Patent Application: US-22101398-A

Abstract:
the invention relates generally to isolated genes which encode polypeptides involved in cellulose biosynthesis in plants and transgenic plants expressing same in sense or antisense orientation , or as ribozymes , co - suppression or gene - targeting molecules . more particularly , the present invention is directed to a nucleic acid molecule isolated from arabidopsis thaliana , oryza sativa , wheat , barley , maize , brassica ssp ., gossypium hirsutum ssp . and eucalyptus ssp . which encode an enzyme which is important in cellulose biosynthesis , in particular the cellulose synthase enzyme and homologues , analogues and derivatives thereof and uses of same in the production of transgenic plants expressing altered cellulose biosynthetic properties .

Description:
the derived amino acid sequences encoded by the cdnas listed in table 8 , is provided in fig8 and 9 and seq id nos : 8 and 10 herein . fig1 a schematic representation of the important features of the rsw1 polypeptide which are conserved within a . thaliana and between a . thaliana and other plant species . in addition to the species indicated in fig1 , the present inventors have also identified maize , wheat , barley and brassica ssp . cellulose biosynthetic genes by homology search . accordingly , the present invention extends to cellulose genes and cellulose biosynthetic polypeptides as hereinbefore defined , derived from any plant species , including a . thaliana , cotton , rice , wheat , barley , maize , eucalyptus ssp ., brassica ssp . pinus ssp ., populus ssp ., picea ssp ., hemp , jute and flax , amongst others . arabidopsis thaliana double - stranded cdna and cdna libraries were prepared using the capfinder cdna kit ( clontech ). rna was isolated from arabidopsis thaliana columbia rsw1 mutant plants grown in sterile conditions for 21 days . the full - length rsw1 mutant nucleotide sequence was generated by sequencing two amplified dna fragments spanning the rsw1 mutant gene . the 5 ′- end sequence of the cdna ( comprising the 5 ′- untranslated region and exons 1 - 11 ) was amplified using the primer combination 2280 - f / csp1 - r ( example 7 ). the 3 ′- end sequence was amplified using the primers est1 - f and cs3 - r set forth below : wherein primer est1 - f corresponds to nucleotide positions 1399 - 1420 of seq id no : 5 ( within exon 8 ) and primer cs3 - r is complementary to nucleotides 3335 - 3359 of seq id no : 5 ( within the 3 ′- untranslated region of the wild - type transcript ). the full - length sequence of the mutant rsw1 transcript is set forth herein as seq id no : 11 . whilst not being bound by any theory or mode of action , a single nucleotide substitution in the rsw1 mutant nucleotide sequence ( nucleotide position 1716 in seq id no : 11 ), relative to the wild - type rsw1 nucleotide sequence ( nucleotide position 1646 in seq id no : 5 ), resulting in ala549 being substituted with val549 in the mutant polypeptide , may contribute to the altered activity of the rsw1 polypeptide at non - permissive temperatures such as 31 ° c . additional amino acid substitutions are also contemplated by the present invention , to alter the activity of the rsw1 polypeptide , or to make the polypeptide temperature - sensitive . one example of transgenic plants in which cellulose production is inhibited is provided by the expression of an antisense genetic construct therein . antisense technology is used to target expression of a cellulose gene ( s ) to reduce the amount of cellulose produced by transgenic plants . by way of exemplification , an antisense plant transformation construct has been engineered to contain the t20782 cdna insert ( or a part thereof ) in the antisense orientation and in operable connection with the camv 35s promoter present in the binary plasmid prd410 ( datla et al . 1992 ). more particularly , the t20782 cdna clone , which comprises the 3 ′- end of the wild - type rsw1 gene , was digested with xbai and kpni and cloned into the kanamycin - resistant derivative of pgem3zf (−), designated as plasmid , pjkkmf (−). the rsw1 sequence was sub - cloned , in the antisense orientation , into the binary vector prd410 as a xbai / saci fragment , thereby replacing the β - glucuronidase ( gus or uida ) gene . this allows the rsw1 sequence to be transcribed in the antisense orientation under the control of the camv 35s promoter . the antisense rsw1 binary plasmid vector was transferred to agrobacterium tumefaciens strain agl1 , by triparental mating and selection on rifampicin and kanamycin , as described by lazo et al . ( 1991 ). the presence of the rsw1 insert in transformed a . tumefaciens cells was confirmed by southern hybridization analysis ( southern , 1975 ). the construct was shown to be free of deletion or rearrangements prior to transformation of plant tissues , by back - transformation into escherichia coli strain jm101 and restriction digestion analysis . eight pots , each containing approximately 16 a . thaliana ecotype columbia plants , were grown under standard conditions . plant tissue was transformed with the antisense rsw1 binary plasmid by vacuum infiltration as described by bechtold et al ( 1993 ). infiltration media contained 2 . 5 % ( w / v ) sucrose and plants were infiltrated for 2 min until a vacuum of approximately 400 mm hg was obtained . the vacuum connection was shut off and plants allowed to sit under vacuum for 5 min . approximately 34 , 000 t1 seed was screened on ms plates containing 50 μg / ml kanamycin , to select for plants containing the antisense rsw1 construct . of the t1 seed sown , 135 kanamycin - resistant seedlings were identified , of which 91 were transferred into soil and grown at 21 ° c . under a long - day photoperiod ( 16 hr light ; 8 hr dark ). of the 91 transgenic lines , 19 lines were chosen for further analysis which had anther filaments in each flower which were too short to deposit pollen upon the stigma and , as a consequence , required hand - pollination to obtain t2 seed therefrom . t2 seed from 14 of these 19 lines was plated out onto vertical hoaglands plates containing kanamycin to determine segregation ratios . between five and ten seed were plated per transgenic line . control seeds , including a . thaliana columbia containing the binary vector pbin19 ( bevan , 1984 ) and segregating 3 : 1 for kanamycin resistance , and the rsw1 mutant transformed with the nptii gene , also segregating 3 : 1 for kanamycin resistance , were grown under the same conditions . kanamycin - resistant plants were transferred to soil and grown at 21 ° c . under long days , until flowering . untransformed arabidopsis thaliana columbia plants were also grown under similar conditions . in the absence of kanamycin . a comparison of the morphology of antisense rsw1 plants grown at 21 ° c ., to mutant rsw1 plants grown at the non - permissive temperature ( i . e . 31 ° c .) has identified a number of common phenotypes . for example , the antisense plants exhibit reduced fertility , inflorescence shortening and have short anthers , compared to wild - type plants , when grown at 21 ° c . these phenotypes are also observed in mutant rsw1 plants grown at 31 ° c . these results suggest that the antisense construct in the transgenic plants may be targeting the expression of the wild - type rsw1 gene at 21 ° c . fig7 shows the reduced inflorescence ( bolt ) height in antisense 35s - rsw1 plants compared to wild - type a . thaliana columbia plants grown under identical conditions . t3 plants which are homozygous for the 35s - rsw1 antisense construct are generated and the content of cellulose therein is determined as described in example 1 . plants expressing the antisense construct are shown to have significantly less cellulose in their cell walls , compared to wild - type plants . additionally , the levels of non - crystalline β - 1 , 4 - glucan and starch are elevated in the cells of antisense plants , compared to otherwise isogenic plant lines which have not been transformed with the antisense genetic construct . total rna was extracted from 0 . 2 g of leaf tissue derived from 33 kanamycin - resistant t1 plants containing the antisense 35s - rsw1 genetic construct , essentially according to longemann et al . ( 1986 ). total rna ( 25 μg ) was separated on a 2 . 2m formaldehyde / agarose gel , blotted onto nylon filters and hybridized to a riboprobe comprising the sense strand sequence of the cdna clone t20782 . to produce the riboprobe , t7 rna polymerase was used to transcribe sense rna from a linearised plasmid template containing t20782 , in the presence of [ α - 32 p ] utp . hybridizations and subsequent washes were performed as described by dolferus et al . ( 1994 ). hybridized membranes were exposed to phosphor screens ( molecular dynamics , usa ). the levels of expression of the rsw1 antisense transcript were determined and compared to the level of fertility observed for the plant lines . as shown in table 9 , the level of antisense gene expression is correlated with the reduced fertility phenotype of the antisense plants . in 13 lines , a very high or high level of expression of the 35s - rsw1 antisense gene was observed and , in 11 of these lines fertility was reduced . only lines 2w and 3e which expressed high to very high levels of antisense mrna , appeared to be fully fertile . in 12 lines which expressed medium levels of antisense mrna , approximately one - half were fertile and one - half appeared to exhibit reduced fertility . in contrast , in 8 plant lines in which only a low or very low level of expression of the antisense 35s - rsw1 genetic construct was observed , a wild - type ( i . e . fertile ) phenotype was observed for all but one transgenic line , line 2r . data presented in table 9 and fig7 indicate that the phenotype of the cellulose - deficient mutant rsw1 may be reproduced by expressing antisense rsw1 genetic constructs in transgenic plants . to confirm reduced cellulose synthesis and / or deposition in transgenic plants expressing the antisense rsw1 gene , the level of cellulose is measured by the 14 c incorporation assay or as acetic / nitric acid insoluble material as described in example 1 and compared to cellulose production in otherwise isogenic wild - type plants . cellulose production in the transgenic plants is shown to be significantly reduced compared to wild - type plants . the severity of phenotype of the transgenic plants thus produced varies considerably , depending to some extent upon the level of inhibition of cellulose biosynthesis . to identify rsw1 related nucleotide sequences in rice , a genetic sequence database was searched for nucleotide sequences which were closely - related to one or more of the arabidopsis thaliana rsw1 nucleotide sequences described in the preceding examples . rice est s0542 ( maff dna bank , japan ) was identified , for which only a partial nucleotide sequences was available . additionally , before the instant invention , there was no probable function attached to the rice est s0542 sequence . the present inventors have obtained the complete nucleotide sequence of clone s0542 and derived the amino acid sequence encoded therefor . the s0542 cdna is only 1741 bp in length and appears to be a partial cdna clone because , although it comprises 100 bp of 5 ′- untranslated sequence and contains the atg start codon , it is truncated at 3 ′- end and , as a consequence encodes only the first 547 amino acid residues of the rice rsw1 or rsw1 - like polypeptide . based upon the length of the corresponding arabidopsis thaliana rsw1 polypeptide ( 1081 amino acids ), the rice rsw1 sequence set forth in seq id no : 14 appears to contain approximately one - half of the complete amino acid sequence . the n - terminal half of the rice rsw1 amino acid sequence is approximately 70 % identical to the arabidopsis thaliana rsw1 polypeptide set forth in seq id no : 6 , with higher homology ( approximately 90 %) occurring between amino acid residues 271 - 547 of the rice sequence . these data strongly suggest that s0542 is the rice homologue of the a . thaliana rsw1 gene . alignments of rice , a . thaliana and cotton rsw1 amino acid sequences are presented in fig9 and 10 . to isolate full - length cdna clones and genomic clone equivalents of s0542 ( this study and maff dna bank , japan ) or d48636 ( pear et al ., 1996 ), cdna and genomic clone libraries are produced using rice mrna and genomic dna respectively , and screened by hybridisation using the s0542 or d48636 cdnas as a probe , essentially as described herein . positive - hybridising plaques are identified and plaque - purified . during further rounds of screening by hybridisation , to single plaques . the rice clones are sequenced as described in the preceding examples to determine the complete nucleotide sequences of the rice rsw1 genes and derived amino acid sequences therefor . those skilled in the art will be aware that such gene sequences are useful for the production of transgenic plants , in particular transgenic cereal plants having altered cellulose content and / or quality , using standard techniques . the present invention extends to all such genetic sequences and applications therefor . a 32 p - labelled rsw1 pcr fragment was used to screen approximately 200 , 000 cdna clones in a cotton fibre cdna library . the rsw1 pcr probe was initially amplified from arabidopsis thaliana wild type cdna using the primers 2280 - f and csp1 - r described in the preceding examples . and then re - amplified using the primer combination 2370 - f / csp1 - r , also described in the preceding examples . hybridisations were carried out under low stringency conditions at 55 ° c . six putative positive - hybridising plaques were identified in the first screening round . using two further rounds of screening by hybridisation , four of these plaques were purified to single plaques . three plaques hybridise very strongly to the rsw1 probe while the fourth plaque hybridises less intensely . we conclude that the positive - hybridising plaques which have been purified are strong candidates for comprising cotton rsw1 gene sequences or rsw1 - like gene sequences . furthermore , the cotton cdnas may encode the catalytic subunit of cellulose synthase , because the subunit protein architecture of cellulose synthase appears to be highly conserved among plants as highlighted in the preceding example . furthermore , a southern blot of cotton genomic dna digested with bglii was hybridised with the 5 ′ end of the rsw1 cdna , under low stringency hybridisation conditions at 55 ° c . results are presented in fig1 . these data demonstrate that rsw1 - related sequences exist in the cotton genome . the cotton cdna clones described herein are sequenced as described in the preceding examples and used to produce transgenic cotton plants having altered fibre characteristics . the cdnas are also used to genetically alter the cellulose content and / or quality of other plants , using standard techniques . putative eucalyptus ssp . cellulose synthase catalytic subunit gene fragments were obtained by amplification using pcr . dna primers were designed to conserved amino acid residues found in the arabidopsis thaliana rsw1 and 12c4 amino acid sequences . three primers were used for pcr . the primers are listed below : using standard pcr conditions ( 50 ° c . annealing temperature ) and solutions , the primer sets pcsf - i / pcsr - ii and pcsf - ii / pcsr - ii were used to amplify genetic sequences from pooled eucalyptus ssp . cdna . in the first reaction primers pcsf - i and pcsr - ii were used to generate a fragment approximately 700 bp in length . in the second pcr reaction , which used primers pcsf - ii and pcsr - ii , a fragment estimated to 700 bp was obtained . the sizes of the pcr fragments are within the size range estimated for the corresponding arabidopsis thaliana sequences . we conclude that the amplified eucalyptus ssp . pcr fragments are likely to be related to the arabidopsis thaliana rsw1 gene and may encode at least a part of the eucalyptus ssp . cellulose synthase catalytic subunit . the eucalyptus ssp . pcr clones described herein are sequenced as described in the preceding examples and used to isolate the corresponding full - length eucalyptus ssp cdnas and genomic gene equivalents . those skilled in the art will be aware that such gene sequences are useful for the production of transgenic plants , in particular transgenic eucalyptus ssp plants having altered cellulose content and / or quality , using standard techniques . the present invention extends to all such genetic sequences and applications therefor . the properties of plant cell walls depend on the carbohydrates , proteins and other polymers of which they are composed and the complex ways in which they interact . increasing the quantities of non - crystalline β - 1 , 4 - glucan in cell walls affects those wall properties which influence mechanical , nutritional and many other qualities as well as having secondary consequences resulting from the diversion of carbon into non - crystalline glucan at the expense of other uses . to illustrate one of these effects , we investigated the ability of the non - crystalline glucan to hydrogen bond to other wall components particularly cellulose in the way that has been shown to be important for wall mechanics . hemicelluloses such as xyloglucans cross - link cellulose microfibrils by hydrogen bonding to the microfibril surface ( levy et al ., 1991 ). since the β - 1 , 4 - glucan backbone of xyloglucan is thought to be responsible for hydrogen bonding ( with the xylose , galactose and fucose substitutions limiting the capacity to form further hydrogen bonds ) we can expect the non - crystalline β - 1 , 4 - glucan also to have a capacity to hydrogen bond and cross link cellulose . the effectiveness of strong alkalis in extracting xyloglucans is thought to relate to their disruption of the hydrogen bonds with cellulose ( hayashi and maclachlan , 1984 ). to demonstrate that the non - crystalline β - 1 , 4 - glucan forms similar associations with the cellulose microfibrils , we examined whether the 4 m koh fraction , extracted from shoots of the rsw1 mutant and from wild type rsw1 plants , contained non - crystalline glucan in addition to xyloglucan . the non - crystalline glucan was separated from xyloglucan in the 4 m koh extract by dialysing the neutralised extract against distilled water and centrifuging at 14000 g for 1 hour . the pellet was shown to be a pure β - 1 , 4 - glucan by using the methods for monosaccharide analysis , methylation analysis and enzyme digestion used to characterise the glucan in the ammonium oxalate fraction ( see example 1 ). table 10 shows the presence of substantial quantities of glucan recovered in pure form in the pellet from 4 m koh fractions extracted from the overproducing rsw1 mutant of arabidopsis thaliana . these data also demonstrate the presence of smaller quantities of non - crystalline β - 1 , 4 - glucan in the 4 m koh fraction from wild type plants , compared to rsw1 , particularly when grown at 31 ° c . the monosaccharide composition of the supernatant remaining after centrifugation was determined after tfa hydrolysis . these data , and data from methylation analysis , are consistent with the supernatant being a relatively pure xyloglucan . the supernatant was free of glucan , because no glucose could be released by the endocellulase / β - glucosidase mixture that released glucose from β - 1 , 4 - glucan . the presence of both non - crystalline β - 1 , 4 - glucan and xyloglucan in the 4 m koh fraction , when taken together with the implications from structural predictions ( levy et al , 1991 ), is consistent with some of the non - crystalline β - 1 , 4 - glucan in the wall hydrogen bonding to cellulose microfibrils in similar fashion to the β - 1 , 4 - glucan backbone of xyloglucan . the cross linking provided when xyloglucans and other hemicelluloses bind to two or more microfibrils is an important determinant of the mechanical properties of cellulosic walls ( hayashi , 1989 ). the effects of increasing the amounts of non - crystalline β - 1 , 4 - glucan in walls are likely to be greatest in walls which otherwise possess relatively low levels of cross linking as a result of high ratios of cellulose : hemicelluloses . such conditions are common in secondary walls including those of various fibres , and the cellulose : hemicellulose ratio is particularly high in cotton fibres . the effects on wall mechanical properties of overproducing non - crystalline glucan are shown by transforming plants with the mutant allele of rsw1 ( seq id no : 11 ) operably under the control of either the rsw1 promoter derived from seq id no : 3 or seq id no : 4 or alternatively , an appropriate constitutive promoter such as the camv 35s promoter . production of non - crystalline glucan is quantified by fractionating the cell walls using the methods described above to show in particular that non - crystalline glucan is recovered in the 4 m koh fraction . mechanical properties of the cell walls are measured using standard methods for fibre analysis to study parameters such as stress - strain curves , and breaking strain , amongst other properties . three strategies are employed to over - express cellulose synthase in arabidopsis thaliana plants . in the first strategy , the camv 35s promoter sequence is operably connected to the full - length cellulose synthase cdna which is obtainable by primer extension of seq id no : 1 . this is achievable by cloning the full - length cdna encoding cellulose synthase , in the sense orientation , between the camv 35s promoter or other suitable promoter operable in plants and the nopaline synthase terminator sequences of the binary plasmid pbi121 . in the second strategy , the coding part of the genomic gene is cloned , in the sense orientation , between the camv 35s promoter and the nopaline synthase terminator sequences of the binary plasmid pbi121 . in the third strategy , the 23h12 binary cosmid clone or the derivative prsw1 , containing the cellulose synthase gene sequence operably under the control of the cellulose synthase gene promoter and terminator sequences is prepared in a form suitable for transformation of plant tissue . for agrobacterium - mediated tissue transformation , binary plasmid constructs discussed supra are transformed into agrobacterium tumefaciens strain agl1 or other suitable strain . the recombinant dna constructs are then introduced into wild type arabidopsis thaliana plants ( columbia ecotype ), as described in the preceding examples . alternatively , plant tissue is directly transformed using the vacuum infiltration method described by beshtold et al . ( 1993 ). the transgenic plants thus produced exhibit a range of phenotypes , partly because of position effects and variable levels of expression of the cellulose synthase transgene . cellulose content in the transgenic plants and isogenic untransformed control plants is determined by the 14 c incorporation assay or as acetic / nitric acid insoluble material as described in example 1 . in general , the level of cellulose deposition and rates of cellulose biosynthesis in the transgenic plants are significantly greater than for untransformed control plants . furthermore , in some cases , co - supression leads to mimicry of the rsw1 mutant phenotype . the nucleotide sequence of the rsw1 gene contained in 23h12 is mutated using site - directed mutagenesis , at several positions to alter its catalytic activity or substrate affinity or glucan properties . in one example , the rsw1 gene is mutated to comprise one or more mutations present in the mutant rsw1 allele . the mutated genetic sequences are cloned into binary plasmid described in the preceding examples , in place of the wild - type sequences . plant tissue obtained from both wild - type arabidopsis thaliana ( columbia ) plants and a . thaliana rsw1 plants is transformed as described herein and whole plants are regenerated . control transformations are performed using the wild - type cellulose synthase gene sequence . plants transformed with genetic constructs described in example 15 ( and elsewhere ) are categorised initially on the basis of number of transgene copies , to eliminate variability arising therefrom . plants expressing single copies of different transgenes are analysed further for cell wall components , including cellulose , non - crystalline β - 1 , 4 - glucan polymer , starch and carbohydrate content . cellulose content in the transgenic plants is determined by the 14 c incorporation assay as described in example 1 . cell walls are prepared fractionated and the monosaccharide composition of individual fractions determined as in example 1 . transgenic plants expressing the rsw1 mutant allele exhibit a higher level of non - crystalline , and therefore extractable , β - 1 , 4 - glucan in cell walls compared to plants expressing an additional copy of the wild - type rsw1 allele . thus , it is possible to change the crystallinity of the β - 1 , 4 - glucan chains present in the cell wall by mutation of the wild - type rsw1 allele . transgenic plants are also analysed to determine the effect of mutagenesis of the rsw1 gene on the level of starch deposited in their roots . the quantity of starch present in material prepared from the crude wall fraction is determined using the anthrone / h 2 so 4 method described in example 1 . the data show that mutating the rsw1 gene to the mutant rsw1 allele increases starch deposition . this demonstrates that the gene can be used to alter the partitioning of carbon into carbohydrates other than cellulose . the cell wall composition of transgenic plant material is also analysed . wild type and rsw1 and transgenic seedlings are grown for 2 d at 21 ° c . and then kept for a further 5 d at either 21 ° c . or 31 ° c . with transfer to 31 ° c . when the seed has scarcely germinated , the wall composition at final harvest largely reflects the operation of the mutated rsw1 gene product at its restrictive temperature . cell wall fractionation is carried out in similar fashion to that described for the 14 c - experiment ( example 1 ) and the monosaccharide composition of each fraction is quantified by gc / ms after hydrolysis with trifluoroacetic acid or , in the case of crystalline cellulose . h 2 so 4 . in some transgenic plants in which the rsw1 gene is mutated , the monosaccharide composition is comparable to that observed for homozygous rsw1 plants , at least in some cases , confirming that there is a major reduction in the quantity of crystalline cellulose in the final , acid insoluble fraction . thus , mutation of the rsw1 gene can be performed to produce changes in the composition of plant cell walls . chemical modification of the rsw1 gene to manipulate cellulose production and plant cell wall content as demonstrated in the preceding examples , the rsw1 gene is involved in cellulose production and the manipulation of cell wall content . in the present example , to identify novel phenotypes and gene sequences important for the normal functioning of the cellulose synthase gene , the rsw1 gene is modified in planta , using the chemical mutagen ems . the mutant plants are identified following germination and the modified rsw1 genes are isolated and characterised at the nucleotide sequence level . a sequence comparison between the mutant gene sequences and the wild type sequence reveals nucleotides which encode amino acids important to the normal catalytic activity of the cellulose synthase enzyme , at least in arabidopsis thaliana plants . this approach thus generates further gene sequences of utility in the modification of cellulose content and properties in plants . five pieces of evidence make a compelling case that the rsw1 gene product encodes the catalytic subunit of cellulose synthase : 1 . the rsw1 mutation selectively inhibits cellulose synthesis and promotes accumulation of a non - crystalline β - 1 , 4 - glucan ; 2 . the rsw1 mutation removes cellulose synthase complexes from the plasma membrane , providing a plausible mechanism for reduced cellulose accumulation and placing the rsw1 product either in the complexes or interacting with them ; 3 . the d , d , d , qxxrw ( seq id no : 37 ) signature identifies the rsw1 gene product as a processive glycosyl transferase enzyme ( saxena , 1995 ); 4 . the wild type allele corrects the temperature sensitive phenotype of the rsw1 mutant ; and 5 . antisense expression of the rsw1 in transgenic plants grown at 21 ° c . reproduces some of the phenotype of rsw1 which is observed following growth at 31 ° c . consistent with the plasma membrane location expected for a catalytic subunit , the putative 122 kda rsw1 product contains 8 predicted membrane - spanning regions . six of these regions cluster near the c - terminus ( fig1 ), separated from the other two by a domain that is probably cytoplasmic and has the weak sequence similarities to prokaryotic glycosyl transferases ( wong , 1990 ; saxena , 1990 ; matthyse , 1995 ; sofia , 1994 ; kutish , 1996 ). rsw1 therefore qualifies as a member of the large family of arabidopsis thaliana genes whose members show weak similarities to bacterial cellulose synthase . rsw1 is the first member of that family to be rigorously identified as an authentic cellulose synthase . among the diverse genes in a . thaliana , at least two genes show very strong sequence similarities to the rsw1 gene and are most likely members of a highly conserved sub - family involved in cellulose synthesis . the closely related sequences come from cosmid 12c4 , a partial genomic clone cross - hybridising with est t20782 designated ath - a . and from a full length cdna designated ath - b . ath - a resembles rsw1 ( seq id no : 5 ) at its n - terminus whereas ath - b starts 22 amino acid residues downstream [ fig8 and fig9 ( i ), ( ii ) and ( iii )]. closely related sequences in other angiosperms are the rice est s0542 [ fig9 ( i ), ( ii ) and ( iii )], which resembles the polypeptides encoded by rsw1 and ath - a and the cotton cela1 gene ( pear , 1996 ) at the n - terminus . the arabidopsis thaliana , rice and cotton genes have regions of very high sequence similarity interspersed with variable regions ( fig9 and 10 ). most of the highest conservation among those gene products occurs in their central cytoplasmic domain where the weak similarities to the bacterial cellulose synthase occur . the n - terminal region that precedes the first membrane spanning region is probably also cytoplasmic but shows many amino acid substitutions as well as sequences in rsw1 that have no counterpart in some of the other genes as already noted for cela . an exception to this is a region comprising 7 cysteine residues with highly conserved spacings ( fig1 ). this is reminiscent of regions suggested to mediate protein - protein and protein - lipid interactions in diverse proteins including transcriptional regulators and may account for the striking sequence similarity between this region of rsw1 and two putative soybean bzip transcription factors ( genbank soystf1a and 1b ). in conclusion , the chemical and ultrastructural changes seen in the cellulose - deficient mutant combine with gene cloning and complementation of the mutant to provide strong evidence that the rsw1 locus encodes the catalytic subunit of cellulose synthase . accumulation of non - crystalline β - 1 , 4 - glucan in the shoot of the rsw1 mutant suggests that properties affected by the mutation are required for glucan chains to assemble into microfibrils . whilst not being bound by any theory or mode of action , a key property may be the aggregation of catalytic subunits into plasma membrane rosettes . at the restrictive temperature , mutant synthase complexes disassemble to monomers ( or smaller oligomers ) that are undetectable by freeze etching . at least in the shoot , the monomers seem to remain biosynthetically active but their β - 1 , 4 - glucan products fail to crystallise into microfibrils probably because the chains are growing from dispersed sites . crystallisation into microfibrils , with all its consequences for wall mechanics and morphogenesis , therefore may depend upon catalytic subunits remaining aggregated as plasma membrane rosettes . those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described . it is to be understood that the invention includes all such variations and modifications . the invention also includes all of the steps , features , compositions and compounds referred to or indicated in this specification , individually or collectively , and any and all combinations or any two or more of said steps or features . 2 . ausubel , f . m ., brent , r ., kingston , r . e ., moore , d . d ., seidman , j . g ., smith , j . a . and struhl , k . 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( usa ) 87 : 8130 - 8134 . cga gct atg aag aga gag tat gaa gag ttt aaa gtg agg ata aat gct 48 ctt gtt gcc aaa gca cag aaa atc cct gga gaa ggc tgg aca atg cag 96 leu val ala lys ala gln lys ile pro gly glu gly trp thr met gln gat ggt act ccc tgg cct ggt aac aac act aga gat cat cct gga atg 144 ata cag gtg ttc tta ggc cat agt ggg ggt ctg gat acc gat gga aat 192 ile gln val phe leu gly his ser gly gly leu asp thr asp gly asn gag ctg cct aga ctc atc tat gtt tct cgt gaa aag cgg cct gga ttt 240 glu leu pro arg leu ile tyr val ser arg glu lys arg pro gly phe caa cac cac aaa aag gct gga gct atg aat gca tcg atc cgt gta tct 288 gln his his lys lys ala gly ala met asn ala ser ile arg val ser gct gtt ctt acc aat gga gca tat ctt ttg aac gtg gat tgt gat cat 336 tac ttt aat aac agt aag gct att aaa gaa gct atg tgt ttc atg atg 384 gac ccg gct att gga aag aag tgc tgc tat gtc cag ttc cct caa cgt 432 asp pro ala ile gly lys lys cys cys tyr val gln phe pro gln arg ttt gac ggt att gat ttg cac gat cga tat gcc aac agg aat ata gtc 480 phe asp gly ile asp leu his asp arg tyr ala asn arg asn ile val ttt ttc gat att aac atg aag ggg ttg gat ggt atc cac ggt cca gta 528 phe phe asp ile asn met lys gly leu asp gly ile his gly pro val tat gtg ggt act ggt tgt tgt ttt aat agg cag gct cta tat ggg tat 576 tyr val gly thr gly cys cys phe asn arg gln ala leu tyr gly tyr gat cct gtt ttg acg gaa gaa gat tta gaa cca aat att att gtc aag 624 agc tgt tgc ggg tca agg aag aaa ggt aaa agt agc aag aag tat aac 672 tac gaa aag agg aga ggc atc aac aga agt gac tcc aat gct cca ctt 720 tyr glu lys arg arg gly ile asn arg ser asp ser asn ala pro leu ttc aat atg gag gac atc gat gag ggt ttt gaa ggt tat gat gat gag 768 agg tct att cta atg tcc cag agg agt gta gag aag cgt ttt ggt cag 816 arg ser ile leu met ser gln arg ser val glu lys arg phe gly gln tcg ccg gta ttt att gcg gca acc ttc atg gaa caa ggc ggc att cca 864 ser pro val phe ile ala ala thr phe met glu gln gly gly ile pro cca aca acc aat ccc gct act ctt ctg aag gag gct att cat gtt ata 912 agc tgt ggt tac gaa gac aag act gaa tgg ggc aaa gag att ggt tgg 960 leu val ala lys ala gln lys ile pro gly glu gly trp thr met gln ile gln val phe leu gly his ser gly gly leu asp thr asp gly asn glu leu pro arg leu ile tyr val ser arg glu lys arg pro gly phe gln his his lys lys ala gly ala met asn ala ser ile arg val ser asp pro ala ile gly lys lys cys cys tyr val gln phe pro gln arg phe asp gly ile asp leu his asp arg tyr ala asn arg asn ile val phe phe asp ile asn met lys gly leu asp gly ile his gly pro val tyr val gly thr gly cys cys phe asn arg gln ala leu tyr gly tyr tyr glu lys arg arg gly ile asn arg ser asp ser asn ala pro leu arg ser ile leu met ser gln arg ser val glu lys arg phe gly gln ser pro val phe ile ala ala thr phe met glu gln gly gly ile pro ile tyr gly ser val thr glu asp ile leu thr gly phe lys met his ala arg gly trp ile ser ile tyr cys asn pro pro arg pro ala phe lys gly ser ala pro ile asn leu ser asp arg leu asn gln val leu arg trp ala leu gly ser ile glu ile leu leu ser arg his cys pro ile glu asp trp trp arg asn asn gln phe trp val ile gly gly thr val ser tyr ala val asn ser gly tyr gln ser trp gly pro leu phe gly lys leu phe phe ala leu trp val ile ala his leu tyr pro phe atg gag gcc agt gcc ggc ttg gtt gct gga tcc tac cgg aga aac gag 48 ctc gtt cgg atc cga cat gaa tct gat ggc ggg acc aaa cct ttg aag 96 leu val arg ile arg his glu ser asp gly gly thr lys pro leu lys aat atg aat ggc cag ata tgt cag atc tgt ggt gat gat gtt gga ctc 144 gct gaa act gga gat gtc ttt gtc gcg tgt aat gaa tgt gcc ttc cct 192 gtg tgt cgg cct tgc tat gag tac gag agg aaa gat gga act cag tgt 240 val cys arg pro cys tyr glu tyr glu arg lys asp gly thr gln cys tgc cct caa tgc aag act aga ttc aga cga cac agg ggg agt cct cgt 288 ttc aat tac gcc cag gga gct aac aag gcg aga cac caa cgc cat ggc 384 gaa gag ttt tct tct tcc tct aga cat gaa tct caa cca att cct ctt 432 ctc acc cat ggc cat acg gtt tct gga gag att cgc acg cct gat aca 480 leu thr his gly his thr val ser gly glu ile arg thr pro asp thr caa tct gtg cga act aca tca ggt cct ttg ggt cct tct gac agg aat 528 gct att tca tct cca tat att gat cca cgg caa cct gtc cct gta aga 576 atc gtg gac ccg tca aaa gac ttg aac tct tat ggg ctt ggt aat gtt 624 gac tgg aaa gaa aga gtt gaa ggc tgg aag ctg aag cag gag aaa aat 672 atg tta cag atg act ggt aaa tac cat gaa ggg aaa gga gga gaa att 720 gaa ggg act ggt tcc aat ggc gaa gaa ctc caa atg gct gat gat aca 768 cgt ctt cct atg agt cgt gtg gtg cct atc cca tct tct cgc cta acc 816 cct tat cgg gtt gtg att att ctc cgg ctt atc atc ttg tgt ttc ttc 864 ttg caa tat cgt aca act cac cct gtg aaa aat gca tat cct ttg tgg 912 leu gln tyr arg thr thr his pro val lys asn ala tyr pro leu trp ttg acc tcg gtt atc tgt gag atc tgg ttt gca ttt tct tgg ctt ctt 960 gat cag ttt ccc aaa tgg tac ccc att aac agg gag act tat ctt gac 1008 asp gln phe pro lys trp tyr pro ile asn arg glu thr tyr leu asp cgt ctc gct ata aga tat gat cga gac ggt gaa cca tca cag ctc gtt 1056 arg leu ala ile arg tyr asp arg asp gly glu pro ser gln leu val cct gtt gat gtg ttt gtt agt aca gtg gac cca ttg aaa gag cct ccc 1104 ctt gtt aca gca aac aca gtt ctc tcg att ctt tct gtg gac tac ccg 1152 gta gat aaa gta gcc tgt tat gtt tca gat gat ggt tca gct atg ctt 1200 acc ttt gaa tcc ctt tct gaa acc gct gag ttt gca aag aaa tgg gta 1248 cca ttt tgc aag aaa ttc aac att gaa cct agg gcc cct gaa ttc tat 1296 ttt gcc cag aag ata gat tac ttg aag gac aag atc caa ccg tct ttt 1344 gtt aaa gag cga cga gct atg aag aga gag tat gaa gag ttt aaa gtg 1392 agg ata aat gct ctt gtt gcc aaa gca cag aaa atc cct gaa gaa ggc 1440 arg ile asn ala leu val ala lys ala gln lys ile pro glu glu gly tgg aca atg cag gat ggt act ccc tgg cct ggt aac aac act aga gat 1488 cat cct gga atg ata cag gtg ttc tta ggc cat agt ggg ggt ctg gat 1536 his pro gly met ile gln val phe leu gly his ser gly gly leu asp acc gat gga aat gag ctg cct aga ctc atc tat gtt tct cgt gaa aag 1584 thr asp gly asn glu leu pro arg leu ile tyr val ser arg glu lys cgg cct gga ttt caa cac cac aaa aag gct gga gct atg aat gca ttg 1632 arg pro gly phe gln his his lys lys ala gly ala met asn ala leu atc cgt gta tct gct gtt ctt acc aat gga gca tat ctt ttg aac gtg 1680 gat tgt gat cat tac ttt aat aac agt aag gct att aaa gaa gct atg 1728 asp cys asp his tyr phe asn asn ser lys ala ile lys glu ala met tgt ttc atg atg gac ccg gct att gga aag aag tgc tgc tat gtc cag 1776 cys phe met met asp pro ala ile gly lys lys cys cys tyr val gln ttc cct caa cgt ttt gac ggt att gat ttg cac gat cga tat gcc aac 1824 phe pro gln arg phe asp gly ile asp leu his asp arg tyr ala asn agg aat ata gtc ttt ttc gat att aac atg aag ggg ttg gat ggt atc 1872 cag ggt cca gta tat gtg ggt act ggt tgt tgt ttt aat agg cag gct 1920 gln gly pro val tyr val gly thr gly cys cys phe asn arg gln ala cta tat ggg tat gat cct gtt ttg acg gaa gaa gat tta gaa cca aat 1968 att att gtc aag agc tgt tgc ggg tca agg aag aaa ggt aaa agt agc 2016 aag aag tat aac tac gaa aag agg aga ggc atc aac aga agt gac tcc 2064 aat gct cca ctt ttc aat atg gag gac atc gat gag ggt ttt gaa ggt 2112 tat gat gat gag agg tct att cta atg tcc cag agg agt gta gag aag 2160 tyr asp asp glu arg ser ile leu met ser gln arg ser val glu lys cgt ttt ggt cag tcg ccg gta ttt att gcg gca acc ttc atg gaa caa 2208 arg phe gly gln ser pro val phe ile ala ala thr phe met glu gln ggc ggc att cca cca aca acc aat ccc gct act ctt ctg aag gag gct 2256 att cat gtt ata agc tgt ggt tac gaa gac aag act gaa tgg ggc aaa 2304 ile his val ile ser cys gly tyr glu asp lys thr glu trp gly lys gag att ggt tgg atc tat ggt tcc gtg acg gaa gat att ctt act ggg 2352 ttc aag atg cat gcc cgg ggt tgg ata tcg atc tac tgc aat cct cca 2400 phe lys met his ala arg gly trp ile ser ile tyr cys asn pro pro cgc cct gcg ttc aag gga tct gca cca atc aat ctt tct gat cgt ttg 2448 arg pro ala phe lys gly ser ala pro ile asn leu ser asp arg leu aac caa gtt ctt cga tgg gct ttg gga tct atc gag att ctt ctt agc 2496 asn gln val leu arg trp ala leu gly ser ile glu ile leu leu ser aga cat tgt cct atc tgg tat ggt tac cat gga agg ttg aga ctt ttg 2544 gag agg atc gct tat atc aac acc atc gtc tat cct att aca tcc atc 2592 cct ctt att gcg tat tgt att ctt ccc gct ttt tgt ctc atc acc gac 2640 aga ttc atc ata ccc gag ata agc aac tac gcg agt att tgg ttc att 2688 cta ctc ttc atc tca att gct gtg act gga atc ctg gag ctg aga tgg 2736 leu leu phe ile ser ile ala val thr gly ile leu glu leu arg trp agc ggt gtg agc att gag gat tgg tgg agg aac gag cag ttc tgg gtc 2784 ser gly val ser ile glu asp trp trp arg asn glu gln phe trp val att ggt ggc aca tcc gcc cat ctt ttt gct gtc ttc caa ggt cta ctt 2832 aag gtt ctt gct ggt atc gac acc aac ttc acc gtt aca tct aaa gcc 2880 lys val leu ala gly ile asp thr asn phe thr val thr ser lys ala aca gac gaa gat ggg gat ttt gca gaa ctc tac atc ttc aaa tgg aca 2928 thr asp glu asp gly asp phe ala glu leu tyr ile phe lys trp thr gct ctt ctc att cca cca acc acc gtc cta ctt gtg aac ctc ata ggc 2976 att gtg gct ggt gtc tct tat gct gta aac agt ggc tac cag tcg tgg 3024 ggt ccg ctt ttc ggg aag ctc ttc ttc gcc tta tgg gtt att gcc cat 3072 ctc tac cct ttc ttg aaa ggt ctg ttg gga aga caa aac cga aca cca 3120 acc atc gtc att gtc tgg tct gtt ctt ctc gcc tcc atc ttc tcg ttg 3168 ctt tgg gtc agg atc aat ccc ttt gtg gac gcc aat ccc aat gcc aac 3216 aac ttc aat ggc aaa gga ggt gtc ttt tagaccctat ttatatactt 3263 leu val arg ile arg his glu ser asp gly gly thr lys pro leu lys val cys arg pro cys tyr glu tyr glu arg lys asp gly thr gln cys leu thr his gly his thr val ser gly glu ile arg thr pro asp thr leu gln tyr arg thr thr his pro val lys asn ala tyr pro leu trp asp gln phe pro lys trp tyr pro ile asn arg glu thr tyr leu asp arg leu ala ile arg tyr asp arg asp gly glu pro ser gln leu val arg ile asn ala leu val ala lys ala gln lys ile pro glu glu gly his pro gly met ile gln val phe leu gly his ser gly gly leu asp thr asp gly asn glu leu pro arg leu ile tyr val ser arg glu lys arg pro gly phe gln his his lys lys ala gly ala met asn ala leu asp cys asp his tyr phe asn asn ser lys ala ile lys glu ala met cys phe met met asp pro ala ile gly lys lys cys cys tyr val gln phe pro gln arg phe asp gly ile asp leu his asp arg tyr ala asn gln gly pro val tyr val gly thr gly cys cys phe asn arg gln ala tyr asp asp glu arg ser ile leu met ser gln arg ser val glu lys arg phe gly gln ser pro val phe ile ala ala thr phe met glu gln ile his val ile ser cys gly tyr glu asp lys thr glu trp gly lys phe lys met his ala arg gly trp ile ser ile tyr cys asn pro pro arg pro ala phe lys gly ser ala pro ile asn leu ser asp arg leu asn gln val leu arg trp ala leu gly ser ile glu ile leu leu ser leu leu phe ile ser ile ala val thr gly ile leu glu leu arg trp ser gly val ser ile glu asp trp trp arg asn glu gln phe trp val lys val leu ala gly ile asp thr asn phe thr val thr ser lys ala thr asp glu asp gly asp phe ala glu leu tyr ile phe lys trp thr atg aat act ggt ggt cgg ctc att gct ggc tct cac aac aga aac gaa 286 met asn thr gly gly arg leu ile ala gly ser his asn arg asn glu ttc gtt ctc att aac gcc gat gag agt gcc aga ata cga tca gta caa 334 phe val leu ile asn ala asp glu ser ala arg ile arg ser val gln gaa ctg agt ggg caa aca tgt caa atc tgt gga gat gaa atc gaa tta 382 acg gtt agc agt gag ctc ttt gtt gct tgc aac gaa tgc gca ttc ccg 430 gtt tgt aga cca tgc tat gag tat gaa cgt aga gaa gga aat caa gct 478 tgt cct cag tgc aaa act cga tac aaa agg att aaa ggt agt cca cgg 526 gtt gat gga gat gat gaa gaa gaa gaa gac att gat gat ctt gag tat 574 gag ttt gat cat ggg atg gac cct gaa cat gcc gct gaa gcc gca ctc 622 tct tca cgc ctt aac acc ggt cgt ggt gga ttg gat tca gct cca cct 670 ggc tct cag att cct ctt ttg act tat tgt gat gaa gat gct gat atg 718 gly ser gln ile pro leu leu thr tyr cys asp glu asp ala asp met tat tct gat cgt cat gct ctt atc gtg cct cct tca acg gga tat ggg 766 tyr ser asp arg his ala leu ile val pro pro ser thr gly tyr gly aat cgc gtc tat cct gca ccg ttt aca gat tct tct gca cct cca cag 814 asn arg val tyr pro ala pro phe thr asp ser ser ala pro pro gln gcg aga tca atg gtt cct cag aaa gat att gcg gaa tat ggt tat gga 862 ala arg ser met val pro gln lys asp ile ala glu tyr gly tyr gly agt gtt gct tgg aag gac cgt atg gaa gtt tgg aag aga cga caa ggc 910 ser val ala trp lys asp arg met glu val trp lys arg arg gln gly gaa aag ctt caa gtc att aag cat gaa gga gga aac aat ggt cga ggt 958 gaa gga aga caa cct ctc tca aga aag cta cct att cgt tca agc aga 1054 ata aat cct tac agg atg tta att ctg tgt cgc ctc gcg att ctt ggt 1102 ctt ttc ttt cat tat aga att ctc cat cca gtc aat gat gca tat gga 1150 leu phe phe his tyr arg ile leu his pro val asn asp ala tyr gly tta tgg tta acg tca gtt ata tgc gaa ata tgg ttt gca gtg tct tgg 1198 att ctt gat caa ttc ccc aaa tgg tat cct ata gaa cgt gaa aca tac 1246 ile leu asp gln phe pro lys trp tyr pro ile glu arg glu thr tyr ctc gat aga ctc tct ctc agg tac gag aag gaa gga aaa ccg tca gga 1294 tta gca cct gtt gat gtt ttt gtt agt aca gtg gat ccg ttg aaa gag 1342 ccc ccc ttg att aca gca aac aca gtt ctt tcc att cta gca gtt gat 1390 tat cct gtg gat aag gtt gcg tgt tat gta tca aac aat ggt gca gct 1438 atg ctt aca ttt gaa gct ctc tct gat aca gct gat ttt gct aca aaa 1486 tgg gtt cct ttt tgt aag aag ttt aat atc gag cca cga gct cct gag 1534 trp val pro phe cys lys lys phe asn ile glu pro arg ala pro glu tgg tat ttt tct cag aag atg gat tac ctg aag aac aaa gtt cat cct 1582 trp tyr phe ser gln lys met asp tyr leu lys asn lys val his pro gct ttt gtc agg gaa cgt cgt gct atg aag aga gat tat gaa gag ttt 1630 aaa gtg aag ata aat gca ctg gtt gct act gca cag aaa gtg cct gag 1678 gaa cgt tgg act atg caa gat gga act cct tgg cct gga aac aac gtc 1726 glu arg trp thr met gln asp gly thr pro trp pro gly asn asn val cgt gac cat cct gga atg att cag gtg ttc ttg ggt cat agt gga gtt 1774 arg asp his pro gly met ile gln val phe leu gly his ser gly val cgt gat acg gat ggt aat gag tta cca cgt cta gtg tat gtt tct cgt 1822 arg asp thr asp gly asn glu leu pro arg leu val tyr val ser arg gag aag cgg cct gga ttt gat cac cac aag aaa gct gga gct atg aat 1870 glu lys arg pro gly phe asp his his lys lys ala gly ala met asn tcc ttg atc cga gtc tct gct gtt cta tca aac gct cct tac ctt ctt 1918 aat gtc gat tgt gat cac tac atc aac aac agc aaa gca att aga gaa 1966 asn val asp cys asp his tyr ile asn asn ser lys ala ile arg glu tct atg tgt ttc atg atg gac ccg caa tcg gga aag aaa gtt tgt tat 2014 ser met cys phe met met asp pro gln ser gly lys lys val cys tyr gtt cag ttt ccg cag aga ttt gat ggg att gat aga cat gat aga tac 2062 tca aac cgt aac gtt gtg ttc ttt gat att aac atg aaa ggt ctt gat 2110 ser asn arg asn val val phe phe asp ile asn met lys gly leu asp ggg ata caa gga ccg ata tat gtc ggg aca ggt tgt gtg ttt aga aaa 2158 gly ile gln gly pro ile tyr val gly thr gly cys val phe arg lys cag gct ctt tat ggt ttt gat gca cca aag aag aag aaa cca cca ggc 2206 aga aag aag agt aaa acg aaa gcc aca gat aag aaa act aac act aaa 2302 gag act tca aag cag att cat gcg cta gag aat gtc gac gaa ggt gtt 2350 glu thr ser lys gln ile his ala leu glu asn val asp glu gly val atc gtc cca gtg tca aat gtt gag aag aga tct gaa gca aca caa ttg 2398 ile val pro val ser asn val glu lys arg ser glu ala thr gln leu aaa ttg gag aag aag ttt gga caa tct ccg gtt ttc gtt gcc tct gct 2446 gtt cta cag aac ggt gga gtt ccc cgt aac gca agc ccc gca tgt ttg 2494 tta aga gaa gcc att caa gtt att agc tgc ggg tac caa gat aaa acc 2542 leu arg glu ala ile gln val ile ser cys gly tyr gln asp lys thr gaa tgg gga aaa gag atc ggg tgg att tat gga tcg gtg act gaa gat 2590 atc ctg acg ggt ttc aag atg cat tgc cat gga tgg aga tct gtg tac 2638 ile leu thr gly phe lys met his cys his gly trp arg ser val tyr tgt atg cct aag cgt gca gct ttt aaa gga tct gct cct att aac ttg 2686 cys met pro lys arg ala ala phe lys gly ser ala pro ile asn leu tca gat cgt ctt cat caa gtt cta cgt tgg gct ctt ggc tct gta gag 2734 ser asp arg leu his gln val leu arg trp ala leu gly ser val glu att ttc ttg agc aga cat tgt ccg ata tgg tat ggt tat ggt ggt ggt 2782 ile phe leu ser arg his cys pro ile trp tyr gly tyr gly gly gly tta aaa tgg ttg gag aga ttc tct tac atc aac tct gtc gtc tat cct 2830 leu lys trp leu glu arg phe ser tyr ile asn ser val val tyr pro tgg act tca ctt cca ttg atc gtc tat tgt tct ctc ccc gcg gtt tgt 2878 tta ctc aca gga aaa ttc atc gtc cct gag ata agc aac tac gca ggt 2926 leu leu thr gly lys phe ile val pro glu ile ser asn tyr ala gly ata ctc ttc atg ctc atg ttc ata tcc ata gca gta act gga atc ctc 2974 gaa atg caa tgg gga ggt gtc gga atc gat gat tgg tgg aga aac gag 3022 cag ttt tgg gta atc gga ggg gcc tcc tcg cat cta ttt gct ctg ttt 3070 caa ggt ttg ctc aaa gtt cta gcc gga gtt aac acg aat ttc aca gtc 3118 act tca aaa gca gca gac gat gga gct ttc tct gag ctt tac atc ttc 3166 thr ser lys ala ala asp asp gly ala phe ser glu leu tyr ile phe aag tgg aca act ttg ttg att cct ccg aca aca ctt ctg atc att aac 3214 atc att gga gtt att gtc ggc gtt tct gat gcc att agc aat ggc tat 3262 gac tca tgg gga cct ctc ttt ggg aga ctt ttc ttc gct ctt tgg gtc 3310 att gtt cat tta tac cca ttc ctc aag gga atg ctt ggg aag caa gac 3358 ile val his leu tyr pro phe leu lys gly met leu gly lys gln asp aaa atg cct acg att att gtg gtc tgg tct att ctt cta gct tcg atc 3406 ttg aca ctc ttg tgg gtc aga att aac ccg ttt gtg gct aaa ggg gga 3454 leu thr leu leu trp val arg ile asn pro phe val ala lys gly gly cca gtg ttg gag atc tgt ggt ctg aat tgt gga aac taagatcctc 3500 met asn thr gly gly arg leu ile ala gly ser his asn arg asn glu phe val leu ile asn ala asp glu ser ala arg ile arg ser val gln gly ser gln ile pro leu leu thr tyr cys asp glu asp ala asp met tyr ser asp arg his ala leu ile val pro pro ser thr gly tyr gly asn arg val tyr pro ala pro phe thr asp ser ser ala pro pro gln ala arg ser met val pro gln lys asp ile ala glu tyr gly tyr gly ser val ala trp lys asp arg met glu val trp lys arg arg gln gly leu phe phe his tyr arg ile leu his pro val asn asp ala tyr gly ile leu asp gln phe pro lys trp tyr pro ile glu arg glu thr tyr trp val pro phe cys lys lys phe asn ile glu pro arg ala pro glu trp tyr phe ser gln lys met asp tyr leu lys asn lys val his pro glu arg trp thr met gln asp gly thr pro trp pro gly asn asn val arg asp his pro gly met ile gln val phe leu gly his ser gly val arg asp thr asp gly asn glu leu pro arg leu val tyr val ser arg glu lys arg pro gly phe asp his his lys lys ala gly ala met asn asn val asp cys asp his tyr ile asn asn ser lys ala ile arg glu ser met cys phe met met asp pro gln ser gly lys lys val cys tyr ser asn arg asn val val phe phe asp ile asn met lys gly leu asp gly ile gln gly pro ile tyr val gly thr gly cys val phe arg lys glu thr ser lys gln ile his ala leu glu asn val asp glu gly val ile val pro val ser asn val glu lys arg ser glu ala thr gln leu leu arg glu ala ile gln val ile ser cys gly tyr gln asp lys thr ile leu thr gly phe lys met his cys his gly trp arg ser val tyr cys met pro lys arg ala ala phe lys gly ser ala pro ile asn leu ser asp arg leu his gln val leu arg trp ala leu gly ser val glu ile phe leu ser arg his cys pro ile trp tyr gly tyr gly gly gly leu lys trp leu glu arg phe ser tyr ile asn ser val val tyr pro leu leu thr gly lys phe ile val pro glu ile ser asn tyr ala gly thr ser lys ala ala asp asp gly ala phe ser glu leu tyr ile phe ile val his leu tyr pro phe leu lys gly met leu gly lys gln asp leu thr leu leu trp val arg ile asn pro phe val ala lys gly gly acc gcg gga aag ccg atg aag aac att gtt ccg cag act tgc cag atc 282 thr ala gly lys pro met lys asn ile val pro gln thr cys gln ile tgt agt gac aat gtt ggc aag act gtt gat gga gat cgt ttt gtg gct 330 cys ser asp asn val gly lys thr val asp gly asp arg phe val ala tgt gat att tgt tca ttc cca gtt tgt cgg cct tgc tac gag tat gag 378 agg aaa gat ggg aat caa tct tgt cct cag tgc aaa acc aga tac aag 426 arg lys asp gly asn gln ser cys pro gln cys lys thr arg tyr lys agg ctc aaa ggt agt cct gct att cct ggt gat aaa gac gag gat ggc 474 tta gct gat gaa ggt act gtt gag ttc aac tac cct cag aag gag aaa 522 leu ala asp glu gly thr val glu phe asn tyr pro gln lys glu lys att tca gag cgg atg ctt ggt tgg cat ctt act cgt ggg aag gga gag 570 ile ser glu arg met leu gly trp his leu thr arg gly lys gly glu gaa atg ggg gaa ccc cag tat gat aaa gag gtc tct cac aat cat ctt 618 glu met gly glu pro gln tyr asp lys glu val ser his asn his leu cct cgt ctc acg agc aga caa gat act tca gga gag ttt tct gct gcc 666 pro arg leu thr ser arg gln asp thr ser gly glu phe ser ala ala tca cct gaa cgc ctc tct gta tct tct act atc gct ggg gga aag cgc 714 ser pro glu arg leu ser val ser ser thr ile ala gly gly lys arg ctt ccc tat tca tca gat gtc aat caa tca cca aat aga agg att gtg 762 gat cct gtt gga ctc ggg aat gta gct tgg aag gag aga gtt gat ggc 810 asp pro val gly leu gly asn val ala trp lys glu arg val asp gly tgg aaa atg aag caa gag aag aat act ggt cct gtc agc acg cag gct 858 trp lys met lys gln glu lys asn thr gly pro val ser thr gln ala gct tct gaa aga ggt gga gta gat att gat gcc agc aca gat atc cta 906 gca gat gag gct ctg ctg aat gac gaa gcg agg cag ctt ctg tca agg 954 aaa gtt tca att cct tca tca cgg atc aat cct tac aga atg gtt att 1002 atg ctg cgg ctt gtt atc ctt tgt ctc ttc ttg cat tac cgt ata aca 1050 aac cca gtg cca aat gcc ttt gct cta tgg ctg gtc tct gtg ata tgt 1098 gag atc tgg ttt gcc tta tcc tgg att ttg gat cag ttt ccc aag tgg 1146 glu ile trp phe ala leu ser trp ile leu asp gln phe pro lys trp ttt cct gtg aac cgt gaa acc tac ctc gac agg ctt gct tta aga tat 1194 phe pro val asn arg glu thr tyr leu asp arg leu ala leu arg tyr gat cgt gaa ggt gag cca tca cag tta gct gct gtt gac att ttc gtg 1242 asp arg glu gly glu pro ser gln leu ala ala val asp ile phe val agt act gtt gac ccc ttg aag gag cca ccc ctt gtg aca gcc aac aca 1290 gtg ctc tct att ctg gct gtt gac tac cca gtt gac aag gtg tcc tgt 1338 tat gtt tct gat gat ggt gct gct atg tta tca ttt gaa tca ctt gca 1386 gaa aca tca gag ttt gct cgt aaa tgg gta cca ttt tgc aag aaa tat 1434 glu thr ser glu phe ala arg lys trp val pro phe cys lys lys tyr agc ata gag cct cgt gca cca gaa tgg tac ttt gct gcg aaa ata gat 1482 ser ile glu pro arg ala pro glu trp tyr phe ala ala lys ile asp tac ttg aag gat aaa gtt cag aca tca ttt gtc aaa gat cgt aga gct 1530 tyr leu lys asp lys val gln thr ser phe val lys asp arg arg ala atg aag agg gaa tat gag gaa ttt aaa atc cga atc aat gca ctt gtt 1578 met lys arg glu tyr glu glu phe lys ile arg ile asn ala leu val tcc aaa gcc cta aaa tgt cct gaa gaa ggg tgg gtt atg caa gat ggc 1626 ser lys ala leu lys cys pro glu glu gly trp val met gln asp gly aca ccg tgg cct gga aat aat aca ggg gac cat cca gga atg atc cag 1674 gtc ttc tta ggg caa aat ggt gga ctt gat gca gag ggc aat gag ctc 1722 ccg cgt ttg gta tat gtt tct cga gaa aag cga cca gga ttc cag cac 1770 pro arg leu val tyr val ser arg glu lys arg pro gly phe gln his cac aaa aag gct ggt gct atg aat gca ctg gtg aga gtt tca gca gtt 1818 ctt acc aat gga cct ttc atc ttg aat ctt gat tgt gat cat tac ata 1866 leu thr asn gly pro phe ile leu asn leu asp cys asp his tyr ile aat aac agc aaa gcc tta aga gaa gca atg tgc ttc ctg atg gac cca 1914 asn asn ser lys ala leu arg glu ala met cys phe leu met asp pro aac ctc ggg aag caa gtt tgt tat gtt cag ttc cca caa aga ttt gat 1962 asn leu gly lys gln val cys tyr val gln phe pro gln arg phe asp ggt atc gat aag aac gat aga tat gct aat cgt aat acc gtg ttc ttt 2010 gly ile asp lys asn asp arg tyr ala asn arg asn thr val phe phe gat att aac ttg aga ggt tta gat ggg att caa gga cct gta tat gtc 2058 gga act gga tgt gtt ttc aac aga aca gca tta tac ggt tat gaa cct 2106 gly thr gly cys val phe asn arg thr ala leu tyr gly tyr glu pro cca ata aaa gta aaa cac aag aag cca agt ctt tta tct aag ctc tgt 2154 ggt gga tca aga aag aag aat tcc aaa gct aag aaa gag tcg gac aaa 2202 aag aaa tca ggc agg cat act gac tca act gtt cct gta ttc aac ctc 2250 lys lys ser gly arg his thr asp ser thr val pro val phe asn leu gat gac ata gaa gag gga gtt gaa ggt gct ggt ttt gat gat gaa aag 2298 gcg ctc tta atg tcg caa atg agc ctg gag aag cga ttt gga cag tct 2346 gct gtt ttt gtt gct tct acc cta atg gaa aat ggt ggt gtt cct cct 2394 ala val phe val ala ser thr leu met glu asn gly gly val pro pro tca gca act cca gaa aac ttt ctc aaa gag gct atc cat gtc att agt 2442 ser ala thr pro glu asn phe leu lys glu ala ile his val ile ser tgt ggt tat gag gat aag tca gat tgg gga atg gag att gga tgg atc 2490 tat ggt tct gtg aca gaa gat att ctg act ggg ttc aaa atg cat gcc 2538 tyr gly ser val thr glu asp ile leu thr gly phe lys met his ala cgt gga tgg cga tcc att tac tgc atg cct aag ctt cca gct ttc aag 2586 arg gly trp arg ser ile tyr cys met pro lys leu pro ala phe lys ggt tct gct cct atc aat ctt tca gat cgt ctg aac caa gtg ctg agg 2634 gly ser ala pro ile asn leu ser asp arg leu asn gln val leu arg tgg gct tta ggt tca gtt gag att ctc ttc agt cgg cat tgt cct ata 2682 trp ala leu gly ser val glu ile leu phe ser arg his cys pro ile tgg tat ggt tac aat ggg agg cta aaa ttt ctt gag agg ttt gcg tat 2730 gtg aac acc acc atc tac cct atc acc tcc att cct ctt ctc atg tat 2778 tgt aca ttg cta gcc gtt tgt ctc ttc acc aac cag ttt att att cct 2826 cag att agt aac att gca agt ata tgg ttt ctg tct ctc ttt ctc tcc 2874 att ttc gcc acg ggt ata cta gaa atg agg tgg agt ggc gta ggc ata 2922 ile phe ala thr gly ile leu glu met arg trp ser gly val gly ile gac gaa tgg tgg aga aac gag cag ttt tgg gtc att ggt gga gta tcc 2970 asp glu trp trp arg asn glu gln phe trp val ile gly gly val ser gct cat tta ttc gct gtg ttt caa ggt atc ctc aaa gtc ctt gcc ggt 3018 att gac aca aac ttc aca gtt acc tca aaa gct tca gat gaa gac gga 3066 ile asp thr asn phe thr val thr ser lys ala ser asp glu asp gly gac ttt gct gag ctc tac ttg ttc aaa tgg aca aca ctt ctg att ccg 3114 asp phe ala glu leu tyr leu phe lys trp thr thr leu leu ile pro cca acg acg ctg ctc att gta aac tta gtg gga gtt gtt gca gga gtc 3162 tct tat gct atc aac agt gga tac caa tca tgg gga cca ctc ttt ggt 3210 ser tyr ala ile asn ser gly tyr gln ser trp gly pro leu phe gly aag ttg ttc ttt gcc ttc tgg gtg att gtt cac ttg tac cct ttc ctc 3258 aag ggt ttg atg ggt cga cag aac cgg act cct acc att gtt gtg gtc 3306 lys gly leu met gly arg gln asn arg thr pro thr ile val val val tgg tct gtt ctc ttg gct tct atc ttc tcg ttg ttg tgg gtt agg att 3354 gat ccc ttc act agc cga gtc act ggc ccg gac att ctg gaa tgt gga 3402 asp pro phe thr ser arg val thr gly pro asp ile leu glu cys gly met glu ser glu gly glu thr ala gly lys pro met lys asn ile val pro gln thr cys gln ile cys ser asp asn val gly lys thr val asp pro cys tyr glu tyr glu arg lys asp gly asn gln ser cys pro gln cys lys thr arg tyr lys arg leu lys gly ser pro ala ile pro gly tyr pro gln lys glu lys ile ser glu arg met leu gly trp his leu ile ala gly gly lys arg leu pro tyr ser ser asp val asn gln ser lys glu arg val asp gly trp lys met lys gln glu lys asn thr gly pro val ser thr gln ala ala ser glu arg gly gly val asp ile asp leu his tyr arg ile thr asn pro val pro asn ala phe ala leu trp asp gln phe pro lys trp phe pro val asn arg glu thr tyr leu asp ala val asp ile phe val ser thr val asp pro leu lys glu pro pro pro phe cys lys lys tyr ser ile glu pro arg ala pro glu trp tyr phe ala ala lys ile asp tyr leu lys asp lys val gln thr ser phe arg ile asn ala leu val ser lys ala leu lys cys pro glu glu gly his pro gly met ile gln val phe leu gly gln asn gly gly leu asp ala glu gly asn glu leu pro arg leu val tyr val ser arg glu lys arg pro gly phe gln his his lys lys ala gly ala met asn ala leu val arg val ser ala val leu thr asn gly pro phe ile leu asn leu asp cys asp his tyr ile asn asn ser lys ala leu arg glu ala met cys phe leu met asp pro asn leu gly lys gln val cys tyr val gln phe pro gln arg phe asp gly ile asp lys asn asp arg tyr ala asn gln gly pro val tyr val gly thr gly cys val phe asn arg thr ala lys arg phe gly gln ser ala val phe val ala ser thr leu met glu asn gly gly val pro pro ser ala thr pro glu asn phe leu lys glu ala ile his val ile ser cys gly tyr glu asp lys ser asp trp gly met glu ile gly trp ile tyr gly ser val thr glu asp ile leu thr gly phe lys met his ala arg gly trp arg ser ile tyr cys met pro lys leu pro ala phe lys gly ser ala pro ile asn leu ser asp arg leu asn gln val leu arg trp ala leu gly ser val glu ile leu phe ser arg his cys pro ile trp tyr gly tyr asn gly arg leu lys phe leu glu arg phe ala tyr val asn thr thr ile tyr pro ile thr ser trp ser gly val gly ile asp glu trp trp arg asn glu gln phe trp leu lys val leu ala gly ile asp thr asn phe thr val thr ser lys his leu tyr pro phe leu lys gly leu met gly arg gln asn arg thr leu leu trp val arg ile asp pro phe thr ser arg val thr gly pro ggtggctgcg atg gag gcc agt gcc ggc ttg gtt gct gga tcc tac cgg 109 aga aac gag ctc gtt cgg atc cga cat gaa tct gat ggc ggg acc aaa 157 arg asn glu leu val arg ile arg his glu ser asp gly gly thr lys cct ttg aag aat atg aat ggc cag ata tgt cag atc tgt ggt gat gat 205 gtt gga ctc gct gaa act gga gat gtc ttt gtc gcg tgt aat gaa tgt 253 gcc ttc cct gtg tgt cgg cct tgc tat gag tac gag agg aaa gat gga 301 ala phe pro val cys arg pro cys tyr glu tyr glu arg lys asp gly act cag tgt tgc cct caa tgc aag act aga ttc aga cga cac agg ggg 349 agt cct cgt gtt gaa gga gat gaa gat gag gat gat gtt gat gat atc 397 gag aat gag ttc aat tac gcc cag gga gct aac aag gcg aga cac caa 445 cgc cat ggc gaa gag ttt tct tct tcc tct aga cat gaa tct caa cca 493 att cct ctt ctc acc cat ggc cat acg gtt tct gga gag att cgc acg 541 cct gat aca caa tct gtg cga act aca tca ggt cct ttg ggt cct tct 589 gac agg aat gct att tca tct cca tat att gat cca cgg caa cct gtc 637 cct gta aga atc gtg gac ccg tca aaa gac ttg aac tct tat ggg ctt 685 pro val arg ile val asp pro ser lys asp leu asn ser tyr gly leu ggt aat gtt gac tgg aaa gaa aga gtt gaa ggc tgg aag ctg aag cag 733 gag aaa aat atg tta cag atg act ggt aaa tac cat gaa ggg aaa gga 781 gga gaa att gaa ggg act ggt tcc aat ggc gaa gaa ctc caa atg gct 829 gat gat aca cgt ctt cct atg agt cgt gtg gtg cct atc cca tct tct 877 cgc cta acc cct tat cgg gtt gtg att att ctc cgg ctt atc atc ttg 925 tgt ttc ttc ttg caa tat cgt aca act cac cct gtg aaa aat gca tat 973 cys phe phe leu gln tyr arg thr thr his pro val lys asn ala tyr cct ttg tgg ttg acc tcg gtt atc tgt gag atc tgg ttt gca ttt tct 1021 pro leu trp leu thr ser val ile cys glu ile trp phe ala phe ser tgg ctt ctt gat cag ttt ccc aaa tgg tac ccc att aac agg gag act 1069 trp leu leu asp gln phe pro lys trp tyr pro ile asn arg glu thr tat ctt gac cgt ctc gct ata aga tat gat cga gac ggt gaa cca tca 1117 cag ctc gtt cct gtt gat gtg ttt gtt agt aca gtg gac cca ttg aaa 1165 gag cct ccc ctt gtt aca gca aac aca gtt ctc tcg att ctt tct gtg 1213 gac tac ccg gta gat aaa gta gcc tgt tat gtt tca gat gat ggt tca 1261 gct atg ctt acc ttt gaa tcc ctt tct gaa acc gct gag ttt gca aag 1309 aaa tgg gta cca ttt tgc aag aaa ttc aac att gaa cct agg gcc cct 1357 lys trp val pro phe cys lys lys phe asn ile glu pro arg ala pro gaa ttc tat ttt gcc cag aag ata gat tac ttg aag gac aag atc caa 1405 ccg tct ttt gtt aaa gag cga cga gct atg aag aga gag tat gaa gag 1453 ttt aaa gtg agg ata aat gct ctt gtt gcc aaa gca cag aaa atc cct 1501 gaa gaa ggc tgg aca atg cag gat ggt act ccc tgg cct ggt aac aac 1549 act aga gat cat cct gga atg ata cag gtg ttc tta ggc cat agt ggg 1597 thr arg asp his pro gly met ile gln val phe leu gly his ser gly ggt ctg gat acc gat gga aat gag ctg cct aga ctc atc tat gtt tct 1645 gly leu asp thr asp gly asn glu leu pro arg leu ile tyr val ser cgt gaa aag cgg cct gga ttt caa cac cac aaa aag gct gga gct atg 1693 aat gca ttg atc cgt gta tct gtt gtt ctt acc aat gga gca tat ctt 1741 ttg aac gtg gat tgt gat cat tac ttt aat aac agt aag gct att aaa 1789 leu asn val asp cys asp his tyr phe asn asn ser lys ala ile lys gaa gct atg tgt ttc atg atg gac ccg gct att gga aag aag tgc tgc 1837 tat gtc cag ttc cct caa cgt ttt gac ggt att gat ttg cac gat cga 1885 tyr val gln phe pro gln arg phe asp gly ile asp leu his asp arg tat gcc aac agg aat ata gtc ttt ttc gat att aac atg aag ggg ttg 1933 tyr ala asn arg asn ile val phe phe asp ile asn met lys gly leu gat ggt atc cag ggt cca gta tat gtg ggt act ggt tgt tgt ttt aat 1981 asp gly ile gln gly pro val tyr val gly thr gly cys cys phe asn agg cag gct cta tat ggg tat gat cct gtt ttg acg gaa gaa gat tta 2029 arg gln ala leu tyr gly tyr asp pro val leu thr glu glu asp leu gaa cca aat att att gtc aag agc tgt tgc ggg tca agg aag aaa ggt 2077 aaa agt agc aag aag tat aac tac gaa aag agg aga ggc atc aac aga 2125 agt gac tcc aat gct cca ctt ttc aat atg gag gac atc gat gag ggt 2173 ser asp ser asn ala pro leu phe asn met glu asp ile asp glu gly ttt gaa ggt tat gat gat gag agg tct att cta atg tcc cag agg agt 2221 phe glu gly tyr asp asp glu arg ser ile leu met ser gln arg ser gta gag aag cgt ttt ggt cag tcg ccg gta ttt att gcg gca acc ttc 2269 val glu lys arg phe gly gln ser pro val phe ile ala ala thr phe atg gaa caa ggc ggc att cca cca aca acc aat ccc gct act ctt ctg 2317 aag gag gct att cat gtt ata agc tgt ggt tac gaa gac aag act gaa 2365 lys glu ala ile his val ile ser cys gly tyr glu asp lys thr glu tgg ggc aaa gag att ggt tgg atc tat ggt tcc gtg acg gaa gat att 2413 ctt act ggg ttc aag atg cat gcc cgg ggt tgg ata tcg atc tac tgc 2461 leu thr gly phe lys met his ala arg gly trp ile ser ile tyr cys aat cct cca cgc cct gcg ttc aag gga tct gca cca atc aat ctt tct 2509 gat cgt ttg aac caa gtt ctt cga tgg gct ttg gga tct atc gag att 2557 asp arg leu asn gln val leu arg trp ala leu gly ser ile glu ile ctt ctt agc aga cat tgt cct atc tgg tat ggt tac cat gga agg ttg 2605 aga ctt ttg gag agg atc gct tat atc aac acc atc gtc tat cct att 2653 aca tcc atc cct ctt att gcg tat tgt att ctt ccc gct ttt tgt ctc 2701 atc acc gac aga ttc atc ata ccc gag ata agc aac tac gcg agt att 2749 ile thr asp arg phe ile ile pro glu ile ser asn tyr ala ser ile tgg ttc att cta ctc ttc atc tca att gct gtg act gga atc ctg gag 2797 ctg aga tgg agc ggt gtg agc att gag gat tgg tgg agg aac gag cag 2845 leu arg trp ser gly val ser ile glu asp trp trp arg asn glu gln ttc tgg gtc att ggt ggc aca tcc gcc cat ctt ttt gct gtc ttc caa 2893 phe trp val ile gly gly thr ser ala his leu phe ala val phe gln ggt cta ctt aag gtt ctt gct ggt atc gac acc aac ttc acc gtt aca 2941 tct aaa gcc aca gac gaa gat ggg gat ttt gca gaa ctc tac atc ttc 2989 ser lys ala thr asp glu asp gly asp phe ala glu leu tyr ile phe aaa tgg aca gct ctt ctc att cca cca acc acc gtc cta ctt gtg aac 3037 ctc ata ggc att gtg gct ggt gtc tct tat gct gta aac agt ggc tac 3085 cag tcg tgg ggt ccg ctt ttc ggg aag ctc ttc ttc gcc tta tgg gtt 3133 att gcc cat ctc tac cct ttc ttg aaa ggt ctg ttg gga aga caa aac 3181 ile ala his leu tyr pro phe leu lys gly leu leu gly arg gln asn cga aca cca acc atc gtc att gtc tgg tct gtt ctt ctc gcc tcc atc 3229 ttc tcg ttg ctt tgg gtc agg atc aat ccc ttt gtg gac gcc aat ccc 3277 phe ser leu leu trp val arg ile asn pro phe val asp ala asn pro aat gcc aac aac ttc aat ggc aaa gga ggt gtc ttt tagaccctat 3323 leu val arg ile arg his glu ser asp gly gly thr lys pro leu lys val cys arg pro cys tyr glu tyr glu arg lys asp gly thr gln cys leu thr his gly his thr val ser gly glu ile arg thr pro asp thr leu gln tyr arg thr thr his pro val lys asn ala tyr pro leu trp asp gln phe pro lys trp tyr pro ile asn arg glu thr tyr leu asp arg leu ala ile arg tyr asp arg asp gly glu pro ser gln leu val arg ile asn ala leu val ala lys ala gln lys ile pro glu glu gly his pro gly met ile gln val phe leu gly his ser gly gly leu asp thr asp gly asn glu leu pro arg leu ile tyr val ser arg glu lys arg pro gly phe gln his his lys lys ala gly ala met asn ala leu asp cys asp his tyr phe asn asn ser lys ala ile lys glu ala met cys phe met met asp pro ala ile gly lys lys cys cys tyr val gln phe pro gln arg phe asp gly ile asp leu his asp arg tyr ala asn gln gly pro val tyr val gly thr gly cys cys phe asn arg gln ala tyr asp asp glu arg ser ile leu met ser gln arg ser val glu lys arg phe gly gln ser pro val phe ile ala ala thr phe met glu gln ile his val ile ser cys gly tyr glu asp lys thr glu trp gly lys phe lys met his ala arg gly trp ile ser ile tyr cys asn pro pro arg pro ala phe lys gly ser ala pro ile asn leu ser asp arg leu asn gln val leu arg trp ala leu gly ser ile glu ile leu leu ser leu leu phe ile ser ile ala val thr gly ile leu glu leu arg trp ser gly val ser ile glu asp trp trp arg asn glu gln phe trp val lys val leu ala gly ile asp thr asn phe thr val thr ser lys ala thr asp glu asp gly asp phe ala glu leu tyr ile phe lys trp thr ggg atg gtg gcg gga tcc cgc aac cgg aac gag ttc gtc atg atc cgc 163 ccc gac ggc gac gcg cca ccg ccg gct aag cca ggg aag agt gtg aat 211 ggt cag gtc tgc cag att tgt ggc gac act gtt ggc gtc tcg gcc acc 259 ggc gac gtc ttt gtt gcc tgc aat gag tgc gcc ttc ccg gtc tgc cgc 307 cct tgc tac gag tac gaa cgc aag gaa ggg aac cag tgc tgc ccc cag 355 tgc aag act aga tac aag agg cac aaa ggt tgc cct aga gtt cag ggc 403 gat gag gaa gaa gaa gat gtt gat gac ctg gac aat gaa ttc cat tat 451 aag cat ggc aat ggc aaa ggt cca gag tgg cag ata cag aga cag ggg 499 gaa gat gtt gac ctg tct tca tct tct cgc cac gaa caa cat cgg att 547 ccc cgt ctg aca agt ggg caa cag atc tca gga gag atc cct gat gct 595 pro arg leu thr ser gly gln gln ile ser gly glu ile pro asp ala tcc ccc gat cgc cat tct atc cgc agc gga aca tca agc tat gtt gat 643 cca agt gtt cca gtt cct gtg agg att gtg gac ccc tcc aag gac ttg 691 aat tcc tat ggg att aac agt gtt gac tgg caa gaa aga gtt gcc agc 739 asn ser tyr gly ile asn ser val asp trp gln glu arg val ala ser tgg agg aac aag cag gac aaa aat atg atg cag gta gct aat aaa tat 787 cca gag gca aga ggg gga gac atg gaa ggg act ggt tca aat ggt gaa 835 gat atc caa atg gtt gat gat gca cgt cta cct ctg agc cgc ata gtg 883 cct atc cct tca aac cag ctc aac ctt tac cgg att gtt atc att ctc 931 cgt ctt atc atc ctg atg ttc ttc ttc caa tat cgt gtc act cat cca 979 arg leu ile ile leu met phe phe phe gln tyr arg val thr his pro gtg cgg gat gct tat gga ttg tgg cta gta tct gtt atc tgt gaa att 1027 val arg asp ala tyr gly leu trp leu val ser val ile cys glu ile tgg ttg ccc tta tcc tgg ctc cta gat caa ttc cca aag tgg tac ccg 1075 ata aac cgt gaa aca tac ctt gac agg ctt gca ttg aga tat gat agg 1123 gag gga gag cca tca cag ctt gct ccc att gat gtc ttt gtc agt acg 1171 glu gly glu pro ser gln leu ala pro ile asp val phe val ser thr gtg gat cca cta aag gaa cct cct ctg atc aca gca aac act gtt ttg 1219 tcc att ctg gct gtg gat tac cct gtt gac aaa gtg tca tgc tat gtt 1267 tct gac gat ggt tca gct atg tta act ttt gag gct ctg tca gaa act 1315 gca gaa ttt gct agg aag tgg gtt ccg ttt tgc aag aag cac aat att 1363 ala glu phe ala arg lys trp val pro phe cys lys lys his asn ile gaa cca cga gct cca gag ttt tac ttt gct caa aaa ata gat tac ctg 1411 glu pro arg ala pro glu phe tyr phe ala gln lys ile asp tyr leu aag gac aaa atc caa cct tcc ttt gtt aaa gaa agg cgg gca atg aag 1459 lys asp lys ile gln pro ser phe val lys glu arg arg ala met lys aga gag tat gaa gaa ttc aag gta cgg atc aat gct ctt gtt gcg aag 1507 gca caa aaa gta cct gaa gag ggg tgg acc atg gct gat ggc act gct 1555 ala gln lys val pro glu glu gly trp thr met ala asp gly thr ala tgg cct ggg aat aac cca agg gat cac cct ggc atg att cag gtg ttc 1603 trp pro gly asn asn pro arg asp his pro gly met ile gln val phe ttg ggg cac agt ggt ggg ctt gac act gat ggt aac gag ttg cca cgg 1651 ctt gtc tac gtc tct cgt gaa aag agg cca gga ttc cag cat cac aag 1699 leu val tyr val ser arg glu lys arg pro gly phe gln his his lys aag gct ggt gca atg aat gca ttg att cgt gta tct gct gtg 1741 pro ser lys asp leu asn ser tyr gly ile asn ser val asp trp gln glu arg val ala ser trp arg asn lys gln asp lys asn met met gln val ala asn lys tyr pro glu ala arg gly gly asp met glu gly thr gly ser asn gly glu asp ile gln met val asp asp ala arg leu pro arg val thr his pro val arg asp ala tyr gly leu trp leu val ser val ile cys glu ile trp leu pro leu ser trp leu leu asp gln phe pro lys trp tyr pro ile asn arg glu thr tyr leu asp arg leu ala leu arg tyr asp arg glu gly glu pro ser gln leu ala pro ile asp ala asn thr val leu ser ile leu ala val asp tyr pro val asp lys val ser cys tyr val ser asp asp gly ser ala met leu thr phe glu ala leu ser glu thr ala glu phe ala arg lys trp val pro phe cys lys lys his asn ile glu pro arg ala pro glu phe tyr phe ala gln lys ile asp tyr leu lys asp lys ile gln pro ser phe val lys glu ala leu val ala lys ala gln lys val pro glu glu gly trp thr met met ile gln val phe leu gly his ser gly gly leu asp thr asp gly phe gln his his lys lys ala gly ala met asn ala leu ile arg val