Patent Application: US-78648001-A

Abstract:
a class of wheat sebii genes , called sbeii - 1 , can be used to influence properties of starch produced by a plant , including the gelatinisation and / or pasting temperature of the starch . such a produced starch is useful , for example , in certain industrial applications and the preparation and / or processing of foodstuffs such as bakery products . one aspect of the present invention provides a nucleotide sequence encoding the amino acid sequence shown in fig . 10 .

Description:
a pcr based cloning strategy was devised for isolating starch branching enzymes from wheat using conserved domains within the known cloned gene sequences . starch branching enzymes have been cloned from a number of plant species and fig2 shows amino acid sequence data , obtained from the european molecular biology laboratory ( embl ) nucleotide database for various known starch branching enzymes as follows : zm sbe2a ( maize ) for zea mays ( seq id no : 13 ) zm sbe2b ( maize ) for zea mays ( seq id no : 14 ) ricbce3 ( rice sbeii type enzyme ) for oryza saliva ( seq id no : 17 ) ricesbe - 1 / 97 ( as above , including transit peptide sequence ) ( seq id no : 18 ) pssbeigen ( pea sbei , which is in fact an sbeii - type sequence ) for pisum sativum ( seq id no : 19 ) stsbe ( potato sbei type ) for solanum tuberosum ( seq id no : 20 ) tasbei ( wheat sbei - 2 ) for triticum aestivum ( seq id no : 21 ) zmsbei ( maize sbei ) for i zea mays ( seq id no : 23 ) ricbei ( rice sbei ) for oryza sativa ( seq id no : 24 ) pssbeiign ( pea sbeii , which is in fact an sbei - type sequence ) for pisum sativum ( seq id no : 25 ) fig2 also shows sequence information for a novel wheat sbeii - 1 sequence of the invention , identified as osbeii - 1all ( seq id no : 11 ). the alignment report of fig2 and also fig3 , 8 and 11 , was prepared using clustal method , with pam 250 residue weight table for amino acid sequences and weighted residue weight table for dna sequences . sequence pair distances expressed as % similarity shown in fig2 a and 3a , 4 a , 8 a and 11 a are determined using a ‘ megalign ’ program of dnastar software , and correspond to sequence homology percentages as specified above . alignment of the sequences shown in fig2 reveals several domains which are highly conserved . one such domain , mdkdmyd ( seq id no : 36 ), was almost completely conserved and it was assumed that this domain would also be present in wheat starch branching enzyme genes . this motif was chosen as a target for an oligonucleotide sense primer ( sbea ). 3 ′ race pcr was carried out on endosperm first strand cdna using the primers ro and sbe a . two populations of pcr products of approximately 1 kb and 1 . 2 kb were cloned into the plasmid vector pt7blue ( novagen ). plasmid dna from 36 putative recombinant clones was purified and the insert size estimated by restriction analysis . fifteen clones harbouring inserts of between approximately 1 kb and 1 . 2 kb were selected for sequencing . alignment of the sequence data obtained , using the megalign program of dnastar , indicated that the 15 selected clones could be divided on the basis of degrees of homology into two different classes , which we have designated sbeii - 1 and sbeii - 2 . furthermore , both the sbei1 - i and sbeii - 2 classes may each be further subdivided into three sub - classes , based on sequence differences ( table 1 ). it is thought the sub - division into three sub - classes probably arises because wheat comprises three homoeologous genomes . comparison between sequences within either of the sbeii - 1 or sbeii - 2 classes showed between 90 and 96 . 8 % similarity . in contrast , sequence similarity between representatives of sbeii - 1 and sbeii - 2 classes only display between 58 . 8 and 60 . 0 % homology in the region of comparison ( fig3 and 3 a ). furthermore , we have compared representative sequences from each sbeii - 1 and sbeii - 2 class with the previously reported wheat sbeii clones , pwbe6 ( mousley , 1994 ) and the very recently published sbeii ( nair et al ., 1997 ). the results showed that each of the previously isolated sbeii clones are highly homologous (& gt ; 90 %) to our sbeii - 2 class ( data not shown ). significantly , neither of the previously reported wheat sequences showed high homology to our sbeii - 1 sequence . the isolation and characterisation of three forms of sbeii - 1 ( sbeii - 1 , sub - classes a , b & amp ; c ) is novel . the sbeii - 2 sub - class b is also novel , sub - classes a and c corresponding to the sequences previously disclosed by mousley ( 1994 ) and nair et al ( 1997 ) respectively . alignment of the predicted amino acid sequences from representative clones , b6 and b11 of the wheat sbeii - 1 and sbeii - 2 sequences ( respectively ) against the corresponding regions of the maize sbeiia and sbeiib amino acid sequences ( fig4 and 4 a ) indicate that the wheat sbeii - 1 sequence ( clone b6 ) is more similar to the maize sbeiib sequence ( 88 . 7 % similarity ) than to the wheat sbeii - 2 sequence and the maize sbeiia sequence ( 82 . 2 % & amp ; 82 . 6 % similarity respectively ) and similarly that the wheat sbeii - 2 sequence is more similar to the maize sbeiia sequence ( 86 . 9 % similarity ) than to the wheat sbeii - 1 and maize sbeiib sequences ( 82 . 2 % and 81 . 7 % similarity respectively ). we thus hypothesise that the wheat sbeii - 1 is phylogenetically more related to the maize sbeiib and that the wheat sbeii - 2 is phylogenetically related to the maize sbeiia sequences and that the corresponding wheat and maize sequences are likely to exhibit similar functional properties . while the coding sequences of clones b2 , b10 and b4 have strong sequence homology to the maize sbeiib gene , there is much greater divergence in the 3 ′ untranslated parts of the sequences . fig5 and 7 show the 3 ′ untranslated sequences of clones b2 , b10 and b4 , respectively , and fig8 compares these sequences with the corresponding sequence of maize sbeiib . triticum aestivum cultivar rialto was grown in a glass house under supplementary lighting and temperature control to maintain a 16 hour day - length at 18 +/− 1 ° c . standard procedures were performed essentially according to sambrook et al ., ( 1989 ). dna sequencing was performed on an abi automated sequencer and sequences analysed using dnastar software for macintosh . rna was extracted from triticum aestivum cultivar rialto endosperm , using a purescript rna isolation kit ( flowgen ) essentially according to the manufacturers recommendations . briefly , endosperm tissue was frozen in liquid nitrogen and ground , for 2 min , to a fine powder using a dismembrenator ( braun biotech international ). the ground tissue was stored in liquid nitrogen prior to extraction . approx . 100 mg of ground tissue was transferred to a 1 . 5 ml microcentrifuge tube and 1 . 2 ml of ‘ lysis buffer ’ was added to the tissue before mixing by inversion and placing on ice for 10 minutes . protein and dna were precipitated from the cell lysate by adding 0 . 4 ml of ‘ protein - dna precipitation solution ’ and mixing by inversion before centrifuging at 13 , 000 × g at 4 ° c . for 20 minutes . the supernatant was divided between two fresh 1 . 5 ml tubes each containing 600 μl of isopropanol . the rna precipitate was pelleted by centrifugation at 13 , 000 × g at 4 ° c . for 10 minutes , the supernatant was discarded and the pellets washed with 70 % ethanol by inverting the tube several times . the ethanol was discarded and the pellet air dried for 15 - 20 minutes before the rna was resuspended in 7 . 5 ml of ‘ rna hydration solution ’. wheat endosperm cdna pool was prepared from total rna , extracted as described above , using superscript ™ reverse transcriptase ( life technologies ) essentially according to manufacturers instructions . briefly , five microgrammes of rna . 10 pmol roridt17 [ aaggatccgtcgacatcgataatacgactcactataggga ( t17 )] ( seq id no : 37 ) and sterile distilled water to a reaction volume of 12 μl , in a 500 μl microcentrifuge tube , was heated to 70 ° c . for 10 minutes before being quick chilled on ice . the contents of the tube were collected by brief centrifugation before adding 4 μl 5 × first strand buffer , 2 μl 0 . 1m dtt and 1 μl 10 mm dntps and , after mixing , incubating at 42 ° c . for 2 min . 1 μl of superscript ™ was added and , after mixing , incubation continued for 1 hour . the reaction was inactivated by heating to 70 ° c . for 15 min . 150 μl of t 10 e 1 was added to the reaction mix and the resulting cdna pool was used as a template for amplification in pcr . sbeii sequences were amplified from the endosperm cdna pool using primers ro [ aaggatccgtcgacatc ] ( seq id no : 38 ), which is complementary to the ro region of the roridt17 primer used to synthesise the cdna pool , and the sbeii specific primer , sbea [ atggacaaggatatgtatga ] ( seq id no : 39 ). sbea was designed to be homologous to the mdkdmyd ( seq id no : 36 ) motif which is situated approx . 1 kb from the 3 ′ end of the mature peptide coding sequence . pcr was carried out in a 50 μl reaction , comprising 5 μl of the cdna pool , 25 pmol ro , 50 pmol sbea , 5 μl 5 × taq buffer , 4 μl 25 mm mg 2 + , 0 . 5 μl 20 mm dntps , and 1 . 25 u taq polymerase . all of the reaction components were mixed , except for the taq polymerase , before being pre - heated to 94 ° c . for 7 min and then cooled to 75 ° c . for 5 min . whilst the reaction mixtures were held at 75 ° c . the taq polymerase was added and , after mixing well , the reactions were thermocycled at ( 94 ° c .— 30 sec , 50 ° c .— 30 sec , 72 ° c .— 1 min )× 30 cycles , followed by a final 10 min extension step at 72 ° c . pcr products were purified by phenol / chloroform and chloroform extraction before ligation with pt7 blue ( novagen ) according to manufacturers recommendations . putative sbe clones were initially characterised by standard plasmid dna purification methods and restriction digestion . representative clones harbouring a range of different sized inserts were selected for sequencing . the chinese spring wheat nullisomic - tetrasomic lines as described in sears ( 1966 ) were used for assignment of the sbe sequences chromosome locations . ditelosomic lines ( sears , 1966 ) were used to determine the chromosome arm location . the betzes barley ditelosomic addition lines in wheat are described in islarn ( 1983 ). the chromosomal location of the two families of sbeii sequences ( sbeii - 1 , sbeii - 2 ) was determined by probing wheat nulli - tetra and ditelosomic stock lines with gel - purified inserts of the various clones . fig9 a shows the hybridisation obtained with an sbeii - 2 ( clone b1 ) probe on hindiii digested dna . the euploid chinese spring gives 3 bands , one of which is missing in turn in the lines nullisomic for chromosomes 2a , 2b and 2d . the same blot was re - probed with a sbeii - 1 specific probe ( clone b2 ). this yields an entirely different hybridisation profile ( fig9 b ), demonstrating the specificity of the probe used . again bands are missing in each of the lines nullisomic for 2a , 2b and 2d the same banding pattern was observed using the sbeii - 1 clones b2 and b4 . thus the sbeii sub - family 1 and 2 gene sequences lie on the wheat group 2 set of homeologous chromosomes . ditelosomic addition lines were used to identify the arm location of these genes ( data not shown ). this revealed that the sbeii - 1 and sbeii - 2 sequences are both located on the long arms of the homeologous group 2 chromosomes of wheat . barley addition lines were used to determine whether homologous sequences are present in barley . these showed that sequences homologous to the wheat sbeii - 1 and sbeii - 2 sequences are located on the long arms of barley chromosome 2h . wheat grains were harvested at appropriate intervals and frozen in liquid nitrogen before grinding to a fine powder using either a braun mikrodismembrator ™ or a pestle and mortar . total rna was isolated using the rnaqueous ™ ( ambion inc ) kit according to the manufacturers instructions , or with the following method . frozen powdered grain was mixed with a 10 × volume of 0 . 2m tris - hcl ph9 , 0 . 4m nacl , 25 mm edta , 1 % sds , 1 % pvpp , 0 . 25 % antifoam a , and 0 . 1m dtt . this mixture was extracted twice with an equal volume of phenol / chloroform / isoamyl alcohol ( 25 : 24 : 1 ), the nucleic acids precipitated from the aqueous phase by the addition of 0 . 8 volumes of isopropanol , and the resulting pellet dissolved in h 2 o . the rna was then selectively precipitated by the addition of 1 volume of 4m licl , incubated at 4 ° c . overnight , and the resulting pellet dissolved in sterile distilled h 2 o . 15 μg of total rna was electrophoresed on a 1 % agarose , 2 . 21m formaldehyde , 40 mm mops ph7 . 0 , 10 mm sodium acetate , 1 mm edta gel , in a 40 mm mops ph7 , 10 mm sodium acetate , 1 mm edta running buffer at 1 v / cm overnight . gels were placed in a 50 ng / ml solution of ethidium bromide in water for 30 minutes , de - stained in water for 2 hours , and visualised and photographs under uv light . the gels were then washed briefly in sterile distilled h 2 o , then blotted onto hybond n + ™ ( amersham international ), according to standard protocols ( sambrook et al , 1989 ) overnight . blots were then dismantled and air - dried before uv fixing at 312 nm for 2 minutes . 5 - 10 μg of the plasmids pun1 and psr98 - 29 were digested with sst1 ( life technologies ltd ) according to the manufacturers instructions , to release fragments of approximately 0 . 8kb ( nptii ) and 1 kb ( sbeii - 1 ) respectively . 5 - 10 μg of the plasmid pvt96 - 54 was digested with bamh1 to release a sbeii - 2 fragment of approximately 1 . 2 kb . digests were electrophoresed on 1 % low melting point agarose gels . the gene specific fragments were excised and the dna purified using a wizard ™ gel purification kit ( promega ). 25 ng of the appropriate probe ( maize waxy promoter , nptii , wheat sbeii - 1 or wheat sbeii - 2 fragments ) were radiolabelled using the rediprime 11 ™ system ( amersham international ) using α 32 pdctp ( amersham international ) according to manufacturers instructions . blots were hybridized overnight at 65 ° c . in 0 . 6m nacl , 20 mm pipes , 4 mm na 2 edta2h 2 o , 0 . 2 % gelatin , 0 . 2 % ficoll 400 , 0 . 2 % pvp - 360 , 10 mm na 4 p 2 o 7 10h 2 o , 0 . 8 % sds , 0 . 5 mg / ml denatured salmon sperm dna . post hybridization washes were carried out in 30 mm nacl , 2 mm nah 2 po 4 . 2h 2 o , 0 . 2 mm na 2 edta . 2h 2 o , 0 . 1 % sds at room temperature for 7 minutes , then 65 ° c . for 10 minutes . filters were exposed to kodak biomax mr ™ ( amersham international ) film at − 70 ° c . blots were stripped by washing in 15 mm nacl , 1 mm nah 2 po 4 . 2h 2 o , 0 . 1 mm edta at 90 ° c . for 10 minutes , or until no counts above background remained . extension of the sbeii - 1 3 ′ sequence towards the 5 ′ end of the mature peptide we have exploited the sequence divergence between our wheat sbeii - 1 and sbeii - 2 sequences to design the sbeii - 1 specific 3 ′ primer , sb4 . this primer was used in conjunction with an sbeii specific 5 ′ primer to extend the novel sbeii - 1 sequence using a pcr - based approach . to extend the sbeii - 1 3 ′ sequence towards the 5 ′ end of the mature peptide , a second conserved domain was identified and an oligonucleotide sense primer , agsbei , designed . pcr amplification from the endosperm first strand cdna pool was carried out using the agsbei - sb4 primer pair . separation of the amplification products by electrophoresis through a 1 % ( w / v ) agarose gel ( data not shown ) showed that the reaction yielded a distinct band of approx . 2 . 2 kb . the approx 2 . 2 kb amplification products were excised from the gel , ligated with pt7blue and transformed into competent novablue e . coli cells . following overnight culture , nine putative recombinant clones were selected for further analysis . screening of each of the selected clones using vector specific primers indicated that clones 5a1 , 5a2 , 5a5 and 5a9 harboured inserts of the predicted size . of these clone 5a1 ( which falls in sub - class c ) was selected for sequencing ( fig1 ). the amino acid sequence of fig1 corresponds to the osbeii - 1all sequence of fig2 . although not full length the predicted open reading frame includes nucleotides 44 through to 1823 and encodes a 593 amino acid peptide . based on similarities with the maize genes , it is estimated that this sequence is missing approximately 230 amino acids out of a predicted total of approximately 830 amino acids . on this basis , the partial sequence represents about 70 % of the coding sequence . multiple sequence alignment of this sbeii - 1 sequence with recently published wheat sbeii - 2 ( nair et al ., 1997 ), sbei ( rapellin et al ., 1997 ) and sbei - d2 ( rahman et al ., 1997 ) sequences showed that the sbeii - 1 sequence has similarity indices of 69 . 6 %, 31 . 2 % and 46 . 7 % to sbeii - 2 , sbei and sbei - d2 respectively ( fig1 and 11 a ). this demonstrates that the sbeii - 1 sequence differs from the published wheat sbe sequences , and confirms the analysis of the 3 ′ sequence alignment ( fig3 ). the increase in relative homology when compared to the values obtained following 3 ′ sequence alignment results from the fact that the central domain of sbes is highly conserved ( burton et al ., 1995 ; gao et al ., 1997 ). however , it is clear that this cloned wheat sbeii - 1 sequence is significantly different from previously published wheat sbe sequences and represents a novel sequence . sbeii - 1 sequences were extended toward the 5 ′ end of the mature peptide by amplification from the endosperm cdna pool using the sbeii - 1 specific primer sb4 [ ttttctcacaacgccctggg ] ( seq id no : 40 ) in conjunction with the primer agsbei [ tgtttgggagatcttcctccc ] ( seq id no : 41 ). agsbei was designed to be homologous to the gvweiflp ( seq id no : 42 ) motif which is conserved in all known sbe sequences and is situated toward the 5 ′ end of the mature peptide coding sequence . pcr was carried out in a 50 μl reaction , comprising 5 μl of the cdna pool , 50 pmol sb4 , 50 pmol sbea1 , 5 μl 5 × taq buffer , 4 μl 25 mm mg 2 + , 0 . 5 μl 20 mm dntps , and 1 . 25 u taq polymerase . all of the reaction components were mixed , before thermocycling at ( 94 ° c .— 45 sec , 55 ° c .— 30 sec , 72 ° c .— 1 min 30 sec )× 30 cycles , followed a final 10 min extension step at 72 ° c . amplification products were separated by electrophoresis through a 1 %( w / v ) agarose gel and specific amplification products of the predicted size were excised from the gel . the dna was eluted from the gel slice using qiagen &# 39 ; s gel extraction kit according to the manufacturers recommendations before ligation with pt7 blue ( novagen ). ligation was carried out in a 10 μl reaction volume comprising 7 . 5 μl purified amplification product , 1 μl 10 × ligation buffer , 1 μl pt7blue and 0 . 5 μl t4 dna ligase ( amersham ). the reaction components were mixed well before being placed at 4 ° c . overnight . following overnight incubation , half of the ligation reaction was used to transform competent novablue e . coli cells ( novagen ). transformed cells were plated out onto lb plates supplemented with x − gal ( 40 μgml − 1 ), iptg ( 0 . 1 mm ), carbenicillin ( 100 μgml − 1 ), and tetracycline ( 12 μgml − 1 ), before placing at 37 ° c . overnight . putative recombinant clones were initially screened for the presence of an insert by colony pcr using the vector specific primers t7b and u19 . insert positive clones were then screened using an insert specific primer in conjunction with either t7b or u19 primers to determine the orientation of the insert within the multiple cloning site prior to sequencing . southern analyses of the pre - made nulli - tetra and ditelosomic blots were carried out essentially as described in jack et al ( 1994 ). the sbeii - 1 clones discussed above have been cloned into transformation vectors for transformation of wheat . northern blots were prepared from total rna from developing wheat grains of the cultivar bobwhite . fig1 shows a northern blot of rna from wheat grains of the cultivar bobwhite grown in the glasshouse as described and harvested between 5 and 29 days after anthesis . the blot was probed with the 1 kb sac1 sbeii - 1 fragment and subsequently ( following blot stripping ) with the 1 . 2 kb bamh1 sbeii - 2 fragment , both fragments purified and labelled as described . in fig1 panel a shows the ethidium bromide - stained rna gel prior to northern transfer . panel b shows the results of probing with the sbeii - 1 probe and panel c shows the results of probing with the sbeii - 2 probe . comparing within and between panels b and c differences can be observed in the relative intensities of the signals at the different time points . in particular a relatively stronger signal intensity is observed with the sbeii - 2 probe for the 5 day time point than with the sbeii - 1 probe , indicating that the transcript profiles for sbeii - 1 and sbeii - 2 are distinct , suggesting that the two gene families ( sbeii - 1 and sbeii - 2 ) are differentially expressed during grain development . the size of the transcripts observed for both sbeii - 1 and sbeii - 2 is approximately 3 . 5 kb . however the sbeii - 2 transcript is slightly smaller than the sbeii - 1 transcript . standard molecular biology procedures ( sambrook et al , 1989 ) were used for plasmid constructions . pwxgs + ( fig1 ) comprising a maize granule bound starch synthase gene ( shure et al 1983 ) promoter - gus - nos fusion was obtained as a gift to unilever research from sue wessler ( university of georgia , athens , usa ) and may be obtained on request from that source . the promoter in pwxgs + is approximately 1 . 5 kb in length and represents a truncated version of a similar , but larger promoter fragment described in russell & amp ; fromm ( 1997 ). the sequence of the promoter ( hindiii - bamh1 fragment ) in pwxgs + is presented in fig1 a ( seq id no : 55 ). psrwxgus1 ( fig1 ) was produced by inserting a sac 1 linker [ d ( pcgagctcg ) 0 ] ( new england biolabs [ neb ]) ( neb catalogue no 1044 ) into the sma1 site in pwxgs +. pvtwxgus2 ( fig1 ) was produced by inserting a bamh1 linker [ d ( pcgggatcccg )] ( seq id no : 43 ) ( neb catalogue no . 1071 ) into the ec1136ii ( an isoschizomer of sac1 which gives blunt ends ) site of pwxgs +. a sac1 linker was inserted at the xbai site ( which had been blunted using klenow + dntps ) of the sbeii - 1 clone b6 in the plasmid pt7blue to produce an intermediate clone . the sbe sequence was then purified from this intermediate clone as a sac1 fragment and ligated into the sac1 sites of psrwxgus1 replacing the gus gene sequence to produce the plasmids psr96 - 26 and psr96 - 29 representing antisense and sense orientations of the sbeii - 1 sequence downstream of the waxy promoter , respectively . a bamh1 linker was inserted at the xbai site ( which had been blunted using klenow + dntps ) of the sbeii - 2 clone b11 in pt7blue to produce an intermediate clone . the sbe sequence was then purified from this intermediate as a bamh1 fragment and inserted into the bamh1 sites of pvtwxgus2 , replacing the gus gene sequence , to produce the plasmids pvt96 - 50 and pvt96 - 53 representing antisense and sense orientations , respectively , of the sbeii - 2 sequence downstream of the waxy promoter . pvt96 - 54 . a bamh1 linker was inserted at the xba1 site ( which had been blunted using klenow + dntps ) of the sbeii - 2 clone b9 ( equivalent to clone b1 ) in pt7blue to produce an intermediate clone . the sbeii - 2 sequence was then purified from this intermediate clone as a bamh 1 fragment and inserted into the bamh1 sites of pvtwxgus2 , replacing the gus gene sequence , to produce the plasmid pvt96 - 54 . the waxy - sbe - nos sequences in the plasmids psr96 - 26 and psr96 - 29 and pvt96 - 50 and pvt96 - 53 were purified as hindiii / ecori fragments and inserted into the ecori / hindiii sites of plasmid ppbi - 97 - 2 ( also known as p97 - 2 ) ( fig1 ). plasmid ppbi - 97 - 2 is described in european patent application no . 97305694 . 8 ( published as wo 99 / 06570 ). following removal of the ampicillin resistance marker gene the resulting plasmids were designated psr97 - 26a − ( clone b6 ( sbeii - 1 , sub - class a ) in antisense orientation ), psr97 - 29a − ( clone b6 in sense orientation ), and psr97 - 50a − ( clone b11 ( sbeii - 2 , sub - class a ) in antisense orientation ) and psr97 - 53a − ( clone b11 in sense orientation ) as illustrated in fig1 , 18 , 19 and 20 , respectively . p97 - 2c ( fig2 ) was produced by digesting the polylinker sites ec1136 ii to smai in the plasmid ppbi97 - 2 ( fig1 ), ligating and selecting recombinants in which the polylinker region from smai to ec1136 ii had reinserted in the opposite orientation . the waxy - nos sequences in psrwxgus1 were transferred as a hindiii / ecori fragment into the hindiii / ecori sites of plasmid p97 - 2c to produce the plasmid p97 - 2cwt1 ( fig2 ). psc98 - 1 and psc98 - 2 . the 5 ′ extended sbeii - 1 clone 5a1 in pt7blue ( comprising sbe sequence from coordinate 43 to 2003 bp in fig1 ) was digested with ecori and xba1 , followed by ‘ in - fill ’ of overhangs using klenow polymerase and dntps . the resulting blunt ended sbe fragment was gel purified and ligated to p97 - 2cwt1 ( fig2 ) which bad been digested with ec1136ii and dephophorylated using calf intestinal phosphatase . the resulting recombinants were screened by restriction digest anaylsis arid clones comprising both orientations of the sbe sequence ( with respect to the waxy promoter ) were identified . psc98 - 1 ( fig2 ) is an antisense version and psc98 - 2 ( fig2 ) is a sense version . following removal of the ampicillin marker gene the resulting plasmids were designated psc98 - 1a − and psc98 - 2a − respectively . a sac1 linker was inserted at the smai site of the plasmid pahc25 ( christensen and quail 1996 ) to produce an intermediate plasmid . the gus gene was removed from this intermediate plasmid by digesting with saci followed by self ligation and identification of recombinant molecules lacking the gus sequence to produce the plasmid ppbi95 - 9 . ppbi95 - 9 was digested with ecori and following self ligation recombinant molecules lacking the ubi - bar sequences were identified . the resulting plasmid is designated ppbi96 - 23 . an nptii sequence was amplified as a pcr product using the primers ag95 - 7 : 5 ′ gatgagctccgtttcgcatgattgaacaagatgg ( seq id no : 44 ) and ag95 - 8 : 5 ′ gtcgagctcagaagaactcgtcaagaaggc ( seq id no : 45 ), using ppbibag3 ( goldsbrough et al 1994 as template for the nptii sequence . the amplified product was cloned into the ssti site of pbluescript ( stratagene ) and sequenced . the sequencing revealed that the npti sequence was of the ‘ mutant ’ form rather than the wild - type as had been expected . the ‘ mutant ’ form carries a single base change which is flanked by unique nco1and sph1 sites . the pbluescript clone was digested with nco1 and sph1 to remove the region containing the single base change . two oligonucleotides , ( npt1 : cccgacggcgaggatctcgtcgtgacc ( seq id no : 46 ) and npt2 : catgggtcacgacgagatcctcgccgtcgggcatg ) ( seq id no : 47 ) were then annealed to each other to form an nco1 / sph1 fragment . this was cloned into the nco1 / sph1 digested bluescript / nptii clone , and the resulting clone was sequenced to confirm that the gene was now of the wild type form . the nptii sequences was then purified as a saci fragment and inserted at the saci site of ppbi96 - 23 to produce pun1 ( fig2 ). pun1 includes the wild - type ubiquitin promoter ( ubi promoter ), which is also referred to as the ubiquitin regulatory system ( abbreviated to urs ). the orientation of the nptii sequence in pun1 was determined by restriction digest analysis . the sequence of the nptii saci fragment is presented in fig2 ( seq id no : 35 ). pusn99 - 1 and pusn99 - 2 . the sbeii - 1 ( clone b6 ) sequence was purified as a sac1 fragment from the plasmid psr96 - 26 and inserted at the sac1 site of ppbi96 - 23 to produce the plasmids pusn99 - 1 and pusn99 - 2 ( fig2 and 28 ) representing sense and antisense orientations of the sbeii - 1 sequences respectively . ppbi97 - 2bdun1 . ppbi92 - 2bdun1 ( also sometimes referred to as p97 - 2bdun1 ) comprises a reconstituted ubiquitin regulatory system ( referred to hereafter as a modified ubiquitin promoter or a modified ubiquitin regulatory system ( murs )) which lacks the two overlapping ‘ consensus heatshock elements ’ discussed in ep 0342926 and u . s . pat . no . 5 , 614 , 399 . the modified ubiquitin promoter was prepared via pcr amplification of two dna fragments using maize genomic dna as template , followed by ligation of the two fragments to produce a single fragment lacking the consensus heatshock ( hs ) elements . a kpn1 restriction site was engineered in place of the hs elements . the primers used were designed from sequence information published by liu et al 1995 ( embl dna database accession zmu29159 ). to delete the hs elements and to replace with a diagnostic kpn1 site the ubiquitin promoter and intron sequences were amplified as two fragments using the primer combinations hs1 + ubi3 - 3 and hs2 + ubi5 - 2 , the sequences of which are given below . primers ubi5 - 2 and ubi3 - 3 are homologous to sequences in the sequence published by liu et al 1995 . primers hs1 and hs2 are homologous to sequences located immediately 3 ′ and 5 ′ respectively of the two overlapping hs elements in the ubiquitin promoter as described in ep 0342926 and u . s . pat . no . 5 , 361 , 399 . both of these primers have a kpn1 tail at their 5 ′ ends . the amplified products were subcloned into pgem teasy ( promega ) to produce the plasmids p97 - u1 and p97 - u2 . the full - length ( approx . 2 kb ) modified ubiquitin promoter was reconstructed by subcloning the kpn1 - sac1 fragment from p97 - u1 into the kpn1 / sac1 sites of p97 - u2 to produce p97 - u3 . a partial restriction map of the predicted sequence ( seq id no : 52 ) of the cloned fragment in p97 - u3 is presented in fig2 . ( the modified ubiquitin promotor ( or murs ) is the subject of a copending european patent application filed by the present applicants on the same day as the present application , under the reference c1235 . 01 / m ). the modified ubiquitin promoter was transferred as a psti fragment from p97 - u3 into plasmid ppbi96 - 36 . the plasmid pbi96 - 36 ( fig3 ) comprises the gus - nos reporter gene fusion under the control of the wild - type ubiquitin promoter ( derived from pahc25 ) in a puc plasmid backbone . the promoter replaces the wild - type ubiquitin regulatory system in ppbi96 - 36 to produce an intermediary plasmid p97 - dug1 ( fig3 ). the ubi - nos sequences in ppbi96 - 23 were transferred as an ecori - hindiii fragment into the ecori and hindiii sites of p97 - 2b ( plasmid p97 - 2b is described in european patent application no . 97305694 . 8 published as wo 99 / 06570 ) to produce the plasmid p97 - 2bubinos . the modified ubiquitin promoter was purified as a hindiii / saci fragment from p97 - dug1 ( fig3 ) and transferred into the hindiii and saci sites of p97 - 2bubinos , replacing the wild - type ubiquitin promoter to produce p97 - 2bdubinos . the nptii sequence in pun1 was purified as a saci fragment and transferred into the saci site of p97 - 2bdubinos to produce ppbi97 - 2bdun1 ( fig3 ). following removal of the ampicillin resistance marker using the method as described in wo 99 / 06570 , the resulting plasmid as used for wheat transformation was designated p97 - 2bdun1a −. pcaineo comprises the nptii gene under control of a camv35s promoter and maize adh1 intron . the plasmid is described in fromm et al 1986 . the following plasmid combinations ( co - bombardments ) have been used in the transformation of wheat plants : embryo wheat plants of the spring cultivar bobwhite and the winter cultivar florida were grown in a glasshouse with 16 hr day length supplemented with lights to maintain a minimum light intensity of 500 umol m − 2 s − 1 at 0 . 5m above flag leaf . glasshouse temperatures were maintained at 19 ° c .+/− 1 ° c . during the day and 14 ° c .+/− 1 ° c . at night . immature embryos of wheat were harvested from developing grain . the seeds were harvested and embryos were cultured at approximately 12 days after anthesis when the embryos were approximately 1 mm in length . seeds were first rinsed in 70 % ethanol for 5 minutes and then sterilised in a 10 % solution of domestos bleach ( domestos is a trade mark ) for 15 minutes followed by 6 washes with sterile distilled water . following removal of the embryonic axis the embryos were placed axis surface face down on agargel ( sigma catalogue no . a - 3301 ) solidified mm1 media . the general recipe for mm1 is given in appendix 1 , and the recipes for the various constituents in appendix 2 . the embryos were maintained in darkness for one to two days at 24 ° c .+/− 1 ° c . prior to bombardment . the plasmids pahc25 , pcaineo , pun1 and p97 - 2bdun1 were used to provide selection markers in the combinations with starch gene constructs as detailed in table 2 . pahc25 ( christensen and quail 1996 ) contains a chimeric ubi - bar gene which provides selection of transformants to phosphinothricin , the active ingredient in herbicides basta ™ and bialophos ( see block , m . de . et al 1987 ). the plasmids pcaineo ( fromm et al ., 1986 ), pun1 and p97 - 2bdun1 contain chimeric promoter - nptii gene fusions and provide selection of transformants against a range of aminoglycoside antibiotics including kanamycin , neomycin , geneticin and paromycin . particle bombardments was used to introduce plasmids into plant cells . the following method was used to precipitate plasmid dna onto 0 . 6 μm gold particles ( bio - rad catalogue number 165 - 2262 ): a total of 5 μg of plasmid dna was added to a 50 μl sonicated for one minute suspension of gold particle @ 10 mg / ml ) in a 1 . 5 ml microfuge tube . following a brief vortex for three seconds 50 μl of a 0 . 5m solution of calcium chloride and 20 μl of a 0 . 05m solution of spermidine free base were added to the opposite sides of the microfuge tube lid . the tube contents were mixed together by closing the lid and tapping the calcium chloride and spermidine to the bottom of the tube . following a vortex for three seconds the suspension was centrifuged at 13 , 000 rpm for 5 seconds . the supernatant was then removed and the pellet resuspended in 150 μl of absolute ethanol . this requires scraping the gold particles off the inside of the tube using a pipette tip . following a further three second vortex , the sample was centrifuged again and the pellet resuspended in a total volume of 85 μl in absolute ethanol . the particles were vortexed briefly and sonicated for 5 seconds in a camlab trisonic t310 water bath sonicator to ensure fine dispersion . an aliquot of 5 μl of the dna coated gold particles were placed in the centre of a macrocarrier ( bio - rad catalogue no . 115 - 2335 ) and allowed to dry for 30 mins . particle bombardment was performed by using a biolisit ™ pds - 1000 / he ( bio - rad instruments , hercules calif .) chamber which is illustrated schematically in fig3 , using helium pressure of 650 and 900 psi ( rupture discs : bio - rad catalogue numbers 165 - 2327 and 165 - 2328 respectively ). referring to fig3 , the illustrated vacuum chamber comprises a housing 10 , the inner side walls of which include a series of recesses 12 for receiving shelves such as sample shelf 14 shown at the fourth level down from the top of the housing . a rupture disc 16 is supported in a he pressure shock tube 18 near the top of the housing . a support 20 , resting in the second set of recesses 12 down from the top of the housing , carries unit 22 that includes a stopping screen and a number of rings 24 , with 11 rings below the support 20 and 3 - 4 rings above the support 20 . macrocarrier 26 is supported at the top of unit 22 . the approximate distance from the rupture disc 16 to the macrocarrier 26 is 25 mm , with the approximate distance from the macrocarrier 26 to the stopping screen being 7 mm , and the approximate distance from the stopping screen to the sample shelf 14 being 67 mm . the top of unit 22 is about 21 mm from the bottom of the shock tube 18 , and the bottom unit 22 is about 31 mm from the top of sample shelf 14 . immature embryos were bombarded between 1 and 2 days after culture . for bombardment the immature embryos were grouped into a circular area of approximately 1 cm in diameter comprising 20 - 100 embryos , axis side face down on the mm1 media . the petri dish ( not shown ) containing the tissue was placed in the chamber on shelf 14 , on the fourth shelf level down from the top , as illustrated in fig3 . the air in the chamber was then evacuated to a vacuum of 28 . 5 inches of hg . the macrocarrier 26 was accelerated with a helium shock wave using rupture membranes that burst when the he pressure in the shock tube 18 reaches 650 or 900 psi . within 1 hour after bombardment the bombarded embryos were plated on mm1 media at 10 embryos per 9 cm petri dish and then maintained in constant darkness at 24 ° c . for 2 - 3 weeks . during this period somatic embryogenic callus was produced on the bombarded embryos . after 2 - 3 weeks the embryos were transferred onto agar - solidified regeneration media , known as r media , and incubated under 16 hr daylength at 24 ° c . the general recipe for r media is given in appendix 1 . embryos were transferred on fresh plates at 2 - 3 week intervals . the composition of the regeneration media varied depending on which selection regime was to be used . for transformants bombarded with the bar gene the 3 amino solution was omitted and ppt ( phosphinothricin ) at 1 mg / l , rising to 3 mg / l over a period of three 2 - 3 week transfers was used for selection . for selection of transformants using the nptii gene three different regimes were used : 1 ) geneticin ( gibco - brl catalogue no . 10131 - 019 ) was incorporated ( at 50 mg / l ) immediately on transfer to regeneration media and maintained at 50 mg / l on subsequent transfers to regeneration media . 2 ) & amp ; 3 ) embryos were first transferred to regeneration media without selection for 12 days and 2 - 3 weeks , respectively , and thereafter transferred on to media containing geneticin at 50 mg / l . after 2 - 3 passages on regeneration media regenerating shoots were transferred to individual culture tubes containing 15 ml of regeneration media at half salt strength with selection at 3 mg / l ppt or 35 mg / l geneticin depending on whether the bar gene of nptii gene had been used in the original bombardments . following root formation the regenerated plants were transferred to soil and the glasshouse . southern analyses of primary transformants and progeny material were carried out as follows : freeze dried leaf tissues were ground briefly in a kontes ™ pestle and mortar , and genomic dna extracted as described in fulton et al , 1995 . 5 μg of dna were digested with an appropriate restriction enzyme according to the manufacturers instructions , and electrophoresed overnight on a 1 % agarose gel , after which the gel was then photographed , washed and blotted onto hybond n +™ ( amersham international ) according to the method of southern using standard procedures ( sambrook et al 1989 ). following blotting , the filters were air dried , baked at 65 ° c . for 1 - 2 hours and uv fixed at 312nm for 2 minutes . probe preparation and labelling for the southern analyses of transformed material was carried out as described above . the plasmid pahc25 ( christensen and quail , 1996 ) was transformed into wheat as described in previous sections . transformants were selected on the basis of resistance to phosphinothricin . southern blot analyses were carried out on the primary transformants to confirm integration of the plasmid sequences ( data not shown ). gus histochemical analyses were also carried out and demonstrated that the ubiquitin promoter is capable of mediating high levels of gus expression in a range of wheat tissues . fig3 a , b , c & amp ; d show histochemical localisation of gus expression in the seed , stem , floral and leaf tissues respectively . southern blot and gus histochemical analyses were also carried out on self progeny from primary transformants to confirm that the transformation system used is capable of producing transgenic plants which stably transmit the integrated plasmid sequences to progeny plants . fig3 shows a southern blot of 26 progeny plants of transformant bw119 which had been transformed with pahc25 . in this example genomic dna from the progeny plants was digested with the restriction enzyme sac1 and the blot was probed with the gus gene coding sequence . the southern blot results are suggestive of the presence of two independently segregating integration loci , each comprising concatamers of pahc25 plasmid sequences . evaluation of the maize waxy promoter for endosperm - specific expression of associated transgenes the plasmids pwxgs + and pun1 were co - transformed into wheat as described in previous sections . transformants were selected on the basis of resistance to geneticin . southern blot analyses were carried out on the primary transformants to confirm integration of the plasmid sequences ( data not shown ). gus histochemical analyses were also carried out to determine the expression profile mediated by the maize waxy promoter . the majority of the transformants that expressed gus exhibited expression specifically in endosperm tissue , demonstrating the suitability of this promoter for mediating endosperm expression of associated transgenes . fig3 a & amp ; b shows endosperm specific expression of gus in seeds from two independent transformants . we did not observe gus expression in pollen grains as was seen by russell and fromm ( 1997 ), however the construct they used also incorporated the maize hsp 70 intron which may conceivably have influenced expression both quantitatively and qualitatively . the various construct combinations detailed in table 2 were co - transformed into wheat using the procedures as described in previous sections . transformants were selected on the basis of resistance to geneticin . the primary transformants were confirmed positive by southern blot analysis . blots were sequentially probed with an nptii coding sequence probe and a sbe coding region probe . fig3 shows an example of a southern blot which comprises 22 putative transformants which had been co - bombarded with psr97 - 29a − or psr97 - 26a − and pun1 or p97 - 2bdun1 . genomic dnas on this blot had been digested with sac1 . the blot was first probed with the nptii probe . lanes marked with an asterisk correspond to transformants which give a positive signal with the nptii probe . the blot shown in fig3 was probed with the sbeii - 1 1 kb saci fragment . the sac1 digest is expected to release a 1 kb sbeii - 1 hybridising band from both psr97 - 29a − and psr97 - 26a − plasmid sequences , and the intensity of this band will vary depending on the copy number of inserted plasmid sequences . as can be seen in fig3 several additional sbeii - 1 hybridising bands are also observed . five of these bands are present in all lanes and result from hybridisation to endogenous wheat sbeii - 1 sequences . the additional bands of varying size which are observed in the majority of lanes which show the 1 kb hybridising band most likely result from integration events in which one or more copies of the plasmid had been linearised within the 1 kb sbeii - 1 sequence prior to integration . in the example shown in fig3 , of the 20 nptii positive plants , 16 were found to be co - transformed with the sbeii - 1 sequences , representing a co - transformation efficiency of 80 %. when heated , an aqueous suspension of starch in excess water undergoes a co - operative endothermic transition known as gelatinisation , as discussed above , entailing a melting of the starch crystallites . differential scanning calorimetry ( dsc ) measures the amount of energy ( heat ) absorbed or released by a sample as it is heated , cooled or held in a constant ( isothermal ) temperature . dsc has been widely used to study the gelatinisation and retrogradation of starch . dsc analyses were carried out on single grains or pools of 5 grains from primary transformants generated through transformation using each of the gene construct combinations detailed in table 2 . individual seed samples were crushed and ground using a pestle and mortar . the resulting bran was then separated and samples weighed into 50 μm aluminium dsc pans . water , three times by weight , was added and the sample pans sealed . analyses were performed using a perkin - elmer dsc - 7 robotic ™ system equipped with an intercooler ii ™, for sub - ambient conditions . samples were heated from 25 ° c . to 80 ° c . at a heating rate of 5 ° c . min − 1 . gelatinisation enthalpy , onset and peak and end temperatures were recorded . the thermograms were analysed using the perkin - elmer software programs ( thermal analysis software 7 ). gelatinisation enthalpy is expressed in joules ( j )/ gram ( g ) of sample . pools of 5 seeds from a single primary transformant , or single seeds from primary transformants , were milled using a cemotec 1090 ™ sample mill . the milled sample was then passed through a 250 micron sieve to separate the bran from endosperm . approximately 5 mg of the sieved samples was then accurately weighed into 50 μl aluminium dsc pans . water , three times by weight , was added and the sample pans sealed . analyses were performed using a perkin - elmer pyris 1 ™ dsc equipped with autosampler and intracooler ip . samples were heated from 40 ° c . to 85 ° c . at a heating rate of 10 ° c . per minute . the thermograms were analysed using the perkin - elmer software programs ( pyris software for windows v 3 . 5 ). gelatinisation enthalpy , onset and peak and end temperatures were recorded . using method 1 , dsc analyses were performed on individual mature grains of primary transformants , transformed with the plasmid combinations psr97 - 26a −/ pun1 , psr97 - 26a −/ p97 - 2bdun1 and psr97 - 29a −/ p97 - 2bdun1 . data obtained were compared to data from control material which had been transformed with one of the nptii selectable marker plasmids , but did not contain any of the ‘ starch ’ plasmids . table 3 summarises the average onset , peak , end and enthalpy values for the selected material . the majority of samples showed similar values to the control material . however , as can be seen from table 3 onset , peak and end temperatures were higher for a number of the transgenic samples compared to the control material . for example , transformant bw 326 exhibits a 6 . 7 ° c ., 4 . 9 ° c . and 4 . 6 ° c . increase in onset , peak and end temperatures ( respectively ) compared to the control sample . using method 2 a further series of dsc analyses were carried out on pools of 5 grains from primary transformants , transformed with the plasmid combinations psc98 - 1a −/ p97 - 2bdun1 , pusn - 1p97 - 2bdun1 , pusn - 2 / p97 - 2bdun1 and pusn - 1 / pusn - 2 / puni . data obtained were compared to data from control material which had been transformed with one of the nptii selectable marker plasmids , but did not contain any of the ‘ starch ’ plasmids . table 4 summaries the onset , peak , end and enthalpy values for the selected pooled samples . in many cases there is evidence that the ‘ starch ’ transgenic material shows onset , peak and end temperatures which are greater than those observed for the control material . for example , transformant bw727 exhibits a 9 . 8 ° c ., 8 . 7 ° c . and 9 . 1 ° c . increase in onset , peak and end temperatures ( respectively ) compared to the bw control sample 3 , and a 7 . 6 ° c ., 6 . 8 ° c . and 7 . 8 ° c . increase in onset , peak and end temperatures ( respectively ) compared to the bw control sample 2 . barcelo p ., and lazzeri , p . 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( 1993 ). branching of amylose by the branching isoenzymes of maize endosperm . carbohydrate res . 240 : 252 - 263 . ile asp gly gln leu arg ala arg tyr pro gly ile arg phe gly val trp glu met phe leu pro asn asn ala asp gly ser pro pro ile pro his gly ser arg val lys val arg met asp thr pro ser gly ile lys asp ser ile pro ala trp ile lys tyr ser val gln thr pro gly asp val phe lys his pro gln pro lys arg pro lys ser leu arg ile tyr glu thr his val gly met ser ser pro glu pro lys ile asn thr tyr ala asn phe arg asp glu val leu pro arg ile lys arg leu gly tyr asn ala val gln ile met ala ile gln glu his ser tyr tyr gly ser phe gly tyr his val thr asn phe phe ala pro ser ser arg phe gly ser pro glu asp leu lys ser leu ile asp arg ala his glu leu gly tyr gly asn lys glu val ile arg phe leu leu ser asn ala arg trp trp leu glu glu tyr lys phe asp gly phe arg phe asp gly ala thr leu met leu met asn asp leu ile his gly phe tyr pro glu ala val thr ile gly glu asp val ser gly met pro thr phe ala 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asp gly his his trp met trp asp ser arg leu phe asn tyr gly ser trp asn asp leu ile arg gly leu tyr pro glu ala val ser ile gly glu asp val ser gly met pro thr phe cys ile pro val gln asp gly gly val gly phe asp tyr arg leu his met ala val pro asp lys trp ile glu leu leu lys gln ser asp glu tyr trp glu met gly asp ile val his thr leu thr asn arg arg trp leu glu lys cys val thr tyr cys glu ser his asp gln ala leu val gly asp lys thr ile ala phe trp thr pro arg ile asp arg gly ile ala leu his lys met ile arg leu glu phe gly his pro glu trp ile asp phe pro arg gly pro gln ser gly met gln glu phe asp gln ala met gln his leu glu gly lys tyr glu phe met thr ser asp his ser tyr val ser arg lys his glu glu phe his trp ser asn ser tyr phe asp tyr arg val gly cys phe lys gly phe ser arg leu asp his asp ala glu tyr phe thr ala asp trp pro his asp asn arg pro cys ser phe ser val tyr ala pro ser arg leu glu val pro asp ile ser glu glu thr thr cys gly ala gly val ile asp glu his glu gly gly leu glu ala phe ser arg ser tyr glu lys phe gly phe asn ala ser ala glu gly ile thr tyr arg glu trp asp pro asn ala asp arg met ser lys asn glu phe gly val trp glu ile phe leu pro asn asn ala asp gly thr ser pro ile pro his gly ile pro ala trp ile lys tyr ser val gln ala pro gly glu ile pro arg his ala gln pro lys arg pro lys ser leu arg ile tyr glu thr his val gly met ser ser pro glu pro lys ile asn thr tyr val asn phe arg asp glu val leu pro arg ile lys lys leu gly tyr asn ala val gln ile met ala ile gln glu his ser tyr tyr gly ser phe gly tyr his val thr asn phe phe ala pro ser ser arg phe gly thr pro pro arg gly his his trp met trp asp ser arg leu phe asn tyr gly glu glu tyr lys phe asp gly phe arg phe asp gly val thr ser met leu val asn asp leu ile his gly leu tyr pro glu ala val thr ile gly glu asp val ser gly met pro thr phe ala leu pro val his asp trp ile asp leu leu lys gln ser asp glu thr trp lys met gly asp ile val his thr leu thr asn arg arg trp leu glu lys cys val thr tyr ala glu ser his asp gln ala leu val gly asp lys thr ile ala pro ser thr pro thr ile asp arg gly ile ala leu his lys met ile gln arg leu pro ser gly lys phe ile pro gly asn asn asn ser tyr lys tyr glu phe met thr ser asp his gln tyr ile ser arg lys his asp cys ser his asp asn arg pro tyr ser phe ser val tyr thr pro ser pro pro ile pro his gly ser arg val lys val arg met asp thr pro ser gly thr lys asp ser ile pro ala trp ile lys phe ser val asp asp tyr gly val trp glu ile phe leu pro asn asn ala asp gly ser pro ala ile pro his gly ser arg val lys ile arg met asp thr lys ile phe gln met asp ser met leu asn gly tyr lys tyr his leu phe asn his ser ala glu gly val thr tyr arg glu trp ala pro gly ala his ser ala ala leu val gly asp phe asn asn trp asn pro asn ala asp arg met ser lys asn glu phe gly val trp glu ile phe leu lys val arg met glu thr pro ser gly ile lys asp ser ile pro ala trp ile lys tyr ser val gln ala ala gly glu ile pro tyr asn gly gln pro lys arg pro lys ser leu arg ile tyr glu thr his val gly met ser ser thr glu pro lys ile asn thr tyr ala asn phe arg asp glu val leu pro arg ile lys lys leu gly tyr asn ala val gln ile met ala ile gln glu his ala tyr tyr gly ser phe gly tyr his val thr asn phe phe ala pro ser ser arg phe gly thr pro glu asp leu lys ser leu ile asp lys ala his glu leu gly leu val val leu met his his trp met trp asp ser arg leu phe asn tyr gly asn trp glu his his gly leu gln val ala phe thr gly asn tyr ser glu tyr phe val ser gly met pro thr phe ala leu pro val gln asp gly gly val gly phe asp tyr arg leu his met ala val pro asp lys trp ile glu leu leu lys gln ser asp glu ser trp lys met gly asp ile val his thr leu thr asn arg arg trp ser glu lys cys val thr tyr ala glu ser his asp gln ala leu val gly asp lys thr ile ala phe trp leu pro ser ile asp arg gly ile ala leu his lys met ile arg leu ile phe gly his pro glu trp ile asp phe pro arg ala pro gln val leu met leu glu phe asp arg ala met gln ser leu glu glu lys tyr gly phe met thr ser asp his gln tyr ile ser arg lys his glu glu asp his trp ser asn ser tyr phe asp tyr arg val gly cys leu lys pro phe gly arg ile his his thr ala glu his phe thr ala asp cys ser gly asp gly gln lys ile phe gln met asp ser met leu asn gly tyr asp ile asp gln tyr glu gly gly leu glu thr phe ser arg gly tyr glu lys phe gly phe asn his ser ala glu gly val thr tyr arg glu trp ala pro gly ala his ser ala ala leu val gly asp phe asn asn trp asn pro asn ala asp arg met ser lys asn glu phe gly val trp glu ile phe leu pro asn asn ala asp gly ser ser pro ile pro his gly ser arg val lys val arg met glu thr pro ser gly ile lys asp ser ile pro ala trp ile lys tyr ser val gln ala ala gly glu ile phe lys his pro gln pro lys arg pro lys ser leu arg ile tyr glu thr his val gly met ser ser thr glu pro lys ile asn thr tyr ala asn phe arg asp glu val leu pro arg ile lys lys leu gly tyr asn ala val gln ile met ala ile gln glu his ala tyr tyr gly ser phe gly tyr his val thr asn phe phe ala pro ser ser arg phe gly thr gly ser arg gly his his trp met trp asp ser arg leu phe asn tyr leu glu glu tyr lys phe asp gly phe arg phe asp gly val thr ser met met tyr thr his his gly leu gln val ala phe thr gly asn tyr met leu val asn asp leu ile his gly leu tyr pro glu ala ile thr ile gly glu asp val ser gly met pro thr phe ala leu pro val gln asp gly gly val gly phe asp tyr arg leu his met ala val pro asp asp ile val his thr leu thr asn arg arg trp ser glu lys cys val thr tyr ala glu ser his asp gln ala leu val gly asp lys thr ile arg pro ala thr pro ser ile asp arg gly ile ala leu his lys met met gly asn glu phe gly his pro glu trp ile asp phe pro arg ala arg tyr arg gly met leu glu phe asp arg ala met gln ser leu glu glu lys tyr gly phe met thr ser asp his gln tyr ile ser arg lys ala asp cys ser his asp asn arg pro tyr ser phe ser val tyr ser lys val leu ile pro glu asp gln asp asn ser val ser leu ala asp asp leu thr met lys asp gly asn lys tyr asn ile asp glu ser thr pro gly thr gly gln lys ile tyr glu ile asp pro leu leu gln ala glu glu ile asp lys tyr glu gly gly leu asp ala phe ser arg gly glu trp ala pro gly ala lys ser ala ala leu val gly asp phe asn asn trp asn pro asn ala asp val met thr lys asp ala phe gly val trp glu ile phe leu pro asn asn ala asp gly ser pro pro ile pro his gly ser arg val lys ile his met asp thr pro ser gly ile lys asp ser ile pro ala trp ile lys phe ser val gln ala pro gly glu glu ser his ile gly met ser ser pro glu pro lys ile asn thr tyr ala asn phe arg asp asp val leu pro arg ile lys lys leu gly tyr phe gly tyr his val thr asn phe phe ala pro ser ser arg phe gly thr pro glu asp leu lys ser leu ile asp arg ala his glu leu gly pro gly ser arg gly tyr his trp met trp asp ser arg leu phe asn trp leu asp glu tyr lys phe asp gly phe arg phe asp gly val thr ser met met tyr thr his his gly leu gln val ser phe thr gly asn met met leu val asn asp leu ile his gly leu phe pro glu ala val ser ile gly glu asp val ser gly met pro thr phe cys leu pro thr gln asp gly gly ile gly phe asn tyr arg leu his met ala val ala gly asp ile val his thr leu thr asn arg arg trp leu glu lys cys val val tyr ala glu ser his asp gln ala leu val gly asp lys thr asp arg pro ser thr pro leu ile asp arg gly ile ala leu his lys phe met gly asn glu phe gly his pro glu trp ile asp phe pro arg leu arg tyr his gly met gln glu phe asp arg ala met gln his leu glu glu arg tyr gly phe met thr ser glu his gln tyr ile ser arg thr ser glu gly trp tyr asp asp arg pro arg ser phe leu val tyr ala pro ser arg thr ala val val tyr ala leu ala asp gly val glu thr met ala pro leu glu glu asp val lys thr glu asn ile gly leu arg glu asp gly cys ile val tyr arg glu trp ala pro ala ala gln met met glu lys asp gln phe gly val trp ser ile arg ile pro asp phe lys his gly asn gly val trp val asp arg ile pro ala trp ile val tyr trp asp pro pro pro ser glu arg tyr his phe lys tyr pro met ser ser ser glu pro arg val asn ser tyr arg glu phe ala asp asp val leu pro arg ile lys ala asn asn tyr asn thr val gln leu thr asn phe phe ala val ser ser arg tyr gly asn pro glu asp leu lys tyr leu ile asp lys ala his ser leu gly leu gln val leu val glu arg gly tyr his lys leu trp asp ser arg leu phe asn tyr ala glu glu tyr asn phe asp gly phe arg phe asp gly ile thr ser met leu ala asn asn leu ile his lys ile phe pro asp ala thr val ile gly gly ile gly phe asp tyr arg leu ala met ala ile pro asp lys glu val thr ser ser leu thr asn arg arg tyr thr glu lys cys ile ala tyr ala glu ser his asp gln ser ile val gly asp lys thr ile ala phe leu leu met asp lys glu met tyr ser gly met ser cys leu thr asp ala ser pro val val asp arg gly ile ala leu his lys met ile his phe phe thr met ala leu gly gly glu gly tyr leu asn phe met gly asn glu phe gly his pro glu trp ile asp phe pro arg glu gly asn asn trp ser tyr asp lys cys arg arg gln trp asn leu ala asp ser glu his leu arg tyr lys phe met asn ala phe asp arg ala met asn ser leu asp glu lys phe ser phe leu ala ser gly lys gln gly asp leu val phe val phe asn phe his pro lys asn thr tyr glu asn phe asn gly arg gln ile pro ser lys cys cys leu leu arg glu his val trp leu ile thr glu leu met asn ala cys gln lys leu lys thr met ala thr ala glu asp gly val gly asp leu pro ile tyr asp leu asp pro lys phe ala gly phe lys glu his phe ser tyr arg met asn asp ala thr val tyr arg glu trp ala pro ala ala met asp ala gln leu ile gly asp phe asn asn trp asn gly ser gly his arg met thr lys asp asn tyr gly val trp ser ile arg ile ser his val asn gly lys pro ala ile pro his asn ser lys val lys phe arg phe his ala thr phe asp ala ser lys phe gly ala pro tyr asp gly val his trp asp pro pro ser gly glu arg tyr val phe lys his pro arg pro arg lys pro asp ala pro arg ile tyr glu ala his val gly met ser gly glu lys pro glu val ser thr tyr arg glu phe ala asp asn val leu pro arg ile lys ala asn asn tyr asn thr val gln leu met ala ile met glu his ser tyr tyr ala ser phe gly tyr his val thr asn phe phe ala val ser ser arg ser gly thr pro glu asp leu lys tyr val his ser his ala ser ser asn lys thr asp gly leu asn gly tyr asp val gly gln asn thr gln glu ser tyr phe his thr gly glu arg gly tyr his lys leu trp asp ser arg leu phe asn tyr ala asn trp glu val leu arg phe leu leu ser asn leu arg tyr trp met asp glu asn his his gly ile asn met ser phe ala gly ser tyr lys glu tyr val gly phe asp tyr arg leu ala met ala ile pro asp arg trp ile asp tyr leu lys asn lys asp asp leu glu trp ser met ser gly ile ala his thr leu thr asn arg arg tyr thr glu lys cys ile ala tyr ala glu ser his asp gln ser ile val gly asp lys thr met ala phe leu leu met asp lys glu met tyr thr gly met ser asp leu gln pro ala ser pro thr ile asp arg gly ile ala leu gln lys met ile his asn trp ser tyr asp lys cys arg arg gln trp ser leu ala asp ile asp his leu arg tyr lys tyr met asn ala phe asp gln ala met asn ser asp met asn glu glu lys lys ile ile val phe glu arg gly asp leu val phe val phe asn phe his pro ser lys thr tyr asp gly tyr gly ala ala ser trp gly lys thr ala leu gly tyr ile asp val glu asn lys ile ala ala thr thr gly tyr gly ser asp his leu pro ile thr glu his gly ala ser val tyr arg glu trp ala pro ala ala glu glu ala gln leu val gly asp phe asn asn trp asn gly ser gly his lys met ala lys asp asn phe gly val trp ser ile arg ile ser his val asn gly lys pro ala ile pro his asn ser lys val lys phe arg phe arg his his gly val trp val glu gln ile pro ala trp ile arg his trp asp pro pro ser ser glu arg tyr val phe asn his pro arg pro pro lys pro asp val pro arg ile tyr glu ala his val gly val ser gly gly lys leu glu ala gly thr tyr arg glu phe pro asp asn val leu pro cys leu arg ala thr asn tyr asn thr val gln leu met gly ile met glu his ser asp ser ala ser phe gly tyr his val thr asn phe phe ala val ser ser arg ser gly thr pro glu asp leu lys tyr leu ile asp lys ala his ser leu gly leu arg val leu met asp tyr asp val gly gln ser ala his glu ser tyr phe tyr thr gly asp trp glu val leu arg phe leu leu ser asn leu arg tyr trp met asp tyr ile gly leu asp thr asn val asp ala phe val tyr met met leu ala asn his leu met his lys leu phe pro glu ala ile val val ala gly leu gly phe asp tyr arg gln ala met thr ile pro asp arg trp ile asp tyr leu glu asn lys gly asp gln gln trp ser met ser ser val ile ser gln thr leu thr asn arg arg tyr pro glu lys phe ile ala tyr ala glu arg gln asn his ser ile ile gly ser lys thr met asp pro asp ser pro thr ile asp arg ala ile ala leu gln lys met ile his phe ile thr met ala phe gly gly asp ser tyr leu lys phe met gly asn glu tyr met asn ala phe val gln ala val asp thr pro ser asp lys cys ser phe leu ser ser ser asn gln thr ala ser his met asn glu glu glu lys gly ser ala leu thr lys gly tyr thr his thr met ala thr ala lys gly asp val asp his leu pro ile tyr asp leu asp pro lys leu glu ile phe lys asp his phe arg tyr arg met glu asp gly thr val tyr arg glu trp ala pro ala ala gln glu ala glu leu ile gly asp phe asn asp trp asn gly ala asn his lys met gly lys pro ala ile pro his asn ser lys val lys phe arg phe leu his gly gly val trp val asp arg ile pro ala leu ile arg tyr ala thr val asp ala ser lys phe gly ala pro tyr asp gly val his trp asp pro pro ala ser glu arg tyr thr phe lys his pro arg pro ser lys pro ala ala pro arg ile tyr glu ala his val gly met ser gly glu lys pro ala val ser thr tyr arg glu phe ala asp asn val leu pro arg ile arg ala asn asn tyr asn thr val gln leu met ala val met glu his ser tyr tyr ala ser phe gly tyr his val thr asn phe phe ala val ser ser arg ser gly thr pro glu asp leu lys tyr leu val gly gln ser thr gln glu ser tyr phe his ala gly asp arg gly tyr his lys leu trp asp ser arg leu phe asn tyr ala asn trp glu met phe asp gly phe arg phe asp gly val thr ser met leu tyr his gly phe asp tyr arg leu ala met ala ile pro asp arg trp ile asp tyr leu lys asn lys asp asp ser glu trp ser met gly glu ile ala his thr leu thr asn arg arg tyr thr glu lys cys ile ala tyr ala glu ser his asp gln ser ile val gly asp lys thr ile ala phe leu leu met asp lys glu met tyr thr gly met ser asp leu gln pro ala ser pro thr ile asp arg gly ile ala leu gln lys met ile his phe trp ser tyr asp lys cys arg arg gln trp ser leu val asp thr asp his leu arg tyr lys tyr met asn ala phe asp gln ala met asn ala asp met asn asp glu glu lys val ile val phe glu arg gly asp leu val gly cys asp leu pro gly lys tyr arg val ala leu asp ser asp lys lys gly trp lys phe ala arg gln pro ser asp gln asp thr lys leu asp pro lys leu glu glu phe lys asp his phe asn tyr arg ile lys arg tyr leu asp gln lys cys leu ile glu lys his glu gly gly leu glu glu phe ser lys gly tyr leu lys phe gly ile asn thr val asp gly ala thr ile tyr arg glu trp ala pro ala ala gln glu ala gln leu ile gly glu phe asn asn trp asn gly ala lys his lys met glu lys asp lys phe gly ile trp ser ile lys ile ser his val asn gly lys pro ala ile pro his asn ser lys val lys phe arg phe arg ala thr phe asp ala ser lys phe gly ala pro tyr asp gly val his trp asp pro pro ala cys glu arg tyr val phe lys his pro arg pro pro lys pro asp ala pro arg ile tyr glu ala his val gly met ser gly glu glu pro glu val ser thr tyr arg glu phe ala asp asn val leu pro arg ile arg ala asn asn tyr asn thr val gln leu met ala ile met glu his ser tyr tyr ala ser phe gly tyr his val thr asn phe phe ala val ser ser arg ser gly thr pro glu asp leu lys tyr asp val gly gln asn thr his glu ser tyr phe his thr gly asp arg gly tyr his lys leu trp asp ser arg leu phe asn tyr ala asn trp glu val leu arg phe leu leu ser asn leu arg tyr trp met asp glu asn his leu met his lys leu leu pro glu ala thr ile val ala glu val gly phe asp phe arg leu ala met ala ile pro asp arg trp ile asp tyr leu lys asn lys glu asp arg lys trp ser met ser glu ile val gln thr leu thr asn arg arg tyr thr glu lys cys ile ala tyr ala glu ser his asp gln ser ile val gly asp lys thr ile ala phe leu leu met asp lys glu met tyr thr gly met ser asp leu gln pro ala ser pro thr ile asn arg gly ile ala leu gln lys met ile his asn trp ser tyr asp lys cys arg arg gln trp ser leu val asp thr asp his leu arg tyr lys tyr met asn ala phe asp gln ala met asn ser asp met asn glu lys asp lys val ile val phe glu arg gly asp lys val gly cys asp leu pro gly lys tyr arg val ala leu asp ser thr met pro ser val glu glu asp phe glu asn ile gly ile leu asn val asp ser ser leu glu pro phe lys asp his phe lys tyr arg leu glu asp gly ile ser tyr arg glu trp ala pro ala ala gln glu ala gln ile ile gly asp phe asn gly trp asn gly ser asn leu his met glu lys asp gln phe gly val trp ser ile gln ile pro asp ala asp gly asn pro ala ile pro his asn ser arg val lys phe arg phe lys his ser asp gly val trp val asp arg ile pro ala trp ile lys tyr trp asp pro pro leu ser glu arg tyr gln phe lys his pro arg pro pro lys pro lys ala pro arg ile tyr glu ala his val gly met ser ser ser glu pro arg ile asn ser tyr arg glu phe ala asp asp val leu pro arg ile arg glu asn asn tyr asn thr val gln leu met ala val met glu his ser tyr tyr ala ser phe trp tyr his val thr lys pro phe phe ala val ser ser arg ser gly ser pro glu asp leu lys tyr leu ile asp lys ala his ser leu gly leu asn val leu met asp arg gly tyr his lys leu trp asp ser arg leu phe asn tyr ala asn tyr lys phe asp gly phe arg phe asp gly val thr ser met leu tyr his his his gly ile asn met ala phe thr gly asp tyr asn glu tyr asn ser leu val his asp ile leu pro asp ala thr asp ile ala glu ile gly phe asp tyr arg leu ala met ala ile pro asp lys trp ile ala glu ser his asp gln ser ile val gly asp lys thr ile ala phe leu ser pro thr ile glu arg gly ile ser leu his lys met ile his gly trp ser tyr glu lys cys arg leu thr gln trp asn leu val asp thr asn his leu arg tyr lys phe met asn ala phe asp arg ala met val ser ser thr asn asn glu asp lys val ile val phe glu arg gly asp leu val phe val phe asn phe his pro glu asn thr tyr glu gly pro asn leu gly ser val glu glu thr phe ala ala ala asp thr asp val ala arg ile pro asp val ser met glu ser glu asp ser asn leu met tyr asp phe met ala leu asp arg pro ser thr pro arg ile asp glu trp ile asp phe pro arg gly pro gln thr leu pro thr gly lys val leu pro gly asn asn asn ser tyr asp lys cys arg arg arg phe asp gln ala met gln his leu glu glu lys tyr gly phe met thr ser phe glu arg gly asp leu val phe val phe asn phe his trp ser asn met tyr asp phe met ala leu asp arg pro ser thr pro thr ile asp glu trp ile asp phe pro arg gly pro gln arg leu pro ser gly lys phe ile pro gly asn asn asn ser tyr asp lys cys arg arg arg phe asp leu gly asp ala asp tyr leu arg tyr his gly met gln glu phe asp gln ala met gln his leu glu gln lys tyr glu phe met thr ser ser tyr phe asp tyr arg ile gly cys arg lys pro gly val tyr lys met tyr asp phe met ala leu asp arg pro ser thr pro arg ile asp glu trp ile asp phe pro arg gly pro gln ser leu pro asn gly ser val ile pro gly asn asn asn ser phe asp lys cys arg arg arg phe asp gln ala met gln his leu glu gly lys tyr glu phe met thr ser phe glu arg gly asp leu val phe val phe asn phe his trp ser asn asp his asp ala glu tyr phe thr ala asp trp pro his asp asn arg lys ile ala ala thr thr gly tyr gly ser asp his leu pro ile tyr ala gln leu val gly asp phe asn asn trp asn gly ser gly his lys met ala lys asp asn phe gly val trp ser ile arg ile ser his val asn gly lys pro ala ile pro his asn ser lys val lys phe arg phe arg his his gly val trp val glu gln ile pro ala trp ile arg tyr ala thr val thr ala ser glu ser gly ala pro tyr asp gly leu his trp asp pro pro ser ser glu arg tyr val phe asn his pro arg pro pro lys pro asp val pro arg ile tyr glu ala his val gly val ser gly gly lys leu glu ala gly thr tyr arg glu phe pro asp asn val leu pro cys leu arg ala thr asn tyr asn thr val gln leu met gly ile met glu his ser asp ser ala ser phe gly tyr his val thr asn phe phe ala val ser ser arg ser gly thr pro glu asp leu lys tyr leu ile asp lys ala his ser leu gly leu arg val leu met asp val asp val gly gln ser ala his glu ser tyr phe tyr thr gly asp lys glu val leu arg phe leu leu ser asn leu arg tyr trp met asp glu ile gly leu asp thr asn val asp ala phe val tyr met met leu ala asn his leu met his lys leu phe pro glu ala ile val val ala val leu gly phe asp tyr arg gln ala met thr ile pro asp arg trp ile asp tyr leu glu asn lys gly asp gln gln trp ser met ser ser val ile ser gln thr leu thr asn arg arg tyr pro glu lys phe ile ala tyr ala glu arg gln asn his ser ile ile gly ser lys thr met ala phe leu leu met glu trp glu thr tyr ser gly met ser ala met asp pro asp ser pro thr ile asp arg ala ile ala leu gln lys met ile his phe ile thr met ala phe gly gly asp ser tyr leu lys phe met gly asn glu tyr met asn ala phe val gln ala val asp thr pro ser asp lys cys ser phe leu ser ser ser asn gln thr ala ser his met pro arg asp tyr thr met ala thr ala glu asp gly val gly asp leu pro ile tyr asp leu asp pro lys phe ala gly phe lys glu his phe ser tyr arg met lys lys tyr leu asp gln lys his ser ile glu lys ile asn thr glu asn asp ala thr val tyr arg glu trp ala pro ala ala met asp ala gln leu ile gly asp phe asn asn trp asn gly ser gly his arg met thr lys asp asn tyr gly val trp ser ile arg ile trp ile arg tyr ala thr phe asp ala ser lys phe gly ala pro tyr asp gly val his trp asp pro pro ser gly glu arg tyr val phe lys val gly met ser gly glu lys pro glu val ser thr tyr arg glu phe ala asp asn val leu pro arg ile lys ala asn asn tyr asn thr val gln leu met ala ile met glu his ser tyr tyr ala ser phe gly tyr his val thr asn phe phe ala val ser ser arg ser gly thr pro glu leu met asp val val his ser his ala ser ser asn lys thr asp gly leu asn gly tyr asp val gly gln asn thr gln glu ser tyr phe his thr gly glu arg gly tyr his lys leu trp asp ser arg leu phe asn val val ala glu asp val ser gly met pro val leu cys arg ser val asp glu gly gly val gly phe asp tyr arg leu ala met ala ile pro met ser gly ile ala his thr leu thr asn arg arg tyr thr glu lys cys ile ala tyr ala glu ser his asp gln ser ile val gly asp lys thr met ala phe leu leu met asp lys glu met tyr thr gly met ser lys met ile his phe ile thr met ala leu gly 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