa acg cag aac gta gac agg aga aac ttg ctt ctg gga 243 ctc gga ggt ctc tac ggc gct gcc aac ttg acg acc att ccg tca gcc 291 ttt ggc att ccc atc gct gct cca gac aat att tca gac tgt gtt gct 339 gcg act tca aac cta agg aac agc aaa gac gct ata agg gga cta gcg 387 tgt tgt cct ccg gtg ctt tca aca aac aaa cca atg gat tac gtc ctt 435 cys cys pro pro val leu ser thr asn lys pro met asp tyr val leu cct tca aac cct gtg att cgt gtt cga cca gct gca cag aaa gcc act 483 gcc gat tac act gct aag tat caa caa gca att caa gcc atg aag gat 531 ctc ccc gag gac cac cca cat agc tgg aag caa caa ggc aag att cac 579 leu pro glu asp his pro his ser trp lys gln gln gly lys ile his tgt gct tat tgc aac ggt ggt tac aat caa gaa caa agt ggt tac ccg 627 aat tta caa ctt cag att cac aac tca tgg ctc ttc ttt cct ttc cac 675 cgg tgg tac ctc tat ttc tac gag aag ata ttg ggg aag ttg att aat 723 gat cca act ttc gct cta cct tac tgg aac tgg gat aac cct act gga 771 atg gtt att cct gcc atg ttc gaa cag aac agc aaa act aac tct ctg 819 met val ile pro ala met phe glu gln asn ser lys thr asn ser leu ttt gac cct tta agg gat gcg aaa cac ctc cca cct tct atc ttt gat 867 gtt gaa tat gct ggt gca gac act ggt gcc act tgt ata gac cag ata 915 gcc att aat ctg tct tca atg tac aga cag atg gtc acc aac tcc act 963 ala ile asn leu ser ser met tyr arg gln met val thr asn ser thr gat aca aaa cga ttc ttc ggt ggc gaa ttt gta gct gga aat gac cct 1011 asp thr lys arg phe phe gly gly glu phe val ala gly asn asp pro ctt gcg agc gag ttc aac gta gct ggg acc gta gaa gct ggg gtt cac 1059 act gcg gct cac cgc tgg gtg ggt aat tct agg atg gcc aac agc gaa 1107 thr ala ala his arg trp val gly asn ser arg met ala asn ser glu gac atg ggg aac ttc tac tcc gca gga tat gat cct ctc ttt tac gtc 1155 asp met gly asn phe tyr ser ala gly tyr asp pro leu phe tyr val cac cat gcg aat gtc gac agg atg tgg caa atc tgg aaa gat att gac 1203 his his ala asn val asp arg met trp gln ile trp lys asp ile asp aag aag aca cac aag gat ccg acc tct ggc gac tgg cta aat gca tca 1251 lys lys thr his lys asp pro thr ser gly asp trp leu asn ala ser tac gtg ttt tac gat gag aat gaa aat ctt gta cgt gtc tac aac cga 1299 gac tgt gta gac att aat cgg atg gga tat gac tac gaa agg tca gca 1347 asp cys val asp ile asn arg met gly tyr asp tyr glu arg ser ala atc cca tgg atc cgt agt cgg ccg act gca cat gcg aag ggg gcg aac 1395 ile pro trp ile arg ser arg pro thr ala his ala lys gly ala asn gtt gct gct aag tct gct gga atc gtg cag aag gtg gag gat atc gta 1443 ttc ccg ctg aag tta aac aag ata gtg aag gtt cta gtg aag agg cca 1491 gct aca aac agg acc aag gag gga aag gag aaa gca aat gag ctg ttg 1539 ttc gtg aat gga atc acg ttt gat gct gag cgg ttt cta aag att gac 1587 phe val asn gly ile thr phe asp ala glu arg phe leu lys ile asp gtg ttt gtc aac gac gtc gac gat gga att cag acc acc gct gct gat 1635 agt gag ttt gct ggt agt ttc gca cag ttg cca cat aac cat ggc gac 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tyr asp tyr glu arg ser ala ile pro trp ile ser ala gly ile val gln lys val glu asp ile val phe pro leu lys ile thr phe asp ala glu arg phe leu lys ile asp val phe val asn gly ser phe ala gln leu pro his asn his gly asp lys met phe met cys asp glu val thr ile gly glu ile lys ile gln leu val pro ile